A composition of isoliquiritigenin for improving depression and a preparation method thereof

Abstract

This invention belongs to the field of biomedical technology, specifically relating to an isoliquiritigenin composition for improving depression and its preparation method. The isoliquiritigenin composition for improving depression comprises the following raw materials in parts by weight: 22-40 parts isoliquiritigenin, 10-17 parts N-acetyl-D-glucosamine, and 15-20 parts β-lapaquinone. Isoliquiritigenin, N-acetyl-D-glucosamine, and β-lapaquinone constitute a multi-target synergistic isoliquiritigenin composition for improving depression. Experimental results show that by taking the isoliquiritigenin composition prepared according to this invention, it can systematically exert its effects and comprehensively improve the pathological state of depression, showing broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an isoliquiritigenin composition for improving depression and a preparation method thereof. Background Art

[0002] Depression is a major mental disorder characterized by persistent low mood, accompanied by cognitive impairment, decreased volitional activity, and somatic symptoms. Some severe patients may present psychotic symptoms such as hallucinations and delusions. This disease is characterized by a high prevalence rate and a low clinical cure rate, posing a severe challenge to global public health. According to the prediction of the World Health Organization, depression may become the disease with the first highest burden in the world in 2030, causing great impact on people's physical and mental health.

[0003] Currently, the clinical treatment of depression in Western medicine mainly relies on chemically synthesized drugs, including monoamine oxidase inhibitors, tricyclic antidepressants, tetracyclic antidepressants, 5-HT reuptake inhibitors, 5-HT-NE reuptake inhibitors, etc. Although these drugs are the current mainstream therapies, they have obvious limitations: relatively single action mechanisms, common toxic and side effects, easy to produce tolerance, high recurrence rate after drug withdrawal, and some drugs are expensive, resulting in poor patient compliance and the overall curative effect needs to be improved.

[0004] In traditional Chinese medicine theory, although there is no disease name of "depression", according to its clinical manifestations, it can be classified into the category of "depressive syndrome". Traditional Chinese medicine believes that depressive syndrome is mostly caused by emotional internal injuries, and the key pathogenesis in the early stage is mainly liver qi stagnation and qi mechanism disorder. As the disease course prolongs, qi stagnation can affect blood circulation, leading to blood stasis; it can also invade the spleen horizontally, causing the spleen to lose its transportation and transformation function, and phlegm dampness is generated internally, forming phlegm qi stagnation; prolonged illness consumes the qi of the heart and spleen, leading to deficiency of both the heart and spleen or deficiency of the liver and kidney yin, and finally presenting a complex pathological state of deficiency and excess, with deficiency in origin and excess in superficiality, which is closely related to the dysfunction of multiple zang-fu organs such as the heart, liver, spleen, and kidney. The treatment of depressive syndrome with traditional Chinese medicine compound emphasizes syndrome differentiation and treatment, and overall regulation, with the potential of multi-target and multi-pathway effects, and relatively few side effects. However, the composition of traditional Chinese medicine prescriptions is complex, with a large number of ineffective or low-harm components. Therefore, it is particularly important to screen out prescriptions with clear active ingredients.

[0005] Licorice is the dried root and rhizome of the leguminous plant Glycyrrhiza uralensis Fisch., and its active ingredients include more than 300 flavonoids, more than 20 triterpenoids, as well as polysaccharides, phenols, alkaloids, amino acids, volatile oils, and minerals. Among them, the flavonoid components show excellent performance in antidepressant effects. Isoliquiritigenin is an important monomer component in licorice flavonoid compounds. Modern pharmacological research reveals that its antidepressant mechanism may include: competitively inhibiting the activity of monoamine oxidase, thereby reducing the degradation of monoamine neurotransmitters; protecting dopaminergic neurons from degeneration and excitotoxicity, and having great potential in the treatment of depression.

[0006] To achieve the above objectives, the present invention provides an isoliquiritigenin composition for improving depression and a method for preparing the same. Summary of the Invention

[0007] The first objective of this invention is to provide an isoliquiritigenin composition for improving depression.

[0008] A second objective of this invention is to provide a method for preparing an isoliquiritigenin composition for improving depression.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: An isoliquiritigenin composition for improving depression, said isoliquiritigenin composition comprising the following raw materials in parts by weight: 22-40 parts isoliquiritigenin, 10-17 parts N-acetyl-D-glucosamine, and 15-20 parts β-lapaquinone.

[0010] Furthermore, the isoliquiritigenin composition comprises the following raw materials in parts by weight: 31 parts isoliquiritigenin, 13 parts N-acetyl-D-glucosamine, and 17 parts β-lapaquinone.

[0011] The preparation method of the isoliquiritigenin composition for improving depression described above specifically includes the following steps: (1) Weigh out isoliquiritin and β-lapaquinone by weight, dissolve them in anhydrous ethanol, and sonicate them to obtain isoliquiritin solution and β-lapaquinone solution; dissolve N-acetyl-D-glucosamine in deionized water by weight to obtain N-acetyl-D-glucosamine solution. (2) The isoliquiritin solution, β-lapaquinone solution and N-acetyl-D-glucosamine solution obtained in step (1) are mixed and stirred, ultrasonically treated, rotary evaporated and dried to obtain a mixed powder, and excipients are added to prepare an oral dosage form.

[0012] Further, in step (1), the concentration of the isoliquiritigenin solution is 100-150 μg / mL; the concentration of the β-lapaquinone solution is 20-30 μg / mL; and the concentration of the N-acetyl-D-glucosamine solution is 75-95 μg / mL.

[0013] Furthermore, the mixing time in step (2) is 20-30 min; the ultrasonic treatment time is 10-20 min.

[0014] Furthermore, the oral dosage form is a tablet.

[0015] Compared with the prior art, the main advantages of the present invention are as follows: (1) This invention provides an isoliquiritigenin composition for improving depression, wherein the active ingredients include isoliquiritigenin, N-acetyl-D-glucosamine, and β-lapaquinone, which together constitute a multi-target synergistic isoliquiritigenin composition for improving depression. Experimental results show that by taking the isoliquiritigenin composition prepared by this invention, it can exert a systematic effect and comprehensively improve the pathological state of depression.

[0016] (2) Isoliquiritin belongs to the flavonoid class of compounds and can directly regulate the monoamine neurotransmitter system. It reduces the degradation of 5-hydroxytryptamine and norepinephrine by inhibiting monoamine oxidase activity and selectively activating 5-HT. 1a Receptors, antagonists of 5-HT 2a Receptors increase the concentration of monoamine neurotransmitters in the synaptic space, rapidly relieving depressive mood.

[0017] (3) N-acetyl-D-glucosamine plays a key role in improving depression. It may effectively activate the autophagy process by effectively inhibiting the overactivated mTOR signaling pathway in depression, thereby enabling the specific removal of accumulated abnormal protein aggregates and repairing damaged synaptic plasticity, which helps to restore the function of dopamine neurons in the substantia nigra of the midbrain.

[0018] (4) β-Lapaquinone plays a key role in improving depression. It may significantly upregulate the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus and prefrontal cortex by targeting the BDNF-TrkB pathway, activate the downstream ERK1 / 2 signaling pathway, promote neuronal synaptic remodeling and neurogenesis, and repair neuroplasticity damage. Attached Figure Description

[0019] Figure 1 The graph shows the effect of isoliquiritigenin combination on sucrose preference behavior in mice. Figure 2 The graph shows the effect of the isoliquiritigenin combination on tail suspension behavior in mice. Figure 3 The graph shows the effect of the isoliquiritigenin combination on the expression of mTOR and p-mTOR proteins. Figure 4 The effect of isoliquiritigenin combination on the inflammatory factor TNF-α in the rat hippocampus; Figure 5 The effect of isoliquiritigenin combination on the anti-inflammatory factor IL-10 in the rat hippocampus. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0021] Example 1 An isoliquiritigenin composition for improving depression, the isoliquiritigenin composition comprising the following raw materials in parts by weight: 31 parts isoliquiritigenin, 13 parts N-acetyl-D-glucosamine, and 17 parts β-lapaquinone.

[0022] A method for preparing an isoliquiritigenin composition for improving depression, specifically comprising the following steps: (1) Weigh 31 parts by weight of isoliquiritigenin and 17 parts by weight of β-lapaquinone, dissolve them separately in anhydrous ethanol, and sonicate to obtain isoliquiritigenin solution and β-lapaquinone solution; the concentration of the isoliquiritigenin solution is 120 μg / mL; the concentration of the β-lapaquinone solution is 25 μg / mL. Weigh 13 parts by weight of N-acetyl-D-glucosamine and dissolve them in deionized water to obtain N-acetyl-D-glucosamine solution; the concentration of the N-acetyl-D-glucosamine solution is 85 μg / mL.

[0023] (2) The isoliquiritigenin solution, β-lapaquinone solution and N-acetyl-D-glucosamine solution obtained in step (1) are mixed and stirred for 25 min, ultrasonically treated for 15 min, and then transferred to a rotary evaporator for rotary evaporation and dried to obtain a mixed powder. Based on the total weight of the mixed powder, 25 wt% lactose, 15 wt% microcrystalline cellulose, 3 wt% sodium carboxymethyl starch and 0.5 wt% magnesium stearate are added to prepare tablets.

[0024] Example 2 An isoliquiritigenin composition for improving depression, the isoliquiritigenin composition comprising the following raw materials in parts by weight: 22 parts isoliquiritigenin, 10 parts N-acetyl-D-glucosamine, and 15 parts β-lapaquinone.

[0025] A method for preparing an isoliquiritigenin composition for improving depression, specifically comprising the following steps: (1) Weigh 22 parts by weight of isoliquiritin and 15 parts by weight of β-lapaquinone, dissolve them separately in anhydrous ethanol, and sonicate to obtain isoliquiritin solution and β-lapaquinone solution; the concentration of the isoliquiritin solution is 100 μg / mL; the concentration of the β-lapaquinone solution is 20 μg / mL. Weigh 10 parts by weight of N-acetyl-D-glucosamine and dissolve them in deionized water to obtain N-acetyl-D-glucosamine solution; the concentration of the N-acetyl-D-glucosamine solution is 75 μg / mL.

[0026] (2) The isoliquiritigenin solution, β-lapaquinone solution and N-acetyl-D-glucosamine solution obtained in step (1) are mixed and stirred for 20 min, ultrasonically treated for 10 min, and then transferred to a rotary evaporator for rotary evaporation and dried to obtain a mixed powder. Based on the total weight of the mixed powder, 25 wt% lactose, 15 wt% microcrystalline cellulose, 3 wt% sodium carboxymethyl starch and 0.5 wt% magnesium stearate are added to prepare tablets.

[0027] Example 3 An isoliquiritigenin composition for improving depression, the isoliquiritigenin composition comprising the following raw materials in parts by weight: 40 parts isoliquiritigenin, 17 parts N-acetyl-D-glucosamine, and 20 parts β-lapaquinone.

[0028] A method for preparing an isoliquiritigenin composition for improving depression, specifically comprising the following steps: (1) Weigh 40 parts by weight of isoliquiritigenin and 20 parts by weight of β-lapaquinone, dissolve them separately in anhydrous ethanol, and sonicate to obtain isoliquiritigenin solution and β-lapaquinone solution; the concentration of the isoliquiritigenin solution is 150 μg / mL; the concentration of the β-lapaquinone solution is 30 μg / mL. Weigh 17 parts by weight of N-acetyl-D-glucosamine and dissolve them in deionized water to obtain N-acetyl-D-glucosamine solution; the concentration of the N-acetyl-D-glucosamine solution is 95 μg / mL.

[0029] (2) The isoliquiritigenin solution, β-lapaquinone solution and N-acetyl-D-glucosamine solution obtained in step (1) are mixed and stirred for 30 min, ultrasonically treated for 20 min, and then transferred to a rotary evaporator for rotary evaporation and dried to obtain a mixed powder. Based on the total weight of the mixed powder, 25 wt% lactose, 15 wt% microcrystalline cellulose, 3 wt% sodium carboxymethyl starch and 0.5 wt% magnesium stearate are added to prepare tablets.

[0030] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that N-acetyl-D-glucosamine was omitted from the isoliquiritigenin composition for improving depression, while all other aspects were the same as in Example 1.

[0031] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that β-lapaquinone was omitted from the isoliquiritigenin composition for improving depression, while all other aspects were the same as in Example 1.

[0032] Experimental Example 1 The following are efficacy tests of the isoliquiritigenin compositions prepared in Examples 1-3 and Comparative Examples 1-2 for improving depression: (1) Ninety male mice weighing 18-20g were selected for the experiment. All mice were kept at a constant temperature of 22±2℃ and a relative humidity of 50-60%, with free access to food and water, and were acclimatized for one week. They were randomly divided into four groups: Example 1 high-dose group, Example 1 medium-dose group, Example 1 low-dose group, Example 2 low-dose group, Example 3 low-dose group, Comparative Example 1 low-dose group, Comparative Example 2 low-dose group, model group, and control group, with 10 mice in each group.

[0033] The control group received no external stimulation and had free access to food and water. The other groups of mice were isolated and subjected to different stimuli daily, with the stimuli being as contrasting as possible. The stressors included 10 minutes of horizontal shaking, 24 hours of fasting and water restriction, 5 minutes of tail clamping, 5 minutes of swimming in 4°C cold water, 5 minutes of drying in a 45°C oven, 12 hours of day-night reversal, and 24 hours of moist bedding (7 types in total). Each group of mice received one randomly assigned stimulus daily; no group could tolerate the same stimulus, and the same stimulus was used a maximum of three times. Modeling and drug administration were performed simultaneously.

[0034] (2) Drug intervention is performed at 10:00 AM every day: Example 1 High-dose group: 4 g / kg of the isoliquiritigenin composition of Example 1 was administered by gavage once a day for three consecutive weeks.

[0035] In Example 1, the dosage group was administered 2 g / kg of the isoliquiritigenin composition of Example 1 by gavage once a day for three consecutive weeks.

[0036] Example 1 Low-dose group: 1 g / kg of the isoliquiritigenin composition of Example 1 was administered by gavage once a day for three consecutive weeks.

[0037] Example 2 Low-dose group: 1 g / kg of the isoliquiritigenin composition of Example 2 was administered by gavage once a day for three consecutive weeks.

[0038] Example 3 Low-dose group: 1 g / kg of the isoliquiritigenin composition of Example 3 was administered by gavage once a day for three consecutive weeks.

[0039] Comparative Example 1 Low-dose group: 1 g / kg of the isoliquiritigenin composition of Comparative Example 1 was administered by gavage once a day for three consecutive weeks.

[0040] Comparative Example 2 Low-dose group: 1 g / kg of the isoliquiritigenin composition of Comparative Example 2 was administered by gavage once a day for three consecutive weeks.

[0041] Model group: The same volume of physiological saline was administered by gavage once a day for three consecutive weeks.

[0042] Control group: Administered an equal volume of normal saline by gavage once daily for three consecutive weeks.

[0043] (3) Sugar Water Preference Test (SPT): Before the test, mice in each group were acclimatized to drinking 1% sucrose water for 48 hours to minimize their fear of new things and to avoid preference for a particular drinking location. The location of the drinking bottles was changed once a day. During the test, the mice were deprived of water for 24 hours and then given two bottles of water of equal volume, A and B (bottle A contained tap water, and bottle B contained 1% sucrose water). The sugar water preference was calculated based on the amount of sugar water and tap water consumed by the mice during the 5-hour period. The sugar water preference was calculated using the following formula: Sugar Water Preference (SPT)% = [B / (A+B)] × 100%.

[0044] The results are as follows Figure 1 The figure shown is a graph illustrating the effect of the isoliquiritigenin combination on the sucrose preference behavior of mice. Figure 1 It can be seen that, compared with the control group, the sucrose intake of mice in the model group was significantly reduced, indicating that the chronic depression model was successfully established. Compared with the model group, comparative example 1, and comparative example 2, the isoliquiritigenin compositions prepared in Examples 1-3 of this invention for improving depression can all improve the reduction in sucrose intake in mice and improve the depressive behavior of mice.

[0045] (4) Tail Suspension Test (TST): The tail of a mouse is fixed with tape at 1 / 3 of its tail and suspended 50 cm above the ground. The total time the mouse remains still in this environment is measured within 5 minutes.

[0046] The results are as follows Figure 2 The figure shown is a graph illustrating the effect of the isoliquiritigenin combination on tail suspension behavior in mice. Figure 2 It can be seen that, compared with the control group, the immobility time of mice in the tail suspension experiment in the model group was significantly increased, indicating that the chronic depression model was successfully established. Compared with the model group, the low-dose group of Comparative Example 1, and the low-dose group of Comparative Example 2, the isoliquiritigenin compositions prepared in Examples 1-3 of this invention for improving depression can all reduce the immobility time of mice and improve the depressive behavior of mice.

[0047] (5) After euthanizing the mice, paraffin sections of melanin, 6 μm thick, were prepared and incubated sequentially with rabbit anti-mTOR (1:600) primary antibody, HRP-streptavidin, and goat anti-rabbit biotinylated secondary antibody (1:1000). DAB staining and hematoxylin counterstaining were then performed. Three fields of view were captured under high magnification (×200), and the positive area density was measured using Image J to quantify the expression levels of mTOR and p-mTOR. Electrophoresis images of mTOR and p-mTOR protein expression using the isoliquiritigenin combination are shown below. Figure 3 As shown.

[0048] The results are as follows Figure 3 The figure shown is a graph illustrating the effect of the isoliquiritigenin combination on the expression of mTOR and p-mTOR proteins. Figure 3 It was found that, compared with the control group, the expression of mTOR and p-mTOR proteins was upregulated in the model group. Compared with the low-dose group of Comparative Example 1, the isoliquiritigenin compositions prepared in Examples 1-3 of this invention for improving depression significantly reduced the expression of mTOR and p-mTOR proteins, indicating that the N-acetyl-D-glucosamine component plays a key role. It may effectively inhibit the overactivated mTOR signaling pathway in depression, thereby effectively activating the autophagy process, enabling the specific clearance of accumulated abnormal protein aggregates and repairing damaged synaptic plasticity, thus helping to restore the function of dopamine neurons in the substantia nigra of the midbrain.

[0049] (6) The brain tissue of the mouse hippocampus was isolated. 30 μg of BCA protein was quantified and transferred to a membrane by SDS-PAGE. The membrane was blocked with 5% skim milk and incubated with BDNF, TrkB primary antibody (1:1000) and HRP secondary antibody (1:10000) in sequence. ECL was used for imaging. GAPDH was used as an internal control. The results of the expression of BDNF and TrkB related proteins in mouse hippocampus tissue are shown in Table 1.

[0050] Table 1. Results of expression determination of mouse hippocampal related proteins BDNF and TrkB The results are shown in Table 1, which presents the results of the measurement of the expression of BDNF and TrkB related proteins in the mouse hippocampus. As shown in Table 1, compared with the control group, the expression levels of BDNF and TrkB related proteins in the model group of mice hippocampus were significantly reduced. Compared with the low-dose group of Comparative Example 2, the isoliquiritigenin compositions prepared in Examples 1-3 of this invention for improving depression significantly increased the expression of BDNF and TrkB proteins in mouse hippocampus, indicating that β-lapaquinone plays a key role. It may significantly upregulate the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus and prefrontal cortex by targeting the BDNF-TrkB pathway, activating the downstream ERK1 / 2 signaling pathway, promoting neuronal synaptic remodeling and neurogenesis, and repairing neuroplasticity damage.

[0051] (7) Detection of inflammatory factors: After the mice were sacrificed, their hippocampal tissue was homogenized with phosphate buffer (10% w / v), the supernatant was collected by centrifugation, and the contents of TNF-α and IL-10 in the hippocampal specimen were detected by ELISA kit. The specific operation steps were strictly followed according to the instructions of the kit.

[0052] The results are as follows Figure 4 The image shows the effect of an isoliquiritigenin combination on the inflammatory cytokine TNF-α in the rat hippocampus. Figure 4 It can be seen that, compared with the control group, the inflammatory factor TNF-α in the model group was significantly increased, indicating that the chronic depression model was successfully established. Compared with the model group, the low-dose group of Comparative Example 1, and the low-dose group of Comparative Example 2, the isoliquiritigenin compositions prepared in Examples 1-3 of this invention for improving depression can significantly reduce the content of the inflammatory factor TNF-α in mice and improve the depressive behavior of mice.

[0053] The results are as follows Figure 5 The image shows the effect of the isoliquiritigenin combination on the anti-inflammatory factor IL-10 in the rat hippocampus. Figure 5 It was found that the anti-inflammatory factor IL-10 was significantly reduced in the model group compared with the control group. Compared with the model group, the low-dose group of Comparative Example 1, and the low-dose group of Comparative Example 2, the isoliquiritigenin compositions prepared in Examples 1-3 of this invention for improving depression all significantly increased the content of the anti-inflammatory factor IL-10 in mice and improved the depressive behavior of mice.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. An isoliquiritigenin composition for improving depression, characterized in that, The isoliquiritigenin composition comprises the following raw materials in parts by weight: 22-40 parts isoliquiritigenin, 10-17 parts N-acetyl-D-glucosamine, and 15-20 parts β-lapaquinone.

2. The isoliquiritigenin composition for improving depression according to claim 1, characterized in that, The isoliquiritigenin composition comprises the following raw materials in parts by weight: 31 parts isoliquiritigenin, 13 parts N-acetyl-D-glucosamine, and 17 parts β-lapaquinone.

3. A method for preparing an isoliquiritigenin composition for improving depression according to claim 1 or 2, characterized in that, Specifically, the following steps are included: (1) Weigh out isoliquiritin and β-lapaquinone by weight, dissolve them in anhydrous ethanol, and sonicate them to obtain isoliquiritin solution and β-lapaquinone solution; dissolve N-acetyl-D-glucosamine in deionized water by weight to obtain N-acetyl-D-glucosamine solution. (2) The isoliquiritin solution, β-lapaquinone solution and N-acetyl-D-glucosamine solution obtained in step (1) are mixed and stirred, ultrasonically treated, rotary evaporated and dried to obtain a mixed powder, and excipients are added to prepare an oral dosage form.

4. A method for preparing an isoliquiritigenin composition for improving depression according to claim 3, characterized in that, In step (1), the concentration of the isoliquiritigenin solution is 100-150 μg / mL; the concentration of the β-lapaquinone solution is 20-30 μg / mL; and the concentration of the N-acetyl-D-glucosamine solution is 75-95 μg / mL.

5. A method for preparing an isoliquiritigenin composition for improving depression according to claim 3, characterized in that, The mixing time in step (2) is 20-30 min; the ultrasonic treatment time is 10-20 min.

6. A method for preparing an isoliquiritigenin composition for improving depression according to claim 3, characterized in that, The oral dosage form is a tablet.

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