A pharmaceutical composition and its preparation method and application

Abstract

The present invention relates to the technical field of neurasthenia, and more specifically to a pharmaceutical composition, its preparation method, and its use. The pharmaceutical composition is prepared using a variety of glycosides as raw materials. The pharmaceutical composition can treat or prevent symptoms of neurasthenia in patients, such as neuralgic headaches and headaches caused by hysteria. By intervening in and activating the ERK-CREB signaling pathway, the composition helps inhibit neurasthenia, enhances neuroprotection, and plays a positive role in antidepressants.

Description

Technical Field

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

[0002] Neurasthenia is a chronic functional disease characterized by long-term fatigue, mood swings, difficulty concentrating, and sleep disturbances. Studies have found that neurasthenia may be related to dysfunction of the hypothalamic-pituitary-adrenal axis (HPA axis), imbalances in neurotransmitters (such as 5-HT, GABA, and dopamine), and chronic inflammatory responses.

[0003] Currently, the main medications used clinically to treat neurasthenia include benzodiazepines and antidepressants. While benzodiazepines can quickly alleviate symptoms such as anxiety and insomnia, long-term use can lead to adverse reactions such as drug dependence, drug tolerance, and cognitive impairment. While antidepressants can be somewhat effective in improving patients' mood symptoms, they are slow to take effect, and some patients may experience side effects such as sexual dysfunction and weight gain, limiting their use in the treatment of neurasthenia.

[0004] In the development of Western medicine, some new drugs also show promising application prospects. For example, agomelatine, a melatonin receptor agonist, can improve sleep quality, combat depression, and regulate mood with minimal adverse reactions. Furthermore, several neuroprotective and neurotrophic drugs are under development, such as neurotrophic factors and their analogs, which are expected to fundamentally improve the symptoms of neurasthenia by promoting the growth, repair, and functional recovery of nerve cells.

[0005] Compound medications offer unique advantages in treating neurasthenia, enhancing efficacy through synergistic effects across multiple targets and pathways. For example, some compound medications can simultaneously regulate neurotransmitters, improve cerebral blood flow, and alleviate neuroinflammation, comprehensively improving patient symptoms. Currently, research on compound medications focuses primarily on traditional Chinese medicine compounds, combined traditional Chinese and Western medicine compounds, and Western medicine compounds. Through modern pharmacological research and clinical trials, compound medications with proven efficacy and a good safety profile are being identified, providing more options for the treatment of neurasthenia. Summary of the Invention

[0006] To address the above technical problems, the present invention provides a pharmaceutical composition, its preparation method, and its use. The pharmaceutical composition is prepared using a variety of glycosides as raw materials. The pharmaceutical composition can treat or prevent symptoms of neurasthenia, such as neuralgic headaches and headaches caused by hysteria, and is beneficial for improving patients' symptoms.

[0007] The present invention provides a pharmaceutical composition, comprising, by weight: 80-120 parts of (8E)-ligustrin, 60-80 parts of fraxinoside, 60-80 parts of oleuropein, 60-80 parts of syringoside, 20-60 parts of (+)-pinoresinol-4-O-β-D-pyranoglucoside, 20-50 parts of oleuropein-4'-O-glucoside, 10-50 parts of coniferin, 10-40 parts of benzyl alcohol-β-vicinyl glycoside, 10- 40 parts of holmoside, 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. The term "parts" refers to any weight and does not indicate an absolute value but merely indicates the weight ratio of the components.

[0008] Specifically, the composition comprises, by weight, 100 parts of (8E)-ligustrin, 70 parts of fraxinoside, 65 parts of oleuropein, 65 parts of syringoside, 35 parts of (+)-pinoresinol-4-O-β-D-pyranoglucoside, 40 parts of oleoresinol-4'-O-glucoside, 20 parts of coniferin, 30 parts of benzyl alcohol-β-vicinyl glycoside, 30 parts of hollyoside, 20 parts of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl)pyranoside, 8 parts of (Z)-3-hexenyl-β-D-pyranoside, 15 parts of 2-hydroxy-1-methoxyanthraquinone, 10 parts of (-)-episyringaresinol, 10 parts of dibutyl terephthalate and 6 parts of (-)-3,4-divanillyltetrahydrofuran.

[0009] Specifically, the composition comprises, by weight, 80 parts of (8E)-ligustrin, 60 parts of fraxinoside, 60 parts of oleuropein, 60 parts of syringoside, 20 parts of (+)-pinoresinol-4-O-β-D-pyranoglucoside, 20 parts of oleoresinol-4'-O-glucoside, 10 parts of coniferin, 10 parts of benzyl alcohol-β-vicinyl glycoside, 10 parts of hollyoside, 10 parts of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl)pyranoside, 5 parts of (Z)-3-hexenyl-β-D-pyranoglucoside, 5 parts of 2-hydroxy-1-methoxyanthraquinone, 5 parts of (-)-episyringaresinol, 5 parts of dibutyl terephthalate and 1 part of (-)-3,4-divanillyltetrahydrofuran.

[0010] Specifically, the composition comprises, by weight, 120 parts of (8E)-ligustrin, 80 parts of fraxinoside, 80 parts of oleuropein, 80 parts of syringoside, 60 parts of (+)-pinoresinol-4-O-β-D-pyranoglucoside, 50 parts of oleoresinol-4'-O-glucoside, 50 parts of coniferin, 40 parts of benzyl alcohol-β-vicinyl glycoside, 40 parts of hollyoside, 30 parts of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl)pyranoside, 10 parts of (Z)-3-hexenyl-β-D-pyranoside, 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 bitter taste and odor of the drug and improve bioavailability.

[0012] In addition, the present invention also provides a method for preparing the preparation. The method comprises:

[0013] (1) Dissolution

[0014] Use 15% ethanol aqueous solution to prepare (8E)-ligustrin solution, use ethanol to prepare fraxinoside solution, use methanol to prepare olive glycoside solution, use ethanol to prepare syringoside solution, use ethanol to prepare (+)-pinoresinol-4-O-β-D-pyranoglucoside solution, use methanol to prepare oleoresin-4'-O-glucoside solution, use ethanol to prepare coniferin solution, use ethanol to prepare benzyl alcohol-β-vicinoside solution, use ethanol to prepare wintergreen glycoside solution, use ethyl acetate to prepare acetic acid solution. Prepare (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl)pyranoside solution with ester, prepare (Z)-3-hexenyl-β-D-pyranoside solution with ethanol, prepare 2-hydroxy-1-methoxyanthraquinone solution with ethanol, prepare (-)-episyringaresinol solution with ethanol, prepare dibutyl terephthalate solution with ethyl acetate, and prepare (-)-3,4-divanillyltetrahydrofuran solution with ethanol;

[0015] (2) Mixed

[0016] Mix equal volumes of the component solutions using ethanol or methanol as a solvent, stirring while adding to ensure uniform mixing to obtain a first solution; mix equal volumes of the component solutions using ethyl acetate as a solvent, stirring while adding to ensure uniform mixing to obtain a second solution; then slowly pour the second solution into the first solution, followed by adding ethanol equivalent to 0.25 times the volume of the mixed solution of the first and second solutions, and stirring thoroughly until uniform; and perform ultrasonic treatment to obtain a third solution containing the formulated amounts of each component;

[0017] (3) Concentration and drying

[0018] The third solution is transferred to a rotary evaporator, and rotary evaporation is performed at an appropriate temperature and pressure to remove the solvent to obtain a concentrated mixture; the concentrated mixture is transferred to a drying oven for drying to obtain a mixed powder;

[0019] (4) Preparation molding

[0020] For tablets, 20% of starch by weight of the total tablet can be added as a filler, 5% of sodium carboxymethyl starch by weight can be added as a disintegrant, and 1% of magnesium stearate by weight can be added as a lubricant. For tablets, the mixed powder is placed in a tablet press and compressed to obtain tablets.

[0021] For capsules, 45% of lactose, based on the total capsule weight, may be added as a filler, and 0.75% of magnesium stearate, based on the total capsule weight, may be added as a lubricant. For capsules, the mixed powder is filled into capsule shells to obtain capsules.

[0022] Specifically, in the third solution, the concentration of (8E)-ligustrin is 100.00 mg / L, the concentration of fraxinus glycoside is 70.00 mg / L, the concentration of oleuropein is 65.00 mg / L, the concentration of syringoside is 65.00 mg / L, the concentration of (+)-pinoresinol-4-O-β-D-pyranoglucoside is 35.00 mg / L, the concentration of oleoresinol-4'-O-glucoside is 40.00 mg / L, the concentration of coniferin is 20.00 mg / L, the concentration of benzyl alcohol-β-vicinyl glycoside is 30.00 mg / L, and the concentration of holly glycoside is 100.00 mg / L. The concentration of the aqueous solution of PEG-100 sodium phosphate was 30.00 mg / L, the concentration of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl)pyranoside was 20.00 mg / L, the concentration of (Z)-3-hexenyl-β-D-pyranoside was 8.00 mg / L, the concentration of 2-hydroxy-1-methoxyanthraquinone was 15.00 mg / L, the concentration of (-)-episyringaresinol was 10.00 mg / L, the concentration of dibutyl terephthalate was 10.00 mg / L, and the concentration of (-)-3,4-divanillyltetrahydrofuran was 6.00 mg / L.

[0023] Specifically, in the third solution, the concentration of (8E)-ligustrin was 116.12 mg / L, the concentration of fraxinoside was 87.09 mg / L, the concentration of oleuropein was 87.09 mg / L, the concentration of syringoside was 87.09 mg / L, the concentration of (+)-pinoresinol-4-O-β-D-pyranoglucopyranoside was 29.03 mg / L, the concentration of oleoresinol-4'-O-glucoside was 29.03 mg / L, the concentration of coniferin was 14.52 mg / L, the concentration of benzyl alcohol-β-vicinyl glycoside was 14.52 mg / L, and the concentration of holmoside was 10. The concentration of the crude drug was 14.52 mg / L, the concentration of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl) glucopyranoside was 14.52 mg / L, the concentration of (Z)-3-hexenyl-β-D-glucopyranoside was 7.26 mg / L, the concentration of 2-hydroxy-1-methoxyanthraquinone was 7.26 mg / L, the concentration of (-)-episyringaresinol was 7.26 mg / L, the concentration of dibutyl terephthalate was 7.26 mg / L, and the concentration of (-)-3,4-divanillyltetrahydrofuran was 1.45 mg / L.

[0024] Specifically, in the third solution, the concentration of (8E)-ligustrin is 87.33 mg / L, the concentration of fraxinus glycoside is 58.22 mg / L, the concentration of oleuropein is 58.22 mg / L, the concentration of syringoside is 58.22 mg / L, the concentration of (+)-pinoresinol-4-O-β-D-pyranoglucopyranoside is 43.67 mg / L, the concentration of oleoresinol-4'-O-glucoside is 36.39 mg / L, the concentration of coniferin is 36.39 mg / L, the concentration of benzyl alcohol-β-vicinyl glycoside is 29.11 mg / L, and the concentration of holly glycoside is 29. The concentration of the raw materials was 29.11 mg / L, the concentration of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl)pyranoside was 21.83 mg / L, the concentration of (Z)-3-hexenyl-β-D-pyranoside was 7.28 mg / L, the concentration of 2-hydroxy-1-methoxyanthraquinone was 21.83 mg / L, the concentration of (-)-episyringaresinol was 14.56 mg / L, the concentration of dibutyl terephthalate was 14.56 mg / L, and the concentration of (-)-3,4-divanillyltetrahydrofuran was 7.28 mg / L.

[0025] In addition, the present invention also provides an application of the above-mentioned pharmaceutical composition, which includes preparing at least one of a preparation for increasing sucrose preference, a preparation for reducing post-exercise immobility time, a preparation for activating the ERK-CREB signaling pathway, and a preparation for reducing beta and theta activity frequencies.

[0026] Beneficial effects:

[0027] This invention uses multiple glycosides as raw materials, and through dissolution, mixing, concentration, and drying steps, combined with a formulation molding process, ultimately produces a novel pharmaceutical composition. This formulation demonstrates significant efficacy in chronic unpredictable mild stress (CUMS) rats, increasing their sucrose preference, shortening post-exercise immobility, activating the ERK-CREB signaling pathway, and effectively reducing beta and theta brainwave activity.

[0028] The pharmaceutical composition provided by the present invention has significant alleviating and therapeutic effects on CUMS rats. Its mechanism is to activate the ERK-CREB signaling pathway, effectively suppressing neurasthenia symptoms and enhancing neuroprotection, thereby exerting an antidepressant effect. The pharmaceutical composition provided by the present invention can reduce the ratio of Beta and Theta, regulate mood disorders in CUMS rats, and significantly improve mood and sleep. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The 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 are shown.

[0030] Figure 2 The immobility time of the rats in the forced swimming test of the normal group, model group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group and Comparative Example 2 group.

[0031] Figure 3 The 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.

[0032] Figure 4 The 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.

[0033] Figure 5 The changes in CREB mRNA levels in the rat hippocampus of 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 are shown.

[0034] Figure 6 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 are shown.

[0035] Figure 7The results show the correlation between the hippocampal theta rhythm oscillation power value and spatial learning and memory ability 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 physiological and pathological conditions as evaluated by the Morris water maze test. DETAILED DESCRIPTION

[0036] In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the following examples. It should be understood that the specific examples described herein are merely illustrative of the present invention and are not intended to limit the present invention. Reagents not described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional test methods and are known in the art.

[0037] 1. Pharmaceutical Compositions and Preparations

[0038] 1. Pharmaceutical composition

[0039] (8E)-Ligustrum lucidum glycoside, CAS: 39011-92-2, Yisheng Biotechnology. Fraxinoside, CAS: 524-30-1, Nanjing Dierge Pharmaceutical. Oleuroside, CAS: 32619-42-4, Nanjing Bingcheng Biotechnology. Syringopicrin, CAS: 29118-80-7, Chengdu Standard Sample Biotechnology. (+)-Pinoresinol-4-O-β-D-glucopyranoside, China Chemical Network. Oleoresinol-4'-O-glucoside, CAS: 76880-93-8, Chengdu Pusi Biotechnology. Coniferoside, 124151-33-3, Shanghai Yihe Biotechnology. Benzyl alcohol-β-vicinyl glycoside, PubChem CID: 157010045, PubChem. Hollyoside, CAS: 490-67-5, Chengdu Puruifa 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, China 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 Pharmaceutical.

[0040] The embodiment provides a pharmaceutical composition, comprising, by weight: 80-120 parts of (8E)-ligustrin, 60-80 parts of fraxinoside, 60-80 parts of oleuropein, 60-80 parts of syringoside, 20-60 parts of (+)-pinoresinol-4-O-β-D-pyranoglucoside, 20-50 parts of oleuropein-4'-O-glucoside, 10-50 parts of coniferin, 10-40 parts of benzyl alcohol-β-vicinyl glycoside, 10- 40 parts of holmoside, 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.

[0041] The composition provided in Example 1 comprises, by weight, 100 parts of (8E)-ligustrin, 70 parts of fraxinoside, 65 parts of oleuropein, 65 parts of syringoside, 35 parts of (+)-pinoresinol-4-O-β-D-glucopyranoside, 40 parts of oleoresinol-4'-O-glucoside, 20 parts of coniferin, 30 parts of benzyl alcohol-β-vicinoside, 30 parts of holmoside, 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.

[0042] The composition provided in Example 2 comprises, by weight, 80 parts of (8E)-ligustrin, 60 parts of fraxinoside, 60 parts of oleuropein, 60 parts of syringoside, 20 parts of (+)-pinoresinol-4-O-β-D-pyranoglucoside, 20 parts of oleoresinol-4'-O-glucoside, 10 parts of coniferin, 10 parts of benzyl alcohol-β-vicinyl glycoside, 10 parts of hollyoside, 10 parts of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl)pyranoside, 5 parts of (Z)-3-hexenyl-β-D-pyranoglucoside, 5 parts of 2-hydroxy-1-methoxyanthraquinone, 5 parts of (-)-episyringaresinol, 5 parts of dibutyl terephthalate and 1 part of (-)-3,4-divanillyltetrahydrofuran.

[0043] The composition provided in Example 3 comprises, by weight, 120 parts of (8E)-ligustrin, 80 parts of fraxinoside, 80 parts of oleuropein, 80 parts of syringoside, 60 parts of (+)-pinoresinol-4-O-β-D-glucopyranoside, 50 parts of oleoresinol-4'-O-glucoside, 50 parts of coniferin, 40 parts of benzyl alcohol-β-vicinoside, 40 parts of hollyoside, 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.

[0044] The composition provided in Comparative Example 1 comprises, by weight: 100 parts of (8E)-ligustrin, 70 parts of fraxinoside, 65 parts of oliveoside, 65 parts of syringoside, 35 parts of (+)-pinoresinol-4-O-β-D-pyranoglucoside, 40 parts of oleoresinol-4'-O-glucoside, 20 parts of coniferin, 30 parts of benzyl alcohol-β-vicinatoside, 30 parts of hollyoside, 20 parts of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl)pyranoglucoside, and 8 parts of (Z)-3-hexenyl-β-D-pyranoglucoside.

[0045] The composition provided in Comparative Example 2 comprises, by weight, 15 parts of 2-hydroxy-1-methoxyanthraquinone, 10 parts of (-)-episyringaresinol, 10 parts of dibutyl terephthalate, and 6 parts of (-)-3,4-divanillyltetrahydrofuran.

[0046] 2. Dissolving and mixing

[0047] The choice of solvent should be based on the solubility characteristics of the compound; ethanol, methanol, or dichloromethane are recommended. Excipients should be flexibly combined based on the formulation type, such as starch and lactose as fillers, sodium carboxymethyl starch as disintegrants, and magnesium stearate as lubricants. Instruments and equipment include a mortar, beaker, magnetic stirrer, rotary evaporator, and drying oven.

[0048] The various compounds in Examples 1 to 3 and Comparative Examples 1 to 2 were dissolved respectively.

[0049] (1) Dissolution step

[0050] Table 1 (mg / L)

[0051]

[0052] Use 15% ethanol aqueous solution to prepare (8E)-ligustrin solution, use ethanol to prepare fraxinoside solution, use methanol to prepare olive glycoside solution, use ethanol to prepare syringoside solution, use ethanol to prepare (+)-pinoresinol-4-O-β-D-pyranoglucoside solution, use methanol to prepare oleoresin-4'-O-glucoside solution, use ethanol to prepare coniferin solution, use ethanol to prepare benzyl alcohol-β-vicinoside solution, use ethanol to prepare wintergreen glycoside solution, use ethyl acetate to prepare acetic acid solution. A solution of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl) glucopyranoside was prepared with ester, a solution of (Z)-3-hexenyl-β-D-glucopyranoside was prepared with ethanol, a solution of 2-hydroxy-1-methoxyanthraquinone was prepared with ethanol, a solution of (-)-episyringaresinol was prepared with ethanol, a solution of dibutyl terephthalate was prepared with ethyl acetate, and a solution of (-)-3,4-divanillyltetrahydrofuran was prepared with ethanol. The weight ratios of the component concentrations in Table 1 are approximate to the weight ratios above and are not mathematically equivalent.

[0053] (2) Mixing step

[0054] The above-mentioned solutions of various components with ethanol or methanol as solvent are mixed, stirred while adding, and uniformly mixed to obtain a first solution. The above-mentioned solutions of various components with ethyl acetate as solvent are mixed in equal volumes, stirred while adding, and uniformly mixed to obtain a second solution.

[0055] The second solution was then slowly poured into the first solution, followed by the addition of ethanol equivalent to 0.25 times the volume of the combined solution of the first and second solutions, and thorough stirring until uniform. Ultrasonic treatment was then performed to further homogenize the solution to obtain a third solution, such that the concentrations of the components in the third solution were as shown in Table 1.

[0056] (3) Concentration and drying

[0057] The third solution is transferred to a rotary evaporator and evaporation is initiated at a preset temperature and pressure until the solvent is completely removed, yielding a concentrated mixture. The concentrated mixture is then transferred to a drying oven and dried within a specified temperature range until a dry mixed powder is obtained. It is important to maintain a drying temperature between 40°C and 60°C to prevent excessive temperatures from causing decomposition or deterioration of the compound.

[0058] (4) Preparation molding

[0059] For tablets, starch can be added as a filler at 20% of the total tablet weight, sodium carboxymethyl starch can be added as a disintegrant at 5% of the total tablet weight, and magnesium stearate can be added as a lubricant at 1% of the total tablet weight. For capsules, lactose can be added as a filler at 45% of the total capsule weight, and magnesium stearate can be added as a lubricant at 0.75% of the total capsule weight. For tablets, the mixed powder is placed in a tablet press and compressed to produce tablets. For capsules, the mixed powder is filled into capsule shells to produce capsules.

[0060] 2. Animal Experiments

[0061] 1. Experimental Animals

[0062] Male Sprague-Dawley rats (160-200 g, VSR10001, Beijing Weishanglide Biotechnology Co., Ltd.) were housed in an environment with a temperature of (24 ± 1)°C and a humidity of (50 ± 10%), with a 12-hour circadian rhythm and free access to food and water. According to the standard operating procedures for Sprague-Dawley rat husbandry, the housing environment was clean, dust-free, and provided with fresh air to prevent respiratory illnesses caused by sensitivity to airborne dust, ammonia, and hydrogen sulfide. Before initiating the CUMS experiment, the rats underwent a one-week acclimatization period.

[0063] 2. Model preparation and processing

[0064] Chronic unpredictable mild stress (CUMS) rats were constructed using the method of “Li, H., Xiang, Y., Zhu, Z., Wang, W., Jiang, Z., Zhao, M., et al. (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.”

[0065] In a pilot study, rats were randomly exposed to one of seven mild stimuli daily for 28 days. These stimuli were: ① 5-minute swimming in 0°C ice water; ② 24-hour day / night reversal; ③ 24-hour food and water deprivation; ④ 1-minute tail clamp; ⑤ 5-minute swimming in 45°C hot water; ⑥ 24-hour cage tilt at 45°C in a humid environment; and 7-hour 24-hour humidity. Following model establishment, rats were subjected to a sucrose preference test (SPT) and a forced swimming test (FST) to confirm successful modeling after 28 days.

[0066] 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 stimulated according to the above-described 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 each gavaged with a powder solution prepared by grinding the tablets of Examples 1 to 3 and Comparative Examples 1 to 2 using physiological saline at a dose of 30 mg / kg body weight for 5 consecutive weeks.

[0067] 3. Sucrose preference consumption test

[0068] After five weeks of treatment, rats were acclimated to a 1% sucrose solution. Each rat was housed individually and provided with two bottles of 1% sucrose solution. After 24 hours, one of the bottles was replaced with pure water for an additional 24 hours. Rats were then deprived of food and water for 12 hours, and allowed to freely choose between two bottles (one 1% sucrose solution, one pure water). After one hour, the bottles were swapped. After two hours, the weight of each bottle was measured, and the sucrose preference ratio was calculated: sucrose preference ratio = sucrose water consumption / (sucrose water consumption + water consumption) × 100%.

[0069] like Figure 1 As shown, there were significant differences in sucrose preference rates among the groups. The sucrose preference rate of the model group rats 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 were significantly higher than those in 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 were significantly higher than those in 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 Example 1 has the best effect.

[0070] 4. Forced swim test

[0071] The experiment was conducted using the FST-100 system, using a 21-cm-diameter transparent cylinder filled with 35-cm-deep water at a temperature of (25 ± 1)°C. Each rat was allowed to swim for 15 minutes before being returned to its home cage. After 24 hours, the rat was individually placed in the cylinder and allowed to swim again. The system was used to record the immobility time for each rat over a 5-minute period.

[0072] like Figure 2 As shown, the immobility time of the rats in the model group was significantly longer 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 respectively reduced the immobility time of the 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 the Example 1 to 3 groups relative to the normal group, while the immobility time of the rats in the Comparative Example 1 and Comparative Example 2 groups were significantly longer than that in the normal group (P<0.05, P<0.001). This shows that the pharmaceutical composition preparations provided in Examples 1 to 3 respectively can significantly reduce the immobility time of the forced swimming test in CUMS-treated rats.

[0073] 5. RT-PCR detection of ERK1, ERK2 and CREB mRNA expression levels

[0074] SYBR® RT-PCR was used to measure mRNA levels. Hippocampal samples stored in liquid nitrogen were weighed, and total RNA was extracted using the TaKaRa MiniBEST Universal RNA Extraction Kit. Total RNA was reverse transcribed into complementary DNA (cDNA) using PrimeScript™ RT Master Mix and then amplified using a PCR instrument. The primer sequences for the target genes ERK1 (Extracellular Signal-Regulated Kinase 1), ERK2 (Extracellular Signal-Regulated Kinase 2), and CREB (cAMP Response Element-Binding Protein) and the reference gene GAPDH (Glyceraldehyde 3-Phosphate Dehydrogenase) were 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. The 7500 real-time fluorescence quantitative PCR system was used, and SYBR® Premix Ex Taq was selected as the reaction reagent. The following real-time quantitative PCR analysis program was performed: first, incubation at 50°C for 2 minutes, followed by incubation at 95°C for 10 minutes; then 40 cycles were performed, each cycle consisting of denaturation at 95°C for 15 seconds and annealing and extension at 60°C for 1 minute. Relative quantification method (2 -ΔΔCT ), GAPDH was used as the internal reference to calculate the relative expression level of the target gene.

[0075] like 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 reduced (P<0.01, P<0.01, P<0.01). Sleep deprivation is believed to have an antidepressant effect, which may be exerted by affecting the content and activity of CREB in the hippocampus. The pharmaceutical composition provided in Example 1 significantly increased the mRNA levels of ERK1, ERK2, and CREB in CUMS rats (compared with the model group, P<0.01, P<0.01, P<0.01), which is consistent with the effects of midazolam and propofol on the phosphorylation levels of ERK1, ERK2, and CREB in the rat hippocampus. The pharmaceutical composition provided in Example 2 significantly increased the mRNA levels of ERK1, ERK2, and CREB in CUMS rats (compared with the model group, P<0.01, P<0.01, P<0.01). The pharmaceutical composition preparation provided in Example 3 significantly increased the mRNA levels of ERK1, ERK2, and CREB in CUMS rats (compared with the model group, P<0.01, P<0.01, P<0.05). This result is consistent with the effect of propofol on the phosphorylation levels of ERK1 / ERK2 and CREB in the rat hippocampus. However, the mRNA levels of ERK1, ERK2, and CREB in rats administered with the pharmaceutical composition preparations provided in Comparative Examples 1 and 2 were not significantly different from those in the model group. In addition, the mRNA levels of ERK1, ERK2, and CREB in rats administered with the pharmaceutical composition preparations provided in Examples 1 to 3 were significantly higher than those in the normal group. This indicates 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.

[0076] 6. Collection and analysis of EEG data

[0077] Rats were anesthetized with 1.5% isoflurane and maintained at 1%. The rats' heads were fixed in a stereotaxic apparatus, and the hair was shaved. The meninges were then stripped to expose the skull. A high-speed cranial drill was used to drill a hole and implant an EEG electrode into the CA3 region of the right hippocampus (AP: −3.6 mm, ML: +4.2 mm, DV: −3.8 mm). The electrode was then securely fixed with glass ionomer cement to ensure a secure fit. After the rats emerged from anesthesia naturally, they were individually housed and allowed adequate recovery for 7 days to facilitate subsequent EEG data acquisition. 24-hour EEG data were collected from each group using the Ponemah acquisition system and analyzed using NeuroScore software. After completion of signal acquisition, the rats were sacrificed, and intact brain tissue was removed to verify the accuracy of the electrode implantation.

[0078] like Figure 6As shown, the Beta / Total frequency ratio of the Beta band in the EEG signals of the model group rats was significantly higher than that of the normal group. The pharmaceutical composition preparations provided in Examples 1 to 3 respectively reduced the Beta / Total frequency ratio of the CUMS rats relative to the model group. Although the pharmaceutical composition preparations provided in Comparative Examples 1 to 2 reduced the Beta / Total frequency ratio of the CUMS rats relative to the model group to a certain extent, this reduction effect was obviously not as good as that of the Example 1 to 3 groups. This shows that the pharmaceutical composition preparations provided in Examples 1 to 3 respectively can significantly reduce the Beta / Total frequency ratio of CUMS-treated rats.

[0079] like Figure 7 As shown, the Theta / Total frequency ratio of the model group rats was significantly higher than that of the normal group. The pharmaceutical composition preparations provided in Examples 1 to 3 respectively reduced the Theta / Total frequency ratio of the CUMS rats relative to the model group. Although the pharmaceutical composition preparations provided in Comparative Examples 1 to 2 can partially reduce the Theta / Total frequency ratio of the CUMS rats relative to the model group, this reduction effect is obviously not as good as that of the Example 1 to 3 groups. This shows that the pharmaceutical composition preparations provided in Examples 1 to 3 respectively can significantly reduce the Theta / Total frequency ratio of CUMS-treated rats.

[0080] The CUMS model induces anhedonia in rodents by subjecting them to a mild, unpredictable stress environment for a long period of time. The success of the model is often assessed using tests such as the FST, OFT, and SPT. This study demonstrated that the pharmaceutical compositions provided in Examples 1-3 significantly increased sucrose preference in CUMS rats and shortened the immobility period after forced swimming, demonstrating a significant effect in alleviating and treating CUMS rats.

[0081] In addition, the ERK-CREB signaling pathway plays a key role in the mechanism of action of the antidepressant drug (R)-ketamine, participating in multiple physiological effects such as intercellular signal transmission and recognition, cell growth and development, and has a potential molecular mechanism in regulating depressive behavior. Phosphorylation of ERK can activate the expression of its downstream signaling molecule CREB, and CREB is believed to be partially involved in the expression of most genes regulated by cyclic adenosine monophosphate (cAMP), including BDNF (brain-derived neurotrophic factor). The target protein subsequently released can promote neuronal survival, thereby exerting an antidepressant effect. The pharmaceutical composition 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 neuroprotective effects, and exhibiting antidepressant efficacy.

[0082] An electroencephalogram (EEG) uses electrodes to record the spontaneous, rhythmic electrical activity of groups of brain cells, thereby reflecting changes in brain function. It is widely used in the study of neurasthenia, depression, and sleep disorders. Beta waves typically occur when individuals are stressed, anxious, or emotionally agitated, and are closely associated with thinking, physical movement, and emotional fluctuations such as depression. Theta waves are key to information processing within the hippocampus and other limbic structures and are even considered a possible biomarker of anxiety. Theta waves can change through changes in cognitive-emotional processing, first affecting the hippocampus and then other structures directly involved in anxiety and memory. Studies have reported that the frequency and proportion of beta and theta activity in patients with depression are significantly increased. Characteristic changes in sleep disorders in patients with depression include disrupted sleep continuity and decreased REM and NREM sleep time. This experiment found that compared with the normal control group, the beta and theta ratios in the model rats were significantly increased, indicating that the CUMS depression model rats exhibited mood regulation disorders. The pharmaceutical composition preparations provided in Examples 1 to 3 can regulate the structure and composition of the intestinal flora and reduce the depressive behavior of CUMS rats, showing a significant improvement.

[0083] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed by the present invention should be covered by the scope of protection of the present invention.

Claims

1. A pharmaceutical composition, characterized in that The components are as follows: 80-120 parts of (8E)-ligustrin, 60-80 parts of fraxinoside, 60-80 parts of oleuropein, 60-80 parts of syringoside, 20-60 parts of (+)-pinoresinol-4-O-β-D-pyranoglucoside, 20-50 parts of oleuropein-4'-O-glucoside, 10-50 parts of coniferin, 10-40 parts of benzyl alcohol-β-vicin, 10-40 parts of hollyoside, 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, characterized in that The invention comprises, by weight, 100 parts of (8E)-ligustrin, 70 parts of fraxinoside, 65 parts of oleuropein, 65 parts of syringoside, 35 parts of (+)-pinoresinol-4-O-β-D-glucopyranoside, 40 parts of oleoresinol-4'-O-glucoside, 20 parts of coniferin, 30 parts of benzyl alcohol-β-vicinoside, 30 parts of hollyoside, 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, characterized in that The invention comprises, by weight, 80 parts of (8E)-ligustrin, 60 parts of fraxinoside, 60 parts of oleuropein, 60 parts of syringoside, 20 parts of (+)-pinoresinol-4-O-β-D-glucopyranoside, 20 parts of oleoresinol-4'-O-glucoside, 10 parts of coniferin, 10 parts of benzyl alcohol-β-vicinoside, 10 parts of hollyoside, 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, characterized in that The invention comprises, by weight, 120 parts of (8E)-ligustrin, 80 parts of fraxinoside, 80 parts of oleuropein, 80 parts of syringoside, 60 parts of (+)-pinoresinol-4-O-β-D-glucopyranoside, 50 parts of oleuropein-4'-O-glucoside, 50 parts of coniferin, 40 parts of benzyl alcohol-β-vicinoside, 40 parts of hollyoside, 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 method for preparing the preparation according to claim 5, characterized in that: The preparation method comprises: preparing (8E)-ligustrin solution with 15% ethanol aqueous solution, preparing fraxinoside solution with ethanol, preparing oleuropein solution with methanol, preparing syringoside solution with ethanol, preparing (+)-pinoresinol-4-O-β-D-pyranoglucoside solution with ethanol, preparing oleoresinol-4'-O-glucoside solution with methanol, preparing coniferin solution with ethanol, preparing benzyl alcohol-β-vicinoside solution with ethanol, preparing holmoside solution with ethanol, preparing (4S)-α-terpineol 8-O- -β-D-(3'-O,4'-O,6'-O-triacetyl)pyranoside solution, (Z)-3-hexenyl-β-D-pyranoside solution prepared with ethanol, 2-hydroxy-1-methoxyanthraquinone solution prepared with ethanol, (-)-episyringaresinol solution prepared with ethanol, dibutyl terephthalate solution prepared with ethyl acetate, (-)-3,4-divanillyltetrahydrofuran solution prepared with ethanol; equal volumes of the component solutions using ethanol or methanol as solvent were mixed, stirring while adding to ensure uniform mixing to obtain a first solution; The solutions of each component with ethyl acetate as the solvent are mixed in equal volumes, stirred while adding to ensure uniform mixing to obtain a second solution; the second solution is then slowly poured into the first solution, and then ethanol equivalent to 0.25 times the volume of the mixed solution of the first and second solutions is added, and stirred thoroughly until uniform; ultrasonic treatment is performed to obtain a third solution containing the formulated amount of each component; the third solution is transferred to a rotary evaporator, and rotary evaporation is performed at an appropriate temperature and pressure to remove the solvent to obtain a concentrated mixture; the concentrated mixture is transferred to a drying oven for drying to obtain a mixed powder; for tablets, starch accounting for 20% of the total tablet weight is added as a filler, sodium carboxymethyl starch accounting for 5% of the total weight is added as a disintegrant, and magnesium stearate accounting for 1% of the total tablet weight is added as a lubricant; for tablets, the mixed powder is placed in a tablet press for tableting to obtain tablets; for capsules, lactose accounting for 45% of the total capsule weight is added as a filler, and magnesium stearate accounting for 0.75% of the total capsule weight is added as a lubricant; for capsules, the mixed powder is filled into capsule shells to obtain capsules.

7. The method according to claim 6, characterized in that In the third solution, the concentration of (8E)-ligustrin is 100.00 mg / L, the concentration of fraxinus glycoside is 70.00 mg / L, the concentration of oleuropein is 65.00 mg / L, the concentration of syringoside is 65.00 mg / L, the concentration of (+)-pinoresinol-4-O-β-D-pyranoglucoside is 35.00 mg / L, the concentration of oleoresin-4'-O-glucoside is 40.00 mg / L, the concentration of coniferin is 20.00 mg / L, the concentration of benzyl alcohol-β-vicinyl glycoside is 30.00 mg / L, and the concentration of holly glycoside is 30. .00 mg / L, the concentration of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl)pyranoside was 20.00 mg / L, the concentration of (Z)-3-hexenyl-β-D-pyranoside was 8.00 mg / L, the concentration of 2-hydroxy-1-methoxyanthraquinone was 15.00 mg / L, the concentration of (-)-episyringaresinol was 10.00 mg / L, the concentration of dibutyl terephthalate was 10.00 mg / L, and the concentration of (-)-3,4-divanillyltetrahydrofuran was 6.00 mg / L.

8. The method according to claim 6, characterized in that In the third solution, the concentration of (8E)-ligustrin was 116.12 mg / L, the concentration of fraxinoside was 87.09 mg / L, the concentration of oleuropein was 87.09 mg / L, the concentration of syringoside was 87.09 mg / L, the concentration of (+)-pinoresinol-4-O-β-D-pyranoglucopyranoside was 29.03 mg / L, the concentration of oleoresinol-4'-O-glucoside was 29.03 mg / L, the concentration of coniferin was 14.52 mg / L, the concentration of benzyl alcohol-β-vicinyl glycoside was 14.52 mg / L, and the concentration of holly glycoside was 14.09 mg / L. The concentration of the raw materials was 14.52 mg / L, the concentration of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl)pyranoside was 14.52 mg / L, the concentration of (Z)-3-hexenyl-β-D-pyranoside was 7.26 mg / L, the concentration of 2-hydroxy-1-methoxyanthraquinone was 7.26 mg / L, the concentration of (-)-episyringaresinol was 7.26 mg / L, the concentration of dibutyl terephthalate was 7.26 mg / L, and the concentration of (-)-3,4-divanillyltetrahydrofuran was 1.45 mg / L.

9. The method according to claim 6, characterized in that In the third solution, the concentration of (8E)-ligustrin is 87.33 mg / L, the concentration of fraxinus glycoside is 58.22 mg / L, the concentration of oleuropein is 58.22 mg / L, the concentration of syringoside is 58.22 mg / L, the concentration of (+)-pinoresinol-4-O-β-D-pyranoglucopyranoside is 43.67 mg / L, the concentration of oleoresin-4'-O-glucoside is 36.39 mg / L, the concentration of coniferin is 36.39 mg / L, the concentration of benzyl alcohol-β-vicinyl glycoside is 29.11 mg / L, and the concentration of holly glycoside is 29. .11 mg / L, the concentration of (4S)-α-terpineol 8-O-β-D-(3'-O,4'-O,6'-O-triacetyl)pyranoside was 21.83 mg / L, the concentration of (Z)-3-hexenyl-β-D-pyranoside was 7.28 mg / L, the concentration of 2-hydroxy-1-methoxyanthraquinone was 21.83 mg / L, the concentration of (-)-episyringaresinol was 14.56 mg / L, the concentration of dibutyl terephthalate was 14.56 mg / L, and the concentration of (-)-3,4-divanillyltetrahydrofuran was 7.28 mg / L.

10. Use of the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a preparation for inhibiting neurasthenia.

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