Application of allyl isothiocyanate to improvement of mental disorder

Abstract

The invention discloses application of allyl isothiocyanate in preparation of drugs or health-care foods for improving mental disorder, and belongs to the technical field of medicines. Allyl isothiocyanate shows rapid, convenient and efficient intervention potential for depression, emotional disorder, social difficulty, biphasic emotional fluctuation, anxiety and autism spectrum symptoms when used as a functional component of a medicine or health food. Specific experiments verify that in a depression model constructed by adopting social contusion stress, the component can obviously reverse typical depression-like behaviors such as social avoidance tendency and behavioral despair states shown by mice. In a chronic unpredictable stress induced model, allyl isothiocyanate effectively relieves anxiety and depression symptoms of mice and recovers pleasant sensation experience ability of the mice. In a drug-induced bipolar affective disorder model, the component obviously stabilizes the manic attack and depression alternating behavior of mice. In an autism model mouse, the allyl isothiocyanate effectively relieves the repetitive engraving behavior of the autism model mouse.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and health food technology, specifically relating to the application of allyl isothiocyanate in improving mental disorders. Background Technology

[0002] Common mental health disorders include depressive disorders, mood disorders, social anxiety disorder, generalized anxiety disorder, bipolar disorder, and autism spectrum repetitive behaviors, posing a significant public health challenge globally. The World Health Organization estimates that depression affects approximately 3.8% of the global population and has become the leading cause of disability. The lifetime prevalence of bipolar disorder remains stable at 1%-2%. Comorbidities such as sleep disorders and cognitive impairment significantly reduce patients' quality of life. Current psychotropic drug treatments generally suffer from delayed onset of action and high rates of side effects, necessitating the development of new treatment paradigms.

[0003] Wasabi (scientific name: *Wasabia japonica*), a cruciferous plant, is a traditional condiment. Its seeds are rich in isothiocyanates, which have recently shown potential for neuromodulation. Isopropyl isothiocyanate, a key active ingredient, can modulate pain perception by targeting and activating the TRPA1 ion channel (a key receptor for pain signal transduction). Studies have shown that this component can effectively alleviate pain-related negative behaviors in animal models of chronic pain, suggesting its potential to improve pain complications. However, its potential for intervention in core psychiatric symptoms such as affective disorders and behavioral abnormalities remains largely unexplored.

[0004] This technical solution aims to establish a novel application of allyl isothiocyanate in the preparation of drugs for improving mental disorders. Experimental verification has shown that this compound possesses rapid onset of action and highly effective relief properties for mental disorders, providing a novel candidate substance for overcoming existing treatment limitations. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides the application of allyl isothiocyanate in improving mental disorders.

[0006] To achieve the above objectives, the present invention provides the following technical solution: the application of allyl isothiocyanate in the preparation of drugs or health foods for improving mental disorders.

[0007] In some embodiments, the mental disorder includes at least one of depression, affective disorder, social disorder, anxiety disorder, bipolar disorder, and autistic stereotyped behaviors.

[0008] In some embodiments, the allyl isothiocyanate is used in the preparation of pain-relieving drugs or health foods, wherein the pain includes chronic pain and / or inflammatory pain.

[0009] In some embodiments, the allyl isothiocyanate is used in the preparation of drugs or health foods that improve pain complications.

[0010] In some embodiments, the dosage of the allyl isothiocyanate is 5-20 mg / kg.

[0011] In some embodiments, the allyl isothiocyanate has a rapid onset of action, exerting its therapeutic effect within 0.5-1 hour after administration.

[0012] In some embodiments, the allyl isothiocyanate is administered orally.

[0013] In some embodiments, the allyl isothiocyanate exerts its therapeutic effect by blocking the SERT-nNOS interaction.

[0014] In some embodiments, the duration of efficacy of the allyl isothiocyanate is 24-48 hours.

[0015] A pharmaceutical composition or health food for improving mental disorders, characterized in that it contains a therapeutically effective amount of allyl isothiocyanate as an active ingredient.

[0016] Compared with the prior art, the present invention provides the application of allyl isothiocyanate in improving mental disorders, which has the following beneficial effects: Allyl isothiocyanate has been used to improve mental disorders. Applying allyl isothiocyanate to social frustration-induced depression significantly improved depressive-like behaviors such as social avoidance and behavioral hopelessness in mice. Applying allyl isothiocyanate to a chronic unpredictable stress model significantly improved anxiety, depression, and anhedonia symptoms in mice. Applying allyl isothiocyanate to an induced bipolar disorder model significantly reduced manic and depressive-like behaviors in mice. Applying allyl isothiocyanate to an autistic mouse model significantly improved stereotyped behaviors. Furthermore, allyl isothiocyanate can improve chronic pain, inflammatory pain, and pain complications. These results suggest that allyl isothiocyanate may be used as a pharmaceutical / health food to improve mental disorders. Attached Figure Description

[0017] Figure 1 This describes the effect of AITC on the immobility time of mice in the tail suspension experiment.

[0018] Figure 2 This describes the effect of AITC on the immobility time of mice during the forced swimming experiment.

[0019] Figure 3 This refers to the effect of AITC on the relative luminescence value of the donor in the Nano BRET SERT-nNOS system.

[0020] Figure 4 This relates to the effect of AITC on the relative luminescence value of the acceptor in the Nano BRET SERT-nNOS system.

[0021] Figure 5 This relates to the effect of AITC on the mBRET ratio in the Nano BRET SERT-nNOS system.

[0022] Figure 6 This refers to the effect of AITC on the social index of mice in the CSDS model.

[0023] Figure 7 This describes the effect of AITC on loss of pleasure in mice in the CSDS model.

[0024] Figure 8 This refers to the effect of AITC on the number of times mice in the CSDS model entered the open arm in the elevated cross maze experiment.

[0025] Figure 9 This refers to the effect of AITC on the open-arm dwell time of mice in the CSDS model during the elevated cross maze experiment.

[0026] Figure 10 This relates to the effect of AITC on the number of grid spans in the open field experiment in a mouse model of bipolar disorder.

[0027] Figure 11 This describes the effect of AITC on the total distance of movement in the open field experiment in a mouse model of bipolar disorder.

[0028] Figure 12 This describes the effect of AITC on the number of times the mouse model of bipolar disorder stands in the open field test.

[0029] Figure 13 This describes the effect of AITC on immobility time in a mouse model of bipolar disorder during the tail suspension test.

[0030] Figure 14 This describes the effect of AITC on immobility time in a forced swimming test in mice with bipolar disorder.

[0031] Figure 15 This describes the effect of AITC on the strong social preference index in autistic mice.

[0032] Figure 16 This describes the effect of AITC on the strong social novelty index in autistic mice.

[0033] Figure 17 This describes the effect of AITC on nasal olfaction time in autistic mice during social interaction testing.

[0034] Figure 18It is a graph showing the effect of AITC on the following time in the social interaction test of autistic mice.

[0035] Figure 19 It is the effect of AITC on the grooming time of autistic mice. Specific implementation manners

[0036] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0037] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "upper", "lower", "inner", "outer", "front end", "rear end", "both ends", "one end", "the other end", etc. is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance.

[0038] In the description of the present invention, it should be noted that unless otherwise clearly specified and limited, the terms "installation", "provided with", "connection", etc. should be understood in a broad sense. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0039] Example 1 Evaluation of the efficacy of allyl isothiocyanate (AITC) for rapid antidepressant efficacy 1. Experimental animals 12 male C57BL / 6J mice at 6 - 8 weeks old, with a body weight of 18 - 20 g (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., license number: SCXK (Beijing) 2019 - 0023). The animals were adaptively raised for 1 week for the experiment. The room temperature in the animal house was 25 ± 2 °C, and the animals could freely eat and drink water.

[0040] 2. Animal grouping and treatment Twelve mice were randomly divided into a control group and an AITC (Adamas, 66481C) group according to their body weight. The AITC group was orally administered AITC 20 mg / kg, while the control group was orally administered the same volume of olive oil. Behavioral tests were performed at 0.5 hours, 1 hour, 24 hours, 48 ​​hours, 72 hours, 1 week, and 2 weeks after administration to observe whether AITC has a rapid and sustained antidepressant effect.

[0041] 3. Tail Suspension Test The tail suspension test is generally used to test depressive mouse models. The experiment requires a quiet environment. At the start, the mouse's tail is fixed to a specially designed tail suspension device and suspended upside down, with the tip of the mouse's nose approximately 15-20 cm from the ground. To prevent the mouse from climbing its tail, a transparent plastic cylinder is placed over the mouse's tail, preventing it from grasping it. Each mouse remains still for 6 minutes, and the immobility time and struggle time in the last 4 minutes are recorded. The entire experiment is video-recorded. After the experiment, the time the mouse spends without struggling and remains immobile is analyzed, and the duration of immobility is calculated to represent the mouse's state of despair.

[0042] 4. Forced swimming experiment Before conducting the experiment, the water temperature in the test chamber must be adjusted to 23±1℃. The experimental animals are placed in a transparent water tank (30 cm in diameter and 50 cm in height) from which they cannot escape. The water depth is adjusted according to the animal's weight to ensure that the animal's tail is at an appropriate distance from the bottom of the test chamber. If multiple animals are being tested, they must be separated by an opaque partition to prevent interference. The swimming time is 6 minutes. The swimming time, immobility time, and struggling time in the last 4 minutes are recorded. The length of immobility time in the last 4 minutes is used to characterize the mice's state of despair. 5. Data Statistics and Analysis Data are expressed as Mean ± SEM. Two-way ANOVA combined with the Sidak test was performed using GraphPad Prism 8.0 statistical analysis software. p <0.05 is considered statistically significant.

[0043] 6.1 Results 6.1 Evaluation of the rapid antidepressant efficacy of AITC - tail suspension test To evaluate the rapid antidepressant efficacy characteristics of AITC, this study used a tail suspension test to examine its onset and duration of action. Figure 1 As shown, AITC significantly affected the immobility time in mice during the tail suspension test [F(1, 10) = 16.52, p=0.0023], while repeated testing at different times did not significantly affect the immobility time of mice [F(3.723, 37.23) = 2.425, p =0.069], there is no interaction between AITC and detection time [F (7, 70) =1.953, p =0.0740]. Sidak test showed that 1 hour after AITC treatment ( p <0.01) and 24 hours ( p When the concentration of AITC is <0.01%, the immobility time of mice is significantly shortened, indicating that AITC can exert its antidepressant effect within 1 hour after administration, and the duration of effect can reach 24 hours. With prolonged time, the antidepressant effect of AITC disappears. The above results indicate that the antidepressant function of AITC takes effect within 1 hour after administration, and the duration of effect is 24 hours.

[0044] 6.2 Evaluation of the rapid antidepressant efficacy of AITC - Forced swimming test To further evaluate the rapid antidepressant efficacy characteristics of AITC, this study also used a forced swimming test to examine its onset and duration of action. Figure 2 As shown, AITC has a significant effect on the immobility time of mice in the forced swimming test [F (1, 10) = 52.00, p <0.0001], repeated detection at different times also has a significant impact on the immobility time [F(4.037, 40.37) = 11.69, p <0.0001], There was no significant interaction between AITC and detection time [F (7,70) = 1.540, p =0.1683]. The Sadik test showed that, compared with the control group, AITC was significantly reduced at 0.5 hours after administration ( p <0.001), 1 hour ( p <0.01), 24 hours ( p <0.01) and 48 hours ( p The immobility time of mice was significantly shortened (<0.01), indicating that AITC can exert its antidepressant effect 0.5 hours after administration, and the effect can last for up to 48 hours.

[0045] Example 2: The effect of AITC on SERT / nNOS interaction 1. Experimental Materials (1) Cells: HEK293T cells (2) Drugs: ZZL-7 (TargetMol, T64317): Dissolved in DMSO to prepare a 10-2 M stock solution; ATIC: Dissolved in DMSO to prepare a 10-2 M stock solution; 2. Experimental Procedure (1) Cell seeding plate HEK293T cells were cultured until more than 80% adhered, then digested with 0.25% trypsin, counted to 4×10⁵ / ml, and seeded into six-well plates. The cells were then incubated overnight at 37°C in a 5% CO₂ incubator.

[0046] (2) Transfection Once the cells have adhered to the culture medium to about 75%, discard the original medium and replace it with Opti-MEM phenol red-free medium. Transfect the HT-PDZ and SERT-NL plasmids at a ratio of 500:1.

[0047] Prepare the transfection mixture with a plasmid:EL transfection reagent ratio of 1:2 dissolved in Opti-MEM phenol red-free medium, 200 μl per well. Specific dosages are as follows:

[0048] Mix the transfection mixture thoroughly, let it stand at room temperature for 15 min, then add it to the well plate and incubate at 37℃ in a 5% CO2 incubator. After 12 h, change the medium to Opti-MEM and repeat the transfection process for a total of 2 days.

[0049] (3) Cell seeding plate Discard the original culture medium, wash twice with PBS, resuspend the cells in Opti-MEM medium containing 4% FBS and 1% penicillin, collect them in centrifuge tubes, centrifuge and discard the supernatant, resuspend the cells in Opti-MEM medium containing 4% FBS and 1% penicillin, count to 2×105 / ml, and seed them into white 96-well plates in groups.

[0050] Control group and drug treatment group cell suspension: 618 ligand was added to the cell suspension at a ratio of 1000:1, mixed well and seeded into 96-well plates, 90 μl per well; No ligand group cell suspension: DMSO was added to the cell suspension at a ratio of 1000:1, mixed well, and seeded into 96-well plates, 90 μl per well; cultured overnight in a 37 ℃, 5% CO2 incubator in the dark.

[0051] (4) Cell-based drug delivery The drug was prepared at 10 times the target concentration using Opti-MEM medium containing 4% FBS and 1% penicillin as solvent and added to each well of a 96-well plate at 10 μl. The plates were then incubated at 37 °C in a 5% CO2 incubator in the dark for 2 h.

[0052] (5) Luminescence value detection Dissolve the substrate in Opti-MEM at a ratio of substrate to culture medium of 1:100, mix well in the dark, add 25 μl of substrate to each well, shake for 30 s, and immediately detect the emission values ​​at 460 nm and 615 nm. Calculate the BRET ratio: mBRET ratio = acceptor emission value (615 nm) × 1000 / donor emission value (460 nm).

[0053] (6) Data statistics and analysis Data are expressed as mean ± SEM. One-way ANOVA combined with Dunnett's post-hoc test was used to analyze the data across multiple groups using GraphPad Prism 8.0 statistical analysis software. p <0.05 is considered statistically significant.

[0054] 3. Results This study used NanoBRET technology to examine the effect of AITC on the interaction between SERT-nNOS (PDZ). The fluorescence values ​​of each donor group were detected using 460 nm excitation light. Figure 3 As shown, there was no significant difference in relative fluorescence values ​​among the groups [F(5, 30) = 2.317, p =0.0681], Furthermore, we used 615nm excitation light to detect the fluorescence values ​​of each group of receptors, such as Figure 4 As shown, drug intervention had a significant effect on receptor fluorescence values ​​[F (5, 30) = 39.66, p <0.0001]. Calculated by the ratio of donor fluorescence value to acceptor fluorescence value. Figure 5 The results showed that drug intervention had a significant impact on the mBRET ratio [F (5,30) = 21.09, p <0.0001]. Compared with the ligand-free group, the mBRET ratio in the blank group was significantly increased ( p <0.001). Compared with the blank group, the mBRET ratio of the positive control drug ZZL-7 group was significantly reduced ( p <0.01), indicating that this experimental system can be used to evaluate the effect of AITC on the mBRET ratio. Compared with the blank group, different concentrations of AITC can significantly reduce the mBRET ratio (1 μM, p <0.01; 10 μM, 100 μM p <0.001), indicating that AITC has the effect of blocking the interaction between SERT-nNOS (PDZ).

[0055] Evaluation of the Improvement of Social Deficiency, Anhedonia and Anxiety Symptoms in Mice by AITC in Example 3 1. Experimental animals: Twenty 7-8-month-old male CD-1 mice and fifty 6-8-week-old male C57BL / 6J mice (all purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., license number: SCXK (Beijing) 2019-0023). The animals were adaptively fed for 1 week for the experiment. The room temperature in the animal house was 25±2°C, and the animals had free access to food and water.

[0056] 2. Experimental materials: Mouse double-chamber housing cage (with transparent plastic baffle) 3. Experimental protocol: Phase ①: Screening of aggressive CD-1 mice, cycle 3 days - After all CD-1 mice were adaptively housed individually for 7 days, once a day, a C57 mouse was placed in the CD-1 cage for 3 minutes and then taken out. The aggression of the CD-1 mice was observed. Different C57 mice were used every day for a total of 3 days. Screening criteria: The number of attacks by CD-1 mice within 3 minutes > 5 times, and the attack latency < 1 min. At least 2 days during the screening period met the above requirements.

[0057] Phase ②: Social defeat experiment, cycle 10 days - Direct contact stress: Each day, a different CD-1 mouse was used to attack the C57 mouse in the same-side cage for 1 minute for a total of 10 days. Indirect psychological stress: After the attack, the CD-1 and C57 mice were housed in the same cage on opposite sides for 24 hours (maintaining visual, olfactory and auditory contact). Control group mice were provided with different C57 roommates every day.

[0058] Phase ③: Behavioral detection - After the experiment ended, all mice were housed individually, and relevant behavioral detections were performed 24 hours later.

[0059] 4. Screening of socially deficient mice (social interaction experiment) The duration of the C57 mice entering the contact area was recorded separately by a camera for 2.5 minutes without a CD-1 mouse (no target) and for 2.5 minutes with a CD-1 mouse (target). The ratio of the two durations (SIT index) was used to determine whether the mice had social deficiency. In this study, SIT index < 1.0 was used as the standard, and 30 C57 mice with social deficiency were selected from 50 mice for the pharmacodynamic evaluation of AITC.

[0060] 5. Animal grouping and treatment Mice with social impairment (SIT index < 1.0) were randomly assigned to control, model, fluoxetine (10 mg / kg), and AITC treatment groups at different doses (5 mg / kg, 10 mg / kg, 20 mg / kg) according to body weight. All mice received the corresponding oral medication as directed, with the blank control and model groups receiving the same volume of olive oil. The following behavioral tests were performed within 24 hours of drug administration.

[0061] 6. Behavioral testing 6.1 Effects of AITC on Social Impairment Behavior in Mice The effects of AITC on social impairment behavior in mice were observed using a social interaction experiment, as described in section 4.

[0062] The effect of AITC on anhedonia behavior in mice The sucrose drinking test is an indicator of whether or not a mouse experiences anhedonia, which refers to a lack of interest in reward stimuli. The sucrose preference test consists of two parts: an adaptation training part and a testing part. In the training part, mice are placed in two bottles of 1% (w / v) sucrose solution in each cage for the first 24 hours. Then, one of the bottles is replaced with pure water for the next 24 hours. After adaptation, the mice are fasted and deprived of water for 24 hours, followed by a 12-hour baseline test. In the testing part, mice can only choose two pre-weighed bottles: one containing 1% (w / v) sucrose solution and the other containing pure water. They are fasted for 4 hours, after which both bottles are removed and weighed, and the total fluid consumption, sucrose solution consumption, and pure water consumption are recorded.

[0063] Formula for calculating the sugar water preference index: Sugar Water Preference Index % = ×100% Effects of AITC on anxiety-like behavior in mice The elevated cross maze utilizes the contradictory behavior of animals—their exploratory nature towards novel environments and their fear of open arms—to examine their anxiety levels. At the start of the experiment, mice are placed into the maze from the central grid towards the closed arms, and their activity is recorded for 5 minutes. Observational indicators include: number of entries into the open arms (two front mice must enter the arms), time spent in the open arms, number of entries into the closed arms, and time spent in the closed arms. The proportion of time spent in the open arms, the proportion of entries into the open arms, and the total number of entries into the elevated cross maze are calculated. After the experiment, the mice are removed, their arms are cleaned, and alcohol is sprayed to remove odor. Finally, the data is analyzed using Xeye animal behavior software.

[0064] 7. Data Statistics and Analysis Data are expressed as mean ± SEM. One-way ANOVA combined with Dunnett's post-hoc test was used to analyze the data across multiple groups using GraphPad Prism 8.0 statistical analysis software. p <0.05 is considered statistically significant.

[0065] 8. Results 8.1 Effects of AITC on the social index of CSDS model mice Social impairment is the most common behavioral abnormality in CSDS model mice. For example... Figure 6 As shown, drug treatment had a significant effect on the social index of mice [F (5, 30) = 28.36, p <0.0001]. Compared with the control group, the social index of CSDS model mice was significantly reduced. Compared with the model group, the social index of the positive control drug fluoxetine was significantly increased, indicating that this system can be used to evaluate the effect of AITC on social impairment in mice. Compared with the model group, AITC at 10 mg / kg and 20 mg / kg significantly improved the social index of mice, indicating that AITC has the function of improving social impairment in CSDS model.

[0066] 8.2 Efficacy of AITC in improving anhedonia in CSDS model mice The sucrose preference test is a classic experimental method for testing anhedonia in rodents. For example... Figure 7 As shown, drug treatment significantly improved the sucrose preference rate in CSDS model mice [F (5, 30) = 5.833, p =0.0007]. Duttent's test revealed that, compared with the control group, the CSDS mice had a significantly lower sugar water preference rate ( p <0.01 indicates that CSDS mice exhibit significant anhedonia. Compared to the model group, fluoxetine significantly increased the sucrose preference rate in mice ( p <0.05, indicating that this experimental system can be used to evaluate the effect of AITC on anhedonia in mice. Compared with the model group, the sugar water preference rate of mice in the medium and high dose AITC groups was significantly increased ( p The value <0.05 indicates that AITC at doses of 10 mg / kg and 20 mg / kg is effective in improving anhedonia in CSDS model mice.

[0067] 8.3 Effects of AITC on anxiety-like behavior in CSDS model mice The elevated cross maze test is a classic experimental method for testing anxiety-like behaviors in rodents. Its indicators include the number of times the mouse enters the open arm and the duration of its stay within the open arm. For example... Figure 8 As shown, in the test of the number of times mice entered the open arms, drug treatment could significantly affect the number of times mice entered the open arms [F (5, 30) = 5.676, p = 0.0008]. Compared with the control group mice, the number of times CSDS model mice entered the open arms was significantly reduced ( p <0.01). Compared with the model group, fluoxetine could significantly increase the number of times mice entered the open arms ( p <0.05), indicating that this system could be used to evaluate the effect of AITC on mice's anxiety-like behaviors. Compared with the model group, the number of times mice in the high-dose AITC group entered the open arms was significantly increased ( p <0.05).

[0068] In the test of the residence time in the open arms, drug treatment also had a significant effect on mice [F (5, 30) = 9.006, p <0.001]. As Figure 9 shown, compared with the control group mice, the residence time of CSDS model mice in the open arms was significantly shortened ( p <0.05). Compared with the model group, fluoxetine could significantly increase the residence time of mice in the open arms ( p <0.05). Compared with the model group, the residence time of mice in the high-dose AITC group in the open arms was significantly increased ( p <0.05). It indicated that AITC had the efficacy of improving the anxiety behaviors of CSDS model mice at the dose of 20 mg / kg.

[0069] Example 4 AITC Improves Bipolar Disorder in Mice Induced by Ouabain Experiment Animals A total of 50 SPF C57BL / 6J mice at 6 - 8 weeks old, weighing 18 - 20 g (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., license number: SCXK (Beijing) 2019 - 0023). The animals were adaptively raised for 1 week for the experiment. The room temperature of the animal house was 25 ± 2 °C, and the animals could freely eat and drink water.

[0070] Experiment Reagents

[0071] Reagent Preparation Preparation of ouabain: Dissolve the white ouabain powder in physiological saline to prepare a clear solution with a concentration of 50 μM. Preparation of sodium valproate: Sodium valproate (VPA) is a drug for treating bipolar disorder, a white powder, and is easily soluble in physiological saline.

[0072] 4. Experimental Methods 4.1 Experimental Grouping and Drug Administration To evaluate the effects of AITC on manic and depressive behaviors in BD mice, 48 adult male C57BL / 6J mice were divided into 6 experimental groups of 8 mice each after ICV injection of OUA, as follows: (1) Sal, (2) OUA + Sal, (3) OUA + Valproate 400 mg / kg, (4) OUA + AITC 5 mg / kg, (5) OUA + AITC 10 mg / kg, (6) OUA + AITC 20 mg / kg. On day 7 after ICV injection of OUA, physiological saline and sodium valproate were administered by gavage, and AITC was administered orally. One hour after administration, an open field test was performed. On day 14 after ICV injection of OUA, physiological saline and sodium valproate were administered intraperitoneally, and AITC was administered orally. One hour after administration, a tail suspension test and a forced swimming test were performed.

[0073] 4.2 Establishment of an ouabain-induced mouse model of bipolar disorder Experimental mice were anesthetized by isoflurane inhalation. The mouse heads were prepared and disinfected with povidone-iodine solution. After anesthesia, the mice were fixed in a stereotaxic apparatus. An ophthalmic scissor was used to make a longitudinal incision of approximately 1 cm in the mouse's head. Forceps were used to dissect and remove the periosteal connective tissue along the skull surface. Blood was removed with sterile cotton balls, and the skull surface was cleaned to expose the anterior and posterior fontanelles. The horizontal and vertical coordinate knobs on the stereotaxic apparatus were adjusted so that the dental micro-drill was positioned above and lightly touching the anterior fontanelle, using the Bregma point as the reference point (origin) of the three-dimensional coordinate system. The X and Y axis knobs were adjusted to move the operating arm to the coordinate position using AP = -0.5, ML = 1.0, and DV = 2.0 mm. A micro-drill was slowly used to create a hole. A micro-injector was fixed in place, and the Z-axis knob was adjusted to insert the needle into the lateral ventricle. A slow injection was performed at a rate of 0.5 μL / min, injecting 0.5 μL into each ventricle.

[0074] 4.3 Manic-like behavior detection - open field test The experimental setup consists of two parts: an open field reaction chamber and an automatic data acquisition and processing system. The open field reaction chamber is 30–40 cm high, with a base length of 100 cm. The inner walls are painted black, and the bottom surface is divided into 25 small squares of 4 cm × 4 cm each. A digital camera is mounted 2 m above the chamber, its field of view covering the entire open field. At the start of the experiment, the animal is gently placed in the center or a corner of the open field. The animal's behavior in the open field is then recorded. The observation period is 5 minutes, recording the animal's behavior, including the time spent in the center and at the edge of the open field, and the distance traveled. The inner walls and bottom of the chamber are cleaned to prevent residual information from the previous test (such as feces, urine, and odor) from affecting the results of the next test. The animal is then replaced, and the experiment continues.

[0075] 4.4 Measurement and detection of depressive-like behaviors Tail suspension test The tail suspension test is generally used to test depressive mouse models. The experiment requires a quiet environment. At the start, the mouse's tail is fixed to a specially designed tail suspension tester or support and suspended upside down, typically at a height of about 15-20 cm from the ground at the tip of the mouse's nose. To prevent the mouse from climbing its tail, a transparent plastic cylinder is placed over the mouse's tail, preventing it from grasping it. Each mouse is suspended for 6 minutes, and the immobility time and struggle time in the last 4 minutes are recorded. The entire experimental process is video-recorded. After the experiment, the time the mouse spends without struggling and remains immobile is analyzed, and the percentage of immobility time is calculated.

[0076] Forced swimming Before conducting the experiment, the water temperature in the test chamber must be adjusted to 23 ± 1℃. The experimental animal should be placed in a transparent water tank (30 cm in diameter and 50 cm in height) from which it cannot escape. The water depth should be adjusted according to the animal's weight to ensure that the animal's tail is at an appropriate distance from the bottom of the test chamber. If multiple animals are being tested, they should be separated by an opaque partition to prevent interference. The swimming time is 6 minutes, and the swimming time, immobility time, and struggling time in the last 4 minutes should be recorded.

[0077] 5. Data Statistics and Analysis Data are expressed as mean ± SEM. GraphPad Prism 8.0 statistical analysis software was used to perform one-way ANOVA combined with Dunnett's post-hoc test on the data among multiple groups. P < 0.05 was considered statistically significant.

[0078] 6. Results 6.1 Effects of AITC on manic behavior in ouabain-induced bipolar disorder mice This study used the open field test to observe the effects of AITC on manic behavior in ouabain-induced bipolar disorder mice. Manic behavior in the mice was characterized by a significant increase in the number of squares crossed and the total distance traveled in the open field test, as well as a significant increase in the number of times they stood and explored. In this study, if... Figure 10 As shown, drug treatment significantly affected the number of grid spans in mice [F (5,30) = 8.006, p <0.0001]. Compared with the control group, the number of spans in the ouabain model group mice was significantly increased ( p <0.001, compared with the model group, the positive control drug sodium valproate significantly reduced the number of spans in mice ( p <0.01 indicates that this system can be used to evaluate the effect of AITC on the number of spans in mice. Compared with the model group, the high-dose AITC group significantly reduced the number of spans in mice (p <0.05, in the detection of total movement distance in mice ( Figure 11 Both medium and high doses of AITC significantly reduced the total distance traveled by mice. p <0.05), which is similar to its effect on improving the number of spans.

[0079] In the test of the number of times mice stand up, such as Figure 12 As shown, drug treatment significantly affected the number of times mice stood up [F(5, 30) = 10.89, p <0.0001]. Compared with the control group, the number of standing times in the ouabain model group mice was significantly increased ( p <0.001, compared with the model group, the positive control drug sodium valproate significantly reduced the number of standing times in mice ( p <0.01 indicates that this system can be used to evaluate the effect of AITC on the number of times mice stand up. Compared with the model group, the medium and high dose groups of AITC significantly reduced the number of times mice stood up (10 mg / kg, p <0.01; 20 mg / kg, p <0.001), the above experimental results indicate that AITC has the effect of improving manic behavior in mice.

[0080] 6.2 Effects of AITC on depressive behavior in ouabain-induced bipolar disorder mice To observe the effects of AITC on depressive behavior in bipolar disorder mice, this study employed the tail suspension test and forced swimming test. Figure 13 As shown, drug treatment significantly affected the immobility time of mice in the tail suspension test [F (5, 30) = 5.532, p =0.0010]. Compared with the control group, the immobility time of mice in the ouabain model group was significantly prolonged ( p <0.05. Compared with the model group, the positive control drug sodium valproate significantly reduced the immobility time of mice ( p <0.05, indicating that this system can be used to evaluate the effect of AITC on immobility time in mice during the tail suspension test. Compared with the model group, the medium and high dose groups of AITC significantly reduced the number of times mice stood up (10 mg / kg, p <0.05; 20 mg / kg, p <0.01), the above experimental results indicate that AITC has the effect of improving depressive behavior in mice.

[0081] Furthermore, this experiment also used a forced swimming test to observe the effect of AITC on depressive behavior in a mouse model of bipolar disorder. Figure 14As shown, drug treatment had a significant effect on the immobility time of mice in the forced swimming test [F (5, 30) = 5.615, p = 0.0009]. Compared with the control group, the immobility time of mice in the ouabain model group was significantly prolonged ( p <0.05). Compared with the model group, the positive drug sodium valproate significantly reduced the immobility time of mice ( p <0.05), indicating that this system can be used to evaluate the effect of AITC on the immobility time of mice in the forced swimming test. Compared with the model group, the medium and high dose groups of AITC significantly reduced the number of standing times of mice (10 mg / kg, p <0.01; 20 mg / kg, p <0.05). The above experimental results indicate that AITC has the effect of improving the depressive behavior of mice.

[0082] From the above research results, it can be seen that AITC can significantly relieve the manic behavior and depressive-like behavior of animals with bipolar disorder model.

[0083] Example 5 Effect of AITC on social impairment in autistic mice 1. Experimental animals A total of 6 8-week-old SPF C57BL / 6J mice (2♂: 4♀), weighing 18 - 20 g (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., license number: SCXK (Beijing) 2019-0023). The animals were adaptively fed for 1 week for the experiment. The room temperature in the animal house was 25±2°C, and the animals could eat and drink freely.

[0084] 2. Model preparation Male mice and female mice were caged together at a ratio of 1:2, and were separated at 9:00 the next day. The presence of a vaginal plug was recorded as half a day of pregnancy. On the 12.5th day of pregnancy of female mice, valproic acid VPA was intraperitoneally injected at 600 mg / kg, and pregnant mice in the normal group were injected with normal saline. After the pups were born for 28 days, they were randomly grouped according to their body weight.

[0085] 3. Animal grouping Except for the normal group, VPA pups were randomly divided into a model group, a low-dose group of AITC (5 mg·kg-1), a medium-dose group (10 mg·kg-1), and a high-dose group (20 mg·kg-1) according to their body weight. All mice were orally administered the corresponding dose of AITC 1 hour before the start of the behavior experiment. The normal group was given an equal amount of olive oil.

[0086] 4. Three-chamber social interaction behavior experiment The three-box social interaction test is a classic animal behavior experiment designed to assess the behavioral performance of mice in a social environment. Also known as the "three-box social interaction test," it is primarily used to explore mice's preferences for social stimuli. Particularly in autism research, it is widely used to assess mice's social interest and social cognitive abilities.

[0087] The experimental setup consisted of three rectangular boxes, each separated by transparent resin glass partitions, with a central passage connecting the three boxes. A uniform metal cage, large enough to hold one mouse, was placed in the center of each of the left and right boxes. The entire experiment was recorded for later data analysis. After each round of experiments, the setup was wiped clean with 75% alcohol and tissue paper to ensure hygiene.

[0088] Experimental steps Phase 1 (Social Skills) In the three boxes, an unfamiliar mouse of the same species (Stranger 1) was placed on one side, and an empty metal cage was placed on the other side, allowing the test mouse to move freely in the three boxes.

[0089] (1) Before the experiment, the mice were placed in the behavior testing room for half an hour to adapt.

[0090] (2) Instead of using transparent glass resin plates to separate the three boxes, the test mice were placed directly into the middle box for 5 minutes to adapt.

[0091] (3) Randomly place Stranger 1 same-sex mice into the metal cage in the left box, while leaving the metal cage in the right box empty.

[0092] (4) Remove the partitions so that the test mice can move freely in the three boxes for 10 minutes.

[0093] (5) Immediately start recording and record the following relevant parameters: Parameter 1: The duration of direct contact between the test mouse and a Stranger 1 or an empty metal cage, with the contact area defined as the 3-5 cm radius around the metal cage.

[0094] Parameter 2: Test the number of times and duration the mouse enters each box. The mouse is considered to be in a box only when its head and limbs are both inside the box.

[0095] Phase Two (Social Novelty) Another unfamiliar mouse of the same species (Stranger 2) was placed on the other side, and the test mouse was allowed to move freely in the three cages. After one hour of the first phase of the experiment, a second unfamiliar mouse of the same species and sex (Stranger 2) was placed in an empty metal cage, and the time and number of interactions between the test mouse and Stranger 1 and Stranger 2 were recorded for 10 minutes.

[0096] Social Interaction Activity Experiment A single test mouse was placed on one side of a 25×25×25cm resin glass enclosure with a diagonal partition, while an unfamiliar mouse (a male mouse of similar age or 1-2 weeks younger than the test mouse, weighing no more than the test mouse) was placed on the other side of the enclosure. After 5 minutes of acclimatization, the partition was removed, allowing the test mouse to interact freely with the unfamiliar mouse. Trained independent observers monitored following and sniffing (nose-to-nose or nose-to-anus) time and self-grooming social behavior.

[0097] 5. Data Analysis Data are expressed as mean ± SEM. One-way ANOVA combined with Dunnett's post-hoc test was used to analyze the data across multiple groups using GraphPad Prism 8.0 statistical analysis software. p <0.05 is considered statistically significant.

[0098] 6. Results 6.1 Effects of AITC on VPA-induced social abilities in mice Social impairment is a typical pathological manifestation in mouse models of autism. For example... Figure 15 As shown, we found that AITC treatment had a significant effect on the social abilities of mice [F (4, 25) = 3.616, p =0.0186]. Compared with the control group, the social preference index of mice in the VPA model group was significantly reduced ( p <0.01). Compared with the model group, the medium-dose group (10 mg / kg) and high-dose group (20 mg / kg) of AITC significantly improved the social preference of mice, indicating that AITC has the efficacy of improving the social ability of autistic mouse models.

[0099] 6.2 Effects of AITC on VPA-induced social novelty in mice Reduced social novelty is also a behavioral characteristic of autistic mouse models, manifested as decreased interest in unfamiliar mice. For example... Figure 16As shown, we found that AITC treatment significantly affected social novelty in mice [F(4, 25) = 4.599, p =0.0064]. Compared with the control group, the social novelty index of mice in the VPA model group was significantly reduced ( p <0.01. Compared with the model group, the high-dose AITC group (20 mg / kg) significantly improved social novelty in mice. p The value <0.05 indicates that AITC has the effect of improving social novelty in autistic mouse models.

[0100] 6.3 Effects of AITC on VPA-induced reduction in social interaction in mice To further observe the effect of AITC on social interaction in an autistic mouse model, we used dynamic detection of social interaction between two mice. Under normal circumstances, the test mice would exhibit interactive behaviors with freely moving unfamiliar mice, including social behaviors such as sniffing and following. Figure 17 As shown, we found that AITC significantly affected the nasal olfaction time induced by VPA in the test mice [F(4, 25) = 8.860, p =0.0001]. Compared with the blank group, the nasal olfaction time of mice in the VPA model group was significantly reduced ( p <0.01). Compared with the model group, the intermediate dose group of AITC (10 mg / kg, p <0.05) and the high-dose group (20 mg / kg, p <0.05) can significantly increase the nasal olfaction time in mice.

[0101] In the follow-up time assay, we found that AITC significantly affected the follow-up time of the test mice induced by VPA [F (4, 25) = 6.472, p =0.0010]. Compared with the blank group, the follow-up time of mice in the VPA model group was significantly reduced ( p <0.01). Compared with the model group, the low-dose AITC group (5 mg / kg, p <0.05) medium-dose group (10 mg / kg, p <0.05) compared to the high-dose group (20 mg / kg, p AITC (<0.01) significantly increased the following time in mice, indicating that AITC has the effect of improving social interaction in autistic mouse models.

[0102] Repetitive self-grooming (grooming behavior) can cause pathological damage and is one of the typical stereotyped behaviors in autism in mice. This study found that AITC significantly affected the time spent grooming induced by VPA [F (4, 25) = 4.756,p =0.0054]. Compared with the blank group, the grooming time of mice in the VPA model group was significantly prolonged ( p <0.01). Compared with the model group, the intermediate dose group of AITC (10 mg / kg, p <0.05) compared to the high-dose group (20 mg / , p Both <0.01) significantly shortened the grooming time in mice, indicating that AITC has the effect of improving stereotyped behaviors in autistic mouse models.

[0103] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of allyl isothiocyanate in the preparation of drugs or health foods for improving mental disorders.

2. The application according to claim 1, characterized in that, The mental disorders include at least one of depression, affective disorder, social disorder, anxiety disorder, bipolar disorder, and autistic stereotyped behaviors.

3. The application according to claim 1, characterized in that, The use of allyl isothiocyanate in the preparation of pain-relieving drugs or health foods, wherein the pain includes chronic pain and / or inflammatory pain.

4. The application according to claim 3, characterized in that, The use of allyl isothiocyanate in the preparation of drugs or health foods that improve pain complications.

5. The application according to any one of claims 1-4, characterized in that, The dosage of the allyl isothiocyanate is 5-20 mg / kg.

6. The application according to any one of claims 1-4, characterized in that, The allyl isothiocyanate has a rapid onset of action, exerting its therapeutic effect within 0.5-1 hour after administration.

7. The application according to any one of claims 1-4, characterized in that, The allyl isothiocyanate is administered orally.

8. The application according to any one of claims 1-4, characterized in that, The allyl isothiocyanate exerts its therapeutic effect by blocking the SERT-nNOS interaction.

9. The application according to any one of claims 1-4, characterized in that, The duration of efficacy of the allyl isothiocyanate is 24-48 hours.

10. A pharmaceutical composition or health food for improving mental disorders, characterized in that, It contains a therapeutically effective amount of allyl isothiocyanate as the active ingredient.

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