Novel monomer MJP6 in black garlic, preparation method of novel monomer MJP6 and application of novel monomer MJP6 in preparation of medicine for treating cerebral hemorrhage

Abstract

The invention discloses a novel monomer MJP6 in black garlic, a preparation method of the novel monomer MJP6 and application of the novel monomer MJP6 in preparation of a medicine for treating cerebral hemorrhage. The application of the compound MJP6 in preparing the medicine for treating cerebral hemorrhage has at least one of the following characteristics: the MJP6 has an intracerebral nerve protection effect; the MJP6 has a repairing effect on brain ultrastructure damage; the MJP6 has the effects of inhibiting intracerebral inflammation and infiltration of microglial cells and neutrophil; mJP6 promotes intracerebral hematoma absorption and blood flow recovery; the MJP6 has the effect of relieving dyskinesia.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical active ingredient technology, specifically relating to a novel monomer MJP6 in black garlic, its preparation method, and its application in the preparation of drugs for treating cerebral hemorrhage. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Cerebral hemorrhage poses a serious threat to human health due to its high mortality and disability rates. Complications of cerebral hemorrhage occur in up to 50% of cases, representing a key factor influencing prognosis. Its mechanisms are complex: hematoma formation and nerve damage trigger an immune response, activating oxidative stress and inflammatory pathways, leading to impaired neuronal function and death. Imbalanced neuronal energy metabolism further exacerbates cell damage and even necrosis, driving the pathological process to worsen. The pathological processes include microglial activation and neutrophil infiltration, which are key pathogenic factors leading to neurological dysfunction caused by cerebral hemorrhage.

[0004] Currently used medications for treating cerebral hemorrhage (such as mannitol to lower intracranial pressure, edaravone as a neuroprotective agent, and vitamin K1 as a hemostatic agent) often have side effects such as electrolyte disturbances, kidney damage, abnormal liver function, and allergic reactions. The core challenge of existing treatment methods lies in the fact that both surgical and drug treatments cannot avoid the high risk of complications; hematoma removal is limited by the trauma, location, and timing, resulting in incomplete removal; and they cannot effectively prevent secondary damage or promote nerve regeneration. Therefore, developing low-risk targeted drugs for neurological injury and repair in cerebral hemorrhage and identifying novel compounds with neuroprotective effects are of great significance for improving the prognosis of patients with cerebral hemorrhage.

[0005] Garlic is an ancient plant used for both medicinal and culinary purposes, offering numerous health benefits. However, its pungent odor and spicy taste can cause gastrointestinal discomfort, limiting its use for many. Black garlic overcomes this limitation. It is a novel specialty product made by fermenting fresh garlic for 60-90 days at a high temperature of 60-80℃ and a relative humidity of 70%-90%. It not only has a sweet and soft texture but also lacks the pungent and spicy odor of fresh garlic. The fermentation process retains the original components of fresh garlic while significantly enhancing its nutritional value and health benefits. Black garlic is rich in various bioactive components, such as polyphenols, polysaccharides, and organosulfur compounds, exhibiting antioxidant, anti-inflammatory, and lipid-lowering physiological activities. Garlic and black garlic extracts have shown neuroprotective effects by inhibiting oxidative stress and inflammatory responses, reducing cerebral edema and blood-brain barrier damage, improving nerve function, and alleviating brain damage. Its neuroprotective effects have been widely validated in stroke, Alzheimer's disease, and other neurological diseases. However, there is still relatively little research on the specific active ingredients in black garlic and their neuroprotective effects in cerebral hemorrhage. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention isolates and identifies a novel small molecule monomer, 2-E-feruloyloxyphenylacetate (MJP6), from black garlic and provides its application in the treatment of cerebral hemorrhage, thus providing a basis for the development of drugs for the treatment of cerebral hemorrhage.

[0007] As a first aspect of the present invention, a novel monomer MJP6 from black garlic is provided. This compound, named according to its known structure, has the chemical name 2-E-feruloyloxybenzeneacetic acid ester and the molecular formula C2. 18 H 16 O6, with a molecular weight of 328. No results were found in existing publicly available literature. Its chemical structure is shown below: .

[0008] As a second aspect of the present invention, a method for preparing the compound MJP6 is provided, including both extraction from black garlic and chemical synthesis.

[0009] One preparation method includes the following steps: S101, Extraction: Grind black garlic, add solvent, and extract ultrasonically at room temperature. Centrifuge the extract to remove suspended matter, collect the supernatant, and concentrate by rotary evaporation to obtain crude black garlic extract. S102, Extraction: Disperse the crude black garlic extract prepared in step S101 with water, add an ester solvent for extraction, and distill the extract under reduced pressure to obtain the extract. S103, Separation and Purification: The extract obtained in step S102 was subjected to silica gel column chromatography under reduced pressure, eluted with a dichloromethane:methanol gradient, to obtain 6 fractions FA-FF; fraction FC was subjected to silica gel column chromatography, eluted isocratically with petroleum ether:ethyl acetate, to obtain 8 fractions FC1-FC8; fraction FC7 was subjected to silica gel column chromatography, eluted isocratically with petroleum ether:ethyl acetate, to obtain 9 fractions FC7-1 to FC7-9; FC7-6 was prepared by TLC, to obtain 12 fractions FC7-6-1 to FC7-6-12; FC7-6-6 was separated and purified by semi-preparative high performance liquid chromatography to obtain MJP6.

[0010] Another preparation method includes the following steps: Add 4-hydroxy-3-methoxycinnamic acid, ω-bromo-4-hydroxyacetophenone, and solvent to a reaction vessel, then add N,N-diisopropylethylamine (DIPEA) and stir at room temperature; then extract, wash the organic phase with saturated brine, dry it, concentrate it to dryness, and purify it to obtain the final product.

[0011] The specific reaction formula is as follows: .

[0012] The molar ratio of 4-hydroxy-3-methoxycinnamic acid to ω-bromo-4-hydroxyacetophenone is 1:1.1~1.2.

[0013] The molar ratio of 4-hydroxy-3-methoxycinnamic acid to N,N-diisopropylethylamine is 1:5~6.

[0014] As a third aspect of the invention, it is to provide the use of compound MJP6 in the preparation of a drug for treating cerebral hemorrhage.

[0015] In the embodiments of the present invention, experiments have demonstrated that compound MJP6, in its application in the preparation of drugs for treating cerebral hemorrhage, has at least one of the following characteristics: (1) MJP6 has a neuroprotective effect on the brain; (2) MJP6 has a repair effect on brain ultrastructural damage; (3) MJP6 has an inhibitory effect on intracranial inflammation and infiltration of microglia and neutrophils; (4) MJP6 promotes the absorption of intracerebral hematoma and the restoration of blood flow; (5) MJP6 has a relieving effect on motor dysfunction.

[0016] Furthermore, feature (1) includes: MJP6 can dose-dependently reduce brain hemorrhage damage, shrink the extent of damage, and protect neuronal morphology; MJP6 can also dose-dependently reduce neuronal apoptosis and improve the tissue microenvironment after brain hemorrhage.

[0017] Furthermore, feature (2) includes: MJP6's repair effect on the ultrastructure of the blood-brain barrier; MJP6 can effectively repair the ultrastructure of cells and reduce mitochondrial damage; MJP6 can repair myelin sheath damage caused by cerebral hemorrhage and restore myelin sheath thickness and integrity.

[0018] Furthermore, feature (3) includes: MJP6 can dose-dependently inhibit the expression of inflammatory factors after cerebral hemorrhage, reducing neuroinflammatory damage. MJP6 can significantly inhibit microglial activation, reducing the number of activated microglia. MJP6 can significantly inhibit the infiltration of neutrophils into brain tissue, helping to alleviate pathological reactions such as inflammation after cerebral hemorrhage, reducing secondary brain injury, and improving prognosis. MJP6 can significantly inhibit the expression of TNF-α and IL-1β inflammatory factors after cerebral hemorrhage, reducing neuroinflammatory damage.

[0019] Furthermore, feature (4) includes: MJP6 can dose-dependently improve tissue morphology after cerebral hemorrhage, reduce the hematoma area, and alleviate swelling; MJP6 can dose-dependently improve local blood flow after cerebral hemorrhage, maintain early perfusion, and promote the recovery of neurological function; MJP6 can significantly promote hematoma absorption and brain injury repair after cerebral hemorrhage, as evidenced by the rapid shrinkage of the damaged area over time and significant absorption of brain tissue hematoma.

[0020] Furthermore, feature (5) includes: MJP6 can effectively reduce neurological deficits in mice after cerebral hemorrhage; MJP6 can effectively improve motor function after cerebral hemorrhage, and the intervention effect gradually increases with the extension of postoperative time; MJP6 can significantly improve the symptoms of motor balance disorder in patients with cerebral hemorrhage; MJP6 can significantly improve the spatial learning and memory function of patients with cerebral hemorrhage: reverse the cognitive impairment caused by ICH; MJP6 can effectively improve this impairment by increasing movement distance and speed, and promoting the recovery of voluntary activity function in patients after cerebral hemorrhage.

[0021] This invention reveals that MJP6 exerts a neuroprotective effect against cerebral hemorrhage through multiple targets. MJP6 reduces extensive brain tissue damage and neuronal degeneration and necrosis caused by cerebral hemorrhage, and can dose-dependently reduce neuronal apoptosis. It improves blood-brain barrier damage, reduces mitochondrial swelling and cristae breakage, and repairs ultrastructural damage such as loosened and lost myelin sheath structures. It reduces the expression of inflammatory factors such as TNF-α and IL-1β, and inhibits microglial activation and neutrophil infiltration. It reduces hematoma volume, accelerates hematoma absorption, and promotes blood flow recovery. MJP6's ability to alleviate motor dysfunction and improve learning and memory abilities has been confirmed through mNSS scoring and the water maze test.

[0022] As a fourth aspect of the invention, it is provided that the compound MJP6 is used in the preparation of a treatment for cardiovascular and cerebrovascular diseases and nervous system diseases; the cardiovascular and cerebrovascular diseases include stroke, cerebral hemorrhage and cerebral edema; the nervous system diseases include neurodegenerative diseases and nerve damage.

[0023] As a fifth aspect of the invention, it is to provide a pharmaceutical composition comprising the novel monomer MJP6 described in the first aspect or a pharmaceutically acceptable salt thereof.

[0024] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable diluents, excipients, or excipients.

[0025] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows: 1. This invention provides a novel natural small molecule monomer, 2-E-feruloyloxyphenylacetate (MJP6), derived from black garlic. MJP6 can inhibit neuronal damage and apoptosis in a mouse model of cerebral hemorrhage, repair damage to the ultrastructure of the brain in this model, inhibit inflammatory responses and immune cell infiltration in the brain, promote hematoma absorption and blood flow recovery, improve motor and cognitive function, and exert neuroprotective effects, alleviating brain tissue damage and promoting brain tissue repair. These effects indicate that MJP6 can be used to treat cerebral hemorrhage and has the potential to develop drugs for treating cerebral hemorrhage and related neurological diseases.

[0026] 2. Compared with known ferulic acid esters, the MJP6 provided in this application has more significant effects in reducing the expression of inflammatory factors after cerebral hemorrhage, reducing neuroinflammatory damage, improving hematoma size, and restoring cerebral blood flow.

[0027] 3. The MJP6 provided by this invention, as a novel small molecule derived from black garlic, has a natural source and health benefits that lay a solid foundation for its application in the treatment of stroke. Attached Figure Description

[0028] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0029] Figure 1 The effect of MJP6 on the pathological morphology of brain tissue in ICH model mice was observed using HE staining.

[0030] Figure 2 TUNEL was used to observe neuronal damage.

[0031] Figure 3 This study investigated the repair effect of MJP6 on the ultrastructure of the blood-brain barrier in a mouse model of cerebral hemorrhage.

[0032] Figure 4 To investigate the protective effect of MJP6 against mitochondrial damage in the brain tissue of a mouse model of cerebral hemorrhage.

[0033] Figure 5 This study demonstrates the effect of MJP6 on alleviating myelin sheath damage in mouse brain tissue following cerebral hemorrhage. In the images, a) is an electron micrograph of the brain tissue ultrastructure, b) shows changes in myelin sheath integrity as revealed by g-ratio analysis, and c) is a quantitative analysis of myelin sheath thickness in the brain tissue.

[0034] Figure 6Western blot analysis was used to quantitatively analyze the expression of inflammatory factors around the hematoma. Specifically, a) represents the Western blot analysis of TNF-α and IL-1β protein levels, b) represents the quantitative analysis of the relative levels of TNF-α protein, and c) represents the quantitative analysis of the relative levels of IL-1β protein.

[0035] Figure 7 MJP6 was used to inhibit the activation of microglia in the brain tissue of a mouse model of cerebral hemorrhage and reduce the number of activated microglia. In the figures, a shows the distribution and activation of microglia observed by Iba-1 immunofluorescence staining, and b shows the density of Iba-1 positive microglia.

[0036] Figure 8 MJP6 was used to inhibit neutrophil infiltration in hematoma and surrounding tissues of a mouse model of cerebral hemorrhage. In the figures, a) shows neutrophil infiltration detected by myeloperoxidase (MPO) immunofluorescence staining, and b) shows quantitative analysis of MPO-positive cell density.

[0037] Figure 9 MJP6 was used to inhibit the expression of the inflammatory factor IL-1β in the brain hematoma and surrounding tissues of a mouse model of cerebral hemorrhage. In the figures, a) shows the distribution of IL-1β in brain tissue observed by IL-1β immunofluorescence staining, and b) shows the quantitative analysis of the density of IL-1β-positive cells.

[0038] Figure 10 Brain tissue sections under different treatments in brain slice experiments.

[0039] Figure 11 MJP6 can dose-dependently improve local blood flow after intracerebral hemorrhage. In this image, a shows infrared thermographic images of brain tissue blood perfusion at different time points, and b shows the dynamic changes in cerebral cortical blood perfusion at different postoperative time points.

[0040] Figure 12 To observe the hematoma morphology on days 1, 3, and 7 after modeling using magnetic resonance imaging. Among them, a) shows MRI images of brain hematoma on postoperative days 1, 3, and 7; b) shows the hematoma volume analysis at different postoperative time points; and c) compares the hematoma absorption rate of the cerebral hemorrhage model.

[0041] Figure 13 The mNSS score MJP6 can effectively reduce neurological deficits in mice after cerebral hemorrhage.

[0042] Figure 14 In a forelimb placement experiment, MJP6 effectively improved forelimb motor function in mice after cerebral hemorrhage.

[0043] Figure 15 In the rotarod experiment, MJP6 significantly improved the symptoms of motor balance disorder in mice with cerebral hemorrhage.

[0044] Figure 16 This study demonstrates that MJP6 significantly improved spatial learning and memory function in mice with cerebral hemorrhage during the Morris water maze experiment. The results show: a) trajectory diagrams of the learning and memory stages in the water maze experiment; b) changes in escape latency at different postoperative time points; c) analysis of the number of times the target quadrant was entered; d) analysis of the time spent in the target quadrant; and e) analysis of swimming speed.

[0045] Figure 17 This study demonstrates the effectiveness of MJP6 in improving spontaneous activity in mice during the open field experiment. In the figures, a represents the movement trajectory during the open field experiment, b represents the total movement distance analysis, and c represents the average movement speed analysis.

[0046] Figure 18 This is a diagram illustrating the structural analysis process of MJP6.

[0047] Figure 19 For MJP6 1 H-NMR spectrum.

[0048] Figure 20 For MJP6 13 C-NMR spectrum.

[0049] Figure 21 This is the mass spectrum of MJP6.

[0050] Figure 22 This is the infrared spectrum of MJP6. Detailed Implementation

[0051] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0052] The present invention will be further described below with reference to the embodiments.

[0053] The preparation method for black garlic is as follows: Raw material selection and cleaning: Select large, fresh garlic cloves that are free from pests, diseases, and damage. Use a cleaning device to wash away the dirt and impurities on the surface of the garlic with purified water.

[0054] Drying: The washed garlic is put into a drying device and dried at a low temperature (40-50℃) to remove some of the moisture, so that the moisture content of the garlic reaches a level suitable for fermentation, generally controlled at about 60%-70%.

[0055] Fermentation: Place the dried garlic into a constant temperature and humidity fermentation chamber, set the temperature to 60-80℃ and the humidity to 70%-90%, and ferment for 60 days.

[0056] Post-processing: After fermentation, the black garlic is removed and cooled to room temperature. Then it is sorted, removing any black garlic that does not meet the appearance standards.

[0057] In some embodiments of the present invention, a novel monomer MJP6 for black garlic is provided, the chemical structure of which is shown below: .

[0058] As is well known to those skilled in the art, the compound can be prepared into a pharmaceutical salt or further developed into a derivative. Pharmaceutical salts and derivatives prepared based on this structure should be considered within the scope of protection of this invention.

[0059] In some embodiments of the present invention, methods for preparing MJP6 are also provided, including separation and extraction from black garlic and chemical synthesis.

[0060] In some embodiments of the present invention, the application of compound MJP6 in the preparation of treatments for cardiovascular and cerebrovascular diseases and nervous system diseases is also provided; the cardiovascular and cerebrovascular diseases include stroke, cerebral hemorrhage and cerebral edema; the nervous system diseases include neurodegenerative diseases and nerve damage.

[0061] In some embodiments of the present invention, the use of MJP6 in the preparation of drugs for treating cerebral hemorrhage is also provided.

[0062] In this embodiment of the invention, the effects of MJP6 provided by the present invention were compared with those of compounds with known structures as shown:

[0063] 4-Hydroxyphenylethyl trans-ferulate.

[0064] In some embodiments of the present invention, a pharmaceutical composition comprising the novel monomer MJP6 described above or a pharmaceutically acceptable salt thereof is also provided.

[0065] The pharmaceutical composition also includes pharmaceutically acceptable diluents, excipients, or excipients.

[0066] The pharmaceutical composition can be produced using conventional formulation processes, either alone or in combination with other drugs, to create different dosage forms suitable for clinical use. These dosage forms include capsules, oral solutions, injections, tablets, powders, and granules.

[0067] Example 1 This embodiment provides a compound MJP6 isolated from black garlic. Based on its known structure, its chemical name is 2-E-feruloyloxybenzeneacetic acid ester, [2-E-feruloyloxybenzeneacetic acidester], with the molecular formula C.18 H 16 O6, with a molecular weight of 328. No results were found in existing publicly available literature. Its chemical structure is shown below: .

[0068] Example 2 Isolation, extraction and purification of MJP6: Extraction: Place the ground black garlic sample (approximately 500 g) into a beaker, add an appropriate amount of 95% ethanol-water mixture, and extract using ultrasound at room temperature for 2 hours. Repeat the extraction process three times, filter, and combine the extracts. Centrifuge the extract (approximately 10,000 rpm, 10 min) to remove suspended solids, collect the supernatant, and remove the solvent from the extract using a rotary evaporator to obtain a concentrated crude black garlic extract.

[0069] Extraction: Disperse 300 g of black garlic extract in 1000 mL of water and add it to a 5000 mL separatory funnel. Measure 1500-2000 mL of ethyl acetate using a graduated cylinder and slowly add it along the inner wall of the separatory funnel. Tighten the stopper, close the stopcock, and hold the funnel at a 45° angle. First, expel the air once, then shake for 10-15 seconds and expel the air again. Repeat this process three times (total shaking time ≤ 2 minutes). Place the separatory funnel on an iron stand and let it stand for 15-30 minutes until the layers are clearly separated. Open the glass stopper and slowly unscrew the stopcock, transferring the lower aqueous phase into a 2500 mL Erlenmeyer flask. When the interface is close to the stopcock, close the stopcock and pour the upper ethyl acetate phase into the 5000 mL Erlenmeyer flask. Record the volume of the ethyl acetate extract. Repeat the extraction three times, combine the ethyl acetate extracts, transfer them to a rotary evaporator, and distill under reduced pressure at 37°C to recover the solvent. Approximately 100 g of ethyl acetate extract is obtained.

[0070] Separation and purification: 100g of ethyl acetate extract was subjected to reduced pressure chromatography on a silica gel column (200-300 mesh) with a gradient elution of dichloromethane:methanol (120:1 to 10:1, i.e., 120:1, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1, v:v) to obtain six fractions FA-FF; fraction FC was subjected to silica gel column chromatography with isocratic elution of petroleum ether:ethyl acetate (7:1, v:v) to obtain eight fractions FC1-FC8; fraction FC7 was subjected to silica gel column chromatography with petroleum ether:ethyl acetate (3:2, v:v)... v) Isocratic elution was performed to obtain nine fractions FC7-1 to FC7-9; FC7-6 was prepared by TLC to obtain twelve fractions FC7-6-1 to FC7-6-12; FC7-6-6 was separated and purified by semi-preparative high performance liquid chromatography (Semi-prep HPLC) to obtain MJP6.

[0071] Identification: MJP6 was identified by 1D-NMR, 2D-NMR, high-resolution mass spectrometry, and infrared spectroscopy. The analysis process is as follows: Figure 18 As shown, 1 H-NMR spectrum 13 The C-NMR spectrum, mass spectrum, and infrared spectrum are shown below. Figures 19-22 As shown. Among them, Figure 22 The functional group types, characteristic peak wavenumbers, vibrational modes, and corresponding molecular structure positions are shown in the table below:

[0072] Example 3 The synthesis method was as follows: 4-hydroxy-3-methoxycinnamic acid (400 mg, 2.06 mmol), ω-bromo-4-hydroxyacetophenone (487 mg, 2.27 mmol), and MeCN (5 mL) were added sequentially to a clean 100 mL three-necked flask. Then, DIPEA (1.33 g, 10.30 mmol) was added, and the mixture was stirred at room temperature for 6 hours. TLC (PE / EA = 4:1) showed that the reaction was essentially complete. Post-treatment: Water (30 mL) was added, and the mixture was extracted using DCM (20 mL × 2). The organic phases were combined, washed with saturated brine, dried over anhydrous Na₂SO₄, and concentrated to dryness. The crude product was purified by column chromatography (eluent ratio PE / EA = 4:1) to give 600 mg of a white solid, with a yield of 90%. The specific reaction formula is as follows:

[0073] The characterization data for MJP6 are as follows: 1 H NMR (DMSO, 600 MHz) δ H 3.82 (3H, s), 5.45 (2H, s), 6.59 (1H, d, J =15.9 Hz), 6.80 (1H, d, J = 8.16 Hz), 6.83 (2H, d, J = 8.58 Hz), 7.15 (1H, d,J = 8.16 Hz), 7.60 (1H, d, J = 15.9 Hz), 7.83 (2H, d, J = 8.58 Hz); 13 C NMR (DMSO, 150 MHz) δ C55.6, 65.7, 111.2, 113.7, 115.4, 115.6, 115.6, 123.3, 124.7, 125.3, 130.2, 130.2, 145.7, 147.9, 149.6, 166.0, 163.7, 190.6。

[0074] HRESIMS m / z 327.0867 [M-H] - (calcd for C 18 H 15 O6, 327.0869)。

[0075] Example 4 Application of MJP6 in Neuroprotection Against Intracerebral Hemorrhage Experimental materials: The drug MJP6 was isolated and identified in the laboratory and prepared.

[0076] Experimental animals: Clean-grade healthy male C57BL / 6J mice (8 weeks old, 25 g ± 3 g), purchased from Shandong Pengyue Laboratory Animal Technology Co., Ltd., with the license number SCXK (Lu) 2022-0006. They were housed in a breeding room at room temperature of 20-25 °C, humidity of 45%-65%, and a 12 h light-dark cycle, and were allowed to eat and drink freely. They were divided into 5 groups by the random number table method: a control group (Control), a model group (ICH), and MJP6 low-dose group (20 mg / kg), MJP6 medium-dose group (50 mg / kg), and MJP6 high-dose group (80 mg / kg) drug-administered groups, with 25 mice in each group. The neurological function was evaluated by behavioral scoring, the size of the hematoma was observed by brain section, the neuronal damage was observed by hematoxylin-eosin staining and TUNEL staining, the local blood flow was analyzed by laser speckle contrast imaging, the damage of the blood-brain barrier, brain tissue mitochondria and myelin in intracerebral hemorrhage mice was observed by transmission electron microscopy, the expression of hematoma inflammatory factors was analyzed by immunohistochemistry, the expression level around the hematoma was quantitatively analyzed by Western blot, and the hematoma morphology was observed by magnetic resonance imaging at 1 day, 3 days and 7 days after modeling. The experimental mice were divided into: normal group mice, intracerebral hemorrhage mice, and intracerebral hemorrhage + MJP6 treatment group.

[0077] Establishment of a brain hemorrhage model: Male SPF-grade C57BL / 6J mice approximately 8 weeks old were selected and randomly divided into 5 groups using a random number table: a control group, a model group, and an MJP6-treated group. The treatment methods were as follows: A brain hemorrhage model was induced by stereotactic collagenase injection. Mice were fasted for 8 hours preoperatively and anesthetized with 25 mg / kg sodium pentobarbital. The mice were fixed in a stereotactic position on a brain stereotactic apparatus. After disinfection and preparation, a longitudinal incision was made in the center of the dorsal side of the head, and the subcutaneous tissue was dissected to expose the anterior fontanelle. Using the anterior fontanelle as the origin, a small hole was drilled into the skull 0.3 mm anteriorly and 2.3 mm to the right. 0.2 μL of type IV collagenase (C2399, Sigma) at a concentration of 0.2 U / μL (dissolved in physiological saline) was placed in a microsyringe and inserted vertically 3.8 mm. The needle was slowly advanced to the injection site and injected at a rate of 0.4 μL / min. After injection, the device was kept in place for 10 minutes to prevent collagenase reflux. The syringe was then slowly withdrawn, and the skull opening was sealed with bone wax. Local pressure was applied for hemostasis, and the scalp was sutured. The mice were then transferred to a 37°C warming pad until they regained consciousness. Post-operatively, the mice had free access to water and solid food under constant light conditions. Within 0.5 hours of collagenase injection, mice were injected via the tail vein with the corresponding concentration of MJP6. Control and model mice were injected with the same volume of physiological saline.

[0078] 1. MJP6 inhibits neuronal injury and apoptosis in a mouse model of cerebral hemorrhage. 1.1 Hematoxylin-eosin (HE) staining method for observing neuronal damage Three concentrations of MJP6 were used to treat a mouse model of cerebral hemorrhage to determine its protective effect on mouse nerve cells. For HE staining of brain tissue, the experimental animals were deeply anesthetized before rapid craniotomy to remove the brain tissue, avoiding damage. The brain tissue was fixed by immersion in 4% paraformaldehyde solution pre-cooled at 4°C. After 6 hours, the solution was changed, and the tissue was cut into small pieces and fixed for approximately 18 hours. After fixation, the tissue was rinsed with running water for one day, followed by dehydration with different concentrations of alcohol, clearing with a mixture of anhydrous ethanol and xylene, and then with xylene. The cleared brain tissue was then embedded in molten paraffin and shaped. The paraffin blocks were cut into thin sections of approximately 5 μm using a microtome, baked, and then dewaxed and hydrated. Afterward, the sections were stained with Harris hematoxylin and eosin for 10 minutes, rinsed with tap water, differentiated with hydrochloric acid solution, bluing with tap water, and then stained with eosin for 5 minutes. Finally, the slide is dehydrated with alcohol, then treated with a mixture of anhydrous ethanol and xylene, and finally cleared with xylene. After the liquid is absorbed, a neutral resin is added for mounting, and the slide can then be observed under a microscope.

[0079] The results are as follows Figure 1As shown, HE staining was used to observe neuronal damage: cerebral hemorrhage leads to extensive brain tissue damage and neuronal degeneration and necrosis, resulting in brain tissue swelling and structural disorder. MJP6 can dose-dependently reduce cerebral hemorrhage damage, shrink the damage area, and protect neuronal morphology. The study included a normal control group, an ICH model group, a low-dose group (20 mg / kg), an intermediate-dose group (50 mg / kg), and a high-dose group (80 mg / kg).

[0080] 1.2 TUNEL staining After deep anesthesia, the experimental animals underwent rapid craniotomy to harvest the brain, avoiding damage. The brain tissue was then fixed by immersing it in 4% paraformaldehyde solution pre-cooled to 4°C for 24-48 hours. Next, the samples were treated with a permeabilizer (0.1% Triton X-100 in PBS) at room temperature for 10 minutes. The TUNEL reaction solution (a mixture of TdT enzyme, biotin-labeled dUTP, and dNTP, diluted with buffer) was prepared according to the kit instructions to allow TdT enzyme and nucleotides to enter the cells. The samples were then co-incubated with the TUNEL reaction solution containing TdT enzyme and biotin-labeled dUTP in a humidified chamber at 37°C in the dark for 60 minutes. The TdT enzyme catalyzes the ligation of dUTP to the broken 3'-OH end of DNA. After incubation, the samples were washed three times with PBS for 5 minutes each time to remove unbound reaction solution. For white light detection, an avidin-horseradish peroxidase (HRP) conjugate is used to bind labeled dUTP. After incubation at room temperature for 30 minutes, the sample is washed three times with PBS, and a DAB (diaminobenzidine) chromogenic substrate is added. HRP catalyzes the oxidation of DAB to form a brown precipitate. The color development is monitored under a microscope for 5-10 minutes, thus revealing the morphological characteristics of apoptotic cells under an optical microscope. Finally, counterstaining and mounting are performed. After dehydration with graded ethanol (70%→80%→95%→100%) and clearing with xylene, the number of apoptotic cells can be observed and counted under a white light microscope.

[0081] The results are as follows Figure 2 As shown, the TUNEL experiment observed neuronal damage: after cerebral hemorrhage, neuronal apoptosis in mouse brain tissue was observed. MJP6 can dose-dependently reduce neuronal damage and apoptosis and improve the tissue microenvironment after cerebral hemorrhage.

[0082] 2. MJP6 repairs brain ultrastructural damage in a mouse model of cerebral hemorrhage. 2.1 Transmission electron microscopy observation of blood-brain barrier, mitochondria, and myelin sheath damage in mice with cerebral hemorrhage Mice were anesthetized and subsequently perfused intracardiacly with 0.1 mol / L Sorensen's buffer (pH 7.4) containing 4% paraformaldehyde and 2.5% glutaraldehyde at 1, 3, and 7 days after intracranial hemorrhage. Brain tissue was obtained by dissection at 4°C. The brain tissue was cut into small pieces (1 mm). 3 Subsequently, the samples were fixed with 1.0% OsO4 solution and dehydrated using a series of ethanol solutions of increasing concentration. After dehydration, the samples were infiltrated with propylene oxide, embedded in Epon resin, and then sectioned. The stained samples were analyzed using a Philips CM 100 transmission electron microscope (Hillsboro, Oregon, USA) and digitally recorded using a Hamamatsu ORCA-HR camera (Hamamatsu, Shizuoka, Japan).

[0083] The results are as follows Figure 3 As shown, transmission electron microscopy revealed damage to the blood-brain barrier in mice with cerebral hemorrhage. Following cerebral hemorrhage, cerebral blood vessels exhibited disordered morphology, luminal dilation, extreme expansion of intercellular spaces between endothelial cells, complete disruption of tight junctions (widened gaps and blurred structure), and rupture of the basement membrane, resulting in leakage of contents. This ultrastructural evidence of blood-brain barrier dysfunction suggests that cerebral hemorrhage leads to severe damage to the blood-brain barrier. In the MJP6 group, partial recovery of tight junctions between endothelial cells was observed (black bands became clearer, gaps narrowed), the continuity of the basement membrane improved, and leakage decreased. This indicates that MJP6 has a repairing effect on the ultrastructure of the blood-brain barrier and ameliorates blood-brain barrier damage in mice with cerebral hemorrhage.

[0084] like Figure 4 As shown, transmission electron microscopy revealed mitochondrial damage in the brain tissue of mice with cerebral hemorrhage. Cerebral hemorrhage caused severe damage to the ultrastructure of brain cells, with severe mitochondrial swelling, cristae rupture, and partial membrane damage. After treatment with MJP6, mitochondrial swelling was reduced, cristae partially recovered, and organelle morphology gradually repaired, verifying the drug's protective effect on cellular ultrastructure. This indicates that MJP6 can effectively repair cellular ultrastructure, reduce mitochondrial damage, and protect against mitochondrial damage in the brain tissue of mice with cerebral hemorrhage.

[0085] like Figure 5 As shown, transmission electron microscopy revealed the degree of myelin sheath damage in mice after cerebral hemorrhage. Significant damage to the myelin sheath was observed, characterized by a loose structure, disordered layering, and even partial myelin loss (see the fragmented morphology and increased gaps in the lower row of images). This suggests that cerebral hemorrhage leads to myelin sheath destruction and exacerbates neurological deficits. MJP6 treatment significantly improved myelin sheath structure, with integrity and density approaching those of the Control group. Myelin sheath layering was clear, and loss was reduced, indicating that MJP6 can repair myelin sheath damage caused by cerebral hemorrhage, restoring myelin sheath thickness and integrity, and demonstrating a protective effect against myelin sheath damage in mouse brain tissue after cerebral hemorrhage.

[0086] Among them, Control: normal control group, ICH group: cerebral hemorrhage model group, MJP6 group: cerebral hemorrhage + MJP6 intervention group. (# compared with control group, * compared with model group, * / #p<0.05, ** / ##p<0.01, *** / ###p<0.001, **** / ####p<0.0001).

[0087] MJP6-induced cerebral hemorrhage model mice showed increased inflammatory response and immune cell infiltration in the brain. 3.1 Western blot analysis of the expression levels of inflammation-related proteins in brain tissue. Three days post-surgery, brain tissue was collected from mice in each group. 150 μL of RIPA lysis buffer was added to every 10 mg of brain tissue, and lysis was performed on ice for 30 min. The cells were then centrifuged at 12000 r / min for 15 min at 4℃, and the supernatant was collected. After adding protease inhibitors and loading buffer, the cells were boiled for 10 min to denature the proteins. SDS-PAGE: 4% stacking gel and 12% separating gel were used. Electrophoresis was performed at constant voltage: 90 V for 30 min, 120 V for 60 min. Transfer was performed at constant current of 300 mA for 50 min. Blocking was performed with 5% skim milk powder blocking solution at room temperature for 1 h. The membrane was incubated overnight at 4℃ with primary antibody (1:1000 concentration), washed with 1XTBST (10 min × 3 times), and incubated with secondary antibody (1:2000 concentration) at room temperature for 1 h. The membrane was washed with 1XTBST (10 min × 3 times). ECL luminescence was performed, and images were taken and the relative gray values ​​of the bands were analyzed using a fully automated chemiluminescence image analysis system.

[0088] The results are as follows Figure 6 As shown, cerebral hemorrhage strongly activates the inflammatory response, leading to a significant increase in the expression of TNF-α and IL-1β proteins. MJP6 can dose-dependently inhibit the expression of inflammatory factors after cerebral hemorrhage and reduce neuroinflammatory damage. Furthermore, compared with the known structure (50 mg / kg), the effect of MJP6 is more significant. Control: normal control group; ICH: cerebral hemorrhage model group; 20 mg / kg: low-dose MJP6 group; 50 mg / kg: intermediate-dose MJP6 group; 80 mg / kg: high-dose MJP6 group. (#Compared with control group, *Compared with model group, * / #p<0.05, ** / ##p<0.01, *** / ###p<0.001, **** / ####p<0.0001).

[0089] 3.2 Immunofluorescence staining analysis of the expression of microglia and inflammatory factors in the hematoma and surrounding tissues. After anesthetizing mice, brain tissue was rapidly extracted after perfusion with 4% paraformaldehyde. The brain tissue was prepared into paraffin sections, dewaxed, and hydrated before being placed in EDTA buffer. Antigen retrieval was performed under high temperature and autoclave or water bath to re-expose fixed and masked antigenic epitopes. After cooling to room temperature, the sections were incubated with 3% hydrogen peroxide solution for 10-15 minutes to inactivate endogenous peroxidase, followed by rinsing with PBS three times for 5 minutes each time. Then, the sections were blocked with 5%-10% bovine serum albumin or goat serum at room temperature for 30-60 minutes to reduce non-specific staining. After blocking, excess liquid was discarded, and primary antibodies against inflammatory factors IL-1β, iba1, and MPO were added, respectively. The sections were incubated overnight in a humidified chamber at 4°C. The next day, the sections were warmed to room temperature for 30 minutes and rinsed with PBS three times for 5 minutes each time. Subsequently, the corresponding secondary antibodies (such as horseradish peroxidase-labeled goat anti-rabbit IgG) were added, and the sections were incubated at room temperature for 30-60 minutes, followed by rinsing with PBS three times for 5 minutes each time. The cells were then developed using a DAB chromogenic kit and observed under a microscope. When a brownish-yellow positive signal appeared, the development was stopped by rinsing with distilled water. Finally, the cell nuclei were counterstained with hematoxylin for 1-2 minutes. After differentiation with hydrochloric acid alcohol and blueing with tap water, the cells were dehydrated with a gradient of alcohols, cleared with xylene, and mounted with neutral resin. The images were then observed and captured under a microscope to analyze the expression intensity and distribution of inflammatory factors and microglia in the hematoma area.

[0090] The results are as follows Figure 7 As shown, cerebral hemorrhage strongly activates microglia (increasing in number and changing from a resting to an activated state). MJP6 can significantly inhibit the activation of microglia and reduce the number of activated microglia.

[0091] like Figure 8 As shown, after cerebral hemorrhage, a large number of neutrophils infiltrate the brain tissue, aggravating neurological dysfunction and promoting the development of secondary brain injury. MJP6 can significantly inhibit this infiltration process, helping to reduce pathological reactions such as inflammation after cerebral hemorrhage, reduce secondary brain injury, and improve prognosis.

[0092] like Figure 9 As shown, cerebral hemorrhage strongly activates the inflammatory response, leading to a significant increase in IL-1β expression. MJP6 can significantly inhibit the expression of IL-1β inflammatory factors after cerebral hemorrhage, reduce neuroinflammatory damage, and exert a neuroprotective effect.

[0093] Control group: normal control group; ICH group: cerebral hemorrhage model group; MJP6 group: cerebral hemorrhage + MJP6 intervention group. (#Compared with control group, *Compared with model group, * / #p<0.05, ** / ##p<0.01, *** / ###p<0.001, **** / ####p<0.0001).

[0094] MJP6 promotes the absorption of intracranial hematoma and restoration of blood flow in a mouse model of cerebral hemorrhage. 4.1 Brain slice experiment. Mice treated with different concentrations of MJP6 were established to observe the inhibitory effect of MJP6 on hematoma formation in mice at different concentrations. Three days after the establishment of the experimental animal model, the animals were euthanized under anesthesia, and intact brain tissue was quickly removed through craniotomy. The removed brain tissue was immediately frozen in liquid nitrogen. The cryostat temperature was adjusted to approximately -20°C, and after a period of pre-cooling, the tissue was removed from the liquid nitrogen and placed on the sample holder of the cryostat. The blade was slowly and evenly advanced, and the cut slices automatically adhered to the slides treated with anti-detachment measures. After each slice was cut, the cooling was paused for about 10 seconds to prevent the tissue from softening and deforming. Five equal-thickness slices were cut along the coronal plane. After sectioning, the slices were immediately transferred to a -80°C cryostat for temporary storage, and each group of brain tissue slices were arranged from front to back along the coronal plane for photographing.

[0095] like Figure 10 As shown in the brain slice experiment, cerebral hemorrhage caused significant hematoma and swelling in the mouse brain tissue. The drug MJP6 improved the tissue morphology after cerebral hemorrhage, reduced the hematoma size, and alleviated swelling. Furthermore, within the range of 20-80 mg / kg, the effect increased with increasing dose. Compared to the known structure (50 mg / kg), MJP6 significantly improved hematoma size. The study included: a normal control group, a cerebral hemorrhage model group (ICH), a low-dose group (20 mg / kg), an intermediate-dose group (50 mg / kg), and a high-dose group (80 mg / kg).

[0096] 4.2 Laser speckle contrast imaging analysis of local blood flow Mice were anesthetized at 2 hours, 1, 3, 5, and 7 days after intracerebral hemorrhage. The mice were then secured in appropriate supports, and the laser speckle imaging system was aimed at the target area, ensuring the laser beam irradiated the sample surface. The camera focus was adjusted for clear imaging. The laser was activated, irradiating the sample surface and exciting the speckle pattern. Laser speckle imaging reflects blood flow dynamics by calculating the contrast of the speckle image. The original speckle images and the processed blood flow images were saved to ensure data reliability and reproducibility. Quantitative blood flow data were further analyzed using data analysis software.

[0097] like Figure 11As shown, intracerebral hemorrhage (ICH) leads to a sudden drop in acute blood flow perfusion in the cerebral cortex, with slow long-term recovery. MJP6 can dose-dependently improve local blood flow after ICH, maintain early perfusion, and promote neurological function recovery. The effect is significant at doses of 50-80 mg / kg, promoting neurological function repair. Furthermore, compared to known structures (50 mg / kg), MJP6's effect on restoring cerebral blood flow is more significant. The study included a normal control group, an ICH model group, a low-dose MJP6 group (20 mg / kg), an intermediate-dose MJP6 group (50 mg / kg), and a high-dose MJP6 group (80 mg / kg). (#Compared with the control group, *Compared with the model group, * / #p<0.05, ** / ##p<0.01, *** / ###p<0.001, **** / ####p<0.0001).

[0098] 4.3 Magnetic resonance imaging observation of hematoma morphology on days 1, 3, and 7 after modeling Brain tissue was harvested from mice anesthetized with sodium pentobarbital at 1, 3, and 7 days post-MRI. All mice were then imaged using T2* gradient echo sequences on an MRI scanner. Twenty coronal sections (1.0 mm thick) were obtained from the frontal pole to the brainstem. T2* lesions were delineated along the boundaries of low-signal (dark) areas, including central iso- or high-signal areas. The total T2* lesion volume was calculated by multiplying the section thickness by the total T2* lesion volume across all sections. The total T2* lesion volume represents the hematoma volume.

[0099] like Figure 12 As shown, the cerebral hemorrhage group had the largest lesion area and mixed signals on postoperative day 1, with significant compression of the brain parenchyma. On postoperative day 3, the cerebral hemorrhage lesion area was still large, but the signal began to "fade" (partial absorption of hematoma). After MJP6 treatment, the lesion area shrank significantly, accelerating hematoma absorption. On postoperative day 7, the cerebral hemorrhage group showed persistent damage to the brain parenchyma (hematoma / edema), indicating slow natural recovery. After MJP6 treatment, the lesion area shrank significantly, and the brain parenchyma signal gradually recovered. MJP6 can significantly promote hematoma absorption and brain injury repair after cerebral hemorrhage, as evidenced by the rapid shrinkage of the lesion area over time, and significant absorption of brain hematoma on postoperative day 7. Control: normal control group, ICH group: cerebral hemorrhage model group, MJP6 group: cerebral hemorrhage + MJP6 intervention group. (#Compared with the control group, *Compared with the model group, * / #p<0.05, ** / ##p<0.01, *** / ###p<0.001, **** / ####p<0.0001).

[0100] MJP6 improved motor and cognitive function in a mouse model of cerebral hemorrhage by 5.1 mNSS score. The Modified Neurological Deficit Score (mNSS) is a comprehensive method for assessing the degree of neurological impairment in rats. The total score is 18 points, with higher scores indicating more severe neurological damage. This method covers multiple dimensions, including motor, sensory, balance, and reflexes: Motor tests include the tail-lift test (assessing forelimb / hindlimb flexion and head tilt, maximum 3 points) and walking on flat ground (from normal walking to tilting towards the paralyzed side, maximum 3 points); Sensory tests include placement tests (visual / tactile tests) and proprioceptive tests (deep sensation of limbs), each scored 1 point; the balance beam test assesses the rat's performance on a 1.5 cm wide balance beam, scored from 0-6 points for maintaining balance to rapid slippage; Reflex tests include absence of the auricle, cornea, and panic reflex, each scored 1 point, with an additional 1 point for the presence of abnormal movements such as epilepsy / myoclonus. The mNSS, by quantifying the degree of neurological deficits, is widely used in evaluating the efficacy of treatments in models of neurological diseases such as cerebral ischemia and brain injury.

[0101] like Figure 13 As shown, the mNSS scores of the three groups were similar at baseline (approximately 1-2 points each), indicating no significant difference in preoperative neurological function. However, MJP6 after ICH effectively reduced neurological deficits in mice following intracerebral hemorrhage, and the intervention effect gradually became more significant with increasing postoperative time, providing a potential treatment strategy for neurological function repair after intracerebral hemorrhage. Control: Normal control group; ICH group: Intracerebral hemorrhage model group; MJP6 group: Intracerebral hemorrhage + MJP6 intervention group. (#Compared with control group, *Compared with model group, * / #p<0.05, ** / ##p<0.01, *** / ###p<0.001, **** / ####p<0.0001).

[0102] 5.2 Forelimb Placement Experiment Hold the mouse by the skin on the back of its neck with one hand, and gently pull its tail with the other hand to extend its body. Slowly bring the whiskers on one side close to the edge of the table. A normal mouse will place its forelimb on the same side at the edge of the table, while this reflex is absent in the brain hemorrhage model group. Test each mouse 10 times on each side, and record the number of times the mouse correctly places its forearm on the table edge after the whiskers are touched. Before the formal test, gently move the animal up and down to reduce resistance.

[0103] like Figure 14 As shown, MJP6 can effectively improve the forelimb motor function of mice after cerebral hemorrhage, and the intervention effect gradually increases with the extension of postoperative time, and can bring the function close to normal by the 7th day, providing a strong basis for the treatment of limb motor disorders after cerebral hemorrhage.

[0104] 5.3 Rotating bar experiment Place the mouse on a rotundus and rotate it at a constant low speed (4 rpm) for 2 minutes to allow it to learn to grasp. Repeat 2-3 times, with 10-minute intervals, ensuring the mouse can remain stationary. Assess basic motor ability at a constant speed. Detect motor deficiencies using an accelerated mode (starting at 4 rpm and increasing by 2 rpm per minute). Gently place the mouse on the rotundus and start the instrument. Record the time it takes for the mouse to fall off the rotundus (latency, in seconds) or the maximum tolerable rotational speed (rpm). Test each mouse 3 times, with at least 10 minutes between each test, and take the average to reduce error. Set a maximum test time (300 seconds); stop recording if the mouse does not fall within the time limit. Wipe the compartment with 75% alcohol after each test.

[0105] like Figure 15 As shown, after surgery, the time mice spent on the rotarod was significantly reduced in mice with cerebral hemorrhage. MJP6 effectively improved the balance and motor coordination of mice after cerebral hemorrhage, and the intervention effect gradually became more significant with the extension of postoperative time. This indicates that MJP6 can significantly improve the symptoms of motor balance disorder in mice with cerebral hemorrhage. Control: normal control group, ICH group: cerebral hemorrhage model group, MJP6 group: cerebral hemorrhage + MJP6 intervention group. (#Compared with control group, *Compared with model group, * / #p<0.05, ** / ##p<0.01, *** / ###p<0.001, **** / ####p<0.0001).

[0106] 5.4 Morris Water Maze Test During water maze training, the platform should be placed in the center of the pool, 1 cm below the water surface, to ensure the animal can perceive its presence. Each animal needs to undergo three consecutive tests: first, place the animal on the platform for 20 seconds. The water maze has four starting points: east, south, west, and north; place the animal randomly at one of these points. When entering the water, support the animal with your hand, ensuring its tail enters first to avoid stress caused by its head entering the water. Allow the animal to swim / search for the platform in the water for a maximum of 60 seconds. Initially, the animal may swim along the pool wall to find the exit, but will eventually learn to locate and climb onto the platform. Stop the timer and record the time immediately when the animal reaches the platform. If the platform is not found within 60 seconds, record it as 1 minute. If the animal fails to reach the platform, do not grab it directly; guide it to the platform with a glass or plastic rod and allow it to rest for 15 seconds.

[0107] like Figure 16As shown, the Morris water maze test was used to assess the learning and memory function of mice, and the effect of MJP6 on improving cognitive impairment after intracerebral hemorrhage was analyzed. MJP6 can significantly improve the spatial learning and memory function of mice with intracerebral hemorrhage: by shortening the escape latency, increasing the number of times the target quadrant is explored and the dwell time, it reverses the cognitive impairment caused by ICH. Control: normal control group, ICH group: intracerebral hemorrhage model group, MJP6 group: intracerebral hemorrhage + MJP6 intervention group. (# compared with the control group, * compared with the model group, * / #p<0.05, ** / ##p<0.01, *** / ###p<0.001, **** / ####p<0.0001).

[0108] 5.5 Open Field Experiment In a square, open enclosure (50×50×35 cm), the enclosure is divided into a central area and a peripheral area. A camera or a behavioral analysis system is installed above the enclosure. Before the experiment, the animal is allowed to acclimatize to the experimental environment for at least 1-2 days, 2-4 hours each day, to minimize the impact of stress on the results. During the experiment, the animal is gently placed in the center of the enclosure, and the recording equipment is activated to observe and record its behavior over 5-10 minutes, including movement distance, speed, time spent in the central and peripheral areas, and number of times it stands upright. After the experiment, the enclosure is cleaned promptly to avoid residual odors affecting subsequent animal testing. Finally, professional software is used to analyze the data to assess the animal's spontaneous activity, anxiety level, or motor coordination.

[0109] like Figure 17 As shown, cerebral hemorrhage significantly inhibited the spontaneous activity ability of mice (shortened total movement distance and decreased average speed); the drug MJP6 can effectively improve this damage by increasing movement distance and speed, thus promoting the recovery of spontaneous activity function in mice after cerebral hemorrhage. Control: normal control group, ICH group: cerebral hemorrhage model group, MJP6 group: cerebral hemorrhage + MJP6 intervention group. (#Compared with control group, *Compared with model group, ** / ## p<0.01, *** / ### p<0.001, **** / #### p<0.0001).

[0110] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A novel monomer MJP6 in black garlic, characterized in that, Its chemical structure is shown below: 。 2. The method for preparing the novel monomer MJP6 in black garlic according to claim 1, characterized in that, The preparation method for separating and extracting from black garlic includes the following steps: S101, Extraction: Grind black garlic, add solvent, extract ultrasonically, remove suspended matter by centrifugation, collect supernatant, and concentrate by rotary evaporation to obtain crude black garlic extract. S102, Extraction: Disperse the crude black garlic extract prepared in step S101 with water, add an ester solvent for extraction, and distill the extract under reduced pressure to obtain the extract. S103, Separation and Purification: The extract obtained in step S102 was subjected to silica gel column chromatography under reduced pressure, eluted with a dichloromethane:methanol gradient, to obtain 6 fractions FA-FF; fraction FC was subjected to silica gel column chromatography, eluted isocratically with petroleum ether:ethyl acetate, to obtain 8 fractions FC1-FC8; fraction FC7 was subjected to silica gel column chromatography, eluted isocratically with petroleum ether:ethyl acetate, to obtain 9 fractions FC7-1 to FC7-9; FC7-6 was subjected to preparative thin-layer chromatography, to obtain 12 fractions FC7-6-1 to FC7-6-12; FC7-6-6 was separated and purified using semi-preparative high-performance liquid chromatography to obtain MJP6; Alternatively, it can be a chemical synthesis, including the following steps: Add 4-hydroxy-3-methoxycinnamic acid, ω-bromo-4-hydroxyacetophenone, and solvent to a reaction vessel, then add N,N-diisopropylethylamine and stir at room temperature; then extract, wash the organic phase with saturated brine, dry it, concentrate it to dryness, and purify it to obtain the final product.

3. The application of the novel monomer MJP6 in black garlic according to claim 1 in the preparation of treatments for cardiovascular and cerebrovascular diseases and nervous system diseases, characterized in that, The cardiovascular and cerebrovascular diseases include stroke, cerebral hemorrhage, and cerebral edema; the nervous system diseases include neurodegenerative diseases and nerve damage.

4. The use of the novel monomer MJP6 in the black garlic of claim 1 in the preparation of a drug for treating cerebral hemorrhage.

5. The application according to claim 4, characterized in that, It has at least one of the following characteristics: (1) It has a neuroprotective effect on the brain; (2) It has a repair effect on brain ultrastructural damage; (3) It has an inhibitory effect on intracranial inflammation and infiltration of microglia and neutrophils; (4) Promotes the absorption of intracerebral hematoma and the restoration of blood flow; (5) It has a relieving effect on motor dysfunction.

6. The application according to claim 5, characterized in that, Feature (1) includes: MJP6 dose-dependently reduces brain hemorrhage damage, shrinks the extent of damage, and protects neuronal morphology; it also dose-dependently reduces neuronal apoptosis and improves the tissue microenvironment after brain hemorrhage. Feature (2) includes: MJP6 has a restorative effect on the ultrastructure of the blood-brain barrier; MJP6 effectively repairs the ultrastructure of cells and reduces mitochondrial damage; MJP6 repairs the myelin sheath damaged by cerebral hemorrhage and restores the thickness and integrity of the myelin sheath.

7. The application according to claim 5, characterized in that, Feature (3) includes: MJP6 dose-dependently inhibits the expression of inflammatory factors after cerebral hemorrhage, reducing neuroinflammatory damage; MJP6 inhibits the activation of microglia, reducing the number of activated microglia; MJP6 inhibits the process of neutrophil infiltration into brain tissue, which helps to reduce pathological reactions such as inflammatory damage after cerebral hemorrhage, reduce secondary brain injury, and improve prognosis; MJP6 inhibits the expression of IL-1β inflammatory factor after cerebral hemorrhage, reducing neuroinflammatory damage.

8. The application according to claim 5, characterized in that, Feature (4) includes: MJP6 dose-dependently improves tissue morphology after cerebral hemorrhage, reduces hematoma size, and alleviates swelling; MJP6 dose-dependently improves local blood flow after cerebral hemorrhage, maintains early perfusion, and promotes neurological function recovery; MJP6 promotes hematoma absorption and brain injury repair after cerebral hemorrhage.

9. The application according to claim 5, characterized in that, Feature (5) includes: MJP6 reduces neurological deficits in mice after cerebral hemorrhage; MJP6 effectively improves motor function after cerebral hemorrhage; MJP6 improves symptoms of motor balance disorder in patients with cerebral hemorrhage; MJP6 improves spatial learning and memory function in patients with cerebral hemorrhage and reverses cognitive impairment caused by ICH.

10. A pharmaceutical composition comprising the novel monomer MJP6 of the black garlic of claim 1 or a pharmaceutically acceptable salt thereof.

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