Use of lycorine b and formulations thereof for the preparation of a medicament for the treatment and / or alleviation of vascular dementia

Abstract

The application discloses application of lycorine B and a preparation thereof in preparation of a medicine for treating and / or relieving vascular dementia. The lycorine B and the preparation thereof can obviously improve neuron reduction in a prefrontal area of a mouse model of chronic cerebral ischemia-induced vascular dementia, inhibit inflammatory reaction in the prefrontal area of the mouse model of vascular dementia, inhibit neuron pyroptosis and apoptosis in the prefrontal area, inhibit demyelination in the brain, inhibit vascular reactivity hyperplasia in the brain, and play a neuron protection role. On a whole animal level, the lycorine B and the preparation thereof can relieve cognitive dysfunction of the mouse of vascular dementia and increase neuron quantity in the brain. Therefore, the lycorine B and the preparation thereof can be used for treating vascular dementia and have a development prospect of a medicine.

Description

Technical Field

[0001] This invention relates to the use of licoriceb and its formulations in the preparation of drugs for treating and / or alleviating vascular dementia, and belongs to the field of vascular dementia treatment. Background Technology

[0002] Vascular dementia is a broad concept encompassing a range of syndromes with cognitive impairment symptoms ranging from mild to severe, and it has become a common cause of chronic progressive cognitive impairment in the elderly. One of the main triggering factors for vascular dementia is chronic cerebral ischemia. Chronic cerebral ischemia is a state of reduced blood supply to the brain at the overall level and is a common outcome of vascular risk factors, heart disease or hemodynamic changes, and alterations in blood composition. Patients often experience varying degrees of cognitive impairment, and vascular cognitive impairment caused by chronic cerebral ischemia accounts for half of all cases.

[0003] Studies have shown that this clinical syndrome has significant intervention value. Although it can be prevented through lifestyle modifications and control of vascular risk factors, its early diagnostic sensitivity is not high, and treatment options are limited. Currently, clinical treatment for vascular dementia mainly includes conservative medical treatment, but there is a lack of drugs and surgical procedures with high-level evidence-based medicine. Therefore, current treatment for vascular dementia mainly focuses on eliminating primary inducing factors such as hypertension, hyperglycemia, and hyperlipidemia, improving brain metabolism, combating cerebral hypoxia, and improving cognitive impairment. Developing new therapeutic targets is an urgent need for both basic research and clinical diagnosis and treatment.

[0004] The chemical formula of lecarmine β (RDK) is C. 20 H 20 O4 is a white crystalline solid. Studies have found that RDK has antibacterial, anti-inflammatory, sedative, antitumor, and parasitic disease prevention effects. However, there are no reports on the use of RDK in the treatment of vascular dementia. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide the application of licoriceb and its preparations in the preparation of drugs for treating vascular dementia.

[0006] Technical solution: To solve the above-mentioned technical problems, the present invention provides the application of licoriceb in the preparation of drugs for treating vascular dementia.

[0007] The treatment and / or relief of vascular dementia includes improving cognitive impairment, improving learning and memory function, inhibiting intracranial inflammatory response, inhibiting intracranial pyroptosis, improving demyelinating changes in the brain, and inhibiting neuronal apoptosis, or one or more of these effects.

[0008] The chemical structural formula of lecarin b is as follows: .

[0009] The dosage forms of the preparations include tablets, capsules, decoctions, pills, granules, drop pills, mixtures, injections, and oral liquids.

[0010] The concentration of Licarin B is 5 mg / ml.

[0011] The dosage of Licarin B is 50 mg / Kg.

[0012] The preparation method of the lecarin b includes the following steps:

[0013] (1) The nutmeg dry extract was suspended in water and extracted with ethyl acetate to obtain the ethyl acetate extract;

[0014] (2) Dissolve the ethyl acetate extract in step (1) with hexane-methanol-water, inject into high-speed countercurrent chromatography, collect the eluent after 35-60 min, dry under low temperature and reduced pressure to obtain crude extract;

[0015] (3) Dissolve the crude extract described in step (2), prepare it using a C18 chromatographic column, collect the eluent for 15-20 min, and dry it under low temperature and reduced pressure to obtain Delicalin B.

[0016] In step (2), the high-speed countercurrent chromatography uses hexane-methanol-water as the upper phase and the lower phase as the mobile phase, with a rotation speed of 1000 r / min, a flow rate of 2.2 ml / min, and a detection wavelength of 254 nm.

[0017] In step (3), the volume ratio of the mobile phase acetonitrile-water solution to the C18 column is gradually increased from 50:50 to 90:10 within 0-60 min, the flow rate is 3 ml / min, and the detection wavelength is 254 nm.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention discloses that RDK can clearly improve neuronal damage in a mouse model of vascular dementia induced by long-term chronic cerebral ischemia, inhibit intracranial inflammatory response, pyroptosis and neuronal apoptosis, inhibit demyelinating changes in the brain, and play a neuroprotective role; 2. At the whole animal level, it shows that it alleviates the learning and memory dysfunction in mice with vascular dementia; 3. It shows that RDK can be used for the treatment of vascular dementia and has the prospect of drug development. Attached Figure Description

[0019] Figure 1 To investigate the effect of RDK on memory and learning impairment in a mouse model of vascular dementia induced by long-term chronic cerebral ischemia, the results showed that ### compared with the control group, p<0.01, *** compared with the model group, p<0.001, and no significant difference in ns.

[0020] Figure 2 To assess the effect of RDK on alleviating memory decline in a Y-maze test in mice with a long-term chronic cerebral ischemia-induced vascular dementia model, ### p<0.001 compared with the control group, *** p<0.001 compared with the model group, ns showed no significant difference;

[0021] Figure 3 To assess the effect of RDK in alleviating cognitive impairment in a social test in a mouse model of vascular dementia induced by long-term chronic cerebral ischemia, *p<0.05 compared with empty cage or stranger1, **p<0.01 compared with empty cage or stranger1, ***p<0.001 compared with empty cage or stranger1, with no significant difference in ns.

[0022] Figure 4 To assess the effect of RDK in alleviating cognitive impairment in a mouse model of vascular dementia induced by long-term chronic cerebral ischemia during the water maze test, ### compared with the control group, p<0.001; *** compared with the model group, p<0.001; no significant difference in ns.

[0023] Figure 5 To determine the effect of RDK on significantly increasing the number of NeuN-positive cells in the prefrontal cortex of mice with vascular dementia induced by long-term chronic cerebral ischemia, ### p<0.001 compared with the control group, *** p<0.001 compared with the model group;

[0024] Figure 6 To investigate the effect of RDK on inhibiting the number of Tunel-positive cells in the brains of mice with a long-term chronic cerebral ischemia-induced vascular dementia model, ### p<0.001 compared with the control group, *** p<0.001 compared with the model group, and no significant difference in ns;

[0025] Figure 7 To assess the effect of RDK on reducing the number of activated Iba-1 positive cells in the prefrontal cortex of mice with a long-term chronic cerebral ischemia-induced vascular dementia model, ### p < 0.001 compared with the control group, *** p < 0.001 compared with the model group, and no significant difference in ns;

[0026] Figure 8 To assess the effect of RDK on reducing the number of activated GFAP-positive cells in the prefrontal cortex of mice with a long-term chronic cerebral ischemia-induced vascular dementia model, ### p<0.001 compared with the control group, *** p<0.001 compared with the model group, and no significant difference in ns;

[0027] Figure 9To assess the effect of RDK on reducing GD protein expression in the prefrontal cortex of mice with a long-term chronic cerebral ischemia-induced vascular dementia model, ### p<0.001 compared with the control group, *** p<0.001 compared with the model group, and no significant difference in ns;

[0028] Figure 10 To assess the effect of RDK on reducing ACE-2 positive cells in the prefrontal cortex of mice with a long-term chronic cerebral ischemia-induced vascular dementia model, ### p<0.001 compared with the control group, *** p<0.001 compared with the model group, and no significant difference in ns;

[0029] Figure 11 To assess the effect of RDK on reducing VEGF-positive cells in the prefrontal cortex of mice with a long-term chronic cerebral ischemia-induced vascular dementia model, ### p<0.001 compared with the control group, *** p<0.001 compared with the model group, and no significant difference in ns;

[0030] Figure 12 To assess the effect of RDK on reducing CD31-positive cells in the prefrontal cortex of mice with a long-term chronic cerebral ischemia-induced vascular dementia model, the results showed that ### p < 0.001 compared with the control group, *** p < 0.001 compared with the model group, and no significant difference in ns.

[0031] Figure 13 To alleviate the loss of nerve fibers in the corpus callosum region of mice with vascular dementia induced by long-term chronic cerebral ischemia, RDK showed the following differences: # compared with the model group (CCH group), p<0.05; ## compared with the model group (CCH group), p<0.01; ### compared with the model group (CCH group), p<0.001; ** compared with the normal control group, p<0.001; no significant difference in ns. Detailed Implementation

[0032] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0033] 1. Experimental materials:

[0034] The drug RDK (Licarin B) was prepared in the laboratory as follows: Nutmeg was crushed and extracted twice with 95% ethanol for 2 hours each time. The extract was then dried to obtain a dry extract. The dry extract was suspended in water and extracted three times with ethyl acetate to obtain an ethyl acetate extract. The ethyl acetate extract was dissolved in hexane-methanol-water (7:6:1, v / v) and injected into a high-speed countercurrent chromatogram. Using hexane-methanol-water (7:6:1, v / v) as the stationary phase and the lower phase as the mobile phase, the chromatogram was run at 1000 r / min, the flow rate was 2.2 ml / min, and the detection wavelength was 254 nm. The eluent was collected after 35-60 min and dried under reduced pressure at low temperature to obtain a crude extract. The crude extract was dissolved in methanol and prepared using a C18 column. Within 0-60 min, the volume ratio of the mobile phase acetonitrile-water solution was gradually increased from 50:50 to 90:10, the flow rate was 3 ml / min, the detection wavelength was 254 nm, and the eluent was collected for 15-20 min. The eluent was then dried under reduced pressure at low temperature to obtain lecarin b (RDK), with the chemical formula:

[0035] .

[0036] 2. Laboratory animals:

[0037] C57 / BL6 mice were purchased from the Comparative Medicine Center of Yangzhou University. They weighed 25-28g, were two months old, and were allowed free access to food.

[0038] The experimental mice were divided into: Sham group mice, vascular dementia (CCH) mice, CCH + RDK group, and RDK group.

[0039] 3. Experimental methods:

[0040] Establishment of a chronic cerebral ischemia model (Bilateral common carotid artery stenosis, CCH)

[0041] Mice were induced to have anesthesia with 3% sevoflurane inhalation, and anesthesia was maintained with 2% sevoflurane. The limbs were fixed to the operating table in a supine position with medical tape, and the incisors were fixed with a conical mask. Hair was removed from the midline of the neck, and the skin was longitudinally incised along the midline of the neck with sterilized ophthalmic scissors. Subcutaneous tissue and muscle were then bluntly dissected layer by layer until the trachea was exposed. The carotid sheaths were located and carefully dissected in the anterior cervical triangle on both sides of the trachea. The left and right common carotid arteries were carefully dissected, taking care to avoid stimulating the vagus nerve.

[0042] In the CCH group mice, a microspring (0.18 mm inner diameter, Wuxi Samini Spring Co., Ltd.) was used to wrap around both common carotid arteries at the bifurcation, causing luminal stenosis. Throughout the procedure, the mice were kept warm on a heating blanket. Postoperatively, the incision was sutured layer by layer, and antibiotics were administered to prevent infection. The mice's respiration, heart rate, and skin color on their limbs were closely monitored during the operation.

[0043] Sham-operated mice: Normal mice underwent the same surgery as described above, but the bilateral common carotid arteries were not wrapped with microsprings. The incisions were sutured after surgery and the mice were returned to an incubator for resuscitation.

[0044] Example 1: The alleviating effect of RDK on cognitive function and learning and memory impairment in mice with vascular dementia.

[0045] 1. Conduct a nesting experiment

[0046] Nesting behavior in mice is a significant survival skill and genetic trait. Nesting experiments do not require special equipment or facilities; they can be conducted simply in mouse cages. Furthermore, the absence of human intervention and training phases virtually eliminates the impact of stress on the mice. On the day of testing, the mice are transferred to the testing area to acclimatize. Each mouse is provided with a 3-gram square piece of cotton and then housed individually in a cage. The nesting process requires coordinated movements of the mouse's mouth, face, and forelimbs; they first tear the cotton piece and then arrange it into a nest. This invention conducts experiments at night, recording the state of the nests 18 hours later to avoid destroying them. Nesting performance is scored on a scale of 1 to 5.

[0047] Following surgery for vascular dementia, mice were administered RDK via intraperitoneal injection for 30 consecutive days (concentration 5 mg / ml, dosage 50 mg / kg mouse). Four hours after the RDK injection on day 30, a nesting test was performed to assess memory function. Results are as follows... Figure 1 As shown, the CCH+RDK group mouse score (3.75±0.95) was significantly higher than that of the CCH group (1.25±0.5), while the RDK group (4.5±0.57) showed no significant change compared with the Sham group (4.3±0.87), indicating that RDK can significantly alleviate memory dysfunction in vascular dementia mice.

[0048] 2. Y-maze experiment

[0049] Utilizing the natural tendency of rodents to explore novel environments, a spontaneous alternation experiment was conducted to assess spatial learning and memory in mice. The Y-maze consisted of three identical arms, each with a 120-degree angle, and a movable partition at the center. The starting arm of the Y-maze was designated A. The mouse was placed face-down on arm A, with the remaining two arms designated B and C. Entering an arm with all four limbs inside was considered entry into that arm. Mice were allowed free exploration for 8 minutes, and the sequence of arms entered was recorded. If a mouse remained on a particular arm for more than 60 seconds, it was moved to the center of the Y-maze, and the experiment continued. After each test, the maze arms were wiped with 75% alcohol to prevent interference from residual animal odors. Successive access to three different arms was considered spontaneous alternation, and the percentage of spontaneous alternation ([(number of alternations) / (total number of arms - 2)] × 100%) was used as the experimental indicator. Figure 2 As shown, after surgery for vascular dementia, mice were continuously injected intraperitoneally with RDK for 30 days (concentration of 5 mg / ml, dosage of 50 mg / Kg mice). Four hours after the injection on day 30, the Y-maze test was performed to detect the memory function of the mice. The correct cycle rate of the CCH+RDK group (78.2% ± 1.67) was significantly higher than that of the CCH group (60.3% ± 5.33). There was no significant change in the RDK group (RDK was continuously injected intraperitoneally for 30 days, and the learning and memory ability of the mice was detected by the Y-maze test 4 hours after the injection on day 30) (81.3% ± 4.49) compared with that of the Sham group (81.2% ± 5.62). This indicates that RDK can significantly alleviate the memory dysfunction in mice with vascular dementia.

[0050] 2.3 Social Experiments

[0051] The social experiment device is a rectangular box made of transparent polycarbonate, measuring 40 cm long, 60 cm wide, and 22 cm high. It has two retractable channels in the middle, allowing mice to freely pass through three compartments (compartment A, compartment B, and compartment C).

[0052] 1) Place the mouse to be tested into the device and allow it to explore and move freely for 5 minutes; remove the mouse, wipe the experimental device with alcohol, and ventilate for 5 minutes;

[0053] 2) First stage: Place a completely unfamiliar, same-age, same-sex C57 / BL6 mouse (Mouse A) in one side of the box (Chamber A), and then place the mouse to be tested in the other side of the box (Chamber C). Turn on the device to record the contact time and number of times between the mouse to be tested and the unfamiliar mouse. The exploration ends after 10 minutes. Take out the mouse, wipe the experimental device with alcohol, and ventilate for 5 minutes.

[0054] 3) Second stage: After 30 minutes, the mouse that had been in contact with the mouse in the previous stage (mouse A) and another completely unfamiliar age- and sex-matched C57 / BL6 mouse (mouse B) were placed on opposite sides of the box. The mouse to be tested was then placed back into the middle compartment (chamber B). The device was turned on to record the contact time and number of times between the mouse to be tested and the unfamiliar mouse. The exploration ended after 10 minutes. Results are as follows: Figure 3 A and Figure 3 As shown in B ( Figure 3In A, Strsnger1 represents the contact time between the test mouse and mouse A in the first stage, Empty represents the time the test mouse spent in an empty cage in the first stage; Familiar represents the contact time between the test mouse and mouse A in the second stage, and Novel represents the contact time between the test mouse and mouse B in the second stage. The contact time between the Sham group mice and mouse A in the first stage was 140.4 ± 18 s, significantly longer than the contact time in an empty cage (47.2 ± 7.3 s); the contact time with mouse B in the second stage was 119 ± 1.87 s, significantly longer than the contact time with mouse A (Familiar) (79 ± 1.79 s). The above results indicate that the social and cognitive functions of the Sham group mice were normal. After chronic cerebral ischemia surgery, the contact time between the CCH group mice and mouse A in the first stage was 75.3±9.7 s, which was not significantly different from the contact time in the empty cage (59.1±8.7 s). In the second stage, the contact time with mouse B was 57 s±13.2 s, which was not significantly different from the contact time with mouse A (familiar) (60.4±1.8 s). These results indicate that the social and cognitive functions of the CCH group mice were significantly impaired. After chronic cerebral ischemia surgery, mice were continuously injected intraperitoneally with RDK for 30 days (concentration 5 mg / ml, dosage 50 mg / Kg mouse). Four hours after the RDK injection on day 30, a social test was performed to assess the mice's memory function. The contact time between the CCH+RDK group mice and mouse A in the first stage was 89.2±12.2 s. The contact time with empty cages (25.3±2.8 s) was significantly increased in the second stage; the contact time with B mice was 85.7±3.9 s, significantly increased compared to the contact time with familiar mice (52.2±1.8 s). Compared to the Sham group, the RDK group (RDK was administered intraperitoneally for 30 days, and the learning and memory abilities of mice were assessed in the social test 4 hours after RDK injection) showed that the contact time with familiar mice in the first stage was 141.2±13.9 s, significantly increased compared to the contact time with empty cages (56.6±6 s); and the contact time with B mice in the second stage was 118.8±1.4 s, significantly increased compared to the contact time with familiar mice (75.8±1.8 s). This indicates that RDK can significantly improve memory impairment in mice with chronic cerebral ischemia-induced vascular dementia.

[0055] 2.4 Morris Water Maze Experiment

[0056] Morris water maze experiment: The water maze was a cylindrical pool filled with water, divided into four quadrants: northwest, northeast, southwest, and southeast. The water temperature was maintained at 23-25℃. A transparent escape platform with a diameter of 10 cm was fixed in the northeast quadrant, kept 1 cm below the water surface. Four visible markers were fixed in different quadrants as spatial positioning landmarks and remained unchanged throughout the experiment. The water maze was surrounded by a monochrome, unpatterned curtain to isolate it from the surrounding environment. After placing the mice in the pool, they immediately stood or sat in fixed positions and remained quiet throughout the experiment. After each experiment, the mice were dried and returned to their cages. A positioning and navigation experiment was conducted for 5 days. The mice were placed in the water facing the wall, and the experiment was performed 4 times a day (at fixed time intervals), entering the water from the same starting point. If the mouse found the platform within 60 seconds, it rested on the platform for 5 seconds before the next experiment. The escape latency was the time required from entering the water to finding the platform. If the mouse could not find the platform within 60 seconds, the escape latency was recorded as 60 seconds. The mouse was then guided to the platform, rested on the platform for 20 seconds, and the next experiment was conducted. The VisuTrack software recorded the mouse's escape latency, swimming speed, and total swimming distance, and used the average of four experiments as the mouse's learning performance for that day. Figure 4 As shown in Figure A, the five-day latency (60, 58±2, 44±12, 49±8, 17±7) s in the CCH+RDK group mice decreased significantly faster than the latency (60, 56±8, 53±10, 59±0.8, 60) s in the CCH group.

[0057] On day 6, a space exploration experiment was conducted. The original platform was removed, and each mouse was randomly placed into the water at one of the designated entry points. All mice were tested sequentially, ensuring they entered the same water point. The number of times each mouse traversed the quadrant where the platform was located during the previous five days' tests and the duration of its stay in that quadrant were recorded within 60 seconds. Figure 4 B and Figure 4 As shown in Figure C, after surgery for vascular dementia, mice received intraperitoneal injections of RDK for 30 consecutive days (concentration 5 mg / ml, dosage 50 mg / Kg mice). Four hours after the RDK injection on day 30, the Morris water maze test was performed to assess the mice's learning and memory abilities. The number of times and the time taken for mice in the CCH+RDK group to enter the platform quadrant (4.8±1.92, 14±2.57s) were significantly increased compared to the CCH group (1.8±0.83, 6.6±3.25s). However, the number of times and the time taken for mice in the RDK group (continuous intraperitoneal injection of RDK for 30 days, with the Morris water maze test performed 4 hours after the RDK injection) to enter the platform quadrant (6.2±1.78, 14.4±3.64s) were not significantly different from the Sham group. This indicates that RDK can significantly improve the learning and memory abilities of mice with vascular dementia.

[0058] Example 2: Protective effect of RDK on neurons in the prefrontal cortex of a mouse model of vascular dementia.

[0059] The experimental animals, experimental groups, and the preparation of the vascular dementia model were the same as in Example 1.

[0060] After the behavioral experiments, all animals were anesthetized, perfused with 30 ml of physiological saline via the left ventricle, and then injected with 4% PFA for systemic fixation. The animals were then decapitated and their entire brains were harvested. The brains were sequentially immersed in 15%, 20%, and 30% sucrose solutions, and then frozen sectioned to obtain 5 μm thick sections. The sections were placed in 1x EDTA (pH 9.0) antigen retrieval solution and microwaved on low-medium heat for 8 min, with an 8-min interval, followed by another 8-min microwave-safe retrieval. After natural cooling, the sections were washed three times with 1x PBS (0.01M, pH 7.4). Incubate with 3% H2O2 for 15 min, wash three times with 1X PBS; incubate with normal blocking serum for 30 min, then add rabbit-derived NeuN primary antibody (Hangzhou Huaan Biotechnology Co., Ltd., ET1602-12) and incubate overnight at 4℃ with shake; wash three times with PBS, incubate with HRP-labeled goat anti-rabbit secondary antibody (abcam, AB6721) at room temperature for 1 h, wash three times with PBS, counterstain and mount. Observe the number of NeuN-positive cells in the prefrontal cortex of mice in each group under a microscope. Figure 5 As shown, the Sham group mice had a large number of NeuN-positive cells (1739±107) in the prefrontal cortex, with intact cell bodies and dendrites; the CCH group mice had a significantly reduced number of NeuN-positive cells (880±25.6) in the prefrontal cortex compared to the Sham group, with incomplete cell axons. After chronic cerebral ischemia surgery, mice received continuous intraperitoneal injections of RDK for 30 days (concentration 5 mg / ml, dosage 50 mg / Kg). Four hours after the RDK injection on day 30, the brain was decapitated and subjected to immunohistochemical staining. The number of NeuN-positive cells in the CCH+RDK group mice was (2130±115), showing a significant increase. Compared with the RDK group (continuous intraperitoneal injection of RDK for 30 days, brain was decapitated and subjected to immunohistochemical staining four hours after the RDK injection on day 30) (2311±49.2), there was no significant difference in the number of NeuN-positive cells in the Sham group mice. These results indicate that RDK can alleviate neuronal damage by inhibiting the inflammatory response in the brains of CCH mice.

[0061] Example 3: Inhibitory effect of RDK on neuronal apoptosis in a mouse model of vascular dementia.

[0062] The experimental animals, experimental groups, and the preparation of the vascular dementia model were the same as in Example 1.

[0063] Tunel detection

[0064] The frozen sections obtained in Example 2 were air-dried on a slide rack at room temperature for 20 minutes. The sections were then soaked in 4% paraformaldehyde and fixed at room temperature for 30 minutes. They were then washed three times for 5 minutes each in 1×PBS using a decolorizing shaker. Liquid around the samples was carefully blotted dry with filter paper. 100 µL of a 20 µg / mL proteinase K solution was added to each sample, ensuring complete coverage, and incubated at room temperature for 10 minutes. Residual proteinase K was then removed by washing three times for 5 minutes each with 1×PBS. 100 µL of TUNEL equilibration buffer (140 mmol / L sodium diarsethate, pH 7.2) was added to each sample, and incubation was performed for 5 minutes. The equilibration buffer was then removed, and excess liquid around the sample was carefully blotted dry with filter paper. Next, 50 µL of TUNEL reaction buffer was added, evenly covering the sample. The sections were then placed flat in a humidified chamber and incubated at 37°C in the dark for 2 hours. Discard the reaction solution and rinse the slides three times for 5 minutes each with 1×PBS. Add 2 μg / mL DAPI staining solution to each sample and incubate at room temperature in the dark for 10 minutes. Then discard the staining solution and rinse the slides three times for 5 minutes each with 1×PBS. Finally, add 50 μL of anti-fluorescence quenching mounting solution to each sample and cover with a coverslip. Observe and acquire images using a Zeiss fluorescence microscope (Axio Vert. A1), and analyze the number of TUNEL-positive cells using ImageJ software. Figure 6 As shown, only a small number (136±34.5) of apoptotic neurons (labeled as green fluorescence) were observed in the brains of Sham group mice, while a large number of Tunel-positive (green fluorescence) cells (apoptotic neurons) (1008±238) were observed in the brains of CCH group mice. Following long-term chronic ischemic surgery, RDK was administered intraperitoneally for 30 consecutive days (concentration 5 mg / ml, dosage 50 mg / Kg mouse). Four hours after RDK injection on day 30, the brains were decapitated for Tunel analysis. The number of Tunel-positive cells in the CCH+RDK group mice was significantly reduced (179±135), while the number in the RDK group (30 consecutive days of intraperitoneal injection, with brain decapitation for Tunel analysis four hours after RDK injection) was 74.3±21.5, which was not significantly different from the number of Tunel-positive cells in the brains of Sham group mice. This indicates that RDK has a significant inhibitory effect on neuronal apoptosis in the brains of mice with vascular dementia induced by chronic cerebral ischemia.

[0065] Example 4: Inhibitory effect of RDK on brain inflammation in a mouse model of vascular dementia.

[0066] The experimental animals, experimental groups, and the preparation of the vascular dementia model were the same as in Example 1.

[0067] Staining analysis of inflammatory proteins such as Iba-1 and GFAP in the prefrontal region of mice with vascular dementia

[0068] After the behavioral experiments, the animals in each group were anesthetized, perfused with 30 ml of physiological saline through the left ventricle, and then injected with 4% PFA for systemic fixation. The brains were then decapitated and collected. The brains were sequentially immersed in 15%, 20%, and 30% sucrose solutions, and then frozen sectioned to obtain 5 μm thick sections. The sections were placed in EDTA (pH 9.0) antigen retrieval solution and microwaved on low to medium heat for 8 min. After natural cooling, they were washed three times with PBS. They were then incubated with 3% H2O2 for 15 min, washed three times with PBS, and incubated with normal blocking serum for 30 min. Rabbit-derived Iba-1 (Huaan, ET1705-78) and rabbit-derived GFAP (Huaan, ET-1601-23) primary antibodies were added and incubated overnight at 4°C with shake. The sections were washed three times with PBS, incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:200) (abcam, AB6721) at room temperature for 1 h, washed three times with PBS, counterstained with hematoxylin, and mounted. The number of Iba-1 and GFAP-positive cells in the prefrontal cortex of mice in each group was observed under a microscope. Figure 7 and Figure 8 As shown, the number of Iba-1 and GFAP positive cells in the prefrontal cortex of mice in the Sham group was relatively low (51047±1373 and 151571±10036, respectively); the number of Iba-1 and GFAP positive cells in the prefrontal cortex of mice in the CCH group was significantly increased (827200±22396 and 8368167±152579, respectively), and the Iba-1 and GFAP positive cells showed an amoeboid morphology, indicating that microglia and astrocytes in the brain were activated and increased in number. Following surgery for chronic cerebral ischemia, mice were administered RDK via intraperitoneal injection for 30 consecutive days (concentration 5 mg / ml, dosage 50 mg / kg mice). Four hours after injection on day 30, the mice were decapitated and subjected to immunohistochemical staining. The ICH+RDK group showed a significant decrease in the number of Iba-1 and GFAP-positive cells (277005±11892 and 60835±2078, respectively), and a significant reduction in the number of activated microglia. Compared to the Sham group, the RDK group (continuous intraperitoneal injection of RDK for 30 days, with brains harvested four hours after injection for immunohistochemical staining) showed a significant reduction in the number of Iba-1 and GFAP-positive neurons in the prefrontal cortex (8720±734.9 and 56199±8842, respectively). These results indicate that RDK can alleviate neuronal damage by inhibiting the inflammatory response in the brains of CCH mice.

[0069] Example 5: Inhibitory effect of RDK on pyroptosis in the brain of a mouse model of vascular dementia.

[0070] Staining analysis of pyroptosis proteins such as GD in the prefrontal region of mice with vascular dementia

[0071] The experimental animals, experimental groups, and the preparation of the vascular dementia model were the same as in Example 1.

[0072] After the behavioral experiments, the animals in each group were anesthetized, perfused with 30 ml of physiological saline through the left ventricle, and then injected with 4% PFA for systemic fixation. The brains were then decapitated and collected. The brains were sequentially immersed in 15%, 20%, and 30% sucrose solutions, and then frozen sectioned to obtain 5 μm thick sections. The sections were placed in EDTA (pH 9.0) antigen retrieval solution and microwaved on low-medium heat for 8 min. After natural cooling, they were washed three times with PBS. They were then incubated with 3% H2O2 for 15 min, washed three times with PBS, incubated with normal blocking serum for 30 min, and then incubated overnight at 4°C with rabbit-derived GSDMD (GD) primary antibody (abcam, EPR20859). After washing three times with PBS, they were incubated at room temperature for 1 h with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:200) (abcam, AB6721), washed three times with PBS, counterstained with hematoxylin, and mounted. The number of GD-positive cells in the prefrontal cortex of mice in each group was observed under a microscope. Figure 9 As shown, the number of GD-positive cells in the prefrontal cortex of mice in the Sham group was lower (28.3±19.6 cells); the number of GD-positive cells in the prefrontal cortex of mice in the CCH group was significantly increased (2865±67.5 cells), indicating that the pyroptosis response in the brain was activated. After chronic cerebral ischemia surgery, RDK was continuously injected intraperitoneally for 30 days (concentration of 5 mg / ml, dosage of 50 mg / Kg mice). Four hours after injection on day 30, the brain was decapitated and subjected to immunohistochemical staining. The number of GD-positive cells in the ICH+RDK group mice was significantly decreased (87±28.8 cells), indicating a significant reduction in the pyroptosis response. Compared with the Sham group, the number of GD-positive cells in the RDK group (RDK was continuously injected intraperitoneally for 30 days, and the brain was decapitated and subjected to immunohistochemical staining four hours after injection on day 30) (429±112 cells) showed no significant change. These results indicate that RDK can alleviate neuronal damage by inhibiting the pyroptosis response in the brain of CCH mice.

[0073] Example 6: Inhibitory effect of RDK on abnormal angiogenesis in a mouse model of vascular dementia

[0074] The experimental animals, experimental groups, and the preparation of the vascular dementia model were the same as in Example 1.

[0075] After the behavioral experiments, all animals were anesthetized, perfused with 30 ml of physiological saline through the left ventricle, and then injected with 4% PFA for systemic fixation. The animals were then decapitated and their entire brains were harvested. The brains were sequentially immersed in 15%, 20%, and 30% sucrose solutions, and then frozen sectioned to obtain 5 μm thick sections. The sections were placed in EDTA (pH 9.0) antigen retrieval solution and microwaved on low to medium heat for 10–15 minutes. After natural cooling, the sections were washed three times with PBS. Incubate with 3% H2O2 for 15 min, wash three times with PBS; incubate with normal blocking serum for 30 min, then add primary antibodies for ACE-2 (cell signaling, #92485), VEGF (abcam, ab1316), and CD31 (abcam, ab28364) and incubate overnight at 4°C with shake; wash three times with PBS, incubate with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:200) (abcam, AB6721) at room temperature for 1 h, wash three times with PBS, counterstain with hematoxylin and mount. Observe the number of ACE-2, VEGF, and CD31 positive cells in the prefrontal cortex of mice in each group under a microscope. Figures 10-12 As shown, the total OD values ​​of ACE-2, VEGF, and CD31 in the prefrontal cortex of mice in the Sham group were lower (55224±2144, 92200±2229, and 15198±188, respectively); while the total OD values ​​of ACE-2, VEGF, and CD31 in the prefrontal cortex of mice in the CCH group were significantly increased (260174±2045, 187060±2797, and 180860±2869, respectively), indicating reactive angiogenesis in the brain. Following surgery for chronic cerebral ischemia, mice were administered RDK via intraperitoneal injection for 30 consecutive days (concentration 5 mg / ml, dosage 50 mg / Kg mice). Four hours after injection on day 30, the mice were decapitated and their brains were harvested for immunohistochemical staining. The total OD values ​​of ACE-2, VEGF, and CD31 in the ICH+RDK group (76733±1637, 65916±2416, and 9753±98, respectively) were significantly lower than those in the Sham group, indicating that RDK significantly alleviated reactive angiogenesis in CCH mice. Compared with the normal control group, there was no significant difference in the total OD values ​​of ACE-2, VEGF, and CD31 in the RDK group (30533±503, 73025±4769, and 67414±5459, respectively) of mice. The above results indicate that RDK can inhibit reactive angiogenesis in the brain of CCH mouse models.

[0076] Example 7: Inhibitory effect of RDK on demyelination in the brain of a mouse model of vascular dementia.

[0077] The experimental animals, experimental groups, and the preparation of the vascular dementia model were the same as in Example 1.

[0078] Paraffin sections were dewaxed to 95% ethanol and stained with LFB stain overnight at room temperature. Excess stain was then washed away with 95% ethanol, rinsed with distilled water, and stained with differentiation solution for 15 seconds, followed by 70% ethanol for 30 seconds. After washing with water, the sections were observed under a microscope until the outlines of gray and white matter were clearly defined. The sections were then counterstained with tar violet stain, washed with water, dehydrated, cleared, and mounted with neutral resin. Grading: Grade 0 is normal; Grade 1 indicates disordered nerve fiber arrangement; Grade 2 indicates significant vacuolation; Grade 3 indicates disappearance of myelinated fibers. Figure 13 As shown, the Sham group mice had a higher density of blue fibers in the corpus callosum (242.3±23.8), indicating relatively intact myelin sheath; the CCH group mice showed a significantly lower density of blue fibers in the corpus callosum (34.7±4.16). Following chronic cerebral ischemia surgery, mice received intraperitoneal injections of RDK for 30 consecutive days (concentration 5 mg / ml, dosage 50 mg / kg mice). Four hours after injection on day 30, the brains were decapitated for LFB staining. The density of blue fibers in the corpus callosum of the CCH+RDK group mice (330.7±22) showed a significant dose-dependent increase. Compared with the normal control group, there was no significant difference in the RDK group (330±34) (RDK received intraperitoneal injections for 30 consecutive days, with LFB staining performed four hours after injection on day 30). These results indicate that RDK can alleviate neuronal damage by inhibiting demyelination in the brains of CCH mice.

Claims

1. The use of licoriceb in the preparation of drugs for the treatment and / or relief of vascular dementia.

2. The use of formulations containing licoriceb in the preparation of drugs for the treatment and / or relief of vascular dementia.

3. The application according to claim 1 or 2, characterized in that, The treatment and / or relief of vascular dementia includes one or more of the following: improving learning impairment, improving memory impairment, or improving depressive mood.

4. The application according to claim 1 or 2, characterized in that, The treatment and / or relief of vascular dementia includes one or more of the following: inhibiting intracranial inflammatory response, inhibiting intracranial pyroptosis, improving demyelinating changes in the brain, or inhibiting neuronal apoptosis.

5. The application according to claim 1 or 2, characterized in that, The chemical structural formula of Licarin B is: .

6. The application according to claim 2, characterized in that, The dosage forms of the preparations include tablets, capsules, decoctions, pills, granules, drop pills, injections, or oral liquids.

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