Application of rhodomyrtus tomentosa fruit extract in preparation of antidepressant drugs

Abstract

The invention discloses application of rhodomyrtus tomentosa fruit extract in preparation of antidepressant drugs, and relates to the technical field of biological medicines. It is found that the myrtle fruit extract has obvious anti-depression activity, by adjusting central nervous inflammation and neurogenesis, the myrtle fruit extract remarkably inhibits excessive activation of microglial cells (such as remarkably reducing the Iba-1 expression level) and reverses excessive activation of hypothalamus-pituitary-adrenal axis (HPA), and the myrtle fruit extract shows the activity of rapidly improving depression-like behaviors. The extract not only can effectively relieve depression core symptoms, but also can reduce abnormal expression of neuroinflammatory factors (such as COX-2, TNF-alpha and IL-6) and promote neurogenesis of the hippocampus. The invention provides a safe and efficient new natural medicine source for depression treatment, overcomes the defects of existing medicines in depression treatment, and provides a new medicine basis for developing anti-depression medicines.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of myrtle fruit extract in the preparation of antidepressant drugs. Background Art

[0002] Depression is a mental illness characterized by persistent low mood, low self-worth, and social avoidance, accompanied by decreased cognitive function and sleep disorders. Some patients have self-harm and suicidal thoughts, which seriously affect human health and quality of life.

[0003] The pathogenesis of depression is complex, with its pathology closely linked to monoamine neurotransmitters, the hypothalamic-pituitary-adrenal (HPA) axis, neurotrophic factors, neuroinflammation, oxidative stress, the intestinal barrier, and mitochondrial function. Currently, medication is the mainstay of treatment for depression. Mainstream antidepressants used clinically include fluoxetine and sertraline. While these medications are effective to some extent, they generally have limitations, such as slow onset of action and significant side effects.

[0004] Natural plant extracts have rich biological activities and relatively few side effects, which can solve the problem of significant side effects of traditional antidepressants. Therefore, the search and development of ideal antidepressants from natural plants has gradually attracted the attention of researchers.

[0005] Myrtle, Rhodomyrtus tomentosa (Ait.) Hassk., also known as mountain myrtle and myrtle fruit, is a shrub of the Myrtaceae family. It is known for its blood-nourishing, hemostatic, and bowel-strengthening properties. Myrtle fruit, a specialty wild fruit of the Lingnan region, has a complex and diverse chemical composition, including polysaccharides, flavonoids, phenols, and terpenes. It exhibits antioxidant, anti-inflammatory, lipid-lowering, anti-allergic, hepatoprotective, and intestinal protective properties, but its antidepressant effects remain unclear. Summary of the Invention

[0006] The present invention aims to provide a method for preparing an antidepressant drug by using a myrtle fruit extract to solve the problems of the prior art. The present invention finds that the myrtle fruit extract has significant antidepressant activity and can therefore be used to prepare an antidepressant drug.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides application of myrtle fruit extract in preparing antidepressant medicines.

[0009] Furthermore, the preparation method of the myrtle fruit extract comprises the following steps:

[0010] The myrtle fruit is crushed, added into an ethanol aqueous solution and subjected to ultrasonic extraction to obtain an extract;

[0011] The extract is filtered to obtain a filtrate, which is then concentrated and freeze-dried to obtain the myrtle fruit extract.

[0012] Furthermore, the volume fraction of the ethanol aqueous solution is 10-100%.

[0013] Furthermore, the solid-liquid ratio of the myrtle fruit to the ethanol aqueous solution is 1 g: (5-50) mL.

[0014] Furthermore, the temperature of the ultrasonic extraction is 20-80°C, the time is 10-180 minutes, and the power is 50-500W.

[0015] Furthermore, the volume fraction of the ethanol aqueous solution is 70%;

[0016] The solid-liquid ratio of the myrtle fruit to the ethanol aqueous solution is 1 g:25 mL;

[0017] The ultrasonic extraction was performed at a temperature of 40° C., a time of 60 min, and a power of 300 W.

[0018] The present invention also provides an antidepressant drug, the active ingredient of which includes myrtle fruit extract;

[0019] The preparation method of the myrtle fruit extract comprises the following steps:

[0020] The myrtle fruit is crushed, added into an ethanol aqueous solution and subjected to ultrasonic extraction to obtain an extract;

[0021] The extract is filtered to obtain a filtrate, which is then concentrated and freeze-dried to obtain the myrtle fruit extract.

[0022] Furthermore, the volume fraction of the ethanol aqueous solution is 10-100%;

[0023] The solid-liquid ratio of the myrtle fruit to the ethanol aqueous solution is 1 g: (5-50) mL;

[0024] The temperature of the ultrasonic extraction is 20-80° C., the time is 10-180 min, and the power is 50-500W.

[0025] Preferably, the volume fraction of the ethanol aqueous solution is 70%;

[0026] The solid-liquid ratio of the myrtle fruit to the ethanol aqueous solution is 1 g:25 mL;

[0027] The ultrasonic extraction was performed at a temperature of 40° C., a time of 60 min, and a power of 300 W.

[0028] Furthermore, the antidepressant drug also includes pharmaceutically acceptable excipients.

[0029] The present invention discloses the following technical effects:

[0030] The present invention discovered that myrtle fruit extract has significant antidepressant activity. By regulating central nervous system inflammation and neurogenesis, it significantly inhibits microglial overactivation (such as significantly reducing Iba-1 expression levels), reverses overactivation of the hypothalamic-pituitary-adrenal axis (HPA), and exhibits rapid improvement in depressive-like behaviors. This extract not only effectively alleviates core depressive symptoms (such as loss of pleasure, loss of desire for autonomous activity, and behavioral despair), but also reduces the abnormal expression of neuroinflammatory factors (such as COX-2, TNF-α, and IL-6) and promotes neurogenesis in the hippocampus.

[0031] The present invention provides a new source of safe and efficient natural medicine for the treatment of depression, fills the deficiencies of existing medicines in the treatment of depression, and provides a new pharmaceutical basis for the development of antidepressant drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 The LC-MS / MS analysis results of the myrtle fruit ethanol extract are shown in Figure 1; (A) is the LC-MS / MS spectrum; (B) is a schematic diagram of the chemical structures of the nine chemical components;

[0034] Figure 2 This is the result of the sugar water preference experiment of CUMS depressed mice;

[0035] Figure 3 This is the result of the sugar water preference experiment on LPS-depressed mice;

[0036] Figure 4 The results of the CUMS open field test on depressed mice are shown in Figure 2. (A) shows the movement trajectory of each group; (B) shows the total distance traveled by each group of mice; (C) shows the average speed of each group of mice; and (D) shows the number of times each group of mice crossed the central area.

[0037] Figure 5 Figures show the results of the open field test on LPS-depressed mice; (A) shows the movement trajectory of each group; (B) shows the total distance traveled by each group of mice; (C) shows the average speed of each group of mice; and (D) shows the number of times each group of mice crossed the central area.

[0038] Figure 6 This is the result of the tail suspension experiment of CUMS depressed mice;

[0039] Figure 7 This is the result of the tail suspension experiment on LPS-depressed mice;

[0040] Figure 8 This is the result of the forced swimming test of CUMS depressed mice;

[0041] Figure 9 This is the result of the forced swimming test in LPS-depressed mice;

[0042] Figure 10 This is the test result of serum corticosterone concentration in CUMS depressed mice;

[0043] Figure 11 Figure 2 shows the detection results of Iba-1 expression in the DG region of CUMS-depressed mice; (A) is an immunofluorescence staining image; (B) is a quantitative analysis of Iba-1 expression in the DG region; the scale bars in (A) are 100 μm;

[0044] Figure 12 The figures show the expression of COX-2, TNF-α and IL-6 proteins in CUMS-depressed mice; (A) shows the Western blot results; (B) shows the quantitative analysis of COX-2 expression; (C) shows the quantitative analysis of TNF-α expression; and (D) shows the quantitative analysis of IL-6 expression.

[0045] Figure 13 Figures 2 and 3 show the results of Iba-1 expression in the DG region of LPS-depressed mice; (A) is an immunofluorescence staining image; (B) is a statistical graph of the number of Iba-1-positive cells in the DG region; the scale bars in (A) are 100 μm;

[0046] Figure 14 Figures 2 and 3 show the results of detecting TUNEL-positive cells in the DG region of the hippocampus of CUMS-depressed mice; (A) is an immunofluorescence staining image; (B) is a statistical graph of the number of TUNEL-positive cells in the DG region; the scale bars in (A) are all 100 μm;

[0047] Figure 15 The results of the detection of DCX-positive cells in the DG region of the hippocampus of CUMS-depressed mice are shown in Figure 2. (A) is an immunofluorescence staining image; (B) is a statistical graph of the number of DCX-positive cells in the DG region; the scale bars in (A) are 200 μm.

[0048] Figure 16Figures 2 and 3 show the detection results of BrdU-positive cells in the DG region of the hippocampus of CUMS-depressed mice; (A) is an immunofluorescence staining image; (B) is a statistical graph of the number of BrdU-positive cells in the DG region; the scale bars in (A) are all 100 μm. DETAILED DESCRIPTION

[0049] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0050] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0051] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0052] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0053] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0054] The present invention has found that myrtle fruit extract has antidepressant properties and can therefore be used to prepare antidepressant drugs. The preparation method of the myrtle fruit extract is as follows:

[0055] Myrtle fruit is crushed, added into an ethanol aqueous solution and ultrasonically extracted to obtain an extract; the extract is filtered to obtain a filtrate, which is then concentrated and freeze-dried to obtain a myrtle fruit extract; wherein the volume fraction of the ethanol aqueous solution is 10-100%; the material-liquid ratio of myrtle fruit to the ethanol aqueous solution is 1g:(5-50)mL; the ultrasonic extraction temperature is 20-80°C, the extraction time is 10-180min, and the ultrasonic power is 50-500W.

[0056] The following is a detailed explanation of the antidepressant function of myrtle fruit extract:

[0057] Example 1

[0058] 1. Experimental Methods

[0059] 1.1 Preparation method of myrtle fruit extract

[0060] 200 g of dried myrtle fruit was crushed and added to a 70% ethanol aqueous solution at a material-liquid ratio of 1 g:25 mL. The mixture was ultrasonically extracted at 40°C and 300W for 60 min to obtain a myrtle fruit extract. The extract was filtered, the filtrates were combined, concentrated, and freeze-dried to obtain a myrtle fruit ethanol extract (RTEE).

[0061] 1.2 Liquid chromatography tandem mass spectrometry (LC-MS / MS) analysis

[0062] After reconstitution, the myrtle fruit ethanol extract was analyzed by LC-MS / MS. Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of acetonitrile. Elution conditions were: 5% B (0-0.5 min); 5% to 80% B (0.5-10 min); 80% B (10-12 min); 80% to 5% B (12-12.5 min); and 5% B (12.5-15 min). The flow rate was 0.3 mL / min. The mass spectrometer scan range was 50–1500 nm, and the electrospray ionization source was operated in positive ion mode. The following mass spectrometer parameters were used: capillary voltage: 1.60 kV; sampling cone voltage: 40 V; source offset voltage: 80 V; source temperature: 120°C; desolvation temperature: 450°C; cone gas flow rate: 50 L / hr; and desolvation gas flow rate: 700 L / hr. The chromatographic column model was an ACQUITY UPLC BEH C18 reversed-phase column (2.1 mm × 50 mm, 1.7 μm). The LC-MS / MS raw data were converted and uploaded to MSDIAL Ver 4.9 software. The precursor ion mass and MS / MS of the compounds were matched against the MSMS_Public_EXP_Pos_VS19 positive ion standard database provided by MSDIAL. This generated a compound feature table containing retention time, precursor ion mass-to-charge ratio, adduct ion type, molecular formula, and MS / MS information. The compound feature table was uploaded to MSFINDER Ver 3.6 software and matched against multiple databases (Pubchem, HMDB, KNApSAcK, ChEBI, COCONUT, etc.) to annotate potential compounds. The annotations were then traced to their biological sources using literature and natural product databases (LOTUS, NPASS, etc.) to verify their validity.

[0063] 1.3 Animal experiments

[0064] The animals used in the present invention are male C57BL / 6J mice (4 weeks old, SPF grade). The animals are raised in an environment with a temperature of 22±2°C, a humidity of 40-70%, 12 hours of light and 12 hours of darkness, and are allowed to eat and drink freely.

[0065] 1.4 Experimental modeling and grouping

[0066] Chronic unpredictable mild stress (CUMS) and lipopolysaccharide (LPS) were used to induce depression in mice.

[0067] Chronic unpredictable mild stressors include: (1) fasting for 12 hours; (2) water deprivation for 12 hours; (3) cage tilting for 12 hours; (4) wet cage for 12 hours; (5) single cage for 12 hours; (6) odor cage for 12 hours; (7) strobe light for 12 hours; (8) light exposure for 24 hours; (9) swimming at 20°C for 10 minutes; (10) restraint for 3 hours; (11) shaking at 150 rpm for 2.5 hours; and (12) hypothermia at 4°C for 3 hours. Two stressors were randomly applied daily for 8 consecutive weeks. Each stressor should be randomly selected, and the interval between the application of the same stressor should be greater than 3 days.

[0068] Acute depression model was established by intraperitoneal injection of 0.83 mg / kg LPS into mice.

[0069] Grouping: After two weeks of adaptive feeding, mice were randomly divided into a control group, a model group, a low-dose myrtle fruit extract group (L-RTEE, 200 mg / kg / day myrtle fruit ethanol extract), a medium-dose myrtle fruit extract group (M-RTEE, 400 mg / kg / day myrtle fruit ethanol extract), and a high-dose myrtle fruit extract group (H-RTEE, 800 mg / kg / day myrtle fruit ethanol extract). Behavioral data such as sugar water preference, open field, tail suspension, and forced swimming were used as test criteria to determine the establishment of the depression model.

[0070] 1.5 Animal Dosing

[0071] In the CUMS depression mouse experiment, the mice were subjected to chronic unpredictable mild stress modeling for 4 weeks, and continuous gavage was administered for 4 weeks starting from the 5th week, during which the modeling was also established. Behavioral experiments were performed after the last administration.

[0072] In the LPS-induced depression mouse experiment, mice were continuously gavaged for one week. After the final dose, 0.83 mg / kg LPS was injected intraperitoneally. Behavioral testing was performed 24 hours later. To avoid contamination from external factors, all behavioral experiments were conducted in a sterile environment.

[0073] 1.6 Sugar water preference experiment

[0074] The principle of the sucrose preference test (SPT) is that depressed mice do not show a preference for sucrose like normal mice do, reflecting a lack of pleasure. To change the mice's drinking habits, they were trained to drink sucrose. The specific method was as follows: on the first day, they were given two bottles of distilled water at the same time for 24 hours; on the second day, they were given two bottles of 1% sucrose solution at the same time for 24 hours; on the third day, they were given one bottle of distilled water and one bottle of 1% sucrose solution for 24 hours, and then the two bottles were swapped after 12 hours; on the fourth day, they were deprived of food and water for 24 hours; on the fifth day, they were given one bottle of distilled water and one bottle of 1% sucrose solution for 4 hours, and then the two bottles were swapped after 2 hours. After the end of the training, the consumption of distilled water and 1% sucrose solution was calculated, and the sucrose preference rate was calculated according to the following formula:

[0075] Sugar water preference rate (%) = 1% sucrose aqueous solution consumption / (1% sucrose aqueous solution consumption + distilled water consumption) × 100%

[0076] 1.7 Open field experiment

[0077] The open field test (OFT) is commonly used to assess mice's exploratory behavior, anxiety levels, and motor skills in novel environments. Mice are placed in the center of the bottom of an open field box in a dark, quiet environment. Each mouse is allowed to freely explore for 5 minutes before the experiment. Infrared video tracking software then records the mouse's activity. The following statistical data are collected based on the experiment: total distance moved, speed, and number of times the mouse crosses the center area. After each experiment, the open field box is cleaned with a 75% ethanol solution.

[0078] 1.8 Tail suspension test

[0079] The tail suspension test (TST) is a classic method for rapidly evaluating antidepressant drugs. A mouse's tail is fixed 1 cm above the ground with a thin rope, and the mouse's immobility time is recorded for 5 minutes.

[0080] 1.9 Forced swimming test

[0081] The forced swim test (FST) is primarily used to assess the emotional state of mice and the effects of antidepressant drugs. Mice are placed in a cylindrical tub (20 cm diameter, 50 cm height) filled with water at 25 ± 1°C, and the duration of immobility is recorded for 5 minutes.

[0082] 1.10 ELISA determination of corticosterone concentration

[0083] Blood was collected from the mouse orbits and serum was collected by centrifugation. Serum corticosterone concentrations were measured using an ELISA kit (Jiangsu Jingmei Biotechnology Co., Ltd., Jiangsu, China). The specific steps were strictly followed according to the manufacturer's instructions, with the following sequence: spike addition, sample addition, antibody addition, incubation, washing, color development, termination, and measurement.

[0084] 1.11 Western blot (WB) detection

[0085] Hippocampal tissue was homogenized in ice-cold protein lysis buffer containing protease inhibitors (phenylmethylsulfonyl fluoride and phosphatase inhibitors), centrifuged, and the supernatant was collected. Protein concentration was determined using a BCA protein assay kit. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane. The membrane was blocked, incubated with a primary antibody, washed with the primary antibody, incubated with a secondary antibody, washed with the secondary antibody, and then chemiluminescent. Images were acquired using a chemiluminescent gel imaging system, and the grayscale of protein bands was analyzed using Image J software and normalized to β-actin.

[0086] 1.12 Immunofluorescence detection

[0087] After anesthesia, mice were perfused thoroughly transcardially with ice-cold PBS and 4% paraformaldehyde (PFA). Brain tissue was isolated and fixed in 4% PFA for 4 hours, dehydrated in 30% sucrose solution, and then embedded in OCT embedding medium. The brain tissue was placed on a freezing microtome, and the hippocampus was located according to a mouse brain atlas. Serial sections of 20 μm thickness were prepared coronally and adhered to anti-smear slides. The brain slices were permeabilized, blocked, incubated with primary antibodies, washed with primary antibodies, incubated with secondary antibodies, washed with secondary antibodies, incubated with DAPI, and mounted. Images were acquired using an upright fluorescence microscope, and fluorescence intensity was analyzed using Image J software.

[0088] 1.13TUNEL apoptosis assay

[0089] Cell apoptosis was detected using a one-step TUNEL apoptosis detection kit (Elerite Biotechnology Co., Ltd., Wuhan, China). The specific steps were carried out strictly according to the manufacturer's instructions, in the following order: fixation, permeabilization, equilibration, addition of spikes, washing, DAPI incubation, and sealing. Images were acquired using an upright fluorescence microscope, and fluorescence intensity was analyzed using Image J software.

[0090] 1.14 Data Analysis

[0091] Statistical analysis and drawing were performed using statistical software. The results were expressed as mean ± standard deviation of the mean. One-way ANOVA was used for comparison of multiple groups. When p < 0.05, statistical significance was determined as (#) or (*). Compared with the model group, # represents p < 0.05; ## represents p < 0.01; ### represents p < 0.001; compared with the control group, * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001.

[0092] 2. Experimental Results and Analysis

[0093] 2.1 Chemical composition analysis of myrtle fruit ethanol extract

[0094] The results of LC-MS / MS analysis of the myrtle fruit ethanol extract are shown in Figure 1 The main chemical components are shown in Table 1. A total of 9 chemical components were identified, including 1 phenolic acid, 1 flavonoid, 3 phloroglucinols and 4 triterpenoids.

[0095] Table 1 Main chemical components of ethanol extract of myrtle fruit

[0096]

[0097] 2.2 Results of the sugar water preference experiment

[0098] In the CUMS depression mouse experiment, the results of the sugar water preference experiment are shown in Figure 2 Compared with the normal control group, the CUMS-induced model group mice showed a significantly decreased preference for sugar water; whereas compared with the model group, the L-RTEE, M-RTEE, and H-RTEE groups mice showed a significantly increased preference for sugar water.

[0099] In the LPS depression mouse experiment, the results of the sugar water preference experiment are shown in Figure 3 Compared with the normal control group, the preference for sucrose in the LPS-induced model group mice was significantly reduced; whereas compared with the model group, the preference for sucrose in the L-RTEE, M-RTEE, and H-RTEE groups mice was significantly increased.

[0100] The results of the sugar water preference experiment showed that RTEE intervention could reverse the anhedonia caused by CUMS and LPS.

[0101] 2.3 Open field test results

[0102] In the CUMS depression mouse experiment, the results of the open field test are shown in Figure 4 Compared with the normal control group, the total moving distance, speed, and number of times crossing the central area of ​​the mice in the CUMS-induced model group were significantly reduced; while compared with the model group, the total moving distance, speed, and number of times crossing the central area of ​​the mice in the L-RTEE, M-RTEE, and H-RTEE groups were significantly increased.

[0103] In the LPS depression mouse experiment, the results of the open field test are shown in Figure 5 Compared with the normal control group, the total moving distance, speed, and number of times crossing the central zone of mice in the LPS-induced model group were significantly reduced; while compared with the model group, the total moving distance, speed, and number of times crossing the central zone of mice in the L-RTEE, M-RTEE, and H-RTEE groups were increased.

[0104] The results of the open field test showed that RTEE intervention could reverse the loss of spontaneous activity intention caused by CUMS and LPS.

[0105] 2.4 Tail suspension test results

[0106] In the CUMS depression mouse experiment, the results of the tail suspension test are shown in Figure 6 Compared with the normal control group, the immobility time of mice in the CUMS-induced model group was significantly increased; whereas compared with the model group, the immobility time of mice in the L-RTEE, M-RTEE, and H-RTEE groups was significantly decreased.

[0107] In the LPS depression mouse experiment, the results of the tail suspension test are shown in Figure 7 Compared with the normal control group, the immobility time of mice in the LPS-induced model group was significantly increased; whereas compared with the model group, the immobility time of mice in the L-RTEE, M-RTEE, and H-RTEE groups was significantly decreased.

[0108] The results of the tail suspension test showed that RTEE intervention could reverse the behavioral despair caused by CUMS and LPS.

[0109] 2.5 Results of the forced swimming test

[0110] In the CUMS depression mouse experiment, the results of the forced swimming test are shown in Figure 8 Compared with the normal control group, the immobility time of mice in the CUMS-induced model group was significantly increased; while compared with the model group, the immobility time of mice in the L-RTEE, M-RTEE, and H-RTEE groups was significantly decreased.

[0111] In the LPS depression mouse experiment, the results of the forced swimming test are shown in Figure 9 Compared with the normal control group, the immobility time of mice in the LPS-induced model group was significantly increased; while compared with the model group, the immobility time of mice in the L-RTEE, M-RTEE, and H-RTEE groups was significantly decreased.

[0112] The results of the forced swimming test showed that RTEE intervention could reverse the behavioral despair induced by CUMS and LPS.

[0113] 2.6 Serum corticosterone concentration test results

[0114] In the CUMS depression mouse experiment, the results of serum corticosterone concentration are shown in Figure 10 Compared with the normal control group, the serum corticosterone concentration of mice in the CUMS-induced model group was significantly increased; however, compared with the model group, the serum corticosterone concentration of mice in the L-RTEE, M-RTEE, and H-RTEE groups was significantly decreased. The experimental results indicate that RTEE intervention can reverse the overactivation of the hypothalamic-pituitary-adrenal axis (HPA) caused by CUMS.

[0115] 2.7 Results of Neuroinflammation-Related Indicators

[0116] In the CUMS depression mouse experiment, Iba-1 expression in the hippocampal DG region was observed. Figure 11 The expression of COX-2, TNF-α and IL-6 proteins in the hippocampus was Figure 12 Compared with the normal control group, the expression of Iba-1 in the DG region of the hippocampus of the mice in the CUMS-induced model group was significantly upregulated, and the expression levels of COX-2, TNF-α, and IL-6 proteins in the hippocampus were significantly increased; compared with the model group, the expression of Iba-1 in the DG region of the hippocampus of the mice in the L-RTEE, M-RTEE, and H-RTEE groups were significantly downregulated, and the expression levels of COX-2, TNF-α, and IL-6 proteins in the hippocampus were significantly decreased.

[0117] In the LPS depression mouse experiment, the Iba-1 fluorescence intensity in the hippocampal DG region was Figure 13 Compared with the normal control group, the expression of Iba-1 in the DG region of the hippocampus of mice in the CUMS-induced model group was significantly upregulated; whereas compared with the model group, the expression of Iba-1 in the DG region of the hippocampus of mice in the L-RTEE, M-RTEE, and H-RTEE groups was significantly downregulated.

[0118] 2.8 TUNEL assay results

[0119] In the CUMS depression mouse experiment, TUNEL-positive cells were found in the hippocampal DG region. Figure 14 Compared with the normal control group, the number of TUNEL-positive cells in the DG region of the hippocampus of the mice in the CUMS-induced model group was significantly increased; while compared with the model group, the number of TUNEL-positive cells in the DG region of the hippocampus of the mice in the L-RTEE, M-RTEE, and H-RTEE groups was significantly decreased.

[0120] 2.9 Results of neurogenesis-related index detection

[0121] In the CUMS depression mouse experiment, DCX positive cells were found in the DG region of the hippocampus. Figure 15 , BrdU positive cells were seen Figure 16 Compared with the normal control group, the number of DCX- and BrdU-positive cells in the DG region of the hippocampus of mice in the CUMS-induced model group was significantly reduced; whereas compared with the model group, the number of DCX- and BrdU-positive cells in the DG region of the hippocampus of mice in the L-RTEE, M-RTEE, and H-RTEE groups was significantly increased.

[0122] In summary, the myrtle fruit extract provided by the present invention can significantly improve the depressive-like behavior of CUMS and LPS mice, mainly manifested in that the sucrose preference of depressed mice can be significantly improved in the sucrose preference experiment; the total moving distance, speed, and number of times crossing the central area of ​​depressed mice can be significantly increased in the open field experiment; the hanging immobility time of depressed mice can be significantly reduced in the tail suspension experiment; the immobility time in water of depressed mice can be significantly reduced in the forced swimming experiment; and the serum corticosterone concentration of depressed mice can be significantly reduced in the corticosterone concentration determination experiment. This shows that the myrtle fruit extract provided by the present invention has the function of preventing or treating depression, and therefore can be used to prepare antidepressant drugs. The present invention provides a new pharmaceutical basis for the development of antidepressant drugs, and has the therapeutic advantages of fast onset and fewer side effects.

[0123] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of myrtle fruit extract in the preparation of antidepressant drugs.

2. The use according to claim 1, characterized in that The preparation method of the myrtle fruit extract comprises the following steps: The myrtle fruit is crushed, added into an ethanol aqueous solution and subjected to ultrasonic extraction to obtain an extract; The extract is filtered to obtain a filtrate, which is then concentrated and freeze-dried to obtain the myrtle fruit extract.

3. The use according to claim 2, characterized in that The volume fraction of the ethanol aqueous solution is 10-100%.

4. The use according to claim 2, characterized in that The solid-liquid ratio of the myrtle fruit to the ethanol aqueous solution is 1 g: (5-50) mL.

5. The use according to claim 2, characterized in that The temperature of the ultrasonic extraction is 20-80° C., the time is 10-180 min, and the power is 50-500W.

6. The use according to claim 2, characterized in that The volume fraction of the ethanol aqueous solution is 70%; The solid-liquid ratio of the myrtle fruit to the ethanol aqueous solution is 1 g:25 mL; The ultrasonic extraction was performed at a temperature of 40° C., a time of 60 min, and a power of 300 W.

7. An antidepressant drug, characterized in that: Active ingredients include myrtle fruit extract; The preparation method of the myrtle fruit extract comprises the following steps: The myrtle fruit is crushed, added into an ethanol aqueous solution and subjected to ultrasonic extraction to obtain an extract; The extract is filtered to obtain a filtrate, which is then concentrated and freeze-dried to obtain the myrtle fruit extract.

8. The antidepressant drug according to claim 7, characterized in that The volume fraction of the ethanol aqueous solution is 10-100%; The solid-liquid ratio of the myrtle fruit to the ethanol aqueous solution is 1 g: (5-50) mL; The temperature of the ultrasonic extraction is 20-80° C., the time is 10-180 min, and the power is 50-500W.

9. The antidepressant drug according to claim 8, characterized in that The volume fraction of the ethanol aqueous solution is 70%; The solid-liquid ratio of the myrtle fruit to the ethanol aqueous solution is 1 g:25 mL; The ultrasonic extraction was performed at a temperature of 40° C., a time of 60 min, and a power of 300 W.

10. The antidepressant drug according to claim 7, characterized in that The antidepressant drug further comprises pharmaceutically acceptable excipients.

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