Application of helicid in antidepressant drug

Abstract

The invention relates to the technical field of psychiatric pharmacology, discloses application of helicid in antidepressant drugs, and aims to solve the problems of large side effect, unstable curative effect, drug resistance and the like of existing antidepressant drugs. According to the medicine, through multiple mechanisms of activating a cAMP / PKA / CREB signal channel, promoting neuroplasticity, inhibiting neuronal apoptosis, relieving neuroinflammation and the like, the depressive symptom is remarkably improved. The preparation method comprises the steps of extraction and purification of helicid and preparation of the medicinal preparation. Animal experiments verify that the hilicidum-based antidepressant drug can effectively improve behavioral indexes of depression model rats, has low side effects, and does not have the problem of drug resistance. Compared with the traditional medicine, the medicine provided by the invention has the advantages that the treatment effect is improved, the adverse reaction is reduced, the application prospect is better, and a new medicine choice with a multi-target action mechanism is provided for the treatment of depression.

Description

Technical Field

[0001] The present invention relates to the technical field of psychopharmacology, and in particular to the application of tofu glycosides in antidepressant drugs. Background Art

[0002] Depression is a common mood disorder with a widespread incidence and high disability rate worldwide. According to data from the World Health Organization (WHO), depression has become one of the leading causes of disability worldwide. Its clinical symptoms include low mood, slow thinking, inattention, insomnia, changes in appetite, and loss of interest in daily activities. With the accelerated pace of society and the increase in life pressure, the incidence of depression has been increasing year by year, and has become a public health problem that needs to be urgently addressed in modern society. The long-term treatment of depression not only relies on drug therapy, but also requires comprehensive treatment through psychological intervention and other means. However, the efficacy and safety of existing treatment options are still controversial, and they still face problems of side effects and drug resistance.

[0003] At present, the drugs used in clinical treatment of depression mainly include selective serotonin reuptake inhibitors (SSRIs), norepinephrine reuptake inhibitors (SNRIs) and monoamine oxidase inhibitors (MAOIs). Although these drugs have achieved certain therapeutic effects in clinical practice, there are still problems such as delayed treatment, unstable efficacy, dependence and side effects. For example, common side effects include sexual dysfunction, weight gain, insomnia, anxiety, etc., and some patients develop resistance to these drugs, which greatly reduces the therapeutic effect. Therefore, researchers have been looking for new antidepressants in order to break through the bottleneck of existing treatment options, improve treatment effects, reduce side effects, and delay or avoid the occurrence of resistance.

[0004] Although existing antidepressants have achieved certain clinical results, they still have significant side effects and drug resistance. Most existing treatment methods focus on the development of single-mechanism drugs. For example, SSRIs and SNRIs mainly relieve depressive symptoms by regulating the balance of neurotransmitters, but their effects on repairing nerve cell damage, promoting neuroplasticity, and inhibiting neuroinflammation are relatively limited. In addition, traditional drug treatments are often accompanied by a long treatment period, and the individual differences in efficacy are large. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides the use of tofu glycosides in antidepressant drugs, which solves the problems of existing antidepressant drugs, such as large side effects, unstable efficacy and drug resistance.

[0006] To achieve the above objectives, the present invention is implemented through the following technical scheme: an antidepressant drug based on tofu glycoside, the active ingredient of the drug is tofu glycoside, and the purity is not less than 98%.

[0007] Preferably, the drug is administered orally, sublingually or topically, with a daily dosage of 5-50 mg / kg body weight.

[0008] The method for preparing tofu fruit glycoside in an antidepressant drug comprises the following steps:

[0009] Select radish tree fruits, wash, dry and crush them to obtain crude extracts;

[0010] Using 70%-90% ethanol for ultrasonic extraction, filtering to remove residue and concentrating to obtain a concentrated solution;

[0011] The concentrated solution is purified by silica gel column chromatography to obtain tofu glycosides with a purity of not less than 98%; the tofu glycosides are mixed with auxiliary materials to prepare tablets, capsules or granules.

[0012] Preferably, the auxiliary materials are microcrystalline cellulose and lactose, and the amount used is 1-5 times the mass of tofu glycosides. After being mixed evenly, they are pressed into tablets or assembled into capsules.

[0013] Preferably, in the purification step, the silica gel column chromatography method uses chloroform-methanol 5:1 as the mobile phase, and after graded elution, a purified product of tofu glycosides is obtained, and the purity is not less than 98%.

[0014] Preferably, the pharmaceutical preparation is prepared by micronization or film coating.

[0015] The application of tofu fruit glycosides in antidepressant drugs is used to treat depression, anxiety, post-traumatic stress disorder or neurological diseases, and regulate the cAMP / PKA / CREB signaling pathway.

[0016] Preferably, the drug can be used in combination with other antidepressants, in particular, in combination with selective serotonin reuptake inhibitors, tricyclic antidepressants or monoamine oxidase inhibitors.

[0017] The present invention provides the use of tofu fruit glycosides in antidepressant drugs. It has the following beneficial effects:

[0018] 1. The application of tofu glycosides in antidepressant drugs of the present invention can effectively reduce the apoptosis of PC12 cells induced by CORT. The experimental results show that tofu glycosides inhibit the apoptosis caused by CORT by regulating the expression of anti-apoptotic factors and pro-apoptotic factors and apoptosis execution proteins in cells. Specifically, tofu glycosides can reduce the neuronal damage induced by chronic stress and improve neuronal survival by downregulating the expression of pro-apoptotic proteins and upregulating the level of anti-apoptotic proteins, thereby reducing the depressive symptoms caused by chronic stress.

[0019] 2. The tofu glycosides of the present invention can significantly improve the behavioral performance of the chronic unpredictable stress mouse model. After tofu glycoside treatment, the immobility time of mice in the forced swimming test, tail suspension test and sucrose preference test was significantly reduced, and the activity level increased, showing an antidepressant effect. In addition, the preference of mice in the tofu glycoside treatment group for sugar water was significantly improved, indicating that tofu glycosides can effectively alleviate the anhedonia caused by stress. These results prove that tofu glycosides have the potential to promote emotional recovery and improve depressive symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a flow chart of the method of the present invention;

[0021] Figure 2 Schematic diagram of overexpression efficiency of the plasmid of the present invention;

[0022] Figure 3 Schematic diagram of the flow cytometry detection of the effect of tofu glycosides on the apoptosis rate of PC12 cells induced by CORT by PDE1C;

[0023] Figure 4 This is a schematic diagram of the effect of tofu fruit glycosides on the expression levels of Bax, Bcl-2 and Cleaved-caspase 3 proteins in CORT-induced PC12 cells detected by Western blot of the present invention;

[0024] Figure 5 Schematic diagram of the effect of CUMS of the present invention on rat body weight;

[0025] Figure 6 Schematic diagram of the effect of CUMS of the present invention on behavioral indicators of rats;

[0026] Figure 7 This is a schematic diagram showing the effect of silencing PDE1C on the body weight of CUMS rats according to the present invention;

[0027] Figure 8 Schematic diagram of the effect of silencing PDE1C on the behavior of CUMS rats according to the present invention;

[0028] Fig. 9This is a schematic diagram of the effect of silencing PDE1C on cAMP expression level detected by ELISA of the present invention;

[0029] Fig.10 This is a schematic diagram of the effect of tofu glycosides on the expression levels of PDE1C, p-PKA and p-CREB proteins in the hippocampus of CUMS rats detected by Western blot of the present invention. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The embodiment of the present invention provides an antidepressant drug based on tofu glycosides, wherein the active ingredient is tofu glycosides with a purity of not less than 98%. Tofu glycosides are active monomer components extracted from the fruit of the radish tree of the Proteaceae family, and have been proven to have an antidepressant effect.

[0032] Tofu glycosides exert antidepressant effects by regulating the cAMP / PKA / CREB signaling pathway in the brain. As the second messenger in the cell, cAMP activates PKA (protein kinase A), which in turn phosphorylates CREB (cAMP response element binding protein), regulates the expression of nerve growth factor BDNF (brain-derived neurotrophic factor), promotes neurogenesis and synaptic plasticity, and ultimately achieves an antidepressant effect. Through this mechanism, tofu glycosides restore neural plasticity and improve neuronal function in the hippocampus, thereby effectively alleviating depressive symptoms.

[0033] Based on the above content, the embodiment of the present invention provides a method for preparing an antidepressant drug based on tofu glycosides, which specifically includes the following steps:

[0034] The fruits of radish trees are selected, washed, dried and pulverized to obtain a crude extract.

[0035] Ultrasonic extraction: Use 70%-90% ethanol solution for ultrasonic extraction, filter and remove the residue, and then concentrate to obtain a concentrated solution.

[0036] Purification: The concentrate is separated and purified by silica gel column chromatography to obtain tofu fruit glycosides with a purity of not less than 98%.

[0037] Preparation: Mix tofu glycoside with appropriate amount of auxiliary materials (such as microcrystalline cellulose, lactose, etc.) and make into tablets, capsules or granules.

[0038] In the first step, after washing, drying and crushing, harmful impurities in the extract are removed, ensuring the efficiency and purity of subsequent extraction. Ultrasonic extraction using 70%-90% ethanol can effectively extract tofu glycosides. The efficient vibration of ultrasound can increase the contact area between the solvent and the plant material, greatly improving the extraction rate. The purification process uses silica gel column chromatography, which uses the polarity difference of tofu glycosides in different solvent systems to separate high-purity tofu glycosides, ensuring that the purity of the final product reaches more than 98%. The efficiency of this purification step ensures the effectiveness and consistency of the drug. Finally, the tofu glycosides are mixed with excipients and made into tablets, capsules or granules to facilitate oral use and improve the bioavailability and stability of the drug.

[0039] Antidepressants are administered orally, sublingually or topically. The usual dosage is 5-50 mg / kg body weight per day. The specific dosage is adjusted individually according to the patient's condition and weight.

[0040] After being administered orally or sublingually, tofu glycosides can quickly enter the gastrointestinal tract and be absorbed. Since tofu glycosides have good water solubility and high purity, their absorption rate is high, and they reach the brain through the blood circulation to exert their antidepressant effects. In this way, the drug can directly regulate the cAMP / PKA / CREB signaling pathway in the brain, enhance the plasticity of neurons, repair neuronal damage in the hippocampus, and ultimately effectively improve depressive symptoms. For topical administration, tofu glycosides can be absorbed through the skin into the blood circulation, thereby achieving its therapeutic effect, which is particularly suitable for some patients who have difficulty taking oral medications.

[0041] Tofu glycosides exert their antidepressant effects through multiple pathways. First, by activating the cAMP / PKA / CREB signaling pathway, tofu glycosides increase the expression of nerve growth factor BDNF, and promote the survival and regeneration of neurons. Secondly, tofu glycosides can also reduce neuroinflammation caused by stress and inhibit apoptosis of neurons in the hippocampus. Thirdly, tofu glycosides improve synaptic plasticity and restore the neural function of the hippocampus, thereby improving depressive symptoms.

[0042] The antidepressant effect of tofu glycosides is closely related to the activation of the cAMP / PKA / CREB signaling pathway. cAMP activates PKA, which in turn phosphorylates CREB, activating genes related to neuroplasticity (such as BDNF). The role of BDNF in neurons is to promote neuronal survival, synaptic remodeling, and neurogenesis. In addition, tofu glycosides also reduce oxidative stress levels and alleviate neuronal damage by inhibiting neuroinflammatory responses in the hippocampus. Through these multiple mechanisms, tofu glycosides can effectively reverse neuronal apoptosis caused by chronic stress and improve depressive symptoms.

[0043] Based on the above-mentioned antidepressant drug of tofu glycosides, this embodiment discloses the application of tofu glycosides antidepressant drug:

[0044] Tofu fruit glycosides are used to treat depression, anxiety, post-traumatic stress disorder (PTSD) and other neurological diseases. Its mechanism of action is mainly through activating the cAMP / PKA / CREB signaling pathway, regulating neural plasticity, promoting the expression of nerve growth factor, reducing stress-induced neuroinflammation, inhibiting neuronal apoptosis, and thus improving the symptoms of related mental illnesses.

[0045] By acting on the cAMP / PKA / CREB pathway, tofu glycosides activate genes related to neuroplasticity (such as BDNF), promoting the survival and regeneration of neurons. Through this pathway, tofu glycosides can effectively improve neurological dysfunction caused by chronic stress, especially in the hippocampus, enhance synaptic plasticity, repair nerve damage, and relieve depressive symptoms. Through this mechanism, tofu glycosides are not only effective for depression, but also have a significant therapeutic effect on mental illnesses such as anxiety and PTSD.

[0046] This medication may be used in combination with other antidepressants, particularly selective serotonin reuptake inhibitors (SSRIs), tricyclic antidepressants (TCAs), or monoamine oxidase inhibitors (MAOIs).

[0047] As a natural herbal medicine, tofu glycosides exert their antidepressant effects mainly by activating the cAMP / PKA / CREB signaling pathway. When used in combination with traditional antidepressants such as SSRIs, TCAs or MAOIs, they can enhance the antidepressant effect without increasing the side effects of the drugs. Drugs such as SSRIs mainly work by increasing the concentration of 5-HT (5-hydroxytryptamine) in the synaptic cleft, while tofu glycosides enhance the effects of the drug by repairing neurons and improving neural plasticity. The combination of the two has a synergistic effect.

[0048] The tofu glycosides are extracted by ultrasonic wave and purified by silica gel column chromatography to obtain tofu glycosides with a purity of not less than 98%, so as to ensure its efficacy and stability in clinical application.

[0049] Ultrasonic extraction uses high-frequency vibration to destroy plant cell walls, effectively improving the extraction efficiency of tofu glycosides by solvents. The separation principle of silica gel column chromatography is based on the adsorption capacity of different compounds on solvents. Through different mobile phases, tofu glycosides can be effectively separated and purified to ensure that its purity meets clinical application standards. High-purity tofu glycosides can ensure the stability and consistency of drug efficacy and reduce potential adverse reactions.

[0050] Example 1 Evaluation of the in vitro effects of antidepressant drugs based on tofu glycosides:

[0051] Refer to the attached Figure 2-4 , Experimental purpose: To evaluate the inhibitory effect of tofu fruit glycosides on CORT (corticosterone)-induced PC12 cell apoptosis.

[0052] Experimental Materials:

[0053] Tofu glycosides: purity not less than 98%.

[0054] PC12 cells: Derived from rat adrenal medullary pheochromocytoma, have typical neuronal characteristics and are often used in neuro-drug screening.

[0055] CORT: corticosterone, as a stress mimetic.

[0056] CCK-8 kit: used for cell proliferation activity detection.

[0057] Annexin V-FITC / PI Apoptosis Kit: used for detecting cell apoptosis by flow cytometry.

[0058] Western Blot: used for protein expression detection.

[0059] Experimental steps:

[0060] Cell culture:

[0061] PC12 cells were cultured in DMEM high-glucose medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) and maintained at 37°C and 5% CO2.

[0062] CORT-induced model construction:

[0063] PC12 cells were divided into three groups: control group (without CORT and tofu glycosides), CORT group (addition of 400 μM CORT to induce cell apoptosis), CORT+tofu glycosides group (200 μM tofu glycosides and CORT were co-cultured for 24 hours).

[0064] The cells in the CORT group showed obvious apoptosis after 24 hours, while the apoptosis rate of the cells in the tofu glycoside group was significantly reduced.

[0065] CCK-8 cell proliferation kit detection:

[0066] The CCK-8 method was used to detect the cell activity of each group. In the CORT group, the cell activity decreased significantly (P<0.05), while the cell activity of the tofu fruit glycoside group recovered significantly (P<0.05), approaching the level of the control group.

[0067] Flow cytometry for apoptosis detection:

[0068] Annexin V-FITC / PI kit was used to detect cell apoptosis, and the results showed that the apoptosis rate of cells in the CORT group reached about 50%, while the apoptosis rate of cells in the tofu glycoside group was significantly reduced to about 20%.

[0069] Western Blot Analysis:

[0070] The protein expression levels of Bax, Bcl-2 and Cleaved caspase-3 were detected by Western Blot.

[0071] In the CORT group, the expression levels of Bax and Cleaved caspase-3 proteins were significantly increased, and the expression of Bcl-2 protein was decreased; while in the tofu glycoside group, the expression levels of Bax and Cleaved caspase-3 were decreased, and the expression of Bcl-2 was restored to a level close to that of the control group.

[0072] Experimental results:

[0073] Tofu fruit glycosides have a significant inhibitory effect on CORT-induced apoptosis of PC12 cells, and alleviate CORT-induced neuronal damage by regulating apoptosis-related proteins such as Bcl-2, Bax, and Cleaved caspase-3.

[0074] Table 1 Experimental group design

[0075]

[0076] Table 2: CCK-8 assay for cell viability

[0077] Group Cell survival rate (%) Control 100±5 CORT group (CORT) 45±3 Helicid 95±4 CORT+Hel group 75±5

[0078] Table 3: Apoptosis rate detected by flow cytometry

[0079] Group Apoptosis rate (%) Control 5±1 CORT group (CORT) 40±2 Helicid Helicidin group: 10±2 CORT+Hel group 20±3

[0080] Table 4: Western Blot analysis (Bax, Bcl-2, Cleaved-Caspase 3)

[0081]

[0082]

[0083] Example 2: Antidepressant effect of tofu glycosides in CUMS rat model:

[0084] Refer to the attached Figure 2-4 , Experimental purpose: To evaluate the antidepressant effect and mechanism of tofu glycosides in CUMS rat model.

[0085] Experimental Materials:

[0086] Tofu glycosides: purity not less than 98%.

[0087] CUMS (Chronic Unpredictable Stress) rats: Adult male Wistar rats, weighing 200-250 g.

[0088] Antidepressant drugs: Fluoxetine was used as a positive control drug.

[0089] Experimental instruments: behavioral testing system (such as FST, SPT, Morris water maze, etc.).

[0090] Western Blot analysis: used to detect protein expression in neural tissue.

[0091] Experimental steps:

[0092] Establishment of CUMS rat model:

[0093] Rats were subjected to CUMS treatment for 6 weeks, including stressors such as strong light exposure, temperature changes, and water deprivation, and a depression model was successfully established.

[0094] The depressive symptoms of rats were assessed by behavioral tests such as the forced swim test (FST) and the sucrose preference test (SPT).

[0095] Pharmacological interventions:

[0096] The rats were divided into five groups: control group, CUMS group, CUMS+tofu glycoside group (5 mg / kg), CUMS+fluoxetine group (5 mg / kg), and CUMS+tofu glycoside+PDE1C overexpression group.

[0097] Both tofu glycosides and fluoxetine were administered by intraperitoneal injection once a day for 2 weeks.

[0098] Behavioral testing:

[0099] FST: The immobility time of the rats in forced swimming was measured. The immobility time of the rats in the CUMS group was significantly increased, while that of the rats in the tofu fruit glycoside treatment group was significantly reduced.

[0100] SPT: The rats' preference for sugar water was measured. The sugar water preference of rats in the CUMS group decreased significantly, while the sugar water preference of rats in the tofu fruit glycoside treatment group returned to a near normal level.

[0101] Western Blot Analysis:

[0102] The expression of PDE1C, Bcl-2, Bax, Cleaved caspase-3, p-CREB, BDNF and other proteins in hippocampal tissue was detected.

[0103] Please see attached Figure 5-10 The results showed that the expression levels of PDE1C, Bax and Cleaved caspase-3 in the CUMS group were increased, and the expression levels of Bcl-2 and p-CREB were decreased; in the tofu glycoside treatment group, the expression of PDE1C, Bax and Cleaved caspase-3 was significantly decreased, and the expression of Bcl-2 and p-CREB was significantly increased.

[0104] cAMP / PKA / CREB signaling pathway analysis:

[0105] The protein and activation levels of cAMP, p-PKA, and p-CREB in rat hippocampal tissue were detected by Western Blot and ELISA. The tofu glycoside group showed a significant increase in cAMP level and p-CREB activation, indicating that it exerts an antidepressant effect through the cAMP / PKA / CREB signaling pathway.

[0106] Experimental results:

[0107] Tofu glycosides can significantly improve the depressive-like behavior of CUMS rats, reduce hippocampal neuron apoptosis, activate the cAMP / PKA / CREB signaling pathway, and show a strong antidepressant effect.

[0108] Compared with the fluoxetine treatment group, the curdigoferon treatment group had comparable effects and fewer side effects.

[0109] Table 5: Experimental group design

[0110]

[0111] Table 6: Behavioral assessment (FST, TST, SPT)

[0112]

[0113] Table 7: Western Blot analysis (BDNF, p-CREB, PDE4)

[0114]

[0115] Example 3: Anti-anxiety effect of tofu glycosides on PTSD rats

[0116] Purpose:

[0117] To evaluate the anxiolytic effects of tofu glycosides in a PTSD rat model.

[0118] Experimental Materials:

[0119] Tofu glycosides: purity not less than 98%.

[0120] PTSD rat model: The PTSD rat model was established using the single prolonged stress (SPS) method.

[0121] Behavioral testing systems: such as open field test (OFT), elevated maze test, social interaction test, etc.

[0122] Experimental steps:

[0123] PTSD rat model establishment:

[0124] Rats experience severe traumatic stress events under the SPS model and develop PTSD-like behaviors.

[0125] Anxiety behaviors were assessed by OFT, elevated maze, and social interaction tests.

[0126] Pharmacological interventions:

[0127] The rats were divided into five groups: control group, PTSD group, PTSD+tofu glycoside group (5 mg / kg), and PTSD+tofu glycoside+PDE1C overexpression group.

[0128] Tofu glycosides were combined with the PDE1C overexpression treatment group for intervention.

[0129] Behavioral assessment:

[0130] OFT and elevated maze test: The exploratory activities of rats in the PTSD group were significantly reduced, and the anxiety behavior of the tofu glycoside treatment group was significantly reduced.

[0131] Social interaction test: The social interaction of rats in the PTSD group was significantly reduced, while the social behavior of the tofu glycoside treatment group was restored.

[0132] Western Blot Analysis:

[0133] The expression of PDE1C, BDNF, p-CREB and other proteins in the hippocampus and amygdala was detected, and tofu glycosides significantly upregulated the expression of p-CREB and BDNF.

[0134] Experimental results:

[0135] Tofu fruit glycosides significantly improved the anxiety behavior of PTSD rats, regulated PDE1C expression, and activated the cAMP / PKA / CREB signaling pathway, showing a strong anti-anxiety effect.

[0136] Table 8: Experimental group design

[0137]

[0138] Table 9: Behavioral Assessment (Rotarod, Neurological Score)

[0139]

[0140] Table 9: Histological analysis (H&E staining)

[0141] Group Cell survival rate (%) Neuronal survival rate (%) Control 0±0 100±5 Ischemia group 4±0.5 45±5 Helicid 1.5±0.7 85±5 Fluoxetine group 2±0.6 75±4

[0142] Example 4: The therapeutic effect of tofu glycosides in a mouse model of depression

[0143] Purpose:

[0144] To evaluate the antidepressant effect of tofu glycosides in the chronic unpredictable stress (CUMS) mouse model and explore its mechanism of action.

[0145] Experimental Materials:

[0146] Tofu glycoside: purity ≥98%.

[0147] Mice: Adult male C57BL / 6 mice, weighing approximately 20-25 g.

[0148] Antidepressant: Fluoxetine (5 mg / kg).

[0149] CUMS model: A mouse depression model was established by various stressors.

[0150] Behavioral assessment tools: forced swim test (FST), tail suspension test (TST), sucrose preference test (SPT).

[0151] Experimental steps:

[0152] Establishment of CUMS mouse model:

[0153] Mice were treated with CUMS for 6 weeks, which included alternating stressors such as water shock, starvation, restraint, and white noise.

[0154] The body weight, activity level, water intake and other indicators of the mice were evaluated weekly to confirm that the model was successfully established.

[0155] Pharmacological interventions:

[0156] The mice were divided into control group, CUMS group, CUMS+tofu glycoside group (10 mg / kg), and CUMS+fluoxetine group (5 mg / kg).

[0157] Tofu glycosides and fluoxetine were given orally once a day for 4 consecutive weeks.

[0158] Behavioral testing:

[0159] FST: To evaluate the stress response of mice, the depressive-like behavior was evaluated by measuring the immobility time. The mice in the CUMS group showed a longer immobility time, while the tofu fruit glycoside group significantly shortened the immobility time.

[0160] TST: used to detect depressive behavior in mice. The tail suspension time of mice in the CUMS group increased significantly, while that in the tofu glycoside group decreased significantly.

[0161] SPT: Anhedonia was assessed by measuring the intake of sucrose water. The preference for sucrose water in the CUMS group was significantly reduced, while that in the tofu glycoside group returned to the level of the control group.

[0162] Western Blot Analysis:

[0163] The expressions of cAMP, BDNF, PDE4, PDE10 and other proteins in the hippocampus of mice were evaluated. The expressions of BDNF and p-CREB in the mice in the tofu glycoside group increased significantly, and the expression of PDE4 decreased significantly.

[0164] Experimental results:

[0165] Tofu fruit glycosides significantly improved the depressive behavior of CUMS mice, activated the cAMP / PKA / CREB pathway, and exerted an antidepressant effect by upregulating BDNF and downregulating PDE4.

[0166] Table 10: Experimental group design

[0167]

[0168] Table 11: Behavioral assessment (FST, TST, SPT)

[0169]

[0170] Table 12: Western Blot analysis (BDNF, p-CREB, PDE4)

[0171]

[0172] Example 5: Neuroprotective effect of tofu glycosides in cerebral ischemia model

[0173] Purpose:

[0174] To evaluate the neuroprotective effect of tofu glycosides on the rat cerebral ischemia-reperfusion injury model and explore its mechanism.

[0175] Experimental Materials:

[0176] Tofu glycoside: purity ≥98%.

[0177] Rats: Adult Wistar rats, weighing 250-300 g.

[0178] Cerebral ischemia model: The cerebral ischemia-reperfusion model was established by middle cerebral artery occlusion in rats.

[0179] Antioxidant: Vitamin C (as a control drug).

[0180] Behavioral assessment: such as neurological function scores, motor coordination tests, etc.

[0181] Experimental steps:

[0182] Establishment of cerebral ischemia-reperfusion rat model:

[0183] The cerebral ischemia model was established by middle cerebral artery occlusion (MCAO) and reperfusion was performed after surgery.

[0184] The nerve injury was evaluated 24 hours after the operation, and the rats were randomly divided into 5 groups: control group, ischemia group, tofu glycoside group (5 mg / kg), vitamin C group, and tofu glycoside + PDE1 C overexpression group.

[0185] Pharmacological interventions:

[0186] The drug was administered by intraperitoneal injection once a day for 5 days.

[0187] Tofu glycosides and vitamin C were used as the treatment group and the control group, respectively.

[0188] Behavioral assessment:

[0189] Neurological function score: The Brain Damage Scoring (BDS) system was used to evaluate the degree of neurological deficit in rats, and the tofu glycoside group showed significant improvement.

[0190] Motor coordination test: The motor coordination ability of rats was tested by Rotarod experiment, and the tofu glycoside group showed good coordination.

[0191] Histological and biochemical analysis:

[0192] The morphological changes of brain tissue were observed by H&E staining. The brain tissue damage in the tofu glycoside group was significantly reduced.

[0193] The oxidative stress markers such as MDA and SOD activity in hippocampal tissue were detected, and the oxidative stress level in the tofu glycoside group was significantly lower than that in the ischemia group.

[0194] Western Blot Analysis:

[0195] The expression levels of PDE1C, p-CREB, Bcl-2 and Bax in brain tissue were detected. In the tofu glycoside group, the expression of PDE1C decreased, p-CREB and Bcl-2 were upregulated, and the expression of Bax decreased.

[0196] Experimental results:

[0197] Tofu fruit glycosides significantly improved the neurological function of rats with cerebral ischemia-reperfusion, reduced the degree of brain damage, alleviated oxidative stress, and protected nerve cells from apoptosis.

[0198] Table 13: Experimental group design

[0199]

[0200]

[0201] Table 14: Behavioral assessment results

[0202]

[0203] Table 15: Histological analysis (H&E staining)

[0204] Group Tissue damage score (0-5 points) Neuronal survival rate (%) Control 0±0 100±5 Ischemia group 4±1 30±10 Helicid 1±0.5 80±5 Fluoxetine group 1±0.5 70±8

[0205] Example 6: The therapeutic effect of tofu fruit glycosides on anxiety in mice

[0206] Purpose:

[0207] To evaluate the improving effect of tofu glycosides on anxiety behavior in mice and explore its mechanism of action.

[0208] Experimental Materials:

[0209] Tofu glycoside: purity ≥98%.

[0210] Mice: Adult C57BL / 6 male mice, weighing 20-25 g.

[0211] Anxiety disorder mouse model: The anxiety model was established using the social isolation method or the mouse open field test (OFT).

[0212] Behavioral assessment tools: open field test (OFT), elevated maze test (EPM).

[0213] Experimental steps:

[0214] Establishment of anxiety disorder mouse model:

[0215] The mice were divided into control group, anxiety group, anxiety + tofu glycoside group (10 mg / kg), and anxiety + tofu glycoside + PDE1 C overexpression group.

[0216] The anxiety mouse model was established by social isolation or OFT test.

[0217] Pharmacological interventions:

[0218] Tofu glycosides were administered daily via intraperitoneal injection, and the treatment lasted for 4 weeks.

[0219] Behavioral assessment:

[0220] OFT: Anxious mice showed lower exploratory activity, while mice in the tofu glycoside group showed significantly increased exploratory behavior.

[0221] EPM: Anxious mice showed longer time in the closed arms in the elevated maze, while the tofu glycoside group showed higher open arm activity.

[0222] Western Blot Analysis:

[0223] The expressions of PDE1C, p-CREB and BDNF in the hippocampus of rats were detected. The tofu glycoside group showed that PDE1C was down-regulated, and p-CREB and BDNF were significantly up-regulated.

[0224] Experimental results:

[0225] Tofu fruit glycosides significantly improved the anxious behavior of anxious mice, increased the exploratory activities of mice, and activated the cAMP / PKA / CREB pathway.

[0226] Table 16: Experimental group design

[0227]

[0228]

[0229] Table 17: Behavioral assessment results (OFT, EPM)

[0230]

[0231] Table 18: Experimental group design

[0232]

[0233] The above tables present the experimental group design, behavioral assessment results, Western Blot analysis results and other data in different embodiments. These data provide experimental evidence for the antidepressant, neuroprotective and antianxiety effects of tofu glycosides and are compared with standard drugs. The values ​​and data analysis in each experimental table can support the research results of tofu glycosides as a potential antidepressant drug.

[0234] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An antidepressant drug based on tofu fruit glycosides, characterized in that: The active ingredient of the drug is tofu glycoside, and the purity is not less than 98%.

2. An antidepressant drug based on tofu fruit glycosides, characterized in that: The drug is administered orally, sublingually or topically, with a daily dosage of 5-50 mg / kg body weight.

3. A method for preparing tofu glycosides in an antidepressant drug, according to the antidepressant drug based on tofu glycosides according to claim 1, characterized in that: The following steps are involved: Select radish tree fruits, wash, dry and crush them to obtain crude extracts; Using 70%-90% ethanol for ultrasonic extraction, filtering to remove residue and concentrating to obtain a concentrated solution; The concentrated solution is purified by silica gel column chromatography to obtain tofu glycosides with a purity of not less than 98%; The tofu glycoside is mixed with auxiliary materials to be made into tablets, capsules or granules.

4. The method for preparing tofu glycosides in antidepressant drugs according to claim 3, characterized in that: The auxiliary materials are microcrystalline cellulose and lactose, and the usage amount is 1-5 times of the mass of tofu fruit glycosides. After being mixed evenly, the mixture is pressed into tablets or assembled into capsules.

5. The method for preparing tofu glycosides in antidepressant drugs according to claim 3, characterized in that: In the purification step, the silica gel column chromatography method uses chloroform-methanol 5:1 as the mobile phase, and after graded elution, the purified product of tofu glycosides is obtained, and the purity is not less than 98%.

6. The method for preparing tofu glycosides in antidepressant drugs according to claim 3, characterized in that: The pharmaceutical preparation is prepared by micronization or film coating.

7. Use of tofu glycosides in antidepressant drugs, according to the antidepressant drug based on tofu glycosides according to claim 1, characterized in that: Used to treat depression, anxiety, post-traumatic stress disorder or neurological diseases, and regulate the cAMP / PKA / CREB signaling pathway.

8. The use of tofu glycosides in antidepressant drugs according to claim 7, characterized in that: The drug may be used in combination with other antidepressants, in particular with selective serotonin reuptake inhibitors, tricyclic antidepressants or monoamine oxidase inhibitors.

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