CN117257792A

CN117257792A - Application of licarin b and preparation thereof in preparation of medicines for treating and/or relieving vascular dementia

Info

Publication number: CN117257792A
Application number: CN202310948186.2A
Authority: CN
Inventors: 王小洪; 王嘉华; 曹桂云; 江天琳; 周嘉伟; 万小娟
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2023-07-31
Filing date: 2023-07-31
Publication date: 2023-12-22

Abstract

The invention discloses licarin b and application of a preparation thereof in preparing medicaments for treating and/or relieving vascular dementia. The licarpa b and the preparation thereof can definitely improve the reduction of neurons in the forehead lobe area of a mouse model of vascular dementia induced by chronic cerebral ischemia, inhibit inflammatory reaction of the forehead lobe area of the mouse model of vascular dementia, inhibit the scorch and apoptosis of neurons in the forehead lobe area, inhibit demyelination-like changes in brain and inhibit vascular reactive hyperplasia in brain, and play a role in protecting neurons; the whole animal level is expressed as that the cognitive dysfunction of mice with vascular dementia is relieved, and the number of neurons in the brain is increased; so that the licarin b and the preparation thereof can be used for treating vascular dementia and have the prospect of developing medicaments.

Description

Application of licarin b and preparation thereof in preparation of medicines for treating and/or relieving vascular dementia

Technical Field

The invention relates to application of licarin b and a preparation thereof in preparing medicaments for treating and/or relieving vascular dementia, belonging to the field of vascular dementia treatment.

Background

Vascular dementia is a broad concept and is a series of syndromes covering mild to severe symptoms of cognitive impairment, and has become a common cause of chronic progressive cognitive impairment in the elderly population. One of the major contributors to vascular dementia is chronic cerebral ischemia. Chronic cerebral ischemia is a state of reduced blood supply at the overall level of the brain, a common outcome of vascular risk factors, heart disease or hemodynamic changes, and changes in blood composition. Patients often have varying degrees of cognitive impairment, with vascular cognitive impairment caused by chronic cerebral ischemia accounting for half of all cases.

The research shows that the clinical syndrome has great intervention value, can be prevented by changing life style and controlling vascular risk factors, but has low early diagnosis sensitivity and limited treatment means. Currently, the treatment of vascular dementia in clinic mainly comprises medical conservation treatment, but medicines and operation modes of high-level evidence-based medical evidence are not available. Therefore, the current treatment of vascular dementia mainly starts with the treatment in the aspects of eliminating original inducing factors such as antihypertensive, hyperglycemia and hyperlipidemia, improving brain metabolism, resisting cerebral anoxia, improving cognitive dysfunction and the like, and development of new treatment targets is an urgent need for basic research and clinical diagnosis and treatment.

Licaline b (RDK) has the formula C ₂₀ H ₂₀ O ₄ White crystals. The research shows that RDK has antibacterial, antiinflammatory, tranquilizing, antitumor, and parasitic disease preventing effects. RDK has not been reported in the treatment of vascular dementia.

Disclosure of Invention

The invention aims to: the invention aims to solve the technical problem of providing licarin b and application of a preparation thereof in preparing medicaments for treating vascular dementia.

The technical scheme is as follows: in order to solve the technical problems, the invention provides application of licarpae b in preparing medicaments for treating vascular dementia.

Wherein the treatment and/or alleviation of vascular dementia comprises one or more of improving cognitive dysfunction, improving learning and memory functions, inhibiting inflammatory response in the brain, inhibiting pyro-death response in the brain, improving demyelinating changes in the brain, inhibiting neuronal apoptosis.

Wherein, the chemical structural formula of licarin b is:

the preparation comprises tablets, capsules, decoction, pills, medicinal granules, dripping pills, mixture, injection and oral liquid.

Wherein the concentration of the licarin b is 5mg/ml.

Wherein the dosage of the licarpa is 50mg/Kg.

The preparation method of the licarin b comprises the following steps:

(1) Suspending semen Myristicae dry extract in water, extracting with ethyl acetate to obtain ethyl acetate extract;

(2) Dissolving the ethyl acetate extract in n-hexane-methanol-water, injecting into high-speed countercurrent chromatography, collecting effluent for 35-60min, and drying under reduced pressure at low temperature to obtain crude extract;

(3) Dissolving the crude extract in the step (2), preparing by adopting a c18 chromatographic column, collecting effluent liquid for 15-20min, and drying at low temperature and reduced pressure to obtain the licarbazepine b.

Wherein, the high-speed countercurrent chromatography in the step (2) takes n-hexane-methanol-water phase as a fixed Hunan, the lower term is a mobile phase, the rotating speed is 1000r/min, the flow speed is 2.2ml/min, and the detection wavelength is 254nm.

Wherein, the c18 chromatographic column in the step (3) is in 0-60min, and the volume ratio of the acetonitrile-water solution of the mobile phase is 50: the 50 gradient increases to 90:10, flow rate 3ml/min, detection wavelength 254nm.

The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: 1. the invention discloses RDK which can definitely improve neuronal damage of vascular dementia mouse model induced by chronic cerebral ischemia, inhibit inflammatory reaction, cell apoptosis and neuronal apoptosis in brain, inhibit demyelination in brain and play a role of neuronal protection; 2. conditions at the whole animal level that appear to alleviate learning and memory dysfunction in vascular dementia mice; 3. the RDK can be used for treating vascular dementia, and has the prospect of developing medicaments.

Drawings

FIG. 1 is a graph showing the effects of RDK on alleviating memory and learning impairment in a nest-making experiment in mice with vascular dementia induced by chronic cerebral ischemia in a long-term chronic cerebral ischemia model, wherein # # is p <0.01 compared with a control group, and p <0.001 ns is not significantly different from that in the model group;

FIG. 2 is a graph showing the effect of RDK on alleviating the decline of memory capacity in Y maze experiments in mice with vascular dementia induced by chronic cerebral ischemia in a long-term, # compared with control group, p <0.001, ns is not significantly different from that in the model group;

fig. 3 is an effect of RDK on alleviating cognitive dysfunction in social experiments in mice with vascular dementia induced by chronic cerebral ischemia, p <0.05 compared to empty cage or stranger1, p <0.01 compared to empty cage or stranger1, p <0.001 ns is not significantly different;

FIG. 4 is a graph showing the effect of RDK on alleviating cognitive dysfunction in a water maze test in mice with vascular dementia induced by chronic cerebral ischemia in a long-term model, wherein # # is p <0.001 compared with the control group, and p <0.001 is not significantly different in ns compared with the model group;

FIG. 5 shows the effect of RDK on significantly increasing the number of NeuN positive cells in the frontal lobe region of mice with chronic cerebral ischemia-induced vascular dementia, wherein # # is p <0.001 compared with control group and p <0.001 compared with model group;

FIG. 6 is a graph showing the effect of RDK on inhibiting the number of Tunel positive cells in the brain of mice with long-term chronic cerebral ischemia-induced vascular dementia model, wherein # # is p <0.001 compared with control group, p <0.001, ns is not significantly different from model group;

FIG. 7 is a graph showing the effect of RDK on reducing the number of Iba-1 positive cells activated in the prefrontal lobe region of mice model for vascular dementia induced by chronic cerebral ischemia, # # compared to control group with p <0.001, # compared to model group with p <0.001, no significant difference in ns;

FIG. 8 is a graph showing the effect of RDK on reducing the number of GFAP positive cells activated in the prefrontal lobe region of mice model for vascular dementia induced by chronic cerebral ischemia, # compared to control group, p <0.001, ns no significant difference compared to model group;

FIG. 9 shows the effect of RDK on reducing GD protein expression in the prefrontal lobe region of mice in a model of vascular dementia induced by chronic cerebral ischemia, with # # compared to control group p <0.001, ns not significantly different from model group;

FIG. 10 is a graph showing the effect of RDK on reducing ACE-2 positive cells in the prefrontal lobe region of mice in a model of vascular dementia induced by chronic cerebral ischemia, with # # compared to control group p <0.001, # compared to model group p <0.001, ns without significant difference;

FIG. 11 is a graph showing the effect of RDK on reducing VEGF-positive cells in the prefrontal region of mice model for vascular dementia induced by chronic cerebral ischemia, # compared to control group, p <0.001, ns are not significantly different from model group;

FIG. 12 is a graph showing the effect of RDK on reducing CD31 positive cells in the prefrontal lobe region of mice in a model of vascular dementia induced by chronic cerebral ischemia, where # # is p <0.001 compared to control, p <0.001 compared to model, and ns is not significantly different;

fig. 13 shows RDK function of alleviating nerve fiber loss in the callus area of mice with chronic cerebral ischemia induced vascular dementia model, p <0.05 compared to model group (CCH group), p <0.01 compared to model group (CCH group), p <0.001, ns are not significantly different.

Detailed Description

The technical scheme of the invention is further described below with reference to the accompanying drawings.

1. Experimental materials:

the drug licarin b (RDK) was prepared by the laboratory: nutmeg is processed by breaking shell, extracting with 95% ethanol for 2 times and 2 hr each time, and recovering extractive solution to obtain dry extract. Suspending the dry extract with water, and extracting with ethyl acetate for 3 times to obtain ethyl acetate extract. Dissolving ethyl acetate extract with n-hexane-methanol-water (7:6:1, v/v), injecting into high-speed countercurrent chromatography, taking n-hexane-methanol-water (7:6:1, v/v) upper phase as stationary phase, lower phase as mobile phase, rotating at 1000r/min, flowing at 2.2ml/min, detecting wavelength 254nm, collecting 35-60min effluent, and drying under reduced pressure at low temperature to obtain crude extract. The crude extract was dissolved in methanol and prepared using a c18 column. The volume ratio of the acetonitrile-water solution of the mobile phase is 50: the 50 gradient increases to 90:10, detecting wavelength 254nm at flow rate of 3ml/min, collecting effluent liquid of 15-20min, and drying under reduced pressure at low temperature to obtain licarin b (RDK) with chemical formula:

2. experimental animals:

C57/BL6 mice were purchased from the university of Yangzhou comparative medical center, 25-28g, two months old, free diet.

The experimental mice were divided into: sham mice, vascular dementia (CCH) mice, cch+rdk mice, RDK mice.

3. The experimental method comprises the following steps:

establishment of chronic cerebral ischemia model (Bilateral common carotid artery stenosis, CCH)

After the mice are induced by 3% sevoflurane inhalation anesthesia, the anesthesia is maintained by 2% sevoflurane, the limbs are fixed on an operating table in a supine position by using medical adhesive tape, and the incisors are fixed by a conical mask. Removing the median hair before neck, cutting skin longitudinally along the median line before neck with sterilized ophthalmic scissors, then separating subcutaneous tissue and muscle layer by layer, blunt-separating until the trachea is exposed, finding and carefully peeling carotid sheath at the triangle before neck on both sides of the trachea, carefully separating left and right carotid arteries, and taking care to avoid stimulating vagus nerve as much as possible.

CCH group mice were wound around the bilateral common carotid artery at the carotid bifurcation using a micro-spring (Stannless Sami you spring Co., ltd.) with an inner diameter of 0.18mm, resulting in a lumen narrowing. The mice were placed on a heating blanket for heat preservation throughout the procedure. The incision is sutured layer by layer after surgery, and antibiotic treatment is used for preventing infection. The respiration, heartbeat frequency and limb complexion of the mice are closely observed in the operation.

Sham mice (Sham): normal mice were subjected to the same procedure as described above, and bilateral common carotid arteries were not wrapped with a micro-spring, and the incision was sutured after surgery and returned to the incubator for resuscitation.

EXAMPLE 1 alleviation of cognitive function and learning memory dysfunction in vascular dementia mice by RDK

1. Nest making experiment

Nesting behavior in mice is a significant survival skill and genetic trait. The nesting experiment is carried out in a squirrel cage without special instruments and facilities. Furthermore, the effect of stress on mice is almost eliminated as there is no human intervention and training phase. On the day of testing, the mice to be tested were transferred to the test area to be acclimatized. Each mouse was provided with square cotton pieces of 3 gram weight and then individually housed in cages. The nesting process requires coordinated movement of the mouse's mouth and forelimbs, which requires the cotton pieces to be shredded and then placed into a nest. The invention performs experiments at night and records the state of the nest after 18 hours so as to avoid damaging the nest. For nesting scoring, a scoring criteria from 1 to 5 was used.

Following vascular dementia surgery, RDK was continuously injected intraperitoneally for 30 days (concentration 5mg/ml, measured as 50mg/Kg mice), and a littermates experiment was performed 4 hours after the 30 th day of RDK injection to examine the memory function of the mice. The results are shown in figure 1, with a significant increase in the mice score (3.75±0.95) for the cch+rdk group compared to the CCH group (1.25±0.5), and no significant change in the RDK group (4.5±0.57) compared to the Sham group (4.3±0.87), indicating that RDK significantly alleviates vascular dementia in mice with memory dysfunction.

2. Y maze experiment

The spatial learning and memory of mice are evaluated by spontaneous alternation experiments using the natural habit of rodents on new and different environment exploration. The Y maze consisted of 3 identical arms, each arm angled 120 degrees, with a movable partition at the center. The initial arm of the Y maze was designated as a, the mouse was placed facing the maze wall into arm a, the remaining two arms B and C, respectively. All of the mouse's limbs entered one arm were considered to enter that arm. The mice were allowed to explore freely for 8 minutes, record which arm the mice entered in sequence, and if the mice stayed in one arm for more than 60s, the mice were moved to the center of the Y maze and the experiment was continued. After each test, the maze arm was rubbed with 75% alcohol to prevent animal residual odor interference. Three different arms were continuously visited for spontaneous alternation, and the percent spontaneous alternation, i.e., [ (number of alternations)/(total number of arms-2) ]. Times.100%, was used as an experimental index. As shown in FIG. 2, after vascular dementia surgery, RDK was continuously injected intraperitoneally for 30 days (concentration: 5mg/ml, 50mg/Kg mice) and the memory function of the mice was examined by Y maze test 4 hours after the 30 th day of injection of RDK: the correct circulation rate (78.2% 4.5+/-1.67) of mice in the CCH+RDK group is obviously improved compared with that of mice in the CCH group (60.3+/-5.33), the RDK group (RDK is continuously injected intraperitoneally for 30 days, and the study memory capacity of the mice is detected by carrying out Y maze experiments 4 hours after 30 th day of RDK injection) (81.3+/-4.49) is not obviously changed compared with that of mice in the Sham group (81.2+/-5.62), so that the RDK can obviously relieve the condition of vascular dementia.

2.3 social experiments

The social experimental device is a rectangular box made of transparent polycarbonate and having a length of 40 cm x 60 cm x 22 cm, and two channels for separating the telescopic switches are arranged in the middle, so that mice can freely pass through the three compartments (A, B and C).

1) Putting the mice to be tested into a device, and enabling the mice to freely explore and move for 5 minutes; taking out the mice, wiping the experimental device with alcohol, and ventilating for 5 minutes;

2) The first stage: putting a completely strange C57/BL6 mouse (A mouse) with the same age and the same sex on one side (A chamber) of the box, putting the mouse to be detected into the box (C chamber) on the other side again, opening the equipment to record the contact time and the contact times of the mouse to be detected and the strange mouse, and finishing exploration after 10 minutes; the mice were removed, the experimental setup was wiped with alcohol and vented for 5 minutes.

3) And a second stage: after 30 minutes, the mice (A mice) contacted in the previous stage and another completely strange mice (B mice) with the same age and the same sex are placed at the two sides of the box, the mice to be tested are placed in the middle compartment (B chamber) again, the equipment is opened to record the contact time and the contact times of the mice to be tested and the strange mice, and after 10 minutes, the exploration is finished. The results are shown in FIGS. 3A and 3B (Strsgger 1 in FIG. 3A represents the time of contact between the first stage test mice and A mice, empty represents the time of contact between the first stage test mice and A mice, family represents the time of contact between the second stage test mice and A mice, and Novel represents the time of contact between the second stage test mice and B mice). Sham group mice had a contact time of 140.4±18s with a mice at the first stage, which was significantly increased over the contact time (47.2±7.3 s) with Empty cages (Empty); the contact time with the B mice in the second stage is 119s plus or minus 1.87s, which is obviously increased compared with the contact time (79 plus or minus 1.79 s) with the A mice (family), which shows that the social and cognitive functions of the Sham group mice are normal; after chronic cerebral ischemia surgery, the contact time of CCH group mice with A mice in the first stage is 75.3+/-9.7 s, and the CCH group mice have no significant difference compared with the contact time (59.1+/-8.7 s) of an Empty cage (Empty); the contact time with the B mice in the second stage is 57s plus or minus 13.2s, and the contact time with the A mice (family) is not significantly different from that of the A mice (60.4 plus or minus 1.8 s), which indicates that the social and cognitive functions of the CCH mice are significantly impaired; after chronic cerebral ischemia surgery, RDK is continuously injected into abdominal cavity for 30 days (the concentration is 5mg/ml, the mice are measured as 50 mg/Kg), social experiments are carried out 4 hours after the injection of the RDK on the 30 th day to detect the memory function of the mice, the contact time of CCH+RDK group mice with A mice in the first stage is 89.2+/-12.2 s, and the contact time (25.3+/-2.8 s) of the mice is obviously increased compared with that of the mice with Empty cages (Empty); the contact time with B mice in the second stage was 85.7.+ -. 3.9s, which was significantly increased over the contact time with A mice (family) (52.2.+ -. 1.8 s). Compared with the Sham group, the contact time of the first stage of the RDK group (RDK continuous intraperitoneal injection for 30 days, social experiments performed 4 hours after the 30 th day of RDK injection for detecting the learning and memory capacity of mice) with the A mice is 141.2+/-13.9 s, and the contact time of the first stage with the A mice is obviously increased compared with the contact time with an Empty cage (Empty) (56.6+/-6 s); the contact time with B mice in the second stage was 118.8 s.+ -. 1.4s, which was significantly increased over the contact time with A mice (family) (75.8.+ -. 1.8 s). It is shown that RDK can significantly improve the memory dysfunction of mice with vascular dementia induced by chronic cerebral ischemia.

2.4Morris Water maze experiment

Morris water maze experiment: the water maze is a cylindrical pool filled with water and is equally divided into four quadrants of northwest, northeast, southwest and southeast, the water temperature is kept at 23-25 ℃, and a transparent escape platform with the diameter of 10cm is fixedly arranged in the northeast quadrant, and is kept lower than the water surface by 1cm. The four visible markers were fixed as spatial localization markers in different quadrants and remained unchanged throughout the experiment. The water maze is surrounded by a single-color non-pattern cloth curtain, and is isolated from the surrounding environment. The experimenter placed the mice in the pool and immediately stood or sat in a fixed position and remained quiet throughout the experiment, and the mice were wiped dry and returned to the cage after each experiment was completed. A positioning sailing experiment was performed for 5 days, the mouse face wall was put into water, and the experiment was performed 4 times per day (fixed period of time), and water was introduced from the same starting point. If the mouse finds the platform within 60s, it is resting on the platform for 5s and then the next experiment is performed, and the escape latency is the time required from entering water to finding the platform. If the platform cannot be found within 60s, the escape latency is recorded as 60s, the mice are guided to the platform, and the next experiment is carried out after resting on the platform for 20 s. The visual track software records the escape latency, swimming speed and total swimming distance of the mice, and takes the average value of 4 experiments of the mice as the daily learning score of the mice. As shown in fig. 4A, the five-day latency (60, 58±2, 44±12, 49±8, 17±7) s of the cch+rdk group mice decreased significantly faster than the latency (60, 56±8, 53±10, 59±0.8, 60) s of the CCH group.

Space exploration experiments are carried out on the 6 th day, the original platform is removed, 1 water inlet point is selected, all mice are tested according to the sequence, the same water inlet point is needed, and the number of times that the platform is in the quadrant and the time that the platform stays in the quadrant when the tested mice pass through the first 5 days of testing in 60s are recorded. As shown in fig. 4B and 4C, after vascular dementia surgery, RDK was continuously injected intraperitoneally for 30 days (concentration is 5mg/ml, measured as 50mg/Kg mice), the study and memory abilities of mice were tested by Morris water maze test 4 hours after RDK30 days injection, the number and time (4.8±1.92, 14±2.57 s) of the quadrants where the mice of the cch+rdk group entered the platform was significantly increased compared to the number (1.8± 0.83,6.6 ±3.25 s) of the CCH group, RDK group (RDK was continuously injected intraperitoneally for 30 days, and the study memory abilities of mice were tested by Morris water maze test 4 hours after the 30 th day of RDK injection) was not significantly different from that of the Sham group. The RDK can obviously improve the learning and memory capacity of vascular dementia mice.

EXAMPLE 2 protective Effect of RDK on neurons in the prefrontal region of vascular dementia model mice

Experimental animals, experimental groups and vascular dementia models were prepared as in example 1.

After the behavioural experiment was completed, each group of animals was anesthetized, perfused through the left ventricle with 30ml of physiological saline, and then 4% pfa was injected for whole body fixation, and the whole brain was taken after head breakage. The whole brain was immersed in 15%, 20% and 30% sucrose water in sequence, and frozen into slices, to obtain 5 μm thick slices. Placing the slices in 1x EDTA (pH 9.0) antigen retrieval liquid, placing in a microwave oven, and repairing with medium and low fire for 8min at intervals of 8min, and then repairing with medium and low fire for 8min. After natural cooling, 1xPBS (0.01M, PH7.4) was washed 3 times. 3%H by ₂ O ₂ Incubating for 15min, and washing with 1XPBS for 3 times; after 30min incubation with normal blocking serum, rabbit-derived NeuN primary antibody (ET 1602-12, hangzhou Hua's Biotechnology Co., ltd.) was added to the mixture and the mixture was shaken overnight at 4 ℃; washed 3 times with PBS, incubated with HRP-labeled goat anti-rabbit secondary antibody (abcam, AB 6721) for 1h at room temperature, washed 3 times with PBS, counterstained and blocked. The number of NeuN positive cells in the frontal lobe area was observed under the microscope for each group of mice. As shown in FIG. 5, there were a large number of NeuN positive cells (1739.+ -. 107) in the prefrontal region of the Sham group mice, and the cell bodies and dendrites were intact; the dose dependency of the NeuN positive cells (880+/-25.6) in the forehead leaf area of the mice in the CCH group is obviously reduced compared with that of the Sham group, and the cell axons are incomplete. After chronic cerebral ischemia operation, RDK is continuously injected into abdominal cavity for 30 days (the concentration is 5mg/ml, the measurement is 50 mg/Kg), the head breakage and brain line immunohistochemical staining are carried out 4 hours after the 30 th day of injection of RDK, and the number of NeuN positive cells of CCH+RDK mice is 2130+/-115, so that the number is obviously increased. Compared with the RDK group (RDK continuous intraperitoneal injection for 30 days, and brain line immunohistochemical staining detection (2311+/-49.2) by taking a broken head 4 hours after the 30 th day of RDK injection), the NeuN positive cell number of the Sham group mice is not significantly different. The above results indicate that RDK can be inhibited byInflammatory responses in the brains of CCH-producing mice were used to alleviate neuronal damage.

EXAMPLE 3 inhibition of neuronal apoptosis in brain of mice model of vascular dementia by RDK

Tunel detection

The frozen sections obtained in example 2 were left to air dry on a shelf at room temperature for 20 minutes. The sections were immersed in 4% paraformaldehyde, fixed at room temperature for 30 minutes, and then washed in 1×pbs using a decolorizing shaker for 3×5 minutes. The surrounding liquid was carefully blotted using filter paper, and then 100. Mu.L of proteinase K solution at a concentration of 20. Mu.g/mL was added dropwise to each sample, ensuring coverage of the entire sample area and incubation at room temperature for 10 minutes. Followed by 3X 5min washes with 1X PBS to remove residual proteinase K. To each sample, 100. Mu.L TUNEL equilibration buffer (140 mmol/L sodium dipotassium arsonate, pH 7.2) was added, incubated for 5 minutes, then the equilibration buffer was removed, and the excess liquid around the sectioned sample was carefully aspirated using filter paper. Next, 50. Mu.L TUNEL reaction buffer was added to uniformly cover the sample. The sections were placed flat in a wet box and incubated in a 37℃incubator protected from light for 2 hours. The reaction solution was discarded, and immersed and rinsed 3 times for 5 minutes with 1×pbs. DAPI dye was added dropwise to each sample at a concentration of 2. Mu.g/mL, and incubated at room temperature for 10 minutes in the dark. The dye was then decanted and the sections were rinsed by immersing in 1×pbs for 3×5 minutes. Finally, 50. Mu.L of anti-fluorescence quenching coverslip solution was added dropwise to each sample, and covered with a coverslip. Images were observed and collected using a zeiss fluorescence microscope (Axio ver. A1) and the number of TUNEL positive cells was analyzed using Image J software. As shown in fig. 6, only a small number (136±34.5) of apoptotic neurons (labeled green fluorescent) were seen in the brains of Sham group mice, and a large number of Tunel positive (green fluorescent) cells (apoptotic neurons) (1008±238) were present in the brains of CCH group mice. After chronic ischemia surgery, RDK was continuously injected intraperitoneally for 30 days (concentration: 5mg/ml, 50mg/Kg mice) and brains were broken and taken for Tunel test at 4 hours after the 30 th day of RDK injection, and the number of Tunel positive cells was significantly reduced (179.+ -. 135) in CCH+RDK group mice, and was 74.3.+ -. 21.5 (RDK was continuously injected intraperitoneally for 30 days and brains were broken and taken for 4 hours after the 30 th day of RDK injection) without significant difference from the number of Tunel positive cells in brains of Sham group mice. The RDK has obvious inhibiting effect on neuronal apoptosis in the brain of mice with vascular dementia induced by chronic cerebral ischemia.

EXAMPLE 4 inhibition of RDK on brain inflammation in mice with a model of cerebral hemorrhage

The experimental animals, experimental groups and cerebral hemorrhage models were prepared as in example 1.

Staining analysis of inflammatory proteins such as Iba-1 and GFAP in forehead leaf area of vascular dementia mice

After the behavioural experiment was completed, each group of animals was anesthetized, perfused through the left ventricle with 30ml of physiological saline, and then 4% pfa was injected for whole body fixation, and the whole brain was taken after head breakage. The whole brain was immersed in 15%, 20% and 30% sucrose water in sequence, and frozen into slices, to obtain 5 μm thick slices. Placing the slice in EDTA (pH 9.0) antigen retrieval liquid, and placing in a microwave oven for low-fire retrieval for 8min. After natural cooling, PBS was washed 3 times. 3%H by ₂ O ₂ Incubating for 15min, and washing 3 times with PBS; after 30min incubation with normal blocking serum, rabbit-derived Iba-1 (Hua 'an, ET 1705-78) and rabbit-derived GFAP (Hua' an, ET 1601-23) primary antibodies were added to the shaker overnight at 4 ℃; PBS was washed 3 times, incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:200) (abcam, AB 6721) for 1h at room temperature, washed 3 times with PBS, counterstained with hematoxylin and blocked. The number of mice in each group were observed microscopically for the frontal lobe zone Iba-1 and GFAP positive cells. As shown in fig. 7 and 8, sham group mice had a lower amount of Iba-1 and GFAP positive cells in the frontal lobe region (51047 ±1373, 151571 ±10036, respectively); the numbers of the mice in the CCH group in the frontal lobe areas Iba-1 and GFAP positive cells were significantly increased (827200 + -22396, 8368167 + -152579, respectively), and the Iba-1 and GFAP positive cells were in an amoeba-like morphology, indicating that microglia and astrocytes were activated and increased in number in the brain. After chronic cerebral ischemia operation, RDK is continuously injected into abdominal cavity for 30 days (the concentration is 5mg/ml, the mice are measured as 50 mg/Kg), brains are broken and taken out for immunohistochemical staining detection after 4 hours after the 30 th day of RDK injection, and the number of Iba-1 and GFAP positive cells (277005 +/-11892, 60835 +/-2078 respectively) of mice in ICH+RDK group is significantThe number of activated microglia is significantly reduced. Compared with the Sham group, the RDK group (RDK continuous intraperitoneal injection for 30 days, 4 hours after the 30 th day of injection, head breaking and brain taking are performed on immunohistochemical staining detection) has significantly reduced the numbers of the frontal lobe areas Iba-1 and GFAP positive nerve cells (8720+/-734.9, 56199+/-8842 respectively). The above results indicate that RDK can alleviate neuronal damage by inhibiting inflammatory responses in the brain of CCH mice.

EXAMPLE 5 inhibition of brain apoptosis by RDK in mice with brain hemorrhage model

Analysis of staining of focal proteins such as GD in forehead leaf area of vascular dementia mice

After the behavioural experiment was completed, each group of animals was anesthetized, perfused through the left ventricle with 30ml of physiological saline, and then 4% pfa was injected for whole body fixation, and the whole brain was taken after head breakage. The whole brain was immersed in 15%, 20% and 30% sucrose water in sequence, and frozen into slices, to obtain 5 μm thick slices. Placing the slice in EDTA (pH 9.0) antigen retrieval liquid, and placing in a microwave oven for low-fire retrieval for 8min. After natural cooling, PBS was washed 3 times. 3%H by ₂ O ₂ Incubating for 15min, and washing 3 times with PBS; after 30min incubation with normal blocking serum, adding rabbit-derived GSDMD (GD) primary antibody (abcam, EPR 20859) at 4deg.C, shaking overnight; washed 3 times with PBS, incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:200) (abcam, AB 6721) for 1h at room temperature, washed 3 times with PBS, counterstained with hematoxylin and blocked. The number of GD positive cells in the frontal lobe zone of each group of mice was observed under a microscope. As shown in fig. 9, sham group mice had a lower GD positive cell mass in the forehead leaf area (28.3±19.6); the number of GD positive cells in the frontal lobe zone of the mice in the CCH group was significantly increased (2865±67.5), indicating that the brain coke-death response was activated. After chronic cerebral ischemia surgery, RDK is continuously injected into the abdominal cavity for 30 days (the concentration is 5mg/ml, the measurement is 50mg/Kg of mice), brains are broken and taken out for immunohistochemical staining detection after 4 hours after the 30 th day of RDK injection, the number of GD positive cells (87+/-28.8) of mice in ICH+RDK groups is obviously reduced, and the scorching reaction is obviously reduced. Compared with Sham group, RDK group (RDK continuous intraperitoneal injection for 30 days, 4 hours after 30 days of injection, head-broken brain extraction for immunohistochemical staining)Color detection) (429±112) showed no significant change from GD positive cells. The above results indicate that RDK can alleviate neuronal damage by inhibiting the focal death response in the brain of CCH mice.

EXAMPLE 6 inhibition of vascular dysplasia by RDK in mice model of vascular dementia

After the behavioural experiment was completed, each group of animals was anesthetized, perfused through the left ventricle with 30ml of physiological saline, and then 4% pfa was injected for whole body fixation, and the whole brain was taken after head breakage. The whole brain was immersed in 15%, 20% and 30% sucrose water in sequence, and frozen into slices, to obtain 5 μm thick slices. Placing the slices into EDTA (PH 9.0) antigen retrieval liquid, and placing the slices into a microwave oven for low-fire retrieval for 10-15 min. After natural cooling, PBS was washed 3 times. 3%H by ₂ O ₂ Incubating for 15min, and washing 3 times with PBS; adding primary antibodies such as ACE-2 (cell signaling, # 92485), VEGF (abcam, ab 1316), CD31 (abcam, ab 28364) and the like after incubating with normal blocking serum for 30min, and shaking overnight at 4deg.C; washed 3 times with PBS, incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:200) (abcam, AB 6721) for 1h at room temperature, washed 3 times with PBS, counterstained with hematoxylin and blocked. The number of positive cells of ACE-2, VEGF, CD31, etc. was observed microscopically in the frontal lobe area of each group of mice. As shown in fig. 10-12, sham group mice showed less ACE-2, VEGF, CD31 total OD values in the frontal lobe region (55224 ±2144, 92200 ±2229, 15198±188, respectively); the total OD values of the frontal lobe areas ACE-2, VEGF and CD31 of the CCH mice are obviously increased (260174 +/-2045, 187060 +/-2797 and 180860 +/-2869 respectively), which indicates that reactive vascular proliferation occurs in the brain. After chronic cerebral ischemia surgery, RDK is continuously injected into abdominal cavity for 30 days (the concentration is 5mg/ml, the mice are measured as 50 mg/Kg), brains are broken and taken out for immunohistochemical staining detection after 4 hours after the 30 th day of injection of RDK, and the total OD values (76733 +/-1637, 65916 +/-2416 and 9753+/-98 respectively) of mice ACE-2, VEGF and CD31 in ICH+RDK groups are obviously reduced compared with those in Sram groups, so that the RDK can obviously relieve the condition of reactive vascular proliferation of CCH mice. Compared with the normal control group, the RDK group (RDK continuous intraperitoneal injection for 30 days, 4 hours after 30 days of injection, head breaking and brain extraction are carried out for immunohistochemical staining detection) has the total OD values of mice ACE-2, VEGF and CD31 (70 respectively533±503, 73025 ±4769, 67414 ±5459). The results show that RDK can inhibit the cerebral vascular reactivity proliferation of CCH mouse model.

EXAMPLE 7 inhibition of RDK on demyelination in brain of mice in a model of cerebral hemorrhage

Dewaxing paraffin sections until 95% ethanol is added into LFB dye liquor, standing overnight at room temperature, adding 95% ethanol to wash out redundant dye liquor, washing with distilled water, adding differentiation liquor to separate colors for 15s, adding 70% ethanol to separate colors for 30s, washing with water, observing under a microscope until the gray matter and white matter are clear in outline, adding tar violet dye liquor to counterstain, washing with water, dehydrating and transparency, and sealing with neutral resin. Grading: grade 0 is normal; level 1 is a nerve fiber arrangement disorder; grade 2 is significant vacuole formation; grade 3 is the disappearance of the medullary fibers. As shown in FIG. 13, the Sham group mice had a higher density of blue fibers in the callus area (242.3.+ -. 23.8), indicating a more complete myelin sheath; the density of blue fibers in the corpus callosum area was significantly reduced in the CCH group mice (34.7.+ -. 4.16). After chronic cerebral ischemia surgery, RDK is continuously injected into abdominal cavity for 30 days (the concentration is 5mg/ml, and the measurement is 50mg/Kg of mice), the mice are subjected to end-cutting and brain line LFB staining detection at 4 hours after the 30 th day of RDK injection, and the density of blue fibers (330.7 +/-22) of the callus areas of CCH+RDK mice is obviously increased in a dose-dependent manner. There was no significant difference between the RDK group (RDK continuous intraperitoneal injection for 30 days, and 4 hours after day 30 of RDK injection, brain extraction for LFB staining) (330.+ -.34) compared to the normal control group. The above results indicate that RDK can alleviate neuronal damage by inhibiting demyelination in the brain of CCH mice.

Claims

1. Application of licarin b in preparing medicines for treating and/or relieving vascular dementia is provided.

2. Use of a formulation comprising licarin b for the preparation of a medicament for the treatment and/or alleviation of vascular dementia.

3. The use according to claim 1 or 2, wherein the treatment and/or alleviation of vascular dementia comprises one or more of improvement of learning dysfunction, improvement of memory dysfunction or improvement of depressed mood.

4. The use according to claim 1 or 2, wherein the treatment and/or alleviation of vascular dementia comprises one or more of inhibition of inflammatory responses in the brain, inhibition of pyro-lysis responses in the brain, improvement of demyelination in the brain or inhibition of neuronal apoptosis.

5. The use according to claim 1 or 2, characterized in that the concentration of licarin b is 5mg/ml.

6. Use according to claim 1 or 2, characterized in that the licarin b is used in an amount of 50mg/Kg.

7. The use according to claim 1 or 2, wherein the licarin b has the chemical formula:

8. the use according to claim 2, wherein the formulation comprises a tablet, capsule, decoction, pill, granule, dripping pill, mixture, injection or oral liquid.