Application of licalin B, mace isolignan and their preparations in preparing drugs for treating and / or alleviating cerebral hemorrhage

Abstract

The present invention discloses the use of likalin b and its preparation in the preparation of drugs for treating and / or alleviating cerebral hemorrhage. The present invention also discloses the use of mace isolignan and its preparation in the preparation of drugs for treating and / or alleviating cerebral hemorrhage. The likalin b, mace isolignan and its preparation can clearly improve the damage of neurons in the peripheral area of ​​cerebral hemorrhage in a collagenase-induced cerebral hemorrhage mouse model, inhibit the inflammatory response and neuronal apoptosis in the cerebral hemorrhage area and the surrounding brain area, and play a neuron protection role; at the overall animal level, it is manifested as alleviating the motor dysfunction and cognitive impairment of cerebral hemorrhage mice, increasing the number of neurons in the brain, and inhibiting the degeneration and death of neurons; therefore, likalin b, mace isolignan and its preparation can be used for the treatment of cerebral hemorrhage, and have the prospect of developing drugs.

Description

Technical Field

[0001] The invention relates to application of likarin B, maceisolignan and preparations thereof in preparing medicines for treating and / or alleviating cerebral hemorrhage, and belongs to the field of cerebral hemorrhage treatment. Background Art

[0002] Intracerebral hemorrhage (ICH) refers to spontaneous bleeding in the brain parenchyma without trauma, accounting for 10-15% of stroke cases. It is the most common fatal form of stroke, with a mortality rate of 30-50%. About 30% of survivors can achieve functional independence. Its main causes include hypertension and cerebral amyloid degeneration, and the bleeding sites are often located in the basal ganglia, thalamus, cerebral lobes, pons, etc. The pathological process after ICH includes primary damage caused by hematoma formation and secondary damage caused by neuroinflammation, oxidative stress, and neuronal apoptosis. Among them, neuroinflammation is an important factor in brain tissue damage after ICH, and its pathological process includes microglial activation, neutrophil and macrophage infiltration, etc. Neutrophil infiltration is the key pathogenic factor of neurological dysfunction caused by ICH. At present, the treatment of ICH in clinic mainly includes conservative medical treatment and surgical hematoma removal. Hematoma removal is used in patients with surgical indications because it can reduce mortality. However, there is still a lack of drugs and surgical methods with high-level evidence-based medicine. Therefore, the development of new therapeutic targets is an urgent need for basic research and clinical diagnosis and treatment.

[0003] Myristic lignans (MS) have pharmacological effects such as liver protection, anti-inflammatory and immunosuppression. Myristic lignans have good anti-Toxoplasma activity in vivo and in vitro, and can damage the mitochondrial function of Toxoplasma, causing Toxoplasma autophagy and leading to the death of Toxoplasma. In addition, myristic lignans induce BAX, Caspase-3 and Caspase-9 protein expression by inhibiting the PI3K / AKT signaling pathway, promote apoptosis of gastric cancer cells, and play a role in treating gastric cancer. Myristic lignans can alleviate the symptoms of acute liver injury induced by thioacetamide in mice, significantly downregulate the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the plasma of acute liver injury mouse models, and inhibit the damage of liver cell membranes.

[0004] Likalin b (RDK) is a chemical substance with the chemical formula C 20 H 20 O 4, molecular weight is 324.3704, white crystals. RDK is an ingredient extracted from natural Chinese herbal medicine. Studies have found that RDK can effectively prevent and treat non-alcoholic fatty liver disease by reducing blood lipids, body weight, and blood sugar levels in non-alcoholic liver mice, and improving abnormal liver function in non-alcoholic liver mice. RDK can also make Toxoplasma gondii tachyzoites smaller and rounder, promote mitochondrial swelling and degeneration of tachyzoites, and disappearance of organelle membrane structure, effectively inhibiting the invasion and intracellular replication of Toxoplasma tachyzoites, and playing a role in treating or preventing toxoplasmosis. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide the use of licalin b and its preparations in the preparation of drugs for treating and / or alleviating cerebral hemorrhage.

[0006] The technical problem that the present invention also solves is to provide the use of mace lignan and its preparation in the preparation of drugs for treating and / or alleviating cerebral hemorrhage.

[0007] Technical solution: In order to solve the above technical problems, the present invention provides the use of Likaling B in the preparation of drugs for treating and / or alleviating cerebral hemorrhage.

[0008] Wherein, the treatment and / or relief of cerebral hemorrhage includes one or more of improving neurological dysfunction, protecting neurons, inhibiting neuronal apoptosis, inhibiting inflammation, inhibiting cerebral edema, or alleviating cognitive dysfunction.

[0009] Wherein, the chemical structural formula of the likalin b is: .

[0010] The dosage forms of the preparation include tablets, capsules, decoctions, pills, granules, pellets, mixtures, injections, and oral liquids.

[0011] Wherein, the concentration of the lycarin b is 5 mg / ml.

[0012] Wherein, the dosage of the licalin b is 50 mg / Kg.

[0013] Wherein, the preparation method of Likaling B comprises the following steps:

[0014] (1) Suspending nutmeg dry extract in water, and extracting with ethyl acetate to obtain ethyl acetate extract;

[0015] (2) adding n-hexane-methanol-water to the ethyl acetate extract of step (1) to dissolve the extract, injecting the extract into a high-speed countercurrent chromatography, collecting the effluent for 35-60 min, and drying the extract under reduced pressure at low temperature to obtain a crude extract;

[0016] (3) Dissolve the crude extract of step (2), prepare it using a C18 chromatographic column, collect the effluent for 15-20 min, and dry it under reduced pressure at low temperature to obtain delicalin B.

[0017] Wherein, the high-speed countercurrent chromatography in step (2) uses n-hexane-methanol-water as the upper phase as the stationary 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.

[0018] Wherein, in step (3), the volume ratio of the mobile phase acetonitrile-water solution in the C18 chromatographic column is increased from 50:50 to 90:10 in a gradient manner within 0-60 min, the flow rate is 3 ml / min, and the detection wavelength is 254 nm.

[0019] The present invention also provides the use of mace lignan and its preparation in preparing medicine for treating and / or alleviating cerebral hemorrhage.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention discovered for the first time that RDK and MS can clearly improve the damage of neurons in the peri-cerebral hemorrhage area of ​​the collagenase-induced cerebral hemorrhage mouse model, inhibit the inflammatory response and neuronal apoptosis in the cerebral hemorrhage area and the surrounding brain area, and play a neuronal protective role; 2. At the overall animal level, it manifests as alleviating the motor dysfunction and cognitive impairment of mice with cerebral hemorrhage, increasing the number of neurons in the brain, and inhibiting the degeneration and death of neurons; 3. It shows that RDK and MS can be used for the treatment of cerebral hemorrhage, and have the prospect of drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The effect of RDK on alleviating the imbalance of collagenase-induced cerebral hemorrhage model mice, ### compared with the control group, p<0.01, *** compared with the model group, p<0.001, ns, no significant difference;

[0022] Figure 2 The effect of RDK on alleviating forelimb dysfunction in the corner turning experiment of collagenase-induced cerebral hemorrhage model mice, ### compared with the control group, p<0.001, *** compared with the model group, p<0.001, ns, no significant difference;

[0023] Figure 3 The effect of RDK on alleviating forelimb dysfunction in collagenase-induced cerebral hemorrhage model mice, ### compared with the control group, p<0.001, *** compared with the model group, p<0.001, ns, no significant difference;

[0024] Figure 4 A in the figure is the time it takes to pass the balance beam in the balance beam experiment in which RDK alleviates collagenase-induced cerebral hemorrhage model mice. Figure 4B in the figure indicates the number of slips in the balance beam experiment of mice model of cerebral hemorrhage induced by RDK, ### compared with the control group, p<0.001, *** compared with the model group, p<0.001, ns, no significant difference;

[0025] Figure 5 The mNSS scores of the mouse model of collagenase-induced cerebral hemorrhage alleviated by RDK, ### compared with the control group, p<0.001, *** compared with the model group, p<0.001, ns, no significant difference;

[0026] Figure 6 A in the figure shows the cerebral hemorrhage of mice in each group. Figure 6 B in the figure shows the hematoma volume of mice in each group, ### compared with the control group, p<0.001, *** compared with the model group, p<0.001, ns, no significant difference;

[0027] Figure 7 The effect of RDK on inhibiting the apoptosis of neurons in the peri-hemorrhagic area of ​​the mouse model of cerebral hemorrhage induced by collagenase, ### compared with the control group, p<0.001, *** compared with the model group, p<0.001, ns, no significant difference;

[0028] Figure 8 The effect of RDK on improving the survival rate of collagenase-induced cerebral hemorrhage mouse model, ### compared with the control group, p<0.001, *** compared with the model group, p<0.001, ns no significant difference;

[0029] Fig. 9 RDK reduces the Iba-1 positive cells and increases the NeuN positive cells in the peri-cerebral hemorrhage area of ​​mice with collagenase-induced cerebral hemorrhage, ### compared with the control group, p<0.001, *** compared with the model group, p<0.001, ns, no significant difference;

[0030] Fig.10 The effect of RDK on reducing the levels of Iba-1, GFAP, TNF-α, and IL-1β proteins in the peri-cerebral hemorrhage area of ​​mice with collagenase-induced cerebral hemorrhage model, ### compared with the control group, p<0.001, *** compared with the model group, p<0.001, ns, no significant difference;

[0031] Fig.11 The effect of RDK on reducing brain edema in the collagenase-induced intracerebral hemorrhage mouse model, ### compared with the control group, p<0.001, *** compared with the model group, p<0.001, ns, no significant difference;

[0032] Fig.12The effect of RDK on alleviating cognitive dysfunction in the Y-maze test of collagenase-induced cerebral hemorrhage model mice, ### compared with the control group, p<0.001, *** compared with the model group, p<0.001, ns, no significant difference. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0034] Example 1 Effect of RDK on alleviating motor dysfunction in cerebral hemorrhage model mice

[0035] 1 Experimental Materials

[0036] The drug likalin b (RDK) was prepared in the laboratory: the nutmeg medicinal material was broken, and extracted twice with 95% ethanol for 2 hours each time, and the extract was recovered until it was dried into a dry extract. The dry extract was suspended in water and extracted with ethyl acetate three times to obtain an ethyl acetate extract. The ethyl acetate extract was dissolved in n-hexane-methanol-water (7:6:1, v / v) and injected into a high-speed countercurrent chromatography, with n-hexane-methanol-water (7:6:1, v / v) as the stationary phase and the lower phase as the mobile phase. The rotation speed was 1000 r / min, the flow rate was 2.2 ml / min, the detection wavelength was 254 nm, and the effluent of 35-60 min was collected and dried under low temperature and reduced pressure to obtain a crude extract. The crude extract was dissolved in methanol and prepared using a c18 chromatographic column. In 0-60 min, the volume ratio of the mobile phase acetonitrile-water solution was increased from 50:50 to 90:10, the flow rate was 3 ml / min, the detection wavelength was 254 nm, and the effluent was collected for 15-20 min and dried under reduced pressure at low temperature to obtain likalin b (RDK), the chemical formula of which is: .

[0037] A chemical substance similar in structure to RDK, myrisisolignan (MS), was selected for comparative study. The chemical formula of MS is as follows:

[0038]

[0039] The extraction steps of MS are as follows: Nutmeg medicinal material, shell, 95% ethanol extraction twice, each time for 2 hours, and the extract is recovered until dry to obtain a dry extract. The dry extract is suspended in water and extracted with ethyl acetate three times to obtain an ethyl acetate extract. The ethyl acetate extract is dissolved in n-hexane-methanol-water (7:6:1, v / v). Inject into high-speed countercurrent chromatography, with n-hexane-methanol-water (7:6:1, v / v) as the stationary phase and the lower phase as the mobile phase, the rotation speed is 1000 r / min, the flow rate is 2.2 ml / min, the detection wavelength is 254 nm, and the effluent within 35-60 min is collected, and it is dried under low temperature and reduced pressure to obtain a crude extract. The crude extract is dissolved in methanol and prepared using a c18 chromatographic column. Within 0-60 min, the volume ratio of the mobile phase acetonitrile-water solution was increased from 50:50 to 90:10, the flow rate was 3 ml / min, the detection wavelength was 254 nm, the effluent from 15-20 min was collected, and dried under low temperature and reduced pressure to obtain licalin b, and the effluent from 50-55 min was collected to obtain mace lignan (MS).

[0040] 1.2 Experimental animals

[0041] C57 / BL6 mice were purchased from the Comparative Medicine Center of Yangzhou University, weighing 25–28 g and two months old, and fed a free diet.

[0042] The experimental mice were divided into normal group mice, intracerebral hemorrhage (ICH) mice, ICH + RDK group, and intracerebral hemorrhage model + MS similar compound control group.

[0043] 2 Experimental methods

[0044] 2.1 Preparation of intracerebral hemorrhage (ICH) mouse model

[0045] Before surgery, mice were anesthetized with 10% chloral hydrate at 0.25 ml / 100 g intraperitoneally and fixed on a stereotaxic apparatus, with the incisor hook plane 2.4 mm lower than the interaural line plane. The mice were fixed in a prone position, with the anterior and posterior bregmas in the same plane. After disinfection and skin preparation, a longitudinal incision was made in the center of the dorsal side of the head (the distance from the midpoint of the line connecting the two eyes to the posterior bregma), the scalp and subcutaneous tissue were separated, and the anterior bregma was exposed. Take 0.2ul of type IV collagenase (LS004186, Worthington, USA) with a concentration of (0.2U / ul) and place it in a microsyringe; refer to the 3rd edition of the mouse stereotaxic atlas to locate the right caudate putamen: take the bregma coordinates as the origin, 0.3mm forward, 2.3mm to the left, and insert the needle vertically 3.8mm. Insert the needle slowly and inject collagenase after reaching the position. The injection time is 2 minutes. After the injection, keep the needle in place for 3 minutes to prevent the collagenase from refluxing. Slowly withdraw the syringe, seal the skull injury with bone wax, apply local compression to stop bleeding, and suture the scalp.

[0046] Mice in the sham operation group (Control): The surgical procedure was the same as before, except that an equal amount of saline was used instead of collagenase. After the operation, the incision was sutured and the mice were sent back to the incubator for recovery.

[0047] 2.2 Survival rate: The survival status of mice in each group after surgery was recorded, and the number of mouse deaths was recorded daily for 5 consecutive days.

[0048] like Figure 8 As shown, after the intracerebral hemorrhage surgery, RDK was continuously injected intraperitoneally for 4 days (concentration of 5 mg / ml, dosage of 50 mg / Kg), and the survival rate of mice was recorded every day 2 hours after RDK injection. The survival rate of mice in the ICH + RDK group (77.8±6.4%) was significantly higher than that in the ICH group (31.17±5.8%), and was significantly higher than that in the ICH+MS group of the same type of compound (after the intracerebral hemorrhage surgery, MS was continuously injected intraperitoneally for 4 days (concentration of 5 mg / ml, dosage of 50 mg / Kg), and the survival rate of mice was recorded every day 2 hours after MS injection) (60±8.4%). This shows that RDK can significantly improve the survival rate of mice with intracerebral hemorrhage.

[0049] 2.3 Modified Neurological Deficit Score (mNSS)

[0050] The mNSS score includes motor, sensory (vision, touch, proprioception), reflex and balance tests. The full score is 18 points, and the higher the score, the more severe the neurological damage function.

[0051] 2.4 Roller test

[0052] Three days before modeling, C57 / BL6 mice were placed on a rotarod at a speed of 15 r / min. After three days of training, mice with uncoordinated movements were eliminated. The test method is as follows: Place the mouse on a running rotarod and start timing. The time the mouse stays on the rotarod is taken as the latency period, which indicates its motor coordination ability. The measurement time for each mouse is 5 minutes, and a total of 5 tests are conducted at a speed of 30 r / min. Each test is 30 minutes apart, and the average value is taken. The data are expressed as mean ± standard deviation. After the cerebral hemorrhage surgery, RDK was injected intraperitoneally for 4 consecutive days (concentration of 5 mg / ml, dosage of 50 mg / Kg). On the 4th day and 4 hours after the injection, a roller test was performed to detect the mouse's motor balance ability. Figure 1As shown, the time that mice in the ICH+RDK group stayed on the roller (243.9±4.8s) was significantly higher than that in the ICH group (74.1±6.4s), and was also higher than that in the ICH+MS group of the same type (MS was intraperitoneally injected for 4 consecutive days after cerebral hemorrhage surgery, with a concentration of 5 mg / ml and a dosage of 50 mg / Kg. The roller test was performed 4 hours after the 4th day of MS injection to detect the movement balance ability of mice) (218.6±4.6s). This indicates that RDK can significantly improve the symptoms of movement balance disorders in mice with cerebral hemorrhage and enhance their exercise endurance.

[0053] 2.5 Corner experiment

[0054] The above groups of experimental mice were placed in a 30° angle formed by two foam walls placed vertically on a flat ground. When the mouse enters the angle and touches the obstacles on both sides, the mouse will raise its forelimbs and choose to turn left or right. Each mouse repeats the test 10 times a day, with at least 2 minutes interval each time, and the number of right turns in 10 times is recorded. Before modeling, the basal values ​​of C57 / BL6 mice were tested. If the mouse showed a clear preference for turning to one side, it was eliminated and randomly supplemented. The probability of the mice in the group turning left or right should be basically equal. After modeling, the mice mainly turned to the side of brain injury (right side). After cerebral hemorrhage surgery, RDK was injected intraperitoneally for 4 consecutive days (concentration of 5 mg / ml, dosage of 50 mg / Kg), and a corner turning experiment was performed 4 hours after the injection of RDK on the 4th day to detect the forelimb function of the mice. Figure 2 As shown, the turning rate of the healthy side of mice in the ICH+RDK group (58.3±3.2%) was significantly lower than that in the ICH group (81.1±3.4%), and there was no significant difference compared with the same type of compound group of ICH+MS (after cerebral hemorrhage surgery, MS was injected intraperitoneally for 4 consecutive days, with a concentration of 5 mg / ml and a dosage of 50 mg / Kg. The corner turning experiment was performed 4 hours after the 4th day of MS injection to detect the forelimb function of mice) (62.8±3.3%), indicating that RDK can significantly alleviate the forelimb dysfunction on the affected side of mice with cerebral hemorrhage.

[0055] 2.6 Forelimb placement experiment

[0056] During the experiment, the torso of the mouse was fixed, but the freedom of movement of the mouse's forearm was guaranteed. Before the experiment, the mouse was slowly moved up and down to relax its muscles and reduce resistance activities. The test on each forelimb was induced by the ipsilateral whisker touching the corner of the table. Each mouse was tested 10 times on each forelimb, and the number of times the ipsilateral forelimb was correctly placed on the table corner after the whisker was touched was recorded. Normal mice can quickly place the ipsilateral forelimb on the table corner after the whisker was touched. After cerebral hemorrhage, depending on the degree of injury, the contralateral forelimb placement function induced by the whiskers on the opposite side of the injury will be impaired to varying degrees. Figure 3As shown, compared with normal mice (8.2±0.2), the number of times the ICH group mice correctly placed the contralateral forelimb to the table corner (2.67±0.3) was significantly reduced. After the intracerebral hemorrhage surgery, RDK was injected intraperitoneally for 4 consecutive days (concentration of 5 mg / ml, dosage of 50 mg / Kg), and the forelimb placement experiment was performed 4 hours after the injection of RDK on the 4th day to detect the forelimb function of the mice. The number of times the ICH+RDK group mice correctly placed the contralateral forelimb to the table corner (6.3±0.53) was significantly increased compared with the ICH group (2.67±0.3), and there was no significant difference compared with the same type of compound group of ICH+MS (after the intracerebral hemorrhage surgery, MS was injected intraperitoneally for 4 consecutive days, the concentration was 5 mg / ml, the dosage was 50 mg / Kg, and the forelimb placement experiment was performed 4 hours after the 4th day of MS injection to detect the forelimb function of the mice) (6.3±0.4), indicating that RDK can significantly alleviate the contralateral forelimb dysfunction of mice with intracerebral hemorrhage.

[0057] 2.7 Balance beam experiment

[0058] The balance beam used was a round balance beam with a length of 1m and a diameter of 17mm. One side of the balance beam was placed in a black square box, and the mouse was placed on the other side of the balance beam. The time it took for the mouse to pass through the balance beam into the black square box within 60s and the number of times it slipped were recorded. The experiment was divided into a training period and a testing period, which lasted for 3 days. The first day was the training period, using a balance beam to allow the mouse to pass through the balance beam into the small dark box. Each mouse was trained 3 times, with an interval of 2 hours each time. On the second day, the same steps as on the first day were repeated. The third day was the testing phase, allowing the mouse to pass through the balance beam to reach the small dark box, and the time it took to reach the dark box and the number of times it slipped were recorded. If it failed to reach the small wooden box, the time was recorded as 60s. Figure 4 As shown in the figure, the number of slips (5.4±0.94) and the time to pass the balance beam (11.4±1.25s) of mice in the ICH group were significantly increased compared with the number of slips (0.5±0.19) and the time to pass the balance beam (4.5±0.42s) of mice in the normal group. After the intracerebral hemorrhage surgery, RDK was intraperitoneally injected for 4 consecutive days (concentration of 5 mg / ml, dosage of 50 mg / Kg). The balance beam experiment was performed 4 hours after the injection of RDK on the 4th day to detect motor function. The number of slips (2.25±0.37) and the time to pass the balance beam (5.9±0.44s) of mice in the ICH+RDK group were significantly reduced. Compared with the same type of compound group of ICH+MS (MS was intraperitoneally injected for 4 consecutive days after cerebral hemorrhage surgery, the concentration was 5 mg / ml, the dosage was 50 mg / Kg, and the neurological deficit was evaluated 4 hours after the 4th day of MS injection), the time to pass the balance beam (5.25±0.9s) and the number of slips (3.25±0.37) were not significantly different. This shows that RDK can significantly improve the symptoms of motor dysfunction in mice with cerebral hemorrhage.

[0059] In summary, the modified neurological deficit score (mNSS) of each group includes motor, sensory (vision, touch, proprioception), reflex and balance tests. The full score is 18 points, and the higher the score, the more severe the neurological damage. Figure 5 As shown, the neurological deficit score of mice in the ICH group (7.1±0.26) was significantly increased compared with the neurological deficit score of normal (Control) mice (0.22±0.15). After intracerebral hemorrhage surgery, RDK was intraperitoneally injected for 4 consecutive days (concentration of 5 mg / ml, dosage of 50 mg / Kg), and neurological deficit was evaluated 4 hours after RDK injection on the 4th day. The neurological deficit score of mice in the ICH+RDK group (4.7±0.29) was significantly reduced compared with the ICH group (7.1±0.26), and there was no significant difference compared with the same type of compound group of ICH+MS (after intracerebral hemorrhage surgery, MS was intraperitoneally injected for 4 consecutive days, concentration of 5 mg / ml, dosage of 50 mg / Kg, and neurological deficit was evaluated 4 hours after MS injection on the 4th day) (5±0.4), indicating that RDK can significantly improve the neurological deficit of mice with intracerebral hemorrhage.

[0060] Example 2 Protective effect of RDK on neurons in the brain of mice with cerebral hemorrhage model

[0061] The experimental animals, experimental groups and preparation of cerebral hemorrhage model were the same as those in Example 1.

[0062] After anesthesia, the animals were killed by cervical dislocation and decapitation, and the brain tissue was removed and fixed in 4% paraformaldehyde solution for 72 h. The samples were sliced ​​continuously with the modeling pinhole as the center using a high-speed freezing microtome. The hematoma was dark red, and the maximum transverse diameter and maximum longitudinal diameter of the maximum layer of the hematoma were measured. The volume of cerebral hematoma was calculated according to Tada's formula: hematoma volume = (π / 6) × maximum transverse diameter (mm) × maximum longitudinal diameter (mm) × number of hematoma layers × slice thickness (mm).

[0063] like Figure 6 As shown in the figure, no cerebral hematoma was found in normal mouse brain sections (0±0 mm 3 ), mice in the ICH group had obvious intracerebral hemorrhage and hematoma area (26.1±0.84 mm) on the 4th day after surgery. 3 After ICH surgery, RDK was intraperitoneally injected for 4 consecutive days (concentration of 5 mg / ml, dosage of 50 mg / kg). The volume of the hematoma area was measured 4 hours after RDK injection on the 4th day. The volume of the hematoma area of ​​mice in the ICH+RDK group (8.7±0.65 mm 3) was significantly reduced compared with the ICH group and compared with the ICH+MS group of the same type (after intracerebral hemorrhage surgery, MS was intraperitoneally injected for 4 consecutive days at a concentration of 5 mg / ml and a dosage of 50 mg / Kg. The volume of the hematoma area was measured 4 hours after the 4th day of MS injection) (18±1.8 mm 3 ), indicating that RDK can significantly improve the volume of cerebral hemorrhage in mice with cerebral hemorrhage.

[0064] Example 3 Inhibitory effect of RDK on neuronal apoptosis in the brain of a mouse model of cerebral hemorrhage

[0065] The experimental animals, experimental groups and preparation of cerebral hemorrhage model were the same as those in Example 1.

[0066] Fluoro-Jade B (FJB) detection. After the behavioral experiment, each group of mice was anesthetized, 30 ml of normal saline was perfused through the left ventricle, and the brain was quickly cut off on ice to make 5 μm frozen sections. When staining with Fluoro-Jade B, the sections were immersed in a 1% NaOH-80% ethanol mixture for 5 min, 70% ethanol for 2 min, and washed 3 times with distilled water, 2 min / time. The sections were incubated in a 0.06% potassium permanganate solution at room temperature for 20 min, washed 3 times with distilled water, 2 min / time. Reacted in FJB staining solution for 20 min (20℃, protected from light), washed 3 times with distilled water, 2 min / time. Dry, dehydrate, and transparentize with xylene 3 times × 5 min, and then seal the sections. Observe and collect images under a fluorescence microscope. Figure 7 As shown, only a small number of apoptotic neurons (28.5±3.1) were found in the brains of mice in the normal group, while a large number of FJB-positive cells (neuronal apoptosis) (1094.8±74.5) appeared in the brains of mice in the ICH group. After the intracerebral hemorrhage surgery, RDK was intraperitoneally injected for 4 consecutive days (concentration of 5 mg / ml, dosage of 50 mg / Kg), and the brains were cut off and taken for FJB detection 4 hours after the injection of RDK on the 4th day. The number of FJB-positive cells in the ICH+RDK group (585.8±60.0) mice was significantly reduced, which was significantly lower than that in the ICH+MS group of the same type of compound (after the intracerebral hemorrhage surgery, MS was intraperitoneally injected for 4 consecutive days, concentration of 5 mg / ml, dosage of 50 mg / Kg, and the brains were cut off and taken for FJB detection 4 hours after the injection of MS on the 4th day) (698.5±36.7), indicating that RDK has a significant inhibitory effect on neuronal apoptosis in the perihematoma area of ​​mice with intracerebral hemorrhage.

[0067] Example 4 Inhibitory effect of RDK on brain inflammation in mice with cerebral hemorrhage model

[0068] The experimental animals, experimental groups and preparation of cerebral hemorrhage model were the same as those in Example 1.

[0069] 4.1 Iba-1 and NeuN staining analysis in cerebral hemorrhage and surrounding brain areas

[0070] After the behavioral experiment, the animals in each group were anesthetized, perfused through the left ventricle with 30 ml of saline, and then injected with 4% PFA for whole-body fixation. The whole brain was removed by decapitation. The whole brain was immersed in 15%, 20%, and 30% sucrose water in turn, and then frozen and sectioned to obtain 5 μm thick sections. The sections were placed in EDTA (PH 9.0) antigen repair solution and repaired in a microwave oven at medium-low heat for 10-15 minutes. After natural cooling, they were washed 3 times with PBS. 2 O 2 Incubate for 15 min, wash 3 times with PBS; incubate with normal blocking serum for 30 min, add Iba-1 and NeuN primary antibodies and shake overnight at 4°C; wash 3 times with PBS, incubate with fluorescent secondary antibodies (CYTM3-conjugated affinipuregoat anti-rabbit IgG, Jackson ImmunoResearch, USA, 111-165-003; FITC-conjugated affinipure goat anti-mouse IgG, Jackson ImmunoResearch, USA, 115-095-003; Jackson ImmunoResearch, USA, ) at room temperature for 1 h, wash 3 times with PBS, stain the nucleus with DAPI for 5 minutes and seal the slides. The number of Iba-1 and NeuN positive cells in the cerebral hemorrhage area of ​​each group of mice was observed under a fluorescence microscope. Fig. 9As shown, the number of Iba-1 positive cells in the brain of mice in the normal group was small (16±1.9), and there were a large number of NeuN positive cells (5501.7±129.5), with intact cell bodies and dendrites; the number of Iba-1 positive cells in the hematoma area of ​​mice in the ICH group increased significantly (252±6.1), and Iba-1 positive cells showed ameba-like morphology, indicating that microglia in the brain were activated. The number of NeuN positive cells in the brain of mice in the ICH group (3349.3±275.1) was significantly reduced compared with the normal group, and the cell axons were incomplete. After cerebral hemorrhage surgery, RDK was injected intraperitoneally for 4 consecutive days (concentration of 5 mg / ml, dosage of 50 mg / Kg). The brains were removed by decapitation 4 hours after RDK injection on the 4th day for immunohistochemical staining. The number of Iba-1 positive cells (141.4±4.4) in the ICH+RDK group was significantly lower than that in the ICH group, the number of activated microglia was significantly reduced, and the number of NeuN positive cells (4593.3±455.1) was significantly increased compared with that in the ICH group. Compared with the ICH+MS group of the same type of compound (after cerebral hemorrhage surgery, MS was intraperitoneally injected for 4 consecutive days at a concentration of 5 mg / ml and a dosage of 50 mg / Kg. The brain was removed by decapitation and immunohistochemical staining 4 hours after the 4th day of MS injection) (4538.2±350.6), the number of NeuN-positive cells in the ICH+RDK group of mice was not significantly different, but the number of Iba-1-positive neurons in the ICH+MS group of the same type of compound (202.6±9.4) was significantly reduced. The above results show that RDK can alleviate neuronal damage by inhibiting the inflammatory response in the brain of ICH mice.

[0071] 4.2 Western Blotting to detect inflammation-related protein levels

[0072] After the behavioral experiment, the animals in each group were anesthetized and decapitated to remove the brain. 1 mL of protein lysis buffer was added for every 100 mg of brain tissue [phenylmethylsulfonyl fluoride (PMSF) and protease inhibitors were added before use], and the tissue was crushed and homogenized on ice to extract total protein. The total protein concentration was detected by bicinchoninic acid (BCA) method and protein samples were prepared. 25 μg per well was transferred to PVDF by wet transfer method (constant pressure 116 mA, 90 min) on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and blocked with 5% skim milk powder for 1 h. Rabbit anti-mouse Iba-1 antibody (1:1000), GFAP antibody (1:1000), TNF-α antibody (1:1000), IL-1β (1:1000), and β-actin antibody (1:1000) were added and incubated overnight at 4°C. The next day, the cells were washed three times with TBST, each time for 5 min, and incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:5000) at room temperature for 1 h. The ECL chemiluminescent solution was used for color development, and the bands were scanned and recorded by a scanner. β-actin was used as an internal reference, and the bands were processed and analyzed by Image J software. The absorbance ratio of Iba-1, GFAP, TNF-α and IL-1β to β-actin was recorded. Fig.10 As shown in the figure, the levels of inflammation-related proteins such as Iba-1, GFAP, TNF and IL-1β in the brain of mice in the normal group were low, and the levels of the above inflammation-related proteins in mice with ICH were significantly increased. After RDK treatment, the levels of inflammation-related proteins such as Iba-1, GFAP, TNF and IL-1β in mice in the ICH+RDK group were significantly lower than those in the model group, and significantly lower than those in the same type of compound group of ICH+MS. This shows that RDK has a significant effect in inhibiting inflammation in the brain, and the effect is better than the anti-inflammatory effect of MS.

[0073] Example 5 Inhibitory effect of RDK on brain edema in a mouse model of cerebral hemorrhage

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

[0075] Brain water content was measured by wet-dry specific gravity method. After deep anesthesia, mice were killed and the brain was immediately removed. The olfactory bulb, cerebellum, and brain stem were removed using a blade on a brain slice mold, and then the left and right hemispheres were cut symmetrically, and each part was placed in an EP tube (the EP tube was weighed in advance); each sample was immediately weighed using an electric analytical balance to obtain the wet weight; dried at 100 degrees Celsius for 48 hours (finally weighed twice in a row, and the mass difference was less than 1%) to obtain the dry weight; the water content of each sample was calculated using the following formula = [(wet weight − dry weight) / wet weight] x 100%.

[0076] like Fig.11As shown, the brain dry-to-wet ratio of the ICH group mice (0.819±0.016) was significantly increased compared with the normal group (0.755±0.005). After intracerebral hemorrhage surgery, RDK was intraperitoneally injected for 4 consecutive days (concentration of 5 mg / ml, dosage of 50 mg / Kg), and the brain was cut off 4 hours after the injection of RDK on the 4th day to measure the brain water content by dry-wet specific gravity method. The dry-to-wet ratio of the brain of the ICH+RDK group mice (0.745±0.011) was significantly lower than that of the model (ICH) group. Compared with the same type of compound group of ICH+MS (after intracerebral hemorrhage surgery, MS was intraperitoneally injected for 4 consecutive days, with a concentration of 5 mg / ml and a dosage of 50 mg / Kg, and the brain was cut off 4 hours after the injection of MS on the 4th day to measure the brain water content by dry-wet specific gravity method) (0.795±0.015), it was significantly lower. The above results show that RDK treatment has an inhibitory effect on brain edema in the mouse model of intracerebral hemorrhage.

[0077] Example 6 Effect of RDK on Alleviating Cognitive Impairment in Mouse Model of Cerebral Hemorrhage

[0078] The experimental animals, experimental groups and preparation of cerebral hemorrhage model were the same as those in Example 1.

[0079] Maze test memory function. The spontaneous alternation experiment of the Y maze can effectively measure the spatial memory ability of animals. The Y maze consists of three equal long arms (the angle between each two arms is 120 degrees). Place the mouse at the end of any arm of the Y maze and let it explore freely for 8 minutes. The camera system records the changes in animal behavior for 8 minutes and records the following indicators: total number of arm entries, number of turns (entering all three arms of the Y maze once in sequence). Spontaneous alternation score: number of turns / (total number of arm entries-2). Fig.12 As shown in the figure, the correct cycle number of mice in the ICH group (57.8±2.86) was significantly lower than that of mice in the normal group (82.5±1.32). After cerebral hemorrhage surgery, RDK was intraperitoneally injected for 4 consecutive days (concentration of 5 mg / ml, dosage of 50 mg / Kg), and the Y maze test was performed 4 hours after RDK injection on the 4th day to detect cognitive function. The correct cycle number of mice in the ICH+RDK group (76.5±2.03) was significantly higher than that in the model group (ICH group). Compared with the same type of compound group of ICH+MS (after cerebral hemorrhage surgery, MS was intraperitoneally injected for 4 consecutive days, concentration of 5 mg / ml, dosage of 50 mg / Kg, and Y maze test was performed 4 hours after MS injection on the 4th day) (66.3±1.68), it was significantly increased. The above results show that RDK treatment has a significant improvement on the memory and cognitive function of the mouse model of cerebral hemorrhage.

Claims

1. Use of licalin b or its preparation in the preparation of drugs for treating and / or alleviating cerebral hemorrhage.

2. The use according to claim 1, characterized in that: The treatment and / or relief of cerebral hemorrhage includes one or more of improving neurological dysfunction, protecting neurons, inhibiting neuronal apoptosis, inhibiting inflammation, inhibiting cerebral edema, or alleviating cognitive dysfunction.

3. The use according to claim 1, characterized in that: The chemical structural formula of the likalin b is: 。 4. The use according to claim 1, characterized in that: The dosage forms of the preparation include tablets, capsules, decoctions, pills, granules, dripping pills, mixtures, injections and oral liquids.

5. The use according to claim 1, characterized in that: The preparation method of Likaling B comprises the following steps: (1) Suspending nutmeg dry extract in water, and extracting with ethyl acetate to obtain ethyl acetate extract; (2) adding n-hexane-methanol-water to the ethyl acetate extract of step (1) to dissolve the extract, injecting the extract into a high-speed countercurrent chromatography, collecting the effluent for 35-60 min, and drying the extract under reduced pressure at low temperature to obtain a crude extract; (3) Dissolve the crude extract of step (2), prepare it using a C18 chromatographic column, collect the effluent for 15-20 min, and dry it under reduced pressure at low temperature to obtain delicalin B.

6. The use according to claim 5, characterized in that: The high-speed countercurrent chromatography in step (2) uses n-hexane-methanol-water upper phase as the stationary phase and 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.

7. The use according to claim 5, characterized in that: In step (3), the volume ratio of the mobile phase acetonitrile-water solution in the C18 chromatographic column was increased from 50:50 to 90:10 within 0-60 min, the flow rate was 3 ml / min, and the detection wavelength was 254 nm.

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