Tussilago farfara extract and application thereof in mood regulation and depression treatment

Abstract

The invention discloses a tussilago farfara extract and application of the tussilago farfara extract in mood regulation and depression treatment. The tussilago farfara extract is prepared, the activation effect of the tussilago farfara extract on a neurotransmitter receptor is verified through a cell experiment, and the relieving effect of the tussilago farfara extract on depression-like symptoms of mice is verified through an animal experiment. The flos farfarae alcohol extract can improve the depression behavior of a mouse by activating a neurotransmitter receptor, and remarkably improve the content of 5-HT and DA in hippocampus of the depression mouse and the gene expression level of a 5-HT1A receptor and a D2 receptor in a prefrontal lobe. The tussilago farfara extract can be applied to medicines for treating neurotransmitter or neurotransmitter receptor mediated diseases or symptoms, and health care products, foods, cosmetics or daily necessities for regulating emotion.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a coltsfoot flower extract and application thereof. Background Art

[0002] Mood disorders, such as anxiety and depression, severely impact people's quality of life. According to the World Health Organization (WHO), approximately 3.8% of the global population suffers from depression, and anxiety disorders are even more prevalent, affecting over 260 million people. In modern society, factors such as high-stress lifestyles, unstable social environments, and poor sleep quality are contributing to the increasing incidence of mood disorders.

[0003] Mood disorders are closely related to disturbances in neurotransmitter systems. Serotonin (5-HT) and dopamine (DA) are important neurotransmitters for regulating mood. 5-HT is primarily associated with anxiety and depression, while DA plays a key role in motivation, pleasure, and cognitive function. Therefore, modulating these neurotransmitter receptors has become an important strategy for treating mood disorders.

[0004] 5-HT 1A 5-HT receptors and dopamine D2 receptors are important targets for mood regulation. 1A Receptors are widely distributed in the central nervous system and are involved in the regulation of anxiety and depression; D2 receptors play a key role in dopaminergic neurons, regulating motivation, emotion, and cognitive function. 1A D2 receptors have become key targets for the development of new mood regulators.

[0005] Coltsfoot (Tussilago farfara L.) is a perennial herbaceous plant in the Asteraceae family. It is listed as a top-grade medicinal material in the Shennong's Herbal Classic. Coltsfoot as a traditional Chinese medicine is characterized by its holistic regulation, multi-target effects, and synergistic therapeutic effects achieved through the action of multiple active ingredients.

[0006] However, as a traditional Chinese medicine, coltsfoot flower is most commonly used for expectoration, cough relief, and asthma relief. Other medicinal uses, such as regulating the neurotransmitter system or treating mood disorders, have not been reported, and its use cases are relatively limited. Furthermore, there is still an unmet need for new drugs to regulate the neurotransmitter system or treat mood disorders. Summary of the Invention

[0007] In response to the shortcomings of the existing technology, the present invention attempts to prepare coltsfoot extract, verify the activation effect of coltsfoot extract on neurotransmitter receptors through cell experiments, and verify the alleviating effect of coltsfoot extract on depression-like symptoms in mice through animal experiments, providing a new therapeutic means for mood regulation and depression treatment.

[0008] In one aspect, the present invention provides a coltsfoot flower extract, which is obtained by extracting coltsfoot flower with ethanol or an aqueous solution containing 50-99% ethanol.

[0009] Preferably, the coltsfoot flower extract is obtained by extracting coltsfoot flower with an aqueous solution containing 75% ethanol.

[0010] More preferably, the preparation method of the coltsfoot flower extract comprises the following steps:

[0011] S1: drying and pulverization;

[0012] S2: extraction;

[0013] S3: Concentration under reduced pressure;

[0014] S4: Freeze drying.

[0015] Further preferably, step S1 comprises drying the coltsfoot flower and then crushing the coltsfoot flower to obtain coltsfoot flower dry powder.

[0016] Further preferably, step S2 comprises extracting the coltsfoot flower powder with an ethanol-water solution, heating the solution to boiling during extraction, maintaining a slightly boiling state for hot reflux extraction; extracting for a total of 1 to 5 times, each extraction time being 2 to 4 hours. Further preferably, step S2 comprises extracting the coltsfoot flower powder with an ethanol-water solution, heating the solution to boiling during extraction, maintaining a slightly boiling state for hot reflux extraction; extracting for a total of 3 times, each extraction time being 3 hours.

[0017] Further preferably, in step S2, the extract obtained each time is vacuum filtered, and finally the filtrates are combined to obtain an ethanol extract.

[0018] Further preferably, step S3 includes concentrating the ethanol extract under reduced pressure to obtain a crude ethanol extract.

[0019] Further preferably, step S4 comprises freeze-drying the crude ethanol extract to obtain an ethanol extract dry powder.

[0020] In another aspect, the present invention provides a use of a coltsfoot flower extract in the preparation of a drug for activating neurotransmitter receptors or a health product for mood regulation.

[0021] In another aspect, the present invention provides a use of a coltsfoot flower extract in the preparation of a medicine for regulating neurotransmitter synthesis levels or a health product for mood regulation.

[0022] In another aspect, the present invention provides a use of a coltsfoot flower extract in the preparation of a medicine for regulating the expression level of a neurotransmitter receptor or a health product for mood regulation.

[0023] In another aspect, the present invention provides a use of a coltsfoot flower extract in the preparation of a medicament for treating diseases or conditions mediated by neurotransmitters or neurotransmitter receptors, or a health product for mood regulation.

[0024] In one or more embodiments, the disease or condition is selected from one or more of depression, anxiety, and insomnia.

[0025] Preferably, the disease or disorder occurs in a mammal.

[0026] In one or more embodiments, the neurotransmitter is one or both of serotonin and dopamine.

[0027] In one or more embodiments, the neurotransmitter receptor is 5-HT 1A One or both of the D2 receptors.

[0028] In another aspect, the present invention provides a composition comprising the coltsfoot flower extract as described in any embodiment herein; the composition is a pharmaceutical composition, a health product composition, a food composition, a cosmetic composition or a daily necessity composition.

[0029] In another aspect, the present invention provides use of a composition as described in any embodiment herein for the preparation of a medicament for treating a disease or condition mediated by a neurotransmitter or a neurotransmitter receptor.

[0030] In another aspect, the present invention provides use of the composition according to any embodiment herein in health products, foods, cosmetics or daily necessities.

[0031] Compared with the prior art, the application of the coltsfoot flower extract of the present invention has the following beneficial effects:

[0032] 1. When the final concentration of coltsfoot flower extract was 2 mg / mL or below, the survival rate of HEK293T cells was greater than 80%, showing low cytotoxicity.

[0033] 2. In the concentration range of 0.5~2.0mg / mL, the ethanol extract of coltsfoot flower has significant 5-HT 1A Receptor activation effect: within the concentration range of 1.0-2.0 mg / mL, coltsfoot flower ethanol extract has D2 receptor activation effect, and the intensity of the effect shows a certain positive correlation with the dose.

[0034] 3. The ethanol extract of coltsfoot flower can significantly increase the 5-HT and DA content in the hippocampus of depressed mice, as well as the 5-HT in the prefrontal cortex. 1A It can also regulate the gene expression levels of D2 receptors and D-receptors, and has a significant behavioral improvement effect on depressed mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the relative expression level of mRNA of the corresponding receptor gene in stable cells; Figure A is 5-HT 1A 5-HT receptor stably transfected cells 1A Figure 1 is the relative mRNA expression level of the D2 receptor gene. Figure 2 is the relative mRNA expression level of the D2 receptor gene in D2 receptor stably transfected cells.

[0036] Figure 2 5-HT with a FLAG tag 1A The test results of receptor protein or D2 receptor protein.

[0037] Figure 3 Is the effect of coltsfoot flower extract on 5-HT 1A Toxicity test results of receptor stably transfected cells.

[0038] Figure 4 This is the result of the toxicity test of coltsfoot flower extract on D2 receptor stably transfected cells.

[0039] Figure 5 Is the effect of coltsfoot flower extract on 5-HT 1A 5-HT receptor stably transfected cells 1A Receptor activation effect.

[0040] Figure 6 It is the D2 receptor activation effect of coltsfoot flower extract on D2 receptor stably transfected cells.

[0041] Figure 7 These are the results of the effect of coltsfoot flower extract on the behavior of depressed mice; among them, Figure A is the result of the behavioral effect on the open field test of mice; Figure B is the result of the behavioral effect on the forced swimming test of mice; Figure C is the result of the behavioral effect on the tail suspension test of mice.

[0042] Figure 8 Figure 1 is the effect of coltsfoot extract on neurotransmitter levels in the hippocampus of depressed mice; Figure A is the effect on 5-HT levels; Figure B is the effect on DA levels.

[0043] Figure 9 Figure 1 is the effect of coltsfoot extract on the expression of neurotransmitter receptor genes in depressed mice; Figure A is the expression of 5-HT 1A Figure B shows the results of the effects of D2 receptor gene expression. DETAILED DESCRIPTION

[0044] Example 1: Preparation of Coltsfoot Flos Extract

[0045] The coltsfoot herb was dried to a constant weight in a 50°C constant-temperature drying oven, pulverized in a high-speed grinder, and passed through a 40-mesh sieve to obtain a uniform coltsfoot herb powder. Accurately weigh 20g of the coltsfoot herb powder and add 400mL of 75% ethanol or 400mL of pure water at a material-to-liquid ratio of 1:20 (w / v). 0.5g of zeolite was also added to prevent violent boiling. The mixture was transferred to a 1L round-bottom flask, fitted with a reflux condenser. The mixture was then placed in a temperature-controlled electric heating mantle and slowly heated until the solution boiled, maintaining a slight boil for 3 hours of reflux extraction. Each extraction was repeated three times to fully release the active ingredients. After each extraction, vacuum filtration was performed through a double layer of filter paper. The three filtrates were combined to obtain an ethanol extract and an aqueous extract, respectively. The ethanol extract was concentrated under reduced pressure on a rotary evaporator until the alcohol was eliminated, yielding a crude ethanol extract of coltsfoot herb. The crude ethanol extract and aqueous extract were pre-frozen in a -80°C ultra-low temperature freezer.

[0046] The crude ethanol extract and the water extract were placed in a vacuum freeze dryer for drying and dried for 48 hours until constant weight was reached. The water extract was freeze-dried to obtain 10.5 g of brown powder (water extract) (yield 52.5%), and the crude ethanol extract was freeze-dried to obtain 9.2 g of light brown powder (ethanol extract) (yield 46%). All dried samples were sealed and stored in a light-proof glass bottle containing silica gel desiccant and placed in a low-temperature environment of -20°C for use.

[0047] Example 2: Construction of receptor overexpressing cell lines

[0048] This example provides a method for constructing a receptor-overexpressing cell line.

[0049] Human embryonic kidney HEK293T cells were selected as host cells and cultured in high-glucose DMEM medium (containing 10% fetal bovine serum and 1% double antibody) and incubated at 37° C. and 5% CO 2 .

[0050] Human 5-HT 1A The full-length coding sequences of D2 receptor and D5 receptor were obtained as target genes, and the Flag peptide sequence was added and cloned into the lentiviral vector pLVX-puro to construct the recombinant lentiviral expression vector pLVX-5HT. 1A and pLVX-D2. Then the constructed expression vector (pLVX-5HT 1AThe viral vector (pLVX-D2 or pLVX-D2) was co-transfected with packaging plasmids (FV-7433 and FV-7434) into 293T cells, and the virus solution was packaged using a standard three-plasmid co-transfection method. The viral solution was treated with a filter membrane to filter and sterilize, and the filtrate was used to infect HEK293T cells. Polycations (such as 8μg / mL Polybrene) can be added during the infection process to improve transduction efficiency. 48 hours after infection, resistance selection was performed using medium containing 2μg / mL puromycin. Cells that passed the selection were cultured for an additional 7 days and then tested for expression of the target gene.

[0051] 5-HT in transfected cells was detected by qPCR. 1A The mRNA expression levels of D2 receptors and Figure 1 As shown in Figure 2, compared with the untransfected control group, the expression of the two proteins was significantly increased by 32,000 times and 2,400 times, respectively. Figure 2 As shown, compared with the untransfected control group, the expression of Flag-tagged 5-HT in transfected cells was significantly increased. 1A The above results prove that the target receptor protein can be stably expressed in transfected cells, that is, 5-HT 1A The receptor stably transfected cells and D2 receptor stably transfected cells were frozen in liquid nitrogen for future use.

[0052] Example 3: Effect of Coltsfoot Flower Extract on 5-HT 1A Toxicity verification of receptor stably transfected cells

[0053] This example aims to evaluate the effects of water extract and alcohol extract of coltsfoot flower on 5-HT 1A Potential toxicity of cells stably transfected with the receptor.

[0054] The dried powder of water extract and ethanol extract of coltsfoot flower were dissolved in serum-free medium and prepared into sample solutions with final concentrations of 10, 8, 6, 4, 2, and 1 mg / mL. 1A In a 96-well plate containing cells stably transfected with the receptor, three replicate wells were set up for each concentration; a blank control group was set up in which serum-free medium was added instead of sample solution; a positive control group was set up in which 1 μM or 10 μM 8-OH-DPAT (a 5-HT 1A The sample solution was replaced with a solution of a agonist (receptor agonist).

[0055] After 24 hours of treatment, add CCK-8 reagent according to the CCK-8 instructions, continue incubation for 1 to 2 hours, and use a microplate reader to detect the absorbance of each well at 450nm. Figure 3As shown in the figure, when the final concentration of coltsfoot flower extract was 2 mg / mL or below, the cell survival rate was greater than 80%, and no obvious cytotoxic reaction was observed, indicating that the water extract and alcohol extract of coltsfoot flower in this concentration range had an inhibitory effect on 5-HT. 1A The receptor stably transfected cells had no obvious toxic effects and could be used for subsequent testing.

[0056] Example 4: Verification of the toxicity of coltsfoot flower extract on D2 receptor stably transfected cells

[0057] This example aims to evaluate the potential toxicity of water extract and ethanol extract of Coltsfoot Flos on D2 receptor stably transfected cells.

[0058] Dried powders of the aqueous and ethanol extracts of Coltsfoot Flower were dissolved in serum-free medium to prepare sample solutions with final concentrations of 10, 8, 6, 4, 2, and 1 mg / mL, respectively. The sample solutions were added to 96-well plates containing adherent D2 receptor stably transfected cells, with three replicates per well. A blank control group was established, supplemented with serum-free medium instead of the sample solution. A positive control group was established, supplemented with 1 μM or 10 μM bromocriptine (a D2 receptor agonist) dissolved in serum-free medium instead of the sample solution.

[0059] After 24 hours of treatment, add CCK-8 reagent according to the CCK-8 instructions, continue incubation for 1 to 2 hours, and use a microplate reader to detect the absorbance of each well at 450nm. Figure 4 As shown in the data, when the final concentration of coltsfoot flower extract was 2 mg / mL or below, the cell survival rate was greater than 80%, and no obvious cytotoxic reaction was observed, indicating that the water extract and alcohol extract of coltsfoot flower within this concentration range had no obvious toxic effect on D2 receptor stably transfected cells and could be used for subsequent detection.

[0060] Example 5: Effect of Coltsfoot Flower Extract on Cellular 5-HT 1A Receptor activation validation

[0061] The lyophilized powders of coltsfoot flower water extract and alcohol extract were reconstituted with water to prepare sample solutions of 2, 1.5, 1, 0.5 and 0.1 mg / mL. 1A After stably transfected cells were digested with trypsin, the cells were resuspended in DMEM medium and plated evenly at 3×10 5 The cells were plated at a density of 1000 / well in a 12-well plate and incubated for 24 h.

[0062] After the incubation period, all groups were supplemented with DMEM medium containing 10 μM Forskolin for 30 minutes, after which the liquid was discarded. Cells in the sample group were incubated with reconstituted sample solutions of varying concentrations for 30 minutes. A blank control group was supplemented with DMEM medium instead of the sample solution, and a positive control group was supplemented with a 1 μM 8-OH-DPAT solution dissolved in DMEM medium instead of the sample solution.

[0063] After the incubation, the liquid in each well was removed, 200 μL of HCl was added for lysis for 20 min, and the cells were centrifuged at 4000 rpm for 10 min. The supernatant was collected and the cAMP marker content was detected using a cAMP enzyme-linked immunosorbent assay kit to indirectly characterize 5-HT. 1A Receptor activation.

[0064] like Figure 5 As shown in the positive control group, the cAMP level decreased to about 50 μM, which was significantly lower than that of the blank control group (p < 0.05), indicating that 5-HT 1A After the receptor is stimulated, it can inhibit the activity of adenylate cyclase (AC) through Gi protein, thereby reducing the intracellular cAMP content. In the sample group with water extract added, the cAMP level was maintained between about 300 and 350 μM. Except for the 2 mg / mL treatment group, the other groups showed no significant difference compared with the blank control group. In the sample group with alcohol extract added, the cAMP level gradually decreased with increasing concentration, showing an inverse dose correlation. Among them, the cAMP level of the 0.5 mg / mL treatment group decreased to about 290 μM (p < 0.05), and the cAMP level of the 2.0 mg / mL treatment group further decreased to about 100 μM, which was close to the cAMP decrease in the positive control group, and was significantly different from the blank group. In summary, within the concentration range of 0.5 to 2.0 mg / mL, the ethanol extract of coltsfoot flower has a more significant 5-HT than the water extract. 1A The receptor activation effect of coltsfoot flower ethanol extract is dose-dependent.

[0065] Example 6: Verification of the activation of cellular D2 receptors by coltsfoot flower extract

[0066] Lyophilized powders of coltsfoot flower water extract and alcohol extract were added with water to prepare sample solutions of 2, 1.5, 1, 0.5, and 0.1 mg / mL, respectively. D2 receptor stably transfected cells in the logarithmic growth phase were obtained, digested with trypsin, and resuspended in DMEM medium. The cells were evenly distributed at 3×10 5 The cells were plated at a density of 1000 / well in a 12-well plate and incubated for 24 h.

[0067] After incubation, 10 μM forskolin was added to each group, and after 30 min of incubation, the liquid was discarded. The sample group cells were added with different concentrations of sample solution, and incubated for 30 min. The blank control group was set up by adding DMEM medium instead of sample solution. The positive control group was set up by adding 1 μM bromocriptine solution dissolved in DMEM medium instead of sample solution.

[0068] As shown in Figure 6 the positive control group, the cAMP level decreased significantly to about 125 μM, which was significantly lower than that of the blank control group (p<0.05), indicating that D2 receptor activation could inhibit AC activity through the Gi protein pathway, thereby reducing the intracellular cAMP level. In the sample group added with water extract, the cAMP level showed no significant difference compared with the blank control group, indicating that the water extract of Flos Farfarae may not have an activating effect on D2 receptors within the concentration range of 0.1-2.0 mg / mL. In the sample group added with alcohol extract, the alcohol extract at a concentration of 0.1 or 0.5 mg / mL had no significant effect on the cAMP level; when the concentration increased to 1.0 mg / mL, the cAMP level decreased to about 235 μM, which was significantly different from the blank group (p<0.05); when the concentration increased to 1.5-2.0 mg / mL, the cAMP level further decreased. In summary, within the concentration range of 1.0-2.0 mg / mL, the alcohol extract of Flos Farfarae had a D2 receptor activating effect, and the effect intensity showed a dose-dependent manner.

[0069] Example 7: Behavior improvement effect of alcohol extract of Flos Farfarae on depressive mice

[0070] After one week of adaptive feeding of 6-week-old ICR male mice (SPF level, purchased from Shanghai Jesuitze Experimental Animal Co., Ltd.), the mice were randomly divided into groups (normal group, model group, fluoxetine group, alcohol extract group, 5-HT 1A receptor antagonist + alcohol extract group, D2 receptor antagonist + alcohol extract group). Except for the normal group, the remaining experimental mice were induced to form a stable depressive behavior model by using chronic restraint stress (CRS, reference “Xia Tianji. Establishment of mouse sleep disorder model induced by chronic restraint stress and improvement of sleep by Shenyuan extract and its mechanism [D]. Beijing Union Medical College, 2022”) and chronic unpredictable mild stress (CUMS, reference “Fu Xijing. Functional and mechanism study of Leptin / AKT / BDNF signal pathway in improving depressive-like behavior in female mice [D]. Jilin University, 2023”) combined modeling method, and the modeling period was set to 4 weeks.

[0071] After successful modeling, drug administration was initiated for 4 weeks. The drug administration method was daily gavage. The normal group and the model group were gavaged with normal saline daily; the fluoxetine group was gavaged with 10 mg / kg fluoxetine daily; the ethanol extract group was gavaged with 40 mg / kg coltsfoot flower ethanol extract daily; 5-HT 1A The receptor antagonist + ethanol extract group was first gavaged with 1 mg / kg 5-HT 1A The D2 receptor antagonist WAY-100635 was administered orally 30 minutes later, followed by 40 mg / kg of coltsfoot extract. The D2 receptor antagonist + extract group was first administered orally 1 mg / kg of the D2 antagonist haloperidol, followed by 40 mg / kg of coltsfoot extract 30 minutes later. Modeling was performed normally during the gavage treatment. To assess the effects of modeling and drug administration on the emotional state of the mice, behavioral tests were performed after drug administration, including the open field test, forced swim test, and tail suspension test, to evaluate the behavioral improvement effects of coltsfoot extract on depressed mice.

[0072] like Figure 7 As shown in Figure 2, compared with the normal group, the mice in the model group showed obvious center avoidance behavior in the open field test ( Figure 7 , A), showed obvious despair behavior (short struggling time) in the forced swimming test and tail suspension test ( Figure 7 , BC), indicating that the model was successfully established. In the experimental treatment, the ethanol extract of coltsfoot flower significantly improved all behavioral indicators in the three behavioral tests compared with the model group (p<0.05). The improvement in the forced swimming test was comparable to that of the fluoxetine group, and the improvement in the open field test and tail suspension test was better than that of the fluoxetine group. 1A When the receptor antagonist WAY-100635 and D2 receptor antagonist haloperidol were combined, the behavioral improvement effect of the coltsfoot flower alcohol extract group was significantly weakened (p<0.05), indicating that its antidepressant effect is related to 5-HT 1A The receptors are closely related to the D2 receptors.

[0073] Example 8: Effects of Coltsfoot Flower Ethanol Extract on Neurotransmitter Levels in the Hippocampus of Depressed Mice

[0074] Mice were raised according to the method of Example 7, and a stable depression-like behavior model was induced in the mice.

[0075] The content of neurotransmitters in hippocampus was detected by enzyme-linked immunosorbent assay (ELISA). The specific operation was as follows: after the mice were sacrificed, the hippocampus was quickly separated, washed with pre-cooled PBS (0.01M, pH 7.4) to remove residual blood, weighed and cut into pieces, then added into PBS containing protease inhibitors at a mass-volume ratio of 1:9, placed in an enzyme-free grinding bead tube, and grinded at 4°C and 45Hz for 30s, followed by ultrasonic crushing for 10min. The homogenate was centrifuged at 4°C and 10000rpm for 10min, and the supernatant was stored at -20°C. The content of 5-hydroxytryptamine (5-HT) and dopamine (DA) in the sample was quantitatively analyzed by using commercial ELISA kit to evaluate the change of neurotransmitter level in hippocampus after treatment with Chimonanthus praecox alcohol extract.

[0076] As shown in Figure 8 , the level of neurotransmitter in hippocampus of model group mice was significantly reduced (p<0.05), in which the content of 5-HT was reduced to about 8pg / mg, and the content of DA was reduced to about 40pg / mg. The treatment of Chimonanthus praecox alcohol extract could significantly improve the reduction of 5-HT and DA content caused by modeling (p<0.05), and the content of 5-HT was increased to about 20pg / mg Figure 8 (A), and the content of DA was increased to about 60pg / mg Figure 8 (B), which was close to the effect of fluoxetine, indicating that Chimonanthus praecox alcohol extract could significantly increase the content of 5-HT and DA in hippocampus of depressed mice, and further exert the potential of anti-depression. While combined with 5-HT 1A receptor antagonist WAY-100635, the level of 5-HT increased by Chimonanthus praecox alcohol extract was significantly reduced to 15pg / mg (p<0.05), and there was no significant effect on the increase of DA. There was no significant change in the level of two neurotransmitters in the group treated with D2 receptor antagonist haloperidol, indicating that the increase of 5-HT level promoted by Chimonanthus praecox alcohol extract mainly depended on 5-HT 1A receptor.

[0077] Example 9: Effect of Chimonanthus praecox alcohol extract on the expression of neurotransmitter receptors

[0078] The mice were fed according to the method of Example 7, and the mice were induced to form a stable depression model.

[0079] After the mice were bled, they were sacrificed by decapitation, and the brain tissue was quickly separated and the prefrontal cortex was cut, which was placed in an enzyme-free EP tube and immediately frozen in liquid nitrogen, and then transferred to -80°C for frozen storage. The prefrontal cortex tissue was lysed and total RNA was extracted according to the conventional method. The mRNA expression levels of 5-HT 1A receptor and D2 receptor were detected by real-time fluorescent quantitative PCR (qRT-PCR) method, with mouse GAPDH as the internal reference gene. The primer sequences used are shown in Table 1.

[0080] Table 1: Mouse neurotransmitter receptor primer information

[0081]

[0082] like Figure 9 As shown, 5-HT in the prefrontal cortex of model group mice 1A The expression levels of 5-HT and D2 receptor genes were significantly decreased compared with the normal control group (p<0.05). 1A and D2 receptor expression levels (p<0.05), and the improvement effect is close to that of the positive control fluoxetine. This shows that coltsfoot flower ethanol extract can significantly upregulate the expression of the two types of receptors, thereby exerting an antidepressant effect. Combined with 5-HT 1A After treatment with receptor antagonist WAY-100635, the ethanol extract of coltsfoot flower had an effect on 5-HT 1A The promoting effect of 5-HT receptor gene expression was significantly weakened (p < 0.05), and the relative gene expression level dropped to about 0.7, while there was no significant effect on the expression of D2 receptor gene. 1A The expression of D2 receptor genes was not affected, but the expression of D2 receptor genes was significantly increased (p<0.05), with the relative gene expression level rising to about 1.65. This indicates that the changes in the expression of the two receptor genes caused by the ethanol extract of coltsfoot flower are affected by the antagonists of their own receptors.

[0083] Effect of ethanol extract of coltsfoot flower on 5-HT 1A It has significant activation activity on D2 receptors and can significantly improve the depressive behavior of depression-like mouse models, increase the neurotransmitter content in the hippocampus of the brain, and promote the expression of 5-HT in the prefrontal cortex. 1A and D2 receptor expression, indicating that coltsfoot flower ethanol extract has obvious application prospects in regulating emotions and relieving depression, anxiety and sleep.

Claims

1. A coltsfoot flower extract, characterized in that The coltsfoot flower extract is obtained by extracting the coltsfoot flower using ethanol or an aqueous solution containing 50-99% ethanol.

2. Use of the coltsfoot flower extract according to claim 1 in the preparation of a medicine for activating neurotransmitter receptors or a health product for mood regulation.

3. Use of the coltsfoot flower extract according to claim 1 in the preparation of a medicine for regulating the synthesis level of neurotransmitters or a health product for mood regulation.

4. Use of the coltsfoot flower extract according to claim 1 in the preparation of a medicine for regulating the expression level of neurotransmitter receptors or a health product for mood regulation.

5. Use of the coltsfoot flower extract according to claim 1 in the preparation of a medicine for treating diseases or conditions mediated by neurotransmitters or neurotransmitter receptors or a health product for regulating mood.

6. The use according to claim 5, characterized in that The disease or condition is selected from one or more of depression, anxiety and insomnia.

7. The use according to any one of claims 2 to 6, characterized in that The neurotransmitter is one or both of 5-hydroxytryptamine and dopamine.

8. The use according to any one of claims 7, characterized in that The neurotransmitter receptor is 5-HT 1A One or both of the D2 receptors.

9. A composition, characterized in that The composition comprises the coltsfoot flower extract according to claim 1; the composition is a pharmaceutical composition, a health product composition, a food composition, a cosmetic composition or a daily necessity composition.

10. Use of the composition according to claim 9 in the preparation of a medicament for treating diseases or conditions mediated by neurotransmitters or neurotransmitter receptors.

11. Use of the composition according to claim 9 in health products, foods, cosmetics or daily necessities.

Images