Application of isoflavone natural compounds in the preparation of drugs for the prevention or treatment of depression

Abstract

This invention discloses the application of a natural isoflavone compound in the preparation of drugs for the prevention or treatment of depression. The natural isoflavone compound is hematoxylin A, a derivative of hematoxylin A, or a pharmaceutically acceptable salt of hematoxylin A. Hematoxylin A can effectively reduce oxidative damage to HT22 neurons in the hippocampus induced by glutamate and corticosterone, and can significantly improve the behavioral performance of mice in a chronic restraint stress depression model, exhibiting clear antidepressant activity. Hematoxylin A has a well-defined structure and good safety profile, showing promising application prospects in the development of antidepressant drugs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical application technology of organic compounds, and more particularly to the application of an isoflavone natural compound in the preparation of drugs for the prevention or treatment of depression. Background Technology

[0002] Depression is a common mental disorder with high prevalence, high clinical heterogeneity, and complex pathogenesis. It is estimated that more than 300 million people worldwide are affected by depression, and it is projected to become one of the leading causes of global disease burden by 2030.

[0003] Depression is characterized by persistent low mood, loss of interest, cognitive impairment, and sleep disturbances. Its etiology remains incompletely understood. Current research generally suggests that the pathogenesis of depression is multidimensional and multi-pathway-based, involving the neurotransmitter system, endocrine axis, immune inflammation, neural plasticity, and abnormal regulation of various intracellular signaling pathways. Several theoretical hypotheses have been proposed to explain its pathological basis, primarily including imbalance of the monoamine neurotransmitter system, hyperfunction of the hypothalamus-pituitary-adrenal (HPA) axis, decreased expression of neurotrophic factors, and imbalance between neuroinflammation and oxidative stress.

[0004] Currently, first-line antidepressant treatment is mainly based on the "monoamine hypothesis," represented by selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs). Although these drugs have some therapeutic effect on some patients, their clinical application still faces many limitations, including slow onset of action, high incidence of side effects, and high relapse rate after discontinuation. Therefore, based on the multidimensional pathogenesis of depression, developing novel therapies that can intervene in other key pathological stages is of vital importance for improving treatment efficiency and patient prognosis.

[0005] Hematoxylin A is a naturally occurring compound found in plants, belonging to the isoflavone class of natural compounds, and possesses significant antioxidant and anti-inflammatory effects. Chinese patent publication CN107362160A discloses the application of hematoxylin A in the treatment of neurodegenerative diseases, and Chinese patent publication CN109288835B discloses its application in the preparation of drugs for treating acute pulmonary embolism. Chinese patent publication CN111388460A discloses the anti-aging uses of hematoxylin A. However, no reports have been found regarding the application of hematoxylin A in the treatment of depression-related diseases. Summary of the Invention

[0006] This invention provides the application of hematoxylin A in the preparation of drugs for the prevention or treatment of depression. At the cellular level, hematoxylin A can effectively reduce oxidative damage to HT22 neurons in the hippocampus induced by glutamate and corticosterone. At the animal level, hematoxylin A can significantly improve the behavioral performance of mice in a chronic restraint stress depression model, demonstrating clear antidepressant activity. Furthermore, hematoxylin A has a well-defined structure and good safety profile, showing promising application prospects in the development of antidepressant drugs.

[0007] The technical solution of the present invention is as follows: The use of an isoflavone natural compound in the preparation of a drug for the prevention or treatment of depression, wherein the isoflavone natural compound is hematoxylin A, a derivative of hematoxylin A, or a pharmaceutically acceptable salt of hematoxylin A.

[0008] The molecular formula of hematoxylin A is C 18 H 12 O5, its structure is shown in equation (I): .

[0009] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0010] Preferably, the excipients include at least one of pharmaceutically used carriers, excipients, and diluents.

[0011] Preferably, the dosage form of the drug is liquid, powder for injection, tablet, capsule, powder, pill, ointment, granule or dressing.

[0012] Preferably, the concentration of hematoxylin A or its pharmaceutically acceptable salt used on cells is 5-15 μmol / L, and the dosage used on animals is 50-100 mg / kg body weight.

[0013] Preferably, the drug is used to prevent or treat neuronal cell damage caused by excitotoxicity and oxidative stress.

[0014] Preferably, the drug is used to prevent or treat hyperfunction of the hypothalamus-pituitary-adrenal axis and glucocorticoid neurotoxicity caused by chronic stress.

[0015] Preferably, the drug is used for the prevention or treatment of chronic stress-induced depression.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides novel uses of hematoxylin A, its derivatives, or pharmaceutically acceptable salts in the preparation of drugs for the prevention or treatment of depression. Hematoxylin A significantly protects hippocampal neurons from glutamate and corticosterone-induced damage at the cellular level, demonstrating a clear neuroprotective effect. At the animal level, it effectively improves depressive-like behavior in a mouse model of chronic restraint stress depression. Hematoxylin A intervenes in key pathological aspects of depression, such as oxidative stress, providing a structurally well-defined and mechanistically novel candidate compound for the development of novel antidepressants. Attached Figure Description

[0017] Figure 1 The diagram shows the protective effect of hematoxylin A on glutamate-induced HT22 cell damage in Example 1 of this invention. (a)-(d) are microscopic morphological images of the normal control group, the glutamate damage model group (Glu 10 mmol / L), the glutamate and hematoxylin A co-treatment group (Glu + SA 10 μmol / L), and the hematoxylin A alone treatment group (SA 10 μmol / L), respectively. (e)-(h) are flow cytometry apoptosis detection diagrams of the normal control group, the glutamate damage model group, the glutamate and hematoxylin A co-treatment group, and the hematoxylin A alone treatment group, respectively. (i) is a flow cytometry statistical analysis diagram of the apoptosis rate of each group.

[0018] Figure 2 The diagram shows the protective effect of hematoxylin A against corticosterone-induced HT22 cell damage in Example 2 of this invention. (a)-(d) are microscopic morphological images of the normal control group (Control), the corticosterone-induced damage model group (Gort 500 μmol / L), the co-treatment group of corticosterone and hematoxylin A (Gort+SA 10 μmol / L), and the hematoxylin A-only treatment group (SA 10 μmol / L), respectively. (e)-(h) are flow cytometry apoptosis detection diagrams of the normal control group, the corticosterone-induced damage model group, the co-treatment group of corticosterone and hematoxylin A, and the hematoxylin A-only treatment group, respectively. (i) is a flow cytometry statistical analysis diagram of the apoptosis rate of each group.

[0019] Figure 3 This is a statistical graph showing the effect of hematoxylin A on immobility time in a forced swimming experiment in a mouse model of chronic stress-induced depression, as described in Example 4 of this invention.

[0020] Figure 4 This is a statistical graph showing the effect of hematoxylin A on the sucrose preference index in a sucrose preference experiment in mice with chronic stress-induced depression, as described in Example 5 of this invention.

[0021] Figure 5This is a statistical graph showing the effect of hematoxylin A on the time mice spent in the central region and the total distance traveled in an open field experiment in Example 6 of the present invention, in relation to a chronic stress-induced depression model mouse.

[0022] Figure 6 This is a representative diagram of the movement trajectories of mice in each group during the open field experiment in Example 6 of the present invention. Detailed Implementation

[0023] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The embodiments of the present invention include, but are not limited to, the following conditions, and modifications can be made within the scope acceptable to the art. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the protection scope of the present invention. Experimental methods not specifically specified in the embodiments are generally performed under conventional conditions; for example, the conditions described in flow cytometry detection of apoptosis are all according to the manufacturer's recommendations.

[0024] Example 1: Protective effect of hematoxylin A against glutamate-induced HT22 cell damage To evaluate the neuroprotective effect of hematoxylin A, this invention uses mouse hippocampal neurons HT22 to construct a glutamate injury model. This model is used to simulate neuronal damage caused by excitotoxicity and oxidative stress in neurological diseases such as depression. The specific experimental procedure is as follows: (1) After digesting and centrifuging HT22 cells in the logarithmic growth phase and in good condition, discard the supernatant, add a small amount of complete culture medium, mix well by pipetting, and then divide into 1×10⁶ cells per well. 5 Cells were seeded at a density of 1,000 cells per well into 6-well plates and incubated in an incubator. (2) After the plates adhere to the walls, remove the old culture medium from the 6-well plate and add 2 mL of fresh culture medium as follows: complete culture medium (normal control group), culture medium containing 10 mmol / L glutamate, culture medium containing both 10 mmol / L glutamate and 10 μmol / L hematoxylin A, and culture medium containing 10 μmol / L hematoxylin A.

[0025] (3) After 9 hours of treatment with the corresponding drugs, the cell morphology changes of each group of cells were observed under a microscope. When it was observed that more than half of the cells in the glutamate-damaged group had undergone apoptosis, the cell apoptosis rate was detected by flow cytometry to systematically evaluate the protective effect of hematoxylin A on glutamate-induced neuronal damage.

[0026] The experimental results are shown in Figure 1In the glutamate-induced injury model, HT22 cells exhibited significant morphological changes, including cell shrinkage, loss of protrusions, decreased adhesion, and widespread cell death. In contrast, the morphology of cells co-treated with glutamate and hematoxylin A was significantly improved, with intact cell structure and good adhesion, similar to the normal control group, and no obvious signs of cell death were observed. Further apoptosis detection showed that the apoptosis rate in the co-treatment group was significantly lower than that in the glutamate-induced injury group (p < 0.0001), a statistically significant difference. Furthermore, the cell morphology and apoptosis rate in the hematoxylin A single treatment group did not show significant changes. These results indicate that hematoxylin A can effectively maintain cell morphology and inhibit apoptosis in the glutamate-induced HT22 cell injury model, exhibiting significant antioxidant and neuroprotective effects, and these results are minimally affected by the cytotoxicity of hematoxylin A.

[0027] Example 2: Protective effect of hematoxylin A against corticosterone-induced HT22 cell damage This example further evaluates the neuroprotective effect of hematoxylin and eosin A using a corticosteroid (Cort)-induced HT22 cell injury model. This model utilizes prolonged treatment of cells with the stress hormone corticosteroids to simulate hypothalamic-pituitary-adrenal axis hyperfunction and glucocorticoid neurotoxicity induced by chronic stress in patients with depression. The specific experimental procedure is as follows: (1) After digesting and centrifuging HT22 cells in the logarithmic growth phase and in good condition, discard the supernatant, add a small amount of complete culture medium, mix well by pipetting, and then divide into 1×10⁶ cells per well. 5 Cells were seeded at a density of 1,000 cells per well into 6-well plates and incubated in an incubator. (2) After the plates adhere to the walls, remove the old culture medium from the 6-well plate and add 2 mL of fresh culture medium according to the following treatments: complete culture medium (control group), culture medium containing 500 μmol / L corticosterone, culture medium containing both 500 μmol / L corticosterone and 10 μmol / L hematoxylin A, and culture medium containing 10 μmol / L hematoxylin A.

[0028] (3) After 20 hours of treatment with the corresponding drugs, the cell morphology changes of each group of cells were observed under a microscope. When it was observed that more than half of the cells in the corticosterone-damaged group had undergone apoptosis, the cell apoptosis rate was detected by flow cytometry to systematically evaluate the protective effect of hematoxylin A on corticosterone-induced neuronal damage.

[0029] The experimental results are shown in Figure 2Cells in the corticosterone-induced neuronal damage model group exhibited significant morphological abnormalities, characterized by elongated, filamentous cells accompanied by substantial cell death. In contrast, the co-treatment group of corticosterone and hematoxylin A showed significant improvement in cell morphology. Although some cells still exhibited some degree of elongation, they adhered well overall, with no obvious signs of cell death, and their cell state essentially returned to the level of the normal control group. Flow cytometry analysis revealed a significantly lower apoptosis rate in the co-treatment group compared to the corticosterone-induced damage group (p < 0.0001), a statistically significant difference. Furthermore, no significant changes in cell morphology or apoptosis rate were observed in the hematoxylin A single-treatment group. These results indicate that hematoxylin A also has a significant protective effect against corticosterone-induced neuronal damage, and this effect is less affected by the cytotoxicity of hematoxylin A.

[0030] Example 3: Establishment and administration of an experimental animal depression model (a) Laboratory animals SPF-grade C57 / BL6 mice, male, weighing 25-30 g, were purchased from SPF Biotechnology Co., Ltd. and housed in the SPF-grade laboratory of the Experimental Animal Center of the Nanhu Brain-Computer Interdisciplinary Research Institute. The relative temperature was 23±2℃, the relative humidity was 45%-65%, and the animals were fed conventional feed with free access to water and food.

[0031] (II) Experimental Scheme 1. Animal grouping Fifty C57BL / 6 mice were randomly divided into 5 groups of 10 each: control group, model group, positive control group, and low- and high-dose hematoxylin A groups.

[0032] 2. Establishment of a depression model Mice in both the model and drug-treated groups were restrained using adjustable, breathable tubes to ensure they could not move freely but did not experience respiratory pressure. Restraint was performed once daily for 4–6 hours each time, the exact duration determined based on preliminary experiments, aiming to stably induce depressive-like behavior without causing excessive physical harm, for a total of 5 weeks. Control group mice were housed normally in the same laboratory without restraint, but underwent brief daily handling to balance manipulation interference. After the 5-week restraint period, a series of behavioral experiments were conducted to assess the formation of depressive-like behavior and verify the successful establishment of the model.

[0033] 3. Dosing regimen The medication was administered by gavage daily for 28 days. The administration method is shown in Table 1.

[0034] Table 1 Example 4: Forced Swimming Experiment (FST) in Laboratory Animals After the restraint / drug administration in Example 3, the mice were placed in a transparent bucket with a water depth of 20 cm and a diameter of 40 cm for 2 minutes to pre-acclimatize, and the immobility time within the next 4 minutes was recorded.

[0035] The experimental results are shown in Figure 3 The immobility time of mice in the model group during the forced swimming test was significantly increased compared to the control group (p<0.0001), indicating that the mice exhibited a clear state of behavioral despair, confirming the successful establishment of the depression model. Treatment with different doses of hematoxylin A significantly shortened the immobility time of the depression model mice. Compared with the CRS model group, the hematoxylin A treatment groups (50 mg / kg and 100 mg / kg) both showed clear and significant antidepressant activity. Further experimental data showed that the high-dose group (100 mg / kg) of hematoxylin A was slightly better than the low-dose group (50 mg / kg) in shortening immobility time, and the improvement effect of the high-dose group was similar to that of the positive control drug fluoxetine (Flux, 15 mg / kg). These results indicate that hematoxylin A can effectively improve chronic stress-induced depressive-like behavior.

[0036] Example 5: Sucrose Water Experiment in Laboratory Animals (SPT) After the restraint / drug administration in Example 3, mice underwent 48 hours of acclimatization training before the experiment, during which they were simultaneously given 1% sucrose solution and pure water. The bottle positions were changed every 12 hours to eliminate positional preference, after which all mice were deprived of water for 12 hours. During the formal test, each mouse was simultaneously provided with pre-weighed sucrose solution and pure water, and the consumption was recorded after 12 hours. The sucrose preference was calculated using the following formula: Sugar solution preference (%) = [Sucrose solution consumption / (Sucrose solution consumption + Pure water consumption)] × 100%.

[0037] The experimental results are shown in Figure 4 Compared with the control group, the saccharide preference index of mice after modeling showed a significant decrease (p<0.05), indicating successful modeling and the mice exhibiting significant anhedonia behavior. After treatment with hematoxylin A, the saccharide preference index of the model mice showed a significant upward trend, with a significant difference between the treatment group and the model group (p<0.001). The high-dose hematoxylin A group (100 mg / kg) was more effective than the low-dose group (50 mg / kg) in improving the saccharide preference index. The saccharide preference index of the high-dose group was close to that of the blank control group and the positive control group (fluoxetine), showing a good dose-response relationship. These results indicate that hematoxylin A can improve the saccharide preference index in depressed model mice and effectively improve depressive-like behavior.

[0038] Example 6: Experimental Animal Mining Experiment (OFT) After the restraint / administration in Example 3, the mice were placed in a 30×30 cm white square frame, and the distance the mice moved in the central area within 10 minutes was recorded.

[0039] The experimental results are shown in Figure 5 and Figure 6 In the chronic stress model group, the distance the mice moved in the central region was significantly reduced compared to the control group (p<0.05). This was also visually observed in the movement path maps. The tracks of the control group mice were evenly distributed throughout the field, including frequent entry into the central region; while the tracks of the mice after modeling were mostly confined to the peripheral regions, rarely venturing into the center. After group treatment, the depressive behavior of the mice was effectively restored: compared with the CRS group, administration of hematoxylin A significantly increased the distance the mice moved in the central region (p<0.05), with the improvement effect of the 100 mg / kg hematoxylin A group being better than that of the 50 mg / kg group. The movement path maps showed that the distribution density of the movement paths of the mice in the hematoxylin A treatment group was significantly increased in the central region, and the routes were more complex and extensive. This visual result further corroborated the quantitative data.

[0040] In summary, this invention reveals for the first time a novel application of the traditional Chinese medicine monomer hematoxylin A in the treatment of depression. In cell models, hematoxylin A significantly protected against glutamate and corticosterone-induced damage to HT22 neurons. In a mouse model of depression under chronic restraint stress, hematoxylin A dose-dependently improved depressive-like behaviors: in forced swimming, sucrose preference, and open field tests, the 100 mg / kg dose group showed superior overall effects compared to the 50 mg / kg group, and its effects were comparable to those of the positive control drug fluoxetine on multiple behavioral indicators. These results indicate that hematoxylin A exerts its antidepressant effect by alleviating key pathological processes such as oxidative stress, and possesses the potential to be developed into a novel, safe, and effective antidepressant drug.

[0041] The embodiments described above provide a detailed explanation of the technical solutions and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, and equivalent substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of a natural isoflavone compound in the preparation of drugs for the prevention or treatment of depression, characterized in that, The aforementioned isoflavone natural compounds are hematoxylin A, derivatives of hematoxylin A, or pharmaceutically acceptable salts of hematoxylin A.

2. Use according to claim 1, characterized in that, The molecular formula of suavione A is C 18 H 12 O5, which has the structure shown in formula (I): 。 3. Use according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

4. Use according to claim 3, characterized in that, The excipients include at least one of pharmaceutically used carriers, excipients, and diluents.

5. The use according to claim 1, characterized in that, The dosage form of the drug is liquid, powder for injection, tablet, capsule, powder, pill, ointment, granule or dressing.

6. The application according to claim 1, characterized in that, The dosage of hematoxylin A or its pharmaceutically acceptable salt used on cells is 5-15 μmol / L; the dosage used on animals is 50-100 mg / kg body weight.

7. The application according to claim 1, characterized in that, The drug is used to prevent or treat neuronal cell damage caused by excitotoxicity and oxidative stress.

8. The application according to claim 1, characterized in that, The drug is used to prevent or treat hyperfunction of the hypothalamus-pituitary-adrenal axis and glucocorticoid neurotoxicity caused by chronic stress.

9. The application according to claim 1, characterized in that, The aforementioned medication is used to prevent or treat chronic stress-induced depression.

Images