Application of alpha-amyrin in preparation of medicine for treating schizophrenia

Abstract

The invention discloses new application of alpha-amyrin, namely application of alpha-amyrin in preparation of a medicine for treating schizophrenia, experimental results show that alpha-amyrin can effectively relieve MK-801 induced schizophrenia symptoms of mice, reduce HT22 cytotoxicity induced by glutamic acid, and can be used for preparing medicines for treating schizophrenia. The evidences show that the alpha-amyrin can improve the symptoms of schizophrenia.

Description

Technical Field

[0001] This invention relates to the application of α-amyrin in the preparation of drugs for treating schizophrenia, belonging to the field of pharmaceutical technology. Background Technology

[0002] Schizophrenia is a persistent mental disorder primarily characterized by abnormalities in perception, emotion, and behavior, with confused thinking and emotional responses being its typical symptoms. Research indicates that genetics, brain structural abnormalities, pregnancy problems, and environmental factors may all be related to the onset of schizophrenia, but the exact pathogenesis is not fully understood. The main symptoms of schizophrenia include hallucinations, delusions, confused thinking, and abnormal behavior; some patients may exhibit aggression and suicidal behavior. The disease has a long course and often results in varying degrees of mental disability, placing significant pressure on patients, their families, and society. Schizophrenia needs to be differentiated from other mental illnesses such as dissociative identity disorder or multiple personality disorder. Its main characteristic lies in the disruption of thought processes and emotional responses; psychiatrists consider "thought disorder" a key marker.

[0003] α-Amyophylline is a ursane-type pentacyclic triterpenoid compound widely distributed in nature, primarily found in plants such as loquat, apple, and periwinkle. Triterpenoids are plant secondary metabolites with various effects including antitumor, anti-inflammatory, antibacterial, immune-enhancing, sedative, antimalarial, hepatoprotective, and antioxidant properties, and are widely used in the pharmaceutical and health care fields. α-Amyophylline is a needle-like crystal (ethanol) at room temperature, sparingly soluble in water, but soluble in solvents such as ether, benzene, and chloroform. Animal experiments have shown no significant toxic reactions after long-term administration, making it suitable for long-term use. Summary of the Invention

[0004] This invention provides a novel use of α-amyrinol, namely its application in the preparation of drugs for treating schizophrenia.

[0005] The chemical structural formula of the α-amyrin is as follows: ; The pharmaceutical ingredient (or active ingredient) described in this invention is α-amyrin alcohol. One or more pharmaceutically acceptable excipients may also be added to improve the absorption effect of the drug or facilitate its use, such as making it into capsules or pills, powders, tablets, granules, oral liquids and injections, etc., that is, to make it into a suitable dosage form.

[0006] This invention uses glutamate to treat HT22 cells to construct a schizophrenia cell model. After treating the schizophrenia cell model with α-amyrin, we found that α-amyrin improved the viability of HT22 cells and reduced the proportion of apoptotic cells. In a mouse schizophrenia model, we also found through open field and tail suspension experiments that α-amyrin also reversed the behavioral symptoms of schizophrenia mice. The above experimental results indicate that α-amyrin has the function of improving the symptoms of schizophrenia.

[0007] This invention reveals the therapeutic potential of α-amyrin for schizophrenia, providing a new approach to the treatment of schizophrenia symptoms. Attached Figure Description

[0008] Figure 1 To detect the protective effect of α-amyrin against glutamate-induced HT22 cell damage using the CCK8 assay; Figure 2 The results of apoptosis detection of HT22 cells in each group were obtained using flow cytometry; the upper left image is the blank control, the upper right image is the glutamate modeling image, and the lower image is the drug treatment image. Figure 3 Statistical results of apoptosis detection in HT222 cells of each group using flow cytometry; Figure 4 The left image shows the trajectory plot and the right image shows the trajectory heatmap of the mouse open field experiment. Figure 5 The diagram shows the statistical results of the mouse experiment. Figure A shows the total distance traveled by the mice in the mine experiment, Figure B shows the time it took for the mice in the mine to reach the central area, and Figure C shows the time it took for the mice in the tail suspension experiment to remain still. Detailed Implementation

[0009] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following examples. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof. Unless otherwise specified, the instruments, reagents, and materials involved in the following embodiments are all conventional instruments, reagents, and materials already existing in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods involved in the following examples are all conventional experimental methods and detection methods already existing in the prior art.

[0010] Example 1: Protective effect of α-amyrin on glutamate-induced HT22 cells 1. Prepare DMEM medium containing 7.5% fetal bovine serum, and then use this DMEM medium to adjust the HT22 cell density to 5 × 10⁶ cells / year. 4The cells were adjusted to a density of 100 μL and then seeded into each well of a 96-well plate. The cells were cultured at 37°C and 5% CO2 for 24 h to allow the HT22 cells to adhere. The old culture medium was then discarded, and the HT22 cells were incubated with DMEM medium (100 μL per well) containing 50 ng / mL nerve growth factor and 7.5% fetal bovine serum for 24 h. 2. HT22 cells induced in 96-well plates were first treated with DMEM medium containing 5mM glutamate for 24 hours to obtain a schizophrenia cell model. The medium was then removed, and the cells were incubated for 24 hours with DMEM medium containing different concentrations of α-amyrin (5, 10, and 20 μM) (100 μL per well) as the treatment group. The blank control group received only DMEM medium, and the control group received only DMEM medium incubation for 24 hours. Then, 10 μL of CCK8 reagent was added to each well, gently mixed, and incubated at 37°C in the dark for 1-2 hours. The absorbance (OD) was then measured at 450 nm using a microplate reader. Cell viability was calculated as follows: ; See results Figure 1 As can be seen from the figure, compared with the control group, the survival rate of HT22 cells exposed to glutamate for 24 hours was significantly reduced. Treatment with different concentrations of α-amyrin for 24 hours significantly improved the cell survival rate. The results indicate that α-amyrin alleviated the decrease in HT22 cell survival rate caused by glutamate treatment.

[0011] Example 2: The restorative effect of α-amyrin on glutamate-induced apoptosis in HT22 cells HT22 cells were resuspended in culture dishes and seeded into 6-well plates. After culturing for 24 hours and cell adhesion, a blank control group was set up: cells were cultured in DMEM medium for 48 hours. The model group was induced by treating cells in DMEM medium containing 5 mM glutamate for 24 hours. The drug treatment group was treated with DMEM medium containing 5 mM glutamate for 24 hours, the medium was aspirated, and the cells were treated with DMEM medium containing α-amyrin (20 μM). The cells were collected by centrifugation, washed twice with pre-cooled phosphate-buffered saline (PBS) to remove serum from the medium, and the cells were resuspended in phosphate-buffered saline. 100 μL of cell suspension was added to each well of the 6-well plate, followed by 5 μL of Annexin V-FITC. The mixture was gently mixed and incubated at room temperature in the dark for 15 min. Then, 5 μL of PI (final concentration 1-2 μg / mL) was added and incubated in the dark for 5 min. The apoptosis rate of the samples was then detected by flow cytometry. The results are as follows Figure 2-3As shown, glutamate resulted in a cell apoptosis rate as high as 20%, while α-amyrin significantly reduced the apoptosis rate of HT22-mimicked nerve cells.

[0012] Example 3: Therapeutic effect of α-amyrin on MK-801-induced mouse model of schizophrenia Male C57BL / 6J mice, weighing 20-22g, were housed at the Experimental Animal Center of Kunming University of Science and Technology. They had free access to food and water, a room temperature of 22±1℃, a relative humidity of 55-65%, and a 12-hour light cycle. All other reagents were domestically produced and met the experimental requirements.

[0013] Mice were randomly divided into 3 groups of 6 mice each, with the blank control group receiving no treatment. The model group was induced with a mouse model of schizophrenia by intraperitoneal injection of MK-801 (0.4 mg / kg) for 7 consecutive days. In the α-amyrin group, after the model was established, the schizophrenic mouse model was administered α-amyrin (prepared with physiological saline, 20 mg / kg) by gavage daily. One week after gavage, the symptoms of schizophrenia in each group of mice were evaluated using the open field test and the tail suspension test.

[0014] Open field test: This test is used to evaluate the spontaneous exploration activities and anxiety levels of mice in unfamiliar environments. During the test, mice are placed in the center of a clean plastic open field box (40cm × 40cm × 40cm), and their behavior is recorded for 5 minutes. After the test, the box is wiped clean with 75% alcohol. The total distance the mice travel within 5 minutes reflects their spontaneous activity level, while the time spent in the central area of ​​the open field reflects their anxiety level.

[0015] Tail suspension test: Used to evaluate depressive symptoms in mice. During the experiment, approximately 2 cm of the mouse's tail is fixed to the top of the tail suspension test chamber using medical tape. The cumulative immobility time over 5 minutes is recorded. A longer cumulative immobility time indicates more severe despair and a higher level of depression. Figure 4 , 5 Data from the AB open field assay showed that after MK-801 modeling, the model mice exhibited hyperactivity, mimicking the positive symptom of increased excitability in schizophrenia. Simultaneously, the model mice spent significantly less time in the central area of ​​the open field, indicating a fear of the central area and a reduced desire to explore novel environments, reflecting a significant increase in anxiety levels. The drug treatment group significantly reduced the hyperactivity symptoms and increased the time spent exploring the central area, reflecting effective relief of their anxiety symptoms.

[0016] Figure 5The results of the C-tail suspension experiment showed that the cumulative immobility time of the model mice was significantly increased, simulating the despair in the negative symptoms of SCZ patients, and α-amyrin can effectively reverse these abnormalities.

Claims

1. The application of an α-amyrin alcohol in the preparation of a drug for treating schizophrenia.

Images