Anti-depression medicine composition and application thereof

Abstract

The invention provides an anti-depression medicine composition and application thereof, and belongs to the technical field of biological medicine. The anti-depression medicine composition disclosed by the invention is prepared from the following components in parts by mass: 1 to 100 parts of quercetin and 1 to 20 parts of fluoxetine. According to the invention, fluoxetine and quercetin are combined for use, and the depression-like behavior of a mouse is obviously reduced compared with that of the single use of fluoxetine, so that the combined use of fluoxetine and quercetin has obvious advantages in the anti-depression curative effect compared with the single use of fluoxetine, and the fluoxetine and quercetin have a synergistic effect when playing the anti-depression curative effect; the traditional Chinese medicine composition has important significance on treatment of depression and can be widely applied clinically.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an antidepressant drug combination and application thereof. Background Art

[0003] The etiology of depression is complex, closely linked to physiological and psychological factors, and remains largely undetermined. It may be linked to chronic stress and emotional reactions, which trigger metabolic and functional disturbances in monoamine neurotransmitters such as serotonin (5-HT) and norepinephrine (NE) in the brain. This overactivation of the sympathetic adrenal-medullary (SAM) and hypothalamic-pituitary-adrenocortical (HPA) systems, linked to the endocrine and immune systems, mediates related behavioral and physiological effects.

[0004] Currently, a variety of Western medications are used clinically to treat depression. Monoamine oxidase inhibitors (MAO-AIs) such as moclobemide inhibit the degradation of central monoamine neurotransmitters; serotonin reuptake inhibitors (SSRIs) such as fluoxetine selectively inhibit 5-HT reuptake; serotonin-norepinephrine reuptake inhibitors (SNRIs) such as venlafaxine selectively inhibit 5-HT and NE reuptake; dual-acting antidepressants such as nefazodone inhibit both 5-HT reuptake and postsynaptic 5-HT2 receptors; and NE and DA reuptake inhibitors such as bupropin selectively inhibit NE and DA reuptake. Most of these medications, due to their inherent limitations, have drawbacks such as a narrow antidepressant spectrum, significant side effects, high cost, and a high risk of relapse after discontinuation.

[0005] Quercetin is a flavonoid compound widely found in plants with antioxidant and anti-inflammatory properties, but there have been no reports on the combined use of quercetin and Western antidepressant drugs. Summary of the Invention

[0006] In view of this, the present invention provides an antidepressant drug combination and its application. The present invention uses fluoxetine and quercetin in combination, and it is found that the depressive-like behavior of mice is significantly alleviated compared with the use of fluoxetine alone. The two have a synergistic effect in exerting antidepressant therapeutic effects.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides an antidepressant drug combination, which comprises the following components in parts by mass: 1 to 100 parts of quercetin and 1 to 20 parts of fluoxetine.

[0009] Preferably, the drug combination comprises the following components in parts by mass: 20 to 80 parts of quercetin and 5 to 20 parts of fluoxetine.

[0010] Preferably, the antidepressant drug combination comprises the following components in parts by mass: 40 parts of quercetin and 5 parts of fluoxetine.

[0011] Preferably, the antidepressant drug combination comprises the following components in parts by mass: 40 parts of quercetin and 10 parts of fluoxetine.

[0012] Preferably, the dosage form of the antidepressant drug combination is any pharmaceutically acceptable dosage form.

[0013] Preferably, the dosage form of the antidepressant drug combination includes tablets, capsules, granules, powders, pills, oral solutions, injections or films.

[0014] The present invention also provides the use of the antidepressant drug combination in the preparation of antidepressant drugs.

[0015] By adopting the above technical solution, the present invention has the following beneficial effects: The antidepressant drug combination of the present invention comprises the following components by weight: 38-42 parts of quercetin and 5-10 parts of fluoxetine. The present invention uses fluoxetine and quercetin together, and found that the depressive-like behavior of mice after combined use was significantly reduced compared to fluoxetine alone, indicating that the antidepressant efficacy of the combined drug is significantly superior to that of fluoxetine alone. The two drugs have a synergistic effect in exerting antidepressant efficacy, which is of great significance for the treatment of depression and can be widely used in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the flow chart of the antidepressant trial of fluoxetine and quercetin combined therapy.

[0017] Figure 2 The results of body weight, tail suspension immobility time and forced swimming immobility time of mice in the control group and model group 4 weeks after modeling. Note: Compared with the control group, ## P<0.01. ( n 对照 =10,n 模型 =60)

[0018] Figure 3 The results of the weight and behavior changes of mice in each group after 2 weeks of drug intervention are shown in the figure, including weight change, sugar water preference rate, tail suspension immobility time, and forced swimming immobility time. Note: Compared with the control group, ## P<0.01; comparison between model group and drug-treated group, comparison between different drug-treated groups, * P<0.05. ( n=10)

[0019] Figure 4 These are the results of Nissl body staining in different areas of the hippocampal tissue of each group of mice after drug intervention.

[0020] Figure 5 The activity of total superoxide dismutase (T-SOD) and catalase (CAT) in hippocampus tissue after drug intervention. Note: Compared with the control group, ## P<0.01; Comparison between the model group and the drug-treated group, and comparison between different drug-treated groups, *P<0.05. ( n=6)

[0021] Figure 6 is the relative expression of BDNF, TrkB, and 5-HT1A mRNA in hippocampus tissue after drug intervention. Note: Compared with the control group, ## P<0.01; *P<0.05, **P<0.01 compared with each drug group; compared with single drug group and combined drug group, & P<0.05, && P<0.01. ( n=6) DETAILED DESCRIPTION

[0022] The present invention provides an antidepressant drug combination, which comprises the following components in parts by mass: 20 to 80 parts of quercetin and 5 to 20 parts of fluoxetine; the part by mass of the quercetin is preferably 40 to 60 parts, more preferably 50 parts; the part by mass of the fluoxetine is preferably 10 to 16 parts, more preferably 13 parts.

[0023] The dosage form of the antidepressant drug combination of the present invention is any pharmaceutically acceptable dosage form, including but not limited to tablets, capsules, granules, powders, pills, oral solutions, injections or films.

[0024] The present invention also provides the use of the antidepressant drug combination in the preparation of antidepressant drugs.

[0025] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] The mice used in the examples were 6-week-old, SPF-grade C57BL / 6 male mice weighing 18–22 g and purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Mice were housed in an SPF-grade environment with a room temperature of 18–25°C, a relative humidity of 60%–70%, and a 12-h light–dark cycle, with free access to water. Laboratory Animal Ethics Number: 2025B120.

[0027] Fluoxetine hydrochloride was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., F131623; quercetin was purchased from the official website of Sigma-Aldrich Reagent, Q4951; DMSO was purchased from Beijing Solebow Technology Co., Ltd.); normal saline (National Medicine Standard No. H20083400) and sucrose were purchased from Beijing Sugar, Tobacco and Wine Group Co., Ltd.

[0028] The experimental data of the present invention were statistically analyzed and plotted using Graphpad Prism 9.0. One-way ANOVA was used to analyze the data, and Pearson post hoc test was used for comparison between groups. P<0.05 indicated that the difference was statistically significant.

[0029] Example 1. Depression mouse model

[0030] After 7 days of adaptive feeding, 70 mice were randomly divided into a control group (CON) and a model group (CUMS), with 10 mice in the control group and 60 mice in the model group. The control mice were housed normally, while the remaining mice were subjected to 28 days of chronic mild unpredictable stress. The modeling methods included: 24-hour fasting, 24-hour water deprivation, tail clamping (1.5-2 cm from the base of the tail, 5 minutes), swimming in 4°C ice water (4°C, 5 minutes), swimming in 40°C hot water (40°C, 5 minutes), restraint (6 hours), humid cage (water-soaked bedding), day and night reversal, and cage tilt (45°, 12 hours).

[0031] Four weeks after modeling, the model group mice underwent behavioral assessments, including body weight measurement, tail suspension test, and forced swim test. Following behavioral testing, the model group mice were regrouped according to the results: model group, low-dose fluoxetine group, high-dose fluoxetine group, HPS (quercetin) group, HPS + low-dose fluoxetine group, and HPS + high-dose fluoxetine group, for a total of six groups. The grouped mice continued to be subjected to chronic unpredictable stimulation and were gavage-administered 1 hour after the end of the stimulation. The doses of fluoxetine (5 mg / kg), fluoxetine (10 mg / kg), HPS (quercetin) (40 mg / kg), HPS + low-dose fluoxetine (40 mg / kg + 5 mg / kg), and HPS + high-dose fluoxetine (40 mg / kg + 10 mg / kg) were administered. Each drug was dissolved in DMSO and diluted with saline to a final concentration of 0.1% DMSO. The drugs were then gavage-administered for 14 days. All mice were bled 24 h after the last administration and sacrificed, and the hippocampi were quickly isolated on ice, snap-frozen in dry ice, and stored at -80°C until analysis.

[0032] 1.1 Body weight test: Body weight test is performed every week and after the fourth week of stage modeling.

[0033] 1.2 Sucrose Preference Test (SPT)

[0034] Sugar water preference adaptation training: Before the formal experiment, sugar water drinking training was carried out. One bottle of 1% sucrose water and one bottle of pure water were placed in each cage, and the adaptation training lasted for 24 hours. Formal experiment: After the mice were deprived of water and food for 12 hours, they were given one bottle of 1% sucrose water and one bottle of pure water at the same time. After 12 hours, the positions of the water bottles were swapped. The mice continued to drink water freely for 12 hours before the bottles were removed. This lasted for a total of 24 hours. The weight of the two bottles of water before drinking and the remaining weight were weighed respectively to calculate the sugar water preference rate of the mice. Sugar water preference rate (%) = sugar water consumption / (sugar water consumption + pure water consumption) × 100%

[0035] 1.3 Tail Suspension Test (TST): Use medical tape to stick to the hanging hook 1 cm away from the tip of the mouse's tail so that the distance between the ground and the mouse's head is about 15 cm. The total time is 6 minutes. The mouse's immobility time for 4 minutes is recorded and analyzed.

[0036] 1.4 Forced Swim Test (FST): Mice were placed in a cylindrical transparent tube (20 cm diameter, 20 cm height) filled with (24 ± 2)°C water. The water depth was determined to be such that the mouse's tail could not touch the bottom. Timing began when the mouse was placed in the water and lasted for 6 minutes. After a 2-minute acclimatization period, the time the mouse remained floating on the water surface and remained immobile and struggling for 4 minutes was recorded.

[0037] After 4 weeks of modeling, the behavioral changes of mice in the control group and model group were as follows: Figure 2 The results showed that compared with the control group CON (n=10), the weight of mice in the CUMS model group (n=60) decreased significantly, and the tail suspension immobility time and forced swimming immobility time increased significantly, with significant differences, indicating that the mice developed depressive symptoms. All mice in the model groups were divided into groups for drug treatment.

[0038] Experimental Example 2. Drug Experiment in Mice

[0039] All mice with successful depression models were divided into 6 groups: model group (CUMS), fluoxetine low-dose group (FXT-L), fluoxetine high-dose group (FXT-H), quercetin group (HPS), quercetin + fluoxetine low-dose group (HPS + FXT-L), and quercetin + fluoxetine high-dose group (HPS + FXT-H). The mice were then subjected to chronic unpredictable stimulation and gavage 1 hour after the stimulation. The dosages of each group were as follows:

[0040] Fluoxetine low-dose group: 5 mg / kg;

[0041] Fluoxetine high-dose group: 10 mg / kg;

[0042] HPS (quercetin) group: 40 mg / kg;

[0043] HPS+low-dose fluoxetine group: 40 mg / kg HPS+5 mg / kg fluoxetine;

[0044] HPS+high-dose fluoxetine group: 40 mg / kg HPS+10 mg / kg fluoxetine;

[0045] Each group was treated with 0.1% DMSO as a solubilizer and then diluted to the working concentration with normal saline. The drug was then administered orally for 14 days.

[0046] 2.1. Body weight and behavioral assessment of mice after administration

[0047] After 2 weeks of drug intervention, body weight was measured and behavioral tests were performed, including the sugar water preference test, tail suspension test, and forced swimming test. The tail suspension test and forced swimming test were performed in the same manner as above. The results of the sugar water preference test, tail suspension test, and forced swimming test are shown in the figure below. Figure 3 shown.

[0048] Body weight test: After 2 weeks of drug intervention, the behavioral changes of mice in each group were as follows: Figure 3 The results showed that compared with the model group, the body weight of mice in each drug-treated group was significantly increased, with statistically significant differences (P<0.05). Among them, the body weight of the quercetin combined with low-dose fluoxetine group was significantly increased compared with the low-dose fluoxetine group (P<0.05).

[0049] Sugar water preference rate test: The sugar water preference experiment reflects the mouse's preference for sweetness through the sugar water preference rate. This preference of mice in a depressed state will be significantly reduced. By testing the mouse's preference for sugar water, the mouse's depression state can be reflected. The experimental results are as follows Figure 3 As shown, compared with the control group, the CUMS model group had a significantly decreased sugar preference rate (P<0.01); compared with the model group, the sugar preference rate of each drug group was significantly increased (P<0.05). Compared with the fluoxetine and quercetin groups alone, the sugar preference rate of mice in the combination drug group was significantly increased (P<0.05). These results indicate that quercetin and fluoxetine can improve depressive-like behavior in mice, and the effect of the combination drug group is better than that of either drug alone.

[0050] Tail suspension immobility time and forced swimming test: The tail suspension test and forced swimming test simulate the time mice spend struggling under extreme conditions to reflect the state of despair of mice. Mice in a depressed state will show a state of despair, that is, the immobility time will be prolonged. Figure 3As shown in the results: compared with the control group, the tail suspension immobility time and forced swimming time of mice in the CUMS model group were significantly decreased (P<0.01); compared with the model group, the tail suspension immobility time and forced swimming time of mice in each drug group were significantly increased (P<0.05); compared with the fluoxetine and quercetin drug groups alone, the tail suspension immobility time and forced swimming time of mice in the combined drug group were significantly increased (P<0.05).

[0051] In summary, compared with the model group, the body weight of mice in each drug-treated group was significantly increased, with statistically significant differences (P<0.05). Among them, the body weight and sugar water preference rate of the HPS combined with low-dose fluoxetine group were significantly increased compared with low-dose fluoxetine alone (P<0.05), and the tail suspension immobility time and forced swimming time were significantly reduced (P<0.05). The HPS combined with high-dose fluoxetine group and the high-dose fluoxetine group alone also had the above effects; this indicates that the antidepressant effect of the combined drug group is better than that of the single drug group, and can significantly improve the depressive-like behavior of mice.

[0052] 2. Mouse Brain Sample Testing

[0053] After the behavioral experiment, the mice were sacrificed by cervical dislocation after eye blood was collected. The brains were quickly removed from the skull of three mice in each group. The brains were washed with pre-cooled saline and fixed with 4% paraformaldehyde for 72 hours. The hippocampus and cortex of the remaining mice were quickly separated on ice, quickly frozen in dry ice, and then transferred to a -80°C refrigerator for storage. The mouse blood was allowed to stand at room temperature for 2 hours and then rotated at 4°C at 3500 rpm. -1 Centrifuge for 15 minutes and store the supernatant at -80℃ for later use.

[0054] 2.1 Nissl staining of hippocampal tissue

[0055] After the mice were killed by cervical vertebral dislocation, the whole brain was quickly removed on ice, fixed with 4% paraformaldehyde, embedded in paraffin, and then sectioned and dewaxed. The tissue sections were stained with Nissl body stain for 5 minutes, washed with water, and slightly differentiated with 0.1% glacial acetic acid. The degree of differentiation was monitored under a microscope. After terminating the reaction with tap water, the sections were placed in an oven to dry. The sections were transparentized in clean xylene for 10 minutes and sealed with neutral gum. Finally, the sections were examined under a microscope and image acquisition and analysis were performed. The results are shown in the figure below. Figure 4 The experimental results showed that the number of neurons in the DG, CA1, and CA3 regions of the control mice was abundant, with regular morphology and neat arrangement. In the model group, neurons were shrunken and darkly stained, with unclear cytoplasmic boundaries. The cells in each drug group showed improvement compared with the model group, and the combination drug group had the best effect.

[0056] 2.2 Biochemical detection of T-SOD and CAT activity results

[0057] The activity of T-SOD and CAT in mouse hippocampus tissue was detected by total superoxide dismutase (T-SOD) detection kit and catalase (CAT) detection kit. The experimental results are as follows: Figure 5 As shown in the results, compared with the control group, the activities of T-SOD and CAT in the hippocampus of the mice in the model group were significantly decreased (P<0.01); compared with the model group, the activities of T-SOD and CAT in the hippocampus of the HPS group, the high-dose fluoxetine group and the HPS combined with high-dose fluoxetine group were significantly increased (P<0.05), and the activities of T-SOD and CAT in the HPS combined with high-dose fluoxetine group were also significantly different compared with HPS alone and high-dose fluoxetine alone. The above results suggest that the antidepressant effect of HPS and fluoxetine may be related to the increase in the activities of T-SOD and CAT in the hippocampus of mice, and that the combined use of the two is more effective in antidepressant effect than using either of them alone.

[0058] 2.3 Detection of BDNF, TrkB, and 5-HT mRNA Expression by Fluorescence Quantitative PCR

[0059] 20 mg of mouse hippocampal tissue was extracted using RNA extraction solution to obtain a total RNA solution. RNA concentration and purity were determined using a Nanodrop 2000. Any RNA with excessive concentrations was diluted appropriately to a final concentration of 200 ng / μL. 10 μL of the total RNA solution was reverse transcribed into cDNA using a reverse transcription kit on a PCR instrument. PCR reactions were performed using the cDNA as a template. Primer sequences are shown in Table 1.

[0060] Table 1 BDNF, TrkB and 5-HT1A primer sequences

[0061]

[0062]

[0063] The real-time fluorescence quantitative PCR reaction system was 15 μL, including 7.5 μL 2× Universal Blue SYBR Green qPCR MasterMix, 1.5 μL 2.5 μM gene primers (upstream + downstream), 2.0 μL reverse transcription product (cDNA) and 4.0 μL WaterNuclease-Free; 95°C pre-denaturation for 30 seconds, 95°C denaturation for 15 seconds, 60°C annealing for 30 seconds, 40 cycles, and 3 replicates for each sample. Fluorescence signals were collected every 0.5°C increase in temperature to obtain a melting curve. GAPDH was used as an internal control, and 2 -ΔΔCt The relative mRNA expression levels of BDNF, TrkB and 5-HT in the hippocampus were calculated. Figure 6The experimental results showed that compared with the control group, the mRNA levels of BDNF, TrkB, and 5-HT in the hippocampus of mice in the CUMS depression model group were significantly decreased (P<0.01); compared with the model group, the mRNA levels of BDNF, TrkB, and 5-HT in the hippocampus of mice in different drug groups were significantly increased (P<0.05), and the mRNA levels of BDNF, TrkB, and 5-HT in the HPS combined with high-dose fluoxetine group were significantly different from those in the HPS alone group and the high-dose fluoxetine alone group (P<0.05). These results suggest that the antidepressant effect of HPS and fluoxetine may be related to regulating the expression of BDNF, TrkB, and 5-HT, and that the combined use of HPS and fluoxetine is more effective in treating depression than either of them alone.

[0064] As can be seen from the above examples, the present invention provides an antidepressant drug combination and its application. Fluoxetine and quercetin have a synergistic effect in exerting antidepressant therapeutic effects.

[0065] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An antidepressant drug combination, characterized in that: The drug combination comprises the following components in parts by mass: 1 to 100 parts of quercetin and 1 to 20 parts of fluoxetine.

2. An antidepressant drug combination, characterized in that: The drug combination comprises the following components in parts by mass: 20 to 80 parts of quercetin and 5 to 20 parts of fluoxetine.

3. The antidepressant drug combination according to claim 1, characterized in that The antidepressant drug combination comprises the following components in parts by mass: 40 parts of quercetin and 5 parts of fluoxetine.

4. The antidepressant drug combination according to claim 1, characterized in that The antidepressant drug combination comprises the following components in parts by mass: 40 parts of quercetin and 10 parts of fluoxetine.

5. The antidepressant drug combination according to any one of claims 1 to 4, characterized in that: The dosage form of the antidepressant drug combination is any pharmaceutically acceptable dosage form.

6. The antidepressant drug combination according to claim 5, characterized in that The dosage forms of the antidepressant drug combination include tablets, capsules, granules, powders, pills, oral solutions, injections or films.

7. Use of the antidepressant drug combination according to any one of claims 1 to 4 in the preparation of antidepressant drugs.

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