Pharmaceutical composition as well as preparation method and application thereof

Abstract

The invention relates to the technical field of neurasthenia, in particular to a pharmaceutical composition as well as a preparation method and application thereof. The pharmaceutical composition is prepared from various glycoside substances and the like as raw materials. The pharmaceutical composition has the effects of treating or preventing ways beneficial to improving corresponding symptoms of neurasthenia of patients, such as nerve headache, headache caused by hysteria and the like. By intervening and activating an ERK-CREB signal channel, the composition is beneficial for inhibiting neurasthenia and enhancing the neuroprotective effect, and plays a positive role in resisting depression.

Description

Technical Field

[0001] The present invention relates to the technical field of neurasthenia, and particularly relates to a pharmaceutical composition, a preparation method thereof, and an application thereof. Background Art

[0002] Neurasthenia is a chronic functional disease mainly manifested by long-term fatigue, unstable mood, inattentiveness, sleep disorders, etc. Research has found that neurasthenia may be related to the dysfunction of the hypothalamic-pituitary-adrenal axis (HPA axis), the imbalance of neurotransmitters (such as 5-HT, GABA, dopamine), chronic inflammatory responses, etc.

[0003] Currently, the drugs clinically used for the treatment of neurasthenia mainly include benzodiazepines, antidepressants, etc. Although benzodiazepines can quickly relieve symptoms such as anxiety and insomnia in patients, long-term use is likely to cause adverse reactions such as drug dependence, drug resistance, and cognitive function impairment. Although antidepressants have a certain effect on improving the emotional symptoms of patients, they have a slow onset of action, and some patients may experience side effects such as sexual dysfunction and weight gain, which limits their application in the treatment of neurasthenia.

[0004] In the research and development of Western medicines, some new drugs have also shown good application prospects. For example, agomelatine, as a melatonin receptor agonist, can improve sleep quality, antidepressant, regulate mood, and has mild adverse reactions. In addition, some drugs targeting neuroprotection and neurotrophic are also being continuously developed, such as neurotrophic factors and their analogs, which are expected to fundamentally improve the symptoms of neurasthenia patients by promoting the growth, repair, and functional recovery of nerve cells.

[0005] Compound drugs have unique advantages in the treatment of neurasthenia. By acting synergistically through multiple targets and multiple pathways, the efficacy is enhanced. For example, some compound drugs can simultaneously regulate neurotransmitters, improve cerebral blood flow, and reduce nerve inflammation, comprehensively improving the symptoms of patients. Currently, the research on compound drugs mainly focuses on traditional Chinese medicine compounds, integrated traditional Chinese and Western medicine compounds, and Western medicine compounds, etc. Through modern pharmacological research and clinical trials, compound drugs with definite efficacy and good safety are screened out to provide more choices for the treatment of neurasthenia. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a pharmaceutical composition, a preparation method thereof, and an application thereof. The pharmaceutical composition is prepared from various glycosides and other substances as raw materials. The pharmaceutical composition can treat or prevent in a manner beneficial to improving the corresponding symptoms of neurasthenia in patients, such as neural headache, headache caused by hysteria, etc.

[0007] The present invention provides a pharmaceutical composition, which comprises, by weight parts: 80 - 120 parts of (8E)-ligustroside, 60 - 80 parts of fraxin, 60 - 80 parts of oleuropein, 60 - 80 parts of syringopicroside, 20 - 60 parts of (+)-pinoresinol-4-O-β-D-glucopyranoside, 20 - 50 parts of oleocanthal-4'-O-glucoside, 10 - 50 parts of coniferin, 10 - 40 parts of benzyl alcohol-β-vicianoside, 10 - 40 parts of salicin, 10 - 30 parts of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl)glucopyranoside, 5 - 10 parts of (Z)-3-hexenyl-β-D-glucopyranoside, 5 - 30 parts of 2-hydroxy-1-methoxyanthraquinone, 5 - 20 parts of (-)-episyringaresinol, 5 - 20 parts of dibutyl terephthalate and 1 - 10 parts of (-)-3,4-diveratryltetrahydrofuran. Herein, the term "parts" refers to any weight, not an absolute value of weight but only representing the weight ratio relationship between the components.

[0008] Specifically, the composition comprises, by weight parts: 100 parts of (8E)-ligustroside, 70 parts of fraxin, 65 parts of oleuropein, 65 parts of syringopicroside, 35 parts of (+)-pinoresinol-4-O-β-D-glucopyranoside, 40 parts of oleocanthal-4'-O-glucoside, 20 parts of coniferin, 30 parts of benzyl alcohol-β-vicianoside, 30 parts of salicin, 20 parts of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl)glucopyranoside, 8 parts of (Z)-3-hexenyl-β-D-glucopyranoside, 15 parts of 2-hydroxy-1-methoxyanthraquinone, 10 parts of (-)-episyringaresinol, 10 parts of dibutyl terephthalate and 6 parts of (-)-3,4-diveratryltetrahydrofuran.

[0009] Specifically, the composition comprises, by weight parts: 80 parts of (8E)-ligustroside, 60 parts of fraxin, 60 parts of oleuropein, 60 parts of syringopicroside, 20 parts of (+)-pinoresinol-4-O-β-D-glucopyranoside, 20 parts of oleocanthal-4'-O-glucoside, 10 parts of coniferin, 10 parts of benzyl alcohol-β-vicianoside, 10 parts of salicin, 10 parts of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl)glucopyranoside, 5 parts of (Z)-3-hexenyl-β-D-glucopyranoside, 5 parts of 2-hydroxy-1-methoxyanthraquinone, 5 parts of (-)-episyringaresinol, 5 parts of dibutyl terephthalate and 1 part of (-)-3,4-diveratryltetrahydrofuran.

[0010] Specifically, the composition comprises, by weight: 120 parts of (8E)-ligustrin, 80 parts of fraxinoside, 80 parts of olive glycoside, 80 parts of syringoside, 60 parts of (+)-pinoresinol-4-O-β-D-pyranoglucoside, 50 parts of oleoresin-4'-O-glucoside, 50 parts of coniferin, 40 parts of benzyl alcohol-β-vicinyl glycoside, 40 parts of hollyoside, 30 parts of (4S)-α-pinoresinol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl)pyranoglucoside, 10 parts of (Z)-3-hexenyl-β-D-pyranoglucoside, 30 parts of 2-hydroxy-1-methoxyanthraquinone, 20 parts of (-)-episyringaresinol, 20 parts of dibutyl terephthalate and 10 parts of (-)-3,4-divanillyltetrahydrofuran.

[0011] In addition, the present invention also provides a preparation of the above-mentioned pharmaceutical composition, which is a tablet or capsule containing the above-mentioned pharmaceutical composition, wherein the tablet has the characteristics of accurate dosage and convenient storage and transportation, while the capsule can mask the unpleasant bitterness and odor of the drug and improve the bioavailability.

[0012] In addition, the present invention also provides a method for preparing the preparation. The method comprises: (1) Dissolution The (8E)-ligustrin solution was prepared with 15% ethanol in water, the fraxinoside solution was prepared with ethanol, the olive glycoside solution was prepared with methanol, the syringoside solution was prepared with ethanol, the (+)-pinoresinol-4-O-β-D-pyranoglucoside solution was prepared with ethanol, the olive oleoresin-4'-O-glucoside solution was prepared with methanol, the coniferin solution was prepared with ethanol, the benzyl alcohol-β-vicinoside solution was prepared with ethanol, the wintergreen glycoside solution was prepared with ethanol, and the ethyl acetate solution was used to prepare the (+)-pinoresinol-4-O-β-D-pyranoglucoside solution. ester to prepare (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl)pyranoglucoside solution, ethanol to prepare (Z)-3-hexenyl-β-D-pyranoglucoside solution, ethanol to prepare 2-hydroxy-1-methoxyanthraquinone solution, ethanol to prepare (-)-episyringaresinol solution, ethyl acetate to prepare dibutyl terephthalate solution, ethanol to prepare (-)-3,4-divanillyltetrahydrofuran solution; (2) Mixed Mix equal volumes of the component solutions with ethanol or methanol as solvent, stirring while adding to ensure uniform mixing to obtain a first solution; mix equal volumes of the component solutions with ethyl acetate as solvent, stirring while adding to ensure uniform mixing to obtain a second solution; then slowly pour the second solution into the first solution, then add ethanol equivalent to 0.25 times the volume of the mixed solution of the first solution and the second solution, and stir thoroughly until uniform; perform ultrasonic treatment to obtain a third solution containing the formula amount of each component; (3) Concentration and drying Transfer the third solution to a rotary evaporator and perform rotary evaporation at appropriate temperature and pressure to remove the solvent, obtaining a concentrated mixture; transfer the concentrated mixture to a drying oven for drying to obtain a mixed powder; (4)Formulation For tablets, 20% of starch based on the total weight of the tablets can be added as a filler, 5% of sodium carboxymethyl starch based on the total weight as a disintegrant, and 1% of magnesium stearate based on the total weight of the tablets as a lubricant. For tablets, put the mixed powder into a tablet press for tabletting to obtain tablets.

[0013] For capsules, 45% of lactose based on the total weight of the capsules can be added as a filler, and 0.75% of magnesium stearate based on the total weight of the capsules as a lubricant. For capsules, load the mixed powder into capsule shells to obtain capsules.

[0014] Specifically, in the third solution, the concentration of (8E)-ligustroside is 100.00 mg / L, the concentration of fraxin is 70.00 mg / L, the concentration of oleuropein is 65.00 mg / L, the concentration of syringopicroside is 65.00 mg / L, the concentration of (+)-pinoresinol-4-O-β-D-glucopyranoside is 35.00 mg / L, the concentration of oleacein-4'-O-glucoside is 40.00 mg / L, the concentration of coniferin is 20.00 mg / L, the concentration of benzyl alcohol-β-vicianoside is 30.00 mg / L, the concentration of gaultherin is 30.00 mg / L, the concentration of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl) glucopyranoside is 20.00 mg / L, the concentration of (Z)-3-hexenyl-β-D-glucopyranoside is 8.00 mg / L, the concentration of 2-hydroxy-1-methoxyanthraquinone is 15.00 mg / L, the concentration of (-)-episyringaresinol is 10.00 mg / L, the concentration of dibutyl terephthalate is 10.00 mg / L, and the concentration of (-)-3,4-divanillyltetrahydrofuran is 6.00 mg / L.

[0015] Specifically, in the third solution, the concentration of (8E)-ligustroside is 116.12 mg / L, the concentration of fraxin is 87.09 mg / L, the concentration of oleuropein is 87.09 mg / L, the concentration of syringopicroside is 87.09 mg / L, the concentration of (+)-pinoresinol-4-O-β-D-glucopyranoside is 29.03 mg / L, the concentration of oleocanthal-4'-O-glucoside is 29.03 mg / L, the concentration of coniferin is 14.52 mg / L, the concentration of benzyl alcohol-β-vicianoside is 14.52 mg / L, the concentration of gaultherin is 14.52 mg / L, the concentration of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl) glucopyranoside is 14.52 mg / L, the concentration of (Z)-3-hexenyl-β-D-glucopyranoside is 7.26 mg / L, the concentration of 2-hydroxy-1-methoxyanthraquinone is 7.26 mg / L, the concentration of (-)-episyringaresinol is 7.26 mg / L, the concentration of dibutyl terephthalate is 7.26 mg / L, and the concentration of (-)-3,4-divanillyltetrahydrofuran is 1.45 mg / L.

[0016] Specifically, in the third solution, the concentration of (8E)-ligustroside is 87.33 mg / L, the concentration of fraxin is 58.22 mg / L, the concentration of oleuropein is 58.22 mg / L, the concentration of syringopicroside is 58.22 mg / L, the concentration of (+)-pinoresinol-4-O-β-D-glucopyranoside is 43.67 mg / L, the concentration of oleocanthal-4'-O-glucoside is 36.39 mg / L, the concentration of coniferin is 36.39 mg / L, the concentration of benzyl alcohol-β-vicianoside is 29.11 mg / L, the concentration of gaultherin is 29.11 mg / L, the concentration of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl) glucopyranoside is 21.83 mg / L, the concentration of (Z)-3-hexenyl-β-D-glucopyranoside is 7.28 mg / L, the concentration of 2-hydroxy-1-methoxyanthraquinone is 21.83 mg / L, the concentration of (-)-episyringaresinol is 14.56 mg / L, the concentration of dibutyl terephthalate is 14.56 mg / L, and the concentration of (-)-3,4-divanillyltetrahydrofuran is 7.28 mg / L.

[0017] In addition, the present invention also provides the application of the above-mentioned pharmaceutical composition. The application includes at least one of preparing a preparation for increasing the sucrose preference rate, preparing a preparation for reducing the immobility time after exercise, preparing a preparation for activating the ERK-CREB signaling pathway, and preparing a preparation for reducing the Beta and Theta activity frequencies.

[0018] Beneficial effects: The present invention uses various glycoside substances and the like as raw materials, and through steps of dissolution, mixing, concentration and drying, and in combination with a formulation molding process, finally obtains a formulation of a novel pharmaceutical composition. This formulation shows significant effects on chronic unpredictable mild stress (CUMS) rats, can increase their sucrose preference rate, shorten the immobility time after exercise, activate the ERK-CREB signaling pathway, and effectively reduce the frequencies of Beta and Theta brain wave activities.

[0019] The pharmaceutical composition provided by the present invention has significant alleviating and therapeutic effects on CUMS rats. The mechanism is that by activating the ERK-CREB signaling pathway, it effectively inhibits the symptoms of neurasthenia and enhances the neuroprotective effect, thereby exerting an antidepressant effect. The pharmaceutical composition provided by the present invention can reduce the proportion of Beta and Theta, regulate the mood disorder of CUMS rats, and has an obvious effect on improving mood and sleep. Brief Description of the Drawings

[0020] Figure 1 Sucrose preference rates of rats in the normal group, model group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group and Comparative Example 2 group.

[0021] Figure 2 Immobility times of rats in the normal group, model group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group and Comparative Example 2 group in the forced swimming test.

[0022] Figure 3 ERK1 mRNA levels of rats in the normal group, model group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group and Comparative Example 2 group.

[0023] Figure 4 ERK2 mRNA levels of rats in the normal group, model group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group and Comparative Example 2 group.

[0024] Figure 5 Shows the changes in CREB mRNA levels in the hippocampus of rats in the normal group, model group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group and Comparative Example 2 group under the chronic combined stress model.

[0025] Figure 6 Shows the Beta / Total frequency ratios of rats in the normal group, model group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group and Comparative Example 2 group under the chronic unpredictable mild stress model.

[0026] Figure 7It shows the correlation between the hippocampal theta rhythm oscillation power values and spatial learning and memory abilities of rats in the normal group, model group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, and Comparative Example 2 group, which were evaluated by the Morris water maze test under physiological and pathological conditions. Detailed implementation mode

[0027] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. The reagents not specifically described in detail in the present invention are all conventional reagents and can be obtained from commercial channels; the methods not specifically described in detail are all conventional test methods and can be learned from the prior art.

[0028] I. Pharmaceutical composition and its preparation 1. Pharmaceutical composition (8E)-ligustroside, CAS: 39011-92-2, Yeasen Biotech. Fraxin, CAS: 524-30-1, Nanjing Dilger Medicine. Oleuropein, CAS: 32619-42-4, Nanjing Bingcheng Biotech. Syringopicroside, CAS: 29118-80-7, Chengdu Standard Sample Biotech. (+)-pinoresinol-4-O-β-D-glucopyranoside, Huaxia Chemical Network. Oleacein-4'-O-glucoside, CAS: 76880-93-8, Chengdu Pusi Biotech. Coniferin, 124151-33-3, Shanghai Yeehoo Biotech. Benzyl alcohol-β-vicianoside, PubChemCID: 157010045, PubChem. Wintergreen glycoside, CAS: 490-67-5, Chengdu Pufa Technology. (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl) glucopyranoside, CAS: 918803-49-3, Chemical Network. (Z)-3-hexenyl-β-D-glucopyranoside, CAS: 95632-87-4, Huaxia Chemical Network. 2-hydroxy-1-methoxyanthraquinone, CAS: 6170-06-5, MCE. (-)-episyringaresinol, CAS: 6216-82-6, Shanghai Yuanye. Dibutyl terephthalate, CAS: 1962-75-0, Shanghai Yuanye. (-)-3,4-divanillyltetrahydrofuran, CAS: 34730-78-4, Jiangsu Yongjian Medicine.

[0029] The embodiment provides a pharmaceutical composition, which contains, by weight parts: 80 - 120 parts of (8E)-ligustroside, 60 - 80 parts of fraxin, 60 - 80 parts of oleuropein, 60 - 80 parts of syringopicroside, 20 - 60 parts of (+)-pinoresinol-4-O-β-D-glucopyranoside, 20 - 50 parts of oleacein-4'-O-glucoside, 10 - 50 parts of coniferin, 10 - 40 parts of benzyl alcohol-β-vicianoside, 10 - 40 parts of gaultherin, 10 - 30 parts of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl) glucopyranoside, 5 - 10 parts of (Z)-3-hexenyl-β-D-glucopyranoside, 5 - 30 parts of 2-hydroxy-1-methoxyanthraquinone, 5 - 20 parts of (-)-episyringaresinol, 5 - 20 parts of dibutyl terephthalate, and 1 - 10 parts of (-)-3,4-divanillyltetrahydrofuran.

[0030] The composition provided by Example 1 contains, by weight parts: 100 parts of (8E)-ligustroside, 70 parts of fraxin, 65 parts of oleuropein, 65 parts of syringopicroside, 35 parts of (+)-pinoresinol-4-O-β-D-glucopyranoside, 40 parts of oleacein-4'-O-glucoside, 20 parts of coniferin, 30 parts of benzyl alcohol-β-vicianoside, 30 parts of gaultherin, 20 parts of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl) glucopyranoside, 8 parts of (Z)-3-hexenyl-β-D-glucopyranoside, 15 parts of 2-hydroxy-1-methoxyanthraquinone, 10 parts of (-)-episyringaresinol, 10 parts of dibutyl terephthalate, and 6 parts of (-)-3,4-divanillyltetrahydrofuran.

[0031] The composition provided by Example 2 contains, by weight parts: 80 parts of (8E)-ligustroside, 60 parts of fraxin, 60 parts of oleuropein, 60 parts of syringopicroside, 20 parts of (+)-pinoresinol-4-O-β-D-glucopyranoside, 20 parts of oleacein-4'-O-glucoside, 10 parts of coniferin, 10 parts of benzyl alcohol-β-vicianoside, 10 parts of gaultherin, 10 parts of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl) glucopyranoside, 5 parts of (Z)-3-hexenyl-β-D-glucopyranoside, 5 parts of 2-hydroxy-1-methoxyanthraquinone, 5 parts of (-)-episyringaresinol, 5 parts of dibutyl terephthalate, and 1 part of (-)-3,4-divanillyltetrahydrofuran.

[0032] The composition provided by Example 3 contains, by weight parts: 120 parts of (8E)-ligustroside, 80 parts of fraxin, 80 parts of oleuropein, 80 parts of syringopicroside, 60 parts of (+)-pinoresinol-4-O-β-D-glucopyranoside, 50 parts of oleacein-4'-O-glucoside, 50 parts of coniferin, 40 parts of benzyl alcohol-β-vicianoside, 40 parts of gaultherin, 30 parts of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl)glucopyranoside, 10 parts of (Z)-3-hexenyl-β-D-glucopyranoside, 30 parts of 2-hydroxy-1-methoxyanthraquinone, 20 parts of (-)-episyringaresinol, 20 parts of dibutyl terephthalate and 10 parts of (-)-3,4-divanillyltetrahydrofuran.

[0033] The composition provided by Comparative Example 1 contains, by weight parts: 100 parts of (8E)-ligustroside, 70 parts of fraxin, 65 parts of oleuropein, 65 parts of syringopicroside, 35 parts of (+)-pinoresinol-4-O-β-D-glucopyranoside, 40 parts of oleacein-4'-O-glucoside, 20 parts of coniferin, 30 parts of benzyl alcohol-β-vicianoside, 30 parts of gaultherin, 20 parts of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl)glucopyranoside, 8 parts of (Z)-3-hexenyl-β-D-glucopyranoside.

[0034] The composition provided by Comparative Example 2 contains, by weight parts: 15 parts of 2-hydroxy-1-methoxyanthraquinone, 10 parts of (-)-episyringaresinol, 10 parts of dibutyl terephthalate and 6 parts of (-)-3,4-divanillyltetrahydrofuran.

[0035] 2. Dissolution and mixing The solvent should be selected according to the dissolution characteristics of the compounds, and ethanol, methanol, dichloromethane, etc. are recommended. The excipients are flexibly matched according to the preparation type, such as starch, lactose, etc. as fillers, sodium carboxymethyl starch, etc. as disintegrants, and magnesium stearate, etc. as lubricants. Instrument equipment: mortar, beaker, magnetic stirrer, rotary evaporator, drying oven, etc.

[0036] Dissolve various compounds in Examples 1 to 3 and Comparative Examples 1 to 2 respectively.

[0037] (1) Dissolution step Table 1 (mg / L)

[0038] Prepare the (8E)-ligustroside solution with an ethanol aqueous solution containing 15%, prepare the fraxinoside solution with ethanol, prepare the oleuropein solution with methanol, prepare the syringopicroside solution with ethanol, prepare the (+)-pinoresinol-4-O-β-D-glucopyranoside solution with ethanol, prepare the oleacein-4'-O-glucoside solution with methanol, prepare the coniferin solution with ethanol, prepare the benzyl alcohol-β-vicianoside solution with ethanol, prepare the gaultherin solution with ethanol, prepare the (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl) glucopyranoside solution with ethyl acetate, prepare the (Z)-3-hexenyl-β-D-glucopyranoside solution with ethanol, prepare the 2-hydroxy-1-methoxyanthraquinone solution with ethanol, prepare the (-)-episyringaresinol solution with ethanol, prepare the dibutyl terephthalate solution with ethyl acetate, and prepare the (-)-3,4-divanillyltetrahydrofuran solution with ethanol. The weight ratios between the concentrations of the components in Table 1 are approximate ratios to the above weight part ratios and are not equal mathematically.

[0039] (2) Mixing step Mix the solutions of the above components using ethanol or methanol as the solvent while adding and stirring to ensure uniform mixing to obtain the first solution. Mix the solutions of the above components using ethyl acetate as the solvent in equal volumes while adding and stirring to ensure uniform mixing to obtain the second solution.

[0040] Then slowly pour the second solution into the first solution, and subsequently add ethanol in an amount 0.25 times the volume of the mixed solution of the first solution and the second solution, and stir well until uniform. After that, perform ultrasonic treatment to further homogenize the solution to obtain the third solution such that the concentrations of the components in the third solution are as shown in Table 1.

[0041] (3) Concentration and drying Transfer the third solution to a rotary evaporator, start rotary evaporation at a preset appropriate temperature and pressure until the solvent is completely removed to obtain a concentrated mixture. Then, transfer the concentrated mixture into an oven and perform drying treatment within a specified temperature range until a dry mixed powder is obtained. It should be noted that the drying temperature should be maintained at 40 - 60 °C to prevent decomposition or deterioration of the compound due to excessive temperature.

[0042] (4) Forming the preparation For tablets, 20% of starch based on the total tablet weight can be added as a filler, 5% of sodium carboxymethyl starch based on the total weight can be added as a disintegrant, and 1% of magnesium stearate based on the total tablet weight can be added as a lubricant. For capsules, 45% of lactose based on the total capsule weight can be added as a filler, and 0.75% of magnesium stearate based on the total capsule weight can be added as a lubricant. For tablets, the mixed powder is put into a tableting machine for tableting to obtain tablets. For capsules, the mixed powder is filled into capsule shells to obtain capsules.

[0043] II. Animal experiments 1. Experimental animals Male SD rats (160 - 200 g, VSR10001, Beijing Weishang Lide Biotechnology Co., Ltd.) are raised in an environment with a temperature of (24 ± 1) °C and a humidity of (50 ± 10%). The circadian rhythm is 12 h, and they can eat and drink freely. According to the standard operating procedure for the feeding and management of SD rats, it should be ensured that the breeding environment is clean and dust-free and the air is fresh to avoid respiratory diseases caused by the sensitivity of SD rats to dust, ammonia, hydrogen sulfide, etc. in the air. Before starting the CUMS experiment, the rats need to be adaptively raised for one week.

[0044] 2. Model preparation and treatment The method of "Li, H., Xiang, Y., Zhu, Z., Wang, W., Jiang, Z., Zhao, M., etal. (2021) Rifaximin-Mediated Gut Microbiota Regula[1]tion Modulates theFunction of Microglia and Protects against CUMS-Induced Depression-LikeBehaviors in Adoles[1]cent Rat. Journal of Neuroinflammation, 18, Article No.254." is used to construct chronic unpredictable mild stress (CUMS) rats.

[0045] Through pre-experiments, one of the following seven mild stimuli was randomly selected daily to stimulate the rats for 28 days. The 7 stimuli were as follows: ① swimming in ice water at 0°C for 5 min; ② 24-hour circadian rhythm reversal; ③ fasting and water deprivation: fasting and water deprivation for 24 h; ④ tail clamping for 1 min; ⑤ swimming in hot water at 45°C for 5 min; ⑥ tilting the cage at 45° for 24 h; ⑦ humid environment for 24 h. After the modeling, the rats were subjected to the sucrose preference test (SPT) and the forced swimming test (FST) to determine that successful modeling could be achieved in 28 days.

[0046] The rats were divided into a normal group, a model group, an Example 1 group, an Example 2 group, an Example 3 group, a Comparative Example 1 group, and a Comparative Example 2 group. Except for the normal group, the rats in the model group, the Example 1 group, the Example 2 group, the Example 3 group, the Comparative Example 1 group, and the Comparative Example 2 group were all stimulated with reference to the above method. Except for the model group, before each stress stimulation, the rats in the Example 1 group, the Example 2 group, the Example 3 group, the Comparative Example 1 group, and the Comparative Example 2 group were respectively administered a powder solution prepared with physiological saline from the tablets ground in Examples 1 to 3 and Comparative Examples 1 to 2 by gavage at a dose of 30 mg / kg body weight for 5 consecutive weeks.

[0047] 3. Sucrose preference consumption test After 5 weeks of treatment, the rats were allowed to adapt to a 1% sucrose solution. Each rat was housed separately, and two bottles of 1% sucrose solution were provided. After 24 h, one of the bottles of solution was replaced with pure water and continued for another 24 h. Then, the rats were fasted and water-deprived for 12 h, and the rats were allowed to freely choose between two bottles of solution (one bottle of 1% sucrose solution and one bottle of pure water). After 1 h, the positions of the two bottles of solution were exchanged. After 2 h, the weight of each bottle of solution was measured, and the sucrose preference rate was calculated. The sucrose preference rate = sucrose water consumption / (sucrose water consumption + water consumption) × 100%.

[0048] As Figure 1 shown, there were significant differences in the sucrose preference rate among the groups. The sucrose preference rate of the rats in the model group was significantly lower than that of the normal group (P < 0.01). The sucrose preference rates of the rats in Examples 1 to 3 and Comparative Examples 1 to 2 groups were all significantly higher than that of the model group (P values were < 0.01 and < 0.05, respectively). In addition, only the sucrose preference rates of the rats in Examples 1 to 3 groups were significantly higher than that of the normal group (P < 0.05), and there were no significant differences in the other groups. This shows that the pharmaceutical composition preparations provided in Examples 1 to 3 can significantly increase the sucrose preference rate of CUMS-treated rats, and the effect of Example 1 is the best.

[0049] 4. Forced swimming test The experiment was conducted in an FST-100 system using a transparent cylinder with a diameter of 21 cm, which was filled with water at a depth of 35 cm and a temperature of (25 ± 1) °C. During the experiment, each rat was first allowed to swim for 15 minutes and then returned to the breeding cage. After 24 hours, the rat was individually placed in the cylinder to swim again, and the immobility time of each rat within 5 minutes was recorded using the above system.

[0050] As Figure 2 shown, the immobility time of the rats in the model group was significantly higher than that in the normal group (P < 0.005). The pharmaceutical composition preparations provided in Examples 1 to 3 and Comparative Examples 1 to 2 significantly reduced the immobility time of CUMS rats (compared with the model group, P < 0.005, P < 0.005, P < 0.005, P < 0.001, P < 0.05). In addition, there was no significant difference in the immobility time of the rats in Examples 1 to 3 compared with the normal group, while the immobility time of the rats in Comparative Examples 1 and 2 was significantly higher than that in the normal group (P < 0.05, P < 0.001). This indicates that the pharmaceutical composition preparations provided in Examples 1 to 3 can significantly reduce the immobility time of the forced swimming test in CUMS-treated rats.

[0051] 5. Detection of the mRNA expression levels of ERK1, ERK2, and CREB by RT-PCR SYBR RT-PCR analysis was used to detect the mRNA level. The hippocampal samples stored in liquid nitrogen were weighed, and total RNA was extracted using the TaKaRa MiniBEST Universal RNA Extraction Kit. The total RNA was reverse transcribed into complementary DNA (cDNA) using PrimeScript™ RT Master Mix, and then amplified on a PCR instrument. The primer sequences of the target genes ERK1 (Extracellular Signal-Regulated Kinase 1), ERK2 (Extracellular Signal-Regulated Kinase 2), CREB (cAMP Response Element-Binding Protein), and the reference gene GAPDH (Glyceraldehyde 3-Phosphate Dehydrogenase) are as follows: ERK1 forward primer: 5'-GGGCCAAGCTTTTTCCCAAA-3', SEQ ID NO:1; reverse primer: 5'-AGCCACTGGTTCATCTGTCG-3', SEQ ID NO:2. ERK2 forward primer: 5'-ATCTTAAATTGGTCAGGACAAGGG-3', SEQ ID NO:3, reverse primer: 5'-CTCGGAACGGCTCAAAGGAG-3', SEQ ID NO:4. CREB forward primer: 5'-CTGAGGAGCTTGTACCACCG-3', SEQ ID NO:5, reverse primer: 5'-CTGCTGGCATGGATACCTGG-3', SEQ ID NO:6. GAPDH forward primer: 5'-ACAGCAACAGGGTGGTGAC-3', SEQ ID NO:7; reverse primer: 5'-TTTGAGGGGTGCAGCGAACTT-3', SEQ ID NO:8. Using the 7500 Real-Time PCR System and selecting SYBR® Premix Ex Taq as the reaction reagent, the following real-time quantitative PCR analysis procedure was performed: First, incubate at 50 °C for 2 minutes, and then incubate at 95 °C for 10 minutes; then perform 40 cycles, each cycle consisting of denaturation at 95 °C for 15 seconds and annealing and extension at 60 °C for 1 minute. The relative quantification method (2 -ΔΔCT ) was used, with GAPDH as the internal reference to calculate the relative expression level of the target gene.

[0052] Such as Figures 3 - 5As shown, compared with the normal group, the mRNA levels of ERK1, ERK2, and CREB in the hippocampus of rats in the model group were significantly decreased (P<0.01, P<0.01, P<0.01). Sleep deprivation is considered to have an antidepressant effect and may act by affecting the content and activity of CREB in the hippocampal region. The pharmaceutical composition preparation provided in Example 1 significantly increased the mRNA levels of ERK1, ERK2, and CREB in CUMS rats (P<0.01, P<0.01, P<0.01 compared with the model group), which was consistent with the effects of midazolam and propofol on the phosphorylation levels of ERK1, ERK2, and CREB in the rat hippocampus. The pharmaceutical composition preparation provided in Example 2 significantly increased the mRNA levels of ERK1, ERK2, and CREB in CUMS rats (P<0.01, P<0.01, P<0.01 compared with the model group). The pharmaceutical composition preparation provided in Example 3 significantly increased the mRNA levels of ERK1, ERK2, and CREB in CUMS rats (P<0.01, P<0.01, P<0.05 compared with the model group), and this result was consistent with the effect of propofol on the phosphorylation levels of ERK1 / ERK2 and CREB in the rat hippocampus. However, there were no significant differences in the mRNA levels of ERK1, ERK2, and CREB between the rats administered with the pharmaceutical composition preparations provided in Comparative Example 1 and Comparative Example 2 and the model group. Moreover, the mRNA levels of ERK1, ERK2, and CREB in the rats administered with the pharmaceutical composition preparations provided in Examples 1 to 3 were significantly higher than those in the normal group. This shows that the pharmaceutical composition preparations provided in Examples 1 to 3 can significantly increase the mRNA levels of ERK1, ERK2, and CREB in the hippocampus of CUMS-treated rats.

[0053] 6. Collection and analysis of electroencephalogram data After inducing anesthesia in rats with 1.5% isoflurane, anesthesia was maintained at a concentration of 1%. The heads of the rats were fixed on a stereotaxic apparatus, the head hair was shaved off, and then the meninges of the rats were dissected to expose the skull. After drilling a hole with a high-speed skull drill, electroencephalogram electrodes were implanted into the right hippocampal CA3 region of the rats (AP: -3.6 mm, ML: +4.2 mm, DV: -3.8 mm). Then, the electrodes were firmly fixed with glass ionomer cement to ensure stability and accuracy. After the rats naturally woke up from the anesthetized state, they were placed separately in cages for feeding to allow sufficient time for recovery for 7 days for subsequent electroencephalogram data collection. The electroencephalogram data of rats in each group were collected using a Ponemah acquisition system, and the electroencephalogram signals were analyzed using NeuroScore software. After the signal collection was completed, the rats were sacrificed, and the intact brain tissues were taken out to observe whether the implantation positions of the brain electrodes were accurate.

[0054] As Figure 6As shown, the Beta / Total frequency ratio in the Beta band of the electroencephalogram signals of the rats in the model group was significantly higher than that in the normal group. The pharmaceutical composition preparations provided in Examples 1 to 3 significantly reduced the Beta / Total frequency ratio of the CUMS rats compared with the model group. For the pharmaceutical composition preparations provided in Comparative Examples 1 and 2, although they reduced the Beta / Total frequency ratio of the CUMS rats to a certain extent compared with the model group, this reduction effect was obviously inferior to that of the groups in Examples 1 to 3. This shows that the pharmaceutical composition preparations provided in Examples 1 to 3 can significantly reduce the Beta / Total frequency ratio of the rats treated with CUMS.

[0055] As Figure 7 shown, the Theta / Total frequency ratio of the rats in the model group was significantly higher than that in the normal group. The pharmaceutical composition preparations provided in Examples 1 to 3 significantly reduced the Theta / Total frequency ratio of the CUMS rats compared with the model group. Although the pharmaceutical composition preparations provided in Comparative Examples 1 and 2 could partially reduce the Theta / Total frequency ratio of the CUMS rats compared with the model group, this reduction effect was obviously inferior to that of the groups in Examples 1 to 3. This shows that the pharmaceutical composition preparations provided in Examples 1 to 3 can significantly reduce the Theta / Total frequency ratio of the rats treated with CUMS.

[0056] The CUMS model causes anhedonia symptoms by placing rodents in a mild unpredictable stress environment for a long time, and experiments such as FST, OFT, and SPT are often used to evaluate whether the modeling is successful. Through testing, it was found in this experiment that the pharmaceutical compositions provided in Examples 1 to 3 could significantly increase the sucrose preference rate of the CUMS rats and shorten the immobile time after forced swimming, thus showing an obvious effect of alleviating and treating the CUMS rats.

[0057] In addition, the ERK-CREB signaling pathway plays a key role in the mechanism of action of the antidepressant drug (R)-ketamine, participating in various physiological effects such as intercellular signal transduction and recognition, cell growth and development, and having a potential molecular mechanism in regulating depressive behavior. The phosphorylation of ERK can activate the expression of its downstream signaling molecule CREB, and CREB is considered to be partially involved in the expression of most genes regulated by cyclic adenosine monophosphate (cAMP), including BDNF (Brain-Derived Neurotrophic Factor). The subsequently released target protein can promote neuron survival, thereby exerting an antidepressant effect. The pharmaceutical compositions provided in Examples 1 to 3 can significantly increase the mRNA expression levels of ERK1, ERK2, and CREB in the hippocampus of CUMS-treated rats, effectively intervene in and activate the ERK-CREB signaling pathway, thereby inhibiting neurasthenia symptoms, enhancing the neuroprotective effect, and showing an antidepressant effect.

[0058] Electroencephalogram records the spontaneous and rhythmic electrical activities of brain cell groups through electrodes, thereby reflecting the changes in brain function, and is widely used in the research of neurasthenia, depression, and sleep disorders. Beta waves usually appear when an individual is in a state of tension, anxiety, or emotional excitement, and are closely related to human thinking activities, physical movements, and emotional fluctuations such as depression. Theta waves are crucial for information processing within the hippocampus and other limbic structures and are even considered a possible biomarker for anxiety. Theta waves change through changes in cognitive-emotional processing, first affecting the hippocampus and then other structures directly involved in anxiety and memory. It has been reported in some studies that the proportion of Beta and Theta activity frequencies in depressed patients will increase significantly. The characteristic changes in sleep disorders in depression patients include disrupted sleep continuity, reduced REM sleep time, and reduced NREM sleep time. In this experiment, it was found that compared with the normal control group, the proportions of Beta and Theta in the model group of rats were significantly increased, indicating that CUMS-induced depressive model rats had emotional regulation disorders. The pharmaceutical composition preparations provided in Examples 1 to 3 can regulate the structure and composition of the intestinal flora, reduce the depressive behavior of CUMS rats, and have an obvious improvement effect.

[0059] As mentioned above, only the preferred specific embodiments of the present invention are described, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention.

Claims

1. A pharmaceutical composition, characterized in that, Comprising by weight parts: 80 - 120 parts of (8E)-ligustroside, 60 - 80 parts of fraxin, 60 - 80 parts of oleuropein, 60 - 80 parts of syringopicroside, 20 - 60 parts of (+)-pinoresinol-4-O-β-D-glucopyranoside, 20 - 50 parts of oleocanthal-4'-O-glucoside, 10 - 50 parts of coniferin, 10 - 40 parts of benzyl alcohol-β-vicianoside, 10 - 40 parts of gaultherin, 10 - 30 parts of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl)glucopyranoside, 5 - 10 parts of (Z)-3-hexenyl-β-D-glucopyranoside, 5 - 30 parts of 2-hydroxy-1-methoxyanthraquinone, 5 - 20 parts of (-)-episyringaresinol, 5 - 20 parts of dibutyl terephthalate, and 1 - 10 parts of (-)-3,4-divanillyltetrahydrofuran.

2. The pharmaceutical composition according to claim 1, wherein Comprising by weight parts: 100 parts of (8E)-ligustroside, 70 parts of fraxin, 65 parts of oleuropein, 65 parts of syringopicroside, 35 parts of (+)-pinoresinol-4-O-β-D-glucopyranoside, 40 parts of oleocanthal-4'-O-glucoside, 20 parts of coniferin, 30 parts of benzyl alcohol-β-vicianoside, 30 parts of gaultherin, 20 parts of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl)glucopyranoside, 8 parts of (Z)-3-hexenyl-β-D-glucopyranoside, 15 parts of 2-hydroxy-1-methoxyanthraquinone, 10 parts of (-)-episyringaresinol, 10 parts of dibutyl terephthalate, and 6 parts of (-)-3,4-divanillyltetrahydrofuran.

3. The pharmaceutical composition according to claim 1, wherein Comprising by weight parts: 80 parts of (8E)-ligustroside, 60 parts of fraxin, 60 parts of oleuropein, 60 parts of syringopicroside, 20 parts of (+)-pinoresinol-4-O-β-D-glucopyranoside, 20 parts of oleocanthal-4'-O-glucoside, 10 parts of coniferin, 10 parts of benzyl alcohol-β-vicianoside, 10 parts of gaultherin, 10 parts of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl)glucopyranoside, 5 parts of (Z)-3-hexenyl-β-D-glucopyranoside, 5 parts of 2-hydroxy-1-methoxyanthraquinone, 5 parts of (-)-episyringaresinol, 5 parts of dibutyl terephthalate, and 1 part of (-)-3,4-divanillyltetrahydrofuran.

4. The pharmaceutical composition according to claim 1, wherein Comprising, by weight parts: 120 parts of (8E)-ligustroside, 80 parts of fraxin, 80 parts of oleuropein, 80 parts of syringopicroside, 60 parts of (+)-pinoresinol-4-O-β-D-glucopyranoside, 50 parts of oleacein-4'-O-glucoside, 50 parts of coniferin, 40 parts of benzyl alcohol-β-vicianoside, 40 parts of wintergreen glycoside, 30 parts of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl) glucopyranoside, 10 parts of (Z)-3-hexenyl-β-D-glucopyranoside, 30 parts of 2-hydroxy-1-methoxyanthraquinone, 20 parts of (-)-episyringaresinol, 20 parts of dibutyl terephthalate and 10 parts of (-)-3,4-divanillyltetrahydrofuran.

5. A preparation, which is a tablet or capsule comprising the pharmaceutical composition according to any one of claims 1 to 4.

6. A preparation method of the preparation according to claim 5, characterized in that, The preparation method comprises: Preparing a (8E)-ligustroside solution with an ethanol aqueous solution containing 15%, preparing a fraxin solution with ethanol, preparing an oleuropein solution with methanol, preparing a syringopicroside solution with ethanol, preparing a (+)-pinoresinol-4-O-β-D-glucopyranoside solution with ethanol, preparing an oleacein-4'-O-glucoside solution with methanol, preparing a coniferin solution with ethanol, preparing a benzyl alcohol-β-vicianoside solution with ethanol, preparing a wintergreen glycoside solution with ethanol, preparing a (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl) glucopyranoside solution with ethyl acetate, preparing a (Z)-3-hexenyl-β-D-glucopyranoside solution with ethanol, preparing a 2-hydroxy-1-methoxyanthraquinone solution with ethanol, preparing a (-)-episyringaresinol solution with ethanol, preparing a dibutyl terephthalate solution with ethyl acetate, and preparing a (-)-3,4-divanillyltetrahydrofuran solution with ethanol; Mixing the component solutions with ethanol or methanol as solvents in equal volumes, adding while stirring to ensure uniform mixing to obtain a first solution; mixing the component solutions with ethyl acetate as a solvent in equal volumes, adding while stirring to ensure uniform mixing to obtain a second solution; then slowly pouring the second solution into the first solution, and subsequently adding ethanol equivalent to 0.25 times the volume of the mixed solution of the first solution and the second solution, and stirring thoroughly until uniform; performing ultrasonic treatment to obtain a third solution containing the formulation amounts of each component; Transferring the third solution to a rotary evaporator, performing rotary evaporation at appropriate temperature and pressure to remove the solvent, obtaining a concentrated mixture; transferring the concentrated mixture to a drying oven for drying to obtain a mixed powder; For tablets, 20% of starch by weight of the total tablets can be added as a filler, 5% of sodium carboxymethyl starch by weight of the total preparation can be added as a disintegrant, and 1% of magnesium stearate by weight of the total tablets can be added as a lubricant; for tablets, putting the mixed powder into a tableting machine for tableting to obtain tablets; For capsules, lactose accounting for 45% of the total capsule weight can be added as a filler, and magnesium stearate accounting for 0.75% of the total capsule weight can be added as a lubricant; for capsules, the mixed powder is filled into capsule shells to obtain capsules.

7. The method according to claim 6, wherein In the third solution, the concentration of (8E)-ligustroside is 100.00 mg / L, the concentration of fraxin is 70.00 mg / L, the concentration of oleuropein is 65.00 mg / L, the concentration of syringopicroside is 65.00 mg / L, the concentration of (+)-pinoresinol-4-O-β-D-glucopyranoside is 35.00 mg / L, the concentration of oleocanthal-4'-O-glucoside is 40.00 mg / L, the concentration of coniferin is 20.00 mg / L, the concentration of benzyl alcohol-β-vicianoside is 30.00 mg / L, the concentration of gaultherin is 30.00 mg / L, the concentration of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl) glucopyranoside is 20.00 mg / L, the concentration of (Z)-3-hexenyl-β-D-glucopyranoside is 8.00 mg / L, the concentration of 2-hydroxy-1-methoxyanthraquinone is 15.00 mg / L, the concentration of (-)-episyringaresinol is 10.00 mg / L, the concentration of dibutyl terephthalate is 10.00 mg / L, and the concentration of (-)-3,4-divanillyltetrahydrofuran is 6.00 mg / L.

8. The method according to claim 6, wherein In the third solution, the concentration of (8E)-ligustroside is 116.12 mg / L, the concentration of fraxin is 87.09 mg / L, the concentration of oleuropein is 87.09 mg / L, the concentration of syringopicroside is 87.09 mg / L, the concentration of (+)-pinoresinol-4-O-β-D-glucopyranoside is 29.03 mg / L, the concentration of oleocanthal-4'-O-glucoside is 29.03 mg / L, the concentration of coniferin is 14.52 mg / L, the concentration of benzyl alcohol-β-vicianoside is 14.52 mg / L, the concentration of gaultherin is 14.52 mg / L, the concentration of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl) glucopyranoside is 14.52 mg / L, the concentration of (Z)-3-hexenyl-β-D-glucopyranoside is 7.26 mg / L, the concentration of 2-hydroxy-1-methoxyanthraquinone is 7.26 mg / L, the concentration of (-)-episyringaresinol is 7.26 mg / L, the concentration of dibutyl terephthalate is 7.26 mg / L, and the concentration of (-)-3,4-divanillyltetrahydrofuran is 1.45 mg / L.

9. The method according to claim 6, wherein In the third solution, the concentration of (8E)-ligustroside is 87.33 mg / L, the concentration of fraxin is 58.22 mg / L, the concentration of oleuropein is 58.22 mg / L, the concentration of syringopicroside is 58.22 mg / L, the concentration of (+)-pinoresinol-4-O-β-D-glucopyranoside is 43.67 mg / L, the concentration of oleacein-4'-O-glucoside is 36.39 mg / L, the concentration of coniferin is 36.39 mg / L, the concentration of benzyl alcohol-β-vicianoside is 29.11 mg / L, the concentration of wintergreen glycoside is 29.11 mg / L, the concentration of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl) glucopyranoside is 21.83 mg / L, the concentration of (Z)-3-hexenyl-β-D-glucopyranoside is 7.28 mg / L, the concentration of 2-hydroxy-1-methoxyanthraquinone is 21.83 mg / L, the concentration of (-)-episyringaresinol is 14.56 mg / L, the concentration of dibutyl terephthalate is 14.56 mg / L, and the concentration of (-)-3,4-divanillyltetrahydrofuran is 7.28 mg / L.

10. Use of the pharmaceutical composition according to any one of claims 1 to 4, characterized in that, The application includes at least one of preparing a preparation for increasing the sucrose preference rate, preparing a preparation for reducing the immobility time after exercise, preparing an agent for activating the ERK-CREB signaling pathway, and preparing a preparation for reducing the Beta and Theta activity frequencies.

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