Application of lupinus luteus White ketone in preparation of medicine for preventing or treating depression and anxiety

Abstract

The invention discloses application of lupinone (Lup) in preparation of drugs for preventing or treating depression and anxiety, and belongs to the technical field of application of natural drugs. The results of an open field experiment and an elevated cross maze experiment show that the Lup can effectively improve the depression behavior and the accompanying anxiety behavior of a disease model mouse. Meanwhile, a differential gene protein interaction network is obtained by analyzing a transcriptome of an experimental mouse, and a result shows that gene expressions of Acta2, Fos, Tagln, Vwf, Ccl2 and the like have significant differences and can possibly play key biological functions under experimental conditions, so that valuable clues and directions are provided for subsequent in-depth research, and the application prospect is broad. And further analysis of related biological mechanisms and pathological processes is facilitated. The result shows that Lup has potential treatment value in the aspect of improving depression and anxiety symptoms, and an experimental basis is provided for developing related treatment drugs.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to application of yellow lupin whitenerone in the preparation of drugs for preventing or treating depression and anxiety. Background Art

[0002] Depression is a severe mental disorder with high disability and a significant public health burden. Its etiology is highly heterogeneous, involving complex multifactorial interactions, and its core pathophysiological mechanisms remain incompletely elucidated. Current research has explored multiple interrelated hypotheses and pathways, including the classic hypothesis of monoamine neurotransmitter (5-HT, NE, DA) dysfunction, the increasingly popular immune-inflammatory hypothesis (e.g., elevated levels of proinflammatory cytokines), the hypothesis of hypothalamic-pituitary-adrenal (HPA) axis hyperactivity under chronic stress, and the hypothesis of impaired neuroplasticity and neurogenesis in key brain regions such as the hippocampus. Furthermore, glutamatergic system dysregulation (e.g., NMDA receptor dysfunction), endothelial dysfunction, and the interaction between genetic susceptibility and environmental stressors (e.g., early life trauma) are also considered important pathogenic dimensions. In terms of clinical manifestations, the core symptoms of depression are concentrated in persistent low mood and significant loss of interest / pleasure, often accompanied by anxiety, agitation, cognitive impairment (such as decreased attention and executive ability), and various somatic symptoms (such as sleep disorders, changes in appetite, decreased energy, pain, etc.), which together constitute its complex clinical phase.

[0003] Anxiety neurosis is the most common neurotic disorder, characterized by a significant experience of anxiety. This disorder is primarily divided into two clinical types: chronic anxiety, also known as generalized anxiety disorder, and acute anxiety, also known as panic attacks. Typical clinical manifestations include persistent anxiety and motor restlessness (such as fidgeting) in the absence of clear objective triggers, often accompanied by sleep disorders and autonomic dysfunction symptoms such as dizziness, palpitations, shortness of breath, dry mouth, hand tremors, sweating, and frequent and urgent urination. The patient's level of anxiety often does not match the actual threat, or their panic reaction far exceeds the actual situation.

[0004] The etiology of anxiety disorders involves multiple factors. Genetic factors: Individuals with a family history of the disorder are at significantly higher risk than the general population. Neurochemical mechanisms: Research suggests that dysfunction of the gamma-aminobutyric acid (GABA) system is a key biological basis for the onset of anxiety disorders. Furthermore, hyperactivity of the noradrenergic system and dysfunction of the serotonin (5-HT) system are also believed to be involved. Neural circuitry: The neural circuits responsible for emotion regulation in the brain involve key regions such as the prefrontal cortex, amygdala, hippocampus, and hypothalamus. Structural abnormalities, dysfunction, or interconnectedness disorders in these brain regions can lead to impaired emotional control and form the neuropathological basis of anxiety disorders. Psychosocial factors: Psychological factors play a key role in the development and progression of anxiety disorders. For example, the development of generalized anxiety disorder is often associated with long-standing unresolved psychological conflicts or persistent information processing biases; social anxiety disorder often stems from an excessive focus on the evaluations of others, which can be traced back to negative experiences during childhood; and specific phobias are often associated with feared objects and specific traumatic experiences.

[0005] Currently, many types of antidepressants are available on the market, including monoamine oxidase inhibitors, tricyclic drugs, and selective serotonin reuptake inhibitors. However, these drugs all have common problems such as long duration of effect, strong side effects, and different drug sensitivity in different groups of people.

[0006] Lupiwhighteone (Lup) is a natural isoflavone compound with anti-tumor potential. It is primarily isolated from plants such as white lupin seeds and pods, licorice leaves, and broad bean roots. Previous research by the inventors has demonstrated that lupiwhighteone has anti-cancer and anti-tumor effects (e.g., studies on the mechanism of action of lupiwhighteone on SH-SY5Y cells) and lifespan extension and anti-aging effects (CN119280215A). However, there are no reports on the effects of lupiwhighteone on the prevention or treatment of depression and anxiety. Summary of the Invention

[0007] The present invention aims to provide a use of yellow lupin white ketone (Lup) in the preparation of a drug for preventing or treating depression and anxiety.

[0008] Specifically, the technical solutions of the present invention are as follows: The use of Lup in the preparation of drugs for preventing and treating depression and anxiety, wherein Lup is a compound of the following formula: .

[0009] Lupiwhighteone (Lup) is a natural isoflavone compound that can be extracted from the seeds and pods of white lupin, the leaves of licorice, and the roots of broad beans. It can also be prepared by chemical synthesis (such as CN103936706A).

[0010] The invention discloses an application of Lup in the preparation of drugs for treating depression and anxiety. The invention mainly finds that Lup has antidepressant and antianxiety activities through a mine field experiment and an elevated plus maze experiment on mice.

[0011] Experimental data showed that Lup can effectively improve depressive behaviors and accompanying anxiety in disease model mice, and has significant antidepressant and anxiolytic activity: In an open field test, Lup significantly increased the frequency of mice entering the central area and significantly prolonged their stay in the central area, while not affecting the total distance traveled. In an elevated plus maze test, Lup significantly increased the number of mice entering the open arms and the time they spent in the open arms.

[0012] In addition, histological analysis and body weight monitoring results of important organs (heart, liver, spleen, lungs, and kidneys) showed that Lup had no obvious toxic effects or weight loss on major organs such as the heart, liver, spleen, lungs, and kidneys of mice, indicating that it has a high safety profile.

[0013] Transcriptome analysis of the experimental mice revealed a differential gene-protein interaction network, revealing significant differences in the expression of genes such as Acta2, Fos, Tagln, Vwf, and Ccl2. This suggests that these genes may play key biological functions under the experimental conditions, providing valuable clues and directions for subsequent in-depth research and helping to further understand the relevant biological mechanisms and pathological processes.

[0014] The above results suggest that Lup has potential therapeutic value in improving depression and anxiety symptoms, and also provide an experimental basis for the development of therapeutic drugs related to abnormal expression of genes such as Acta2, Fos, Tagln, Vwf, and Ccl2.

[0015] The present invention also provides a pharmaceutical composition comprising Lup, its salt derivatives, solvates or hydrates, and pharmaceutically acceptable carriers or excipients.

[0016] The present invention also provides a pharmaceutical preparation comprising the above pharmaceutical composition, which can be in the form of capsules, tablets, granules, powders, oral liquids, and pills.

[0017] The present invention demonstrates the significant antidepressant and antianxiety effects of Lup, demonstrated through open-field and elevated plus maze experiments. As a widely available, low-toxic Chinese herbal monomer, Lup offers a novel option for the development of new drugs to prevent and treat mental disorders such as depression and anxiety, broadening its application and holding great promise for related drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The effect of Lup on the number of times mice enter the open arm in the elevated plus maze test; Figure 2 The effect of Lup on the time mice spend in the target area in the elevated plus maze test; Figure 3 This is the effect of Lup on the time mice spend in the center of the open field test; Figure 4 This is the effect of Lup on the number of times mice enter the central area in the open field test; Figure 5 H&E staining was used to evaluate the effects of Lup on the heart, liver, spleen, lungs, and kidneys of mice; Figure 6 To evaluate the differential gene-protein interaction network for Lup genes that may play a key role in anxiety and depression; In the figure, ### Indicates p < 0.0001, which means there is a statistically significant difference compared with the blank group; *** It means p<0.0001, that is, there is a statistically significant difference between the drug-treated group and the model group; among them, Lup-L and Lup-H are low concentration and high concentration, respectively. DETAILED DESCRIPTION

[0019] The present invention is further illustrated in detail by the following examples, but it should be noted that the scope of the present invention is not limited by these examples.

[0020] Example 1 Elevated plus maze test Animal grouping and dosing: SPF male C57BL / 6 mice (8 weeks old), weighing approximately 20-25 g, were provided by the Experimental Animal Center of Nantong University. After one week of adaptive feeding, the mice were randomly divided into four groups, each consisting of eight mice. One group served as the normal control group and was injected with PBS alone. The other three groups received daily intraperitoneal injections of reserpine (0.5 mg kg -1 Depression was induced by administering Lup-L2 (dissolved in PBS) for 10 days. Ten days later, the mice were divided into a model group, a low-concentration Lup (2 mg / kg Lup-L), and a high-concentration Lup (8 mg / kg Lup-H) group. The drug-treated groups were administered the corresponding drug by gavage based on body weight, while the normal and model groups were administered saline once daily. Behavioral assessments were performed after two consecutive weeks.

[0021] Elevated plus maze test: (1) Apparatus preparation: The test apparatus consists of four arms, each 25 × 5 cm, extending outward from a 5 × 5 cm central cross platform. Two opposing arms are enclosed by 20 cm opaque walls, forming two closed arms. The entire cross maze is 40 cm above the ground.

[0022] (2) Implementation steps: Each mouse was placed at the central intersection of the elevated plus maze facing the open arm. The mouse's free movement trajectory within 5 minutes was recorded using the ANY-maze analysis system. The number of times the mouse entered the open arm and the closed arm, and the time it stayed in the open and closed arms were analyzed. The open arm stay time, number of entries, etc. were calculated. The proportion of open arm stay time (%) = open arm stay time / (open arm stay time + closed arm stay time) × 100%, and the proportion of open arm stay times (%) = open arm stay times / (open arm stay times + closed arm stay times) × 100%. If the number of times the mouse entered the open arm and the time it stayed in the open arm increased, it showed that the drug had an anxiolytic effect.

[0023] Result analysis: The results of the elevated plus maze test are as follows ( Figure 1 、 2 ) As shown in the data, the model group mice spent significantly less time in the open arms and entered the open arms more frequently than the normal group, with statistical significance. Compared with the model group, the Lup-treated group significantly increased the time the model mice spent in the open arms and the number of times they entered the open arms in a concentration-dependent manner.

[0024] Example 2 Open field experiment

[0025] The experimental animals were grouped and administered the same way as in Example 1.

[0026] The open field test (OFT), also known as the open-box test, is a method for evaluating the locomotor behavior, exploratory behavior, and stress levels of laboratory rodents. It is widely used in the study of psychiatric disorders such as Parkinson's disease, depression, and anxiety. The OFT assesses the anxiety level of experimental animals, with horizontal movement and central dwell time reflecting anxiety. Generally speaking, mice exhibiting anxiety-like behaviors exhibit more pronounced tactile tendencies (preferring to cling to walls), while mice with lower anxiety levels tend to spend more time in the central, open area of ​​the open field.

[0027] Open field test procedures: After setting up the experimental apparatus, remove the experimental mouse from its home cage and place it in the center of the open field, facing away from the experimenter. The experimenter then quickly leaves, allowing the mouse to move freely within the chamber. Using the ANY-maze acquisition and analysis system, the mouse's spontaneous activity, time spent in the central and peripheral zones, and distance traveled are recorded and analyzed over a 15-minute period. At the end of the experimental monitoring period, the mouse is removed and returned to its home cage. The bottom, contents, and side walls of the open field chamber are thoroughly cleaned to prevent any residual information from the current animal from influencing the results of the next test. Most studies use ethanol for cleaning, allowing the ethanol to evaporate completely before testing the next animal.

[0028] The results of the open field test showed that ( Figure 3 、 4 ) showed that compared with the normal group, the model group mice entered the central area less frequently and spent less time there. Lup treatment significantly increased the number of model mice entering the central area and the time depressed mice spent in the central area in a concentration-dependent manner, with the high-dose group showing greater efficacy.

[0029] Example 3 Pathological staining experiment

[0030] H&E staining is a commonly used histological staining method that uses two dyes, hematoxylin and eosin, to stain tissues, clearly showing cell morphology and tissue structure. Hematoxylin makes the cell nucleus appear blue, while eosin makes the cytoplasm appear red, allowing for the location and identification of cells.

[0031] The experimental animals were grouped and dosed as in Example 1. After the initial animal experiments were completed, mice were starved for 12 hours and then sacrificed by dislocation. Organs, including the heart, liver, spleen, lung, and kidney, were collected and rinsed with pre-chilled saline and dried with filter paper. Tissue blocks were fixed in 4% formaldehyde for 24 hours to stabilize their morphology and used for histopathological H&E staining.

[0032] The results of H&E staining are as follows ( Figure 5 ), paraffin sections of the mouse heart, liver, spleen, lung, and kidney, among other organs, were observed through H&E staining, and the tissue structure and cellular morphology of each organ were observed. Among them, the myocardial fibers of the heart were arranged in bundles, the nuclei of hepatocytes were round or oval, the lymphocytes and macrophages of the spleen were densely distributed, the alveolar epithelial cells were arranged in a flat shape within the alveolar cavity, and the glomeruli and renal tubular structures of the kidneys were clearly visible. These results show that compared with the normal control group, the model group mice did not develop obvious lesions in various organs, and there were no significant differences between the Lup-treated group and the model group, indicating that Lup is non-toxic to the heart, liver, spleen, lung, and kidney organs and has a high safety profile.

[0033] Example 4 Gene sequencing and differential gene-protein interaction network analysis Whole-transcriptome sequencing was performed by Gene Denovo Biotechnology Co., Ltd. Total RNA was extracted from the hippocampi of mice in the model and Lup groups (n = 3) using TRIzol reagent. cDNA libraries were sequenced on an Illumina NovaSeq X Plus according to a standard sequencing protocol. Differentially expressed genes (DEGs) between groups were analyzed and identified based on the following criteria: |log2(fold change)|>log2(1.5) and p-value <0.05. DEG enrichment analysis was performed between the model and Lup groups using the GO database (http: / / www.geneontology.org / ) and the KEGG database (https: / / www.kegg.jp / kegg / ). Bioinformatics analysis was performed using Omicsmart, a real-time interactive online data analysis platform (http: / / www.omicsmart.com), and the differentially expressed gene-protein interaction network was drawn using Cytoscape.

[0034] Result analysis: Figure 6 As can be seen, genes such as Acta2, Fos, Tagln, Vwf, and Ccl2 are relatively central or important, suggesting that they may be key nodes in the protein-protein interaction network of the differentially expressed genes. The significant differences in these genes and their central position in the protein-protein interaction network suggest that they may play a key regulatory role in the biological processes studied under the experimental conditions. Key genes and the protein-protein interaction networks they participate in provide potential candidates for drug target screening. For example, in the nervous system, Fos gene expression can be induced by stress and neurotransmitter release. Anxiety and depression are often accompanied by alterations in brain neuronal activity, and changes in Fos gene expression levels reflect neuronal activity. The significant increase in Fos gene expression may indicate that it is involved in changes in gene transcriptional regulation in the neuroplastic changes associated with anxiety and depression. Ccl2 can promote the activation of microglia and astrocytes and participate in neuroinflammatory responses. Anxiety and depression are closely related to neuroinflammation, which affects neurotransmitter metabolism and synaptic plasticity. Drug intervention targeting this gene or its related protein interactions may have potential therapeutic effects in treating anxiety and depression-related diseases.

[0035] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of yellow lupin white ketone in the preparation of drugs for preventing and treating depression and anxiety.

2. The use according to claim 1, characterized in that The depression and anxiety include depression or anxiety caused by abnormal expression of at least one of Acta2, Fos, Tagln, Vwf, and Ccl2 genes.

3. Use of yellow lupin white ketone in the preparation of drugs for preventing and treating diseases related to abnormal expression of Acta2 gene.

4. Application of yellow lupin white ketone in the preparation of drugs for preventing and treating diseases related to abnormal expression of Fos gene.

5. Use of yellow lupin white ketone in the preparation of drugs for preventing and treating diseases related to abnormal expression of Tagln gene.

6. Use of yellow lupin white ketone in the preparation of drugs for preventing and treating diseases related to abnormal expression of Vwf gene.

7. Use of yellow lupin white ketone in the preparation of drugs for preventing and treating diseases related to abnormal expression of Ccl2 gene.

8. A pharmaceutical composition, characterized in that The invention includes yellow lupin white ketone, its salt derivatives, prodrugs, solvates or hydrates.

9. A pharmaceutical preparation, characterized in that The invention comprises the pharmaceutical composition according to claim 8 and pharmaceutically acceptable carriers and excipients.

10. The pharmaceutical preparation according to claim 9, characterized in that The pharmaceutical preparation is in the form of any one of capsules, tablets, granules, powders, oral liquids and pills.

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