Application of gastrodia extract combined with gamma-aminobutyric acid in calming nerves and improving memory

Abstract

The application belongs to the field of biological medicine and food technology, and particularly relates to application of Gastrodia extract combined with gamma-aminobutyric acid in calming nerves and improving memory. The application constructs a memory disorder model mouse, and uses the Gastrodia extract and gamma-aminobutyric acid combined administration, which has the following effects: (1) up-regulating the expression of brain tissue BDNF and PSD95 proteins, and promoting synaptic plasticity repair; (2) balancing the levels of brain tissue / serum 5-HT, NE and GABA neurotransmitters, and stabilizing central nervous function; (3) inhibiting excessive hyperactivity of HPA axis, reducing the content of serum adrenocorticotropic hormone and cortisol, and reducing stress-induced nerve damage; (4) reducing the pathological damage of brain and liver tissues, and playing a whole body protection role; (5) regulating the differential genes and pathways related to neural plasticity and neurotransmitter metabolism. The Gastrodia extract and gamma-aminobutyric acid combined administration can synergistically improve memory and calm nerves.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and food technology, specifically relating to the application of Gastrodia elata extract combined with γ-aminobutyric acid in sedation, tranquilization, and memory improvement. Background Technology

[0002] Learning and memory are core functions of the central nervous system. Disorders of this function are not only common in neurodegenerative diseases such as Alzheimer's and vascular dementia, but also prevalent in sub-health states such as chronic stress and sleep disorders, severely impacting quality of life and cognitive function. Therefore, finding safe and effective interventions to improve learning and memory disorders has significant clinical and applied value. The normal maintenance of learning and memory function depends on the stability of synaptic plasticity, the balance of neurotransmitters, and the homeostatic regulation of the hypothalamus-pituitary-adrenal (HPA) axis. Among these, synaptic plasticity damage, neurotransmitter imbalance, and HPA axis hyperfunction are the core mechanisms leading to memory disorders.

[0003] Drugs reported in existing technologies are typically bioactive peptides. Bioactive peptides undergo digestion in the human or mouse gastrointestinal tract, are absorbed by the small intestine, and reach their target location to exert their physiological effects. However, this complex process, especially the digestion by gastrointestinal proteases, can affect the peptide sequence, thereby influencing its bioactivity. Therefore, developing effective drugs or health supplements adapted to improve memory is essential. Summary of the Invention

[0004] The purpose of this invention is to develop an effective product with sedative and tranquilizing effects, suitable for improving memory, especially for improving memory impairment caused by chlorophenylalanine.

[0005] This invention provides the application of Gastrodia elata extract in combination with γ-aminobutyric acid in the preparation of products for sedation, tranquilization and / or memory improvement, wherein the Gastrodia elata extract includes an aqueous extract of Gastrodia elata.

[0006] Preferably, the improvement of memory refers to the improvement of memory impairment caused by chlorophenylalanine.

[0007] Preferably, the improvement of chlorophenylalanine-induced memory impairment includes one or more of the following: (1) Improves the decrease in 5-hydroxytryptamine content in brain tissue caused by chlorophenylalanine; (2) Improves the increase in norepinephrine levels caused by chlorophenylalanine; (3) Improves the increase in serum adrenocorticotropic hormone caused by chlorophenylalanine; (4) Improves the increase in serum cortisol concentration caused by chlorophenylalanine; (5) It improves the reduced expression levels of BDNF and / or PSD95 proteins in brain tissue caused by chlorophenylalanine, and promotes synaptic plasticity repair; (6) Improves brain tissue damage caused by chlorophenylalanine; (7) Improves liver tissue damage caused by chlorophenylalanine.

[0008] This invention provides the application of Gastrodia elata extract in combination with γ-aminobutyric acid in the preparation of products that regulate HPA axis function.

[0009] Preferably, the mass ratio of Gastrodia elata extract to γ-aminobutyric acid in the product is (0.15~0.60):(10~40), and the mass of the Gastrodia elata extract is based on the mass of gastrodin.

[0010] Preferably, the mass ratio of Gastrodia elata extract to γ-aminobutyric acid in the product is (0.30~0.60):40.

[0011] Preferably, the concentration of gastrodin in the product is 0.15~6.0 mg / mL.

[0012] Preferably, the products include pharmaceuticals, food, and health products.

[0013] Preferably, the preparation method of the Gastrodia elata extract includes: mixing Gastrodia elata and water, heating and refluxing for extraction, collecting the extract and concentrating it to obtain the Gastrodia elata extract.

[0014] This invention provides a product for calming the nerves and / or improving memory, wherein the active ingredients of the product include Gastrodia elata extract and γ-aminobutyric acid, and the Gastrodia elata extract includes a water extract of Gastrodia elata; The mass ratio of Gastrodia elata extract to γ-aminobutyric acid in the product is (0.15~0.60):(10~40), and the mass of Gastrodia elata extract is based on the mass of gastrodin.

[0015] Beneficial effects: This invention constructs a mouse model of memory impairment and uses a combination of Gastrodia elata extract and γ-aminobutyric acid (GABA) for the following effects: (1) upregulating the expression of BDNF and PSD95 proteins in brain tissue and promoting synaptic plasticity repair; (2) balancing the levels of 5-HT, NE, and GABA neurotransmitters in brain tissue and stabilizing central nervous system function; (3) inhibiting excessive hyperactivity of the HPA axis, reducing serum adrenocorticotropic hormone and cortisol levels, and alleviating stress-induced nerve damage; (4) alleviating pathological damage to brain and liver tissues and exerting an overall protective effect on the body; (5) regulating differentially expressed genes and pathways related to neuroplasticity and neurotransmitter metabolism. The combined administration of Gastrodia elata extract and GABA can synergistically improve memory and has a calming and soothing effect. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The total distance traveled in the mine experiment for mice in different treatment groups; compared with the normal control group, * P <0.05,** P <0.01; Figure 2 * Reset time of the mine field experiment results in mice of different treatment groups; compared with the normal control group, * P <0.05,** P <0.01; Figure 3 Results of a navigation experiment in mice with different PCPA-induced memory impairment models; Figure 4 Results of spatial exploration experiments in mice with different PCPA-induced memory impairment models; compared with the model group, * P <0.05, no significant difference in ns; Figure 5 The levels of 5-hydroxytryptamine (5-HT) in the brain tissue of mice with different PCPA-induced memory impairment models; compared with the model group, ** P <0.01, *** P <0.001, no significant difference in ns; Figure 6 The concentrations of norepinephrine (NE) in the brain tissue of mice with different PCPA-induced memory impairment models; compared with the model group, ** P <0.01; Figure 7 The content of γ-aminobutyric acid in the brain tissue of mice with different PCPA-induced memory impairment models; compared with the model group, * P <0.05,** P <0.01, *** P <0.001, no significant difference in ns; Figure 8 Serum ACTH levels in mice with different PCPA-induced memory impairment models; compared with the model group, * P <0.05,** P <0.01, *** P <0.001; Figure 9 Serum cortisol (CORT) concentrations in mice with different PCPA-induced memory impairment models; no significant difference in ns compared to the model group, * P <0.05, *** P <0.001; Figure 10 HE staining results of brain tissue from mice with different PCPA-induced memory impairment models; Figure 11 HE staining results of liver tissue from mice with different PCPA-induced memory impairment models; Figure 12 Immunofluorescence intensity of the expression of neurotrophic factor PSD95 in mice with different PCPA-induced memory impairment models; P <0.01, *** P <0.001, no significant difference in ns; Figure 13 Immunofluorescence intensity of BDNF expression in mice with different PCPA-induced memory impairment models;* P <0.05,** P <0.01, no significant difference in ns; Figure 14 Co-localization results of neurotrophic factors PSD95 and BDNF in different PCPA-induced memory impairment mouse models;* P <0.05, no significant difference in ns. Detailed Implementation

[0018] This invention provides the application of Gastrodia elata extract in combination with γ-aminobutyric acid in the preparation of products for sedation, tranquilization and / or memory improvement, wherein the Gastrodia elata extract includes an aqueous extract of Gastrodia elata.

[0019] In one embodiment, the memory improvement described in this invention is to improve memory impairment caused by chlorophenylalanine. In one embodiment, the improvement of memory impairment caused by chlorophenylalanine in this invention includes one or more of the following: (1) improving the decrease in 5-hydroxytryptamine content in brain tissue caused by chlorophenylalanine; (2) improving the increase in norepinephrine content caused by chlorophenylalanine; (3) improving the increase in serum adrenocorticotropic hormone caused by chlorophenylalanine; (4) improving the increase in serum cortisol concentration caused by chlorophenylalanine; (5) improving the decrease in the expression levels of BDNF protein and / or PSD95 protein in brain tissue caused by chlorophenylalanine, promoting synaptic plasticity repair; (6) improving brain tissue damage caused by chlorophenylalanine; (7) improving liver tissue damage caused by chlorophenylalanine. This invention constructs a PCPA-induced memory impairment mouse model. Compared with memory impairment caused by other factors, the PCPA-induced model has a more complex damage mechanism, exhibiting multiple pathologies such as neurotransmitter metabolism disorders, sleep abnormalities, and decreased synaptic plasticity. It is not easily improved by a single component and can effectively verify the synergistic intervention effect of Gastrodia elata extract combined with γ-aminobutyric acid.

[0020] This invention provides the application of Gastrodia elata extract in combination with γ-aminobutyric acid in the preparation of products that regulate HPA axis function.

[0021] The present invention combines Gastrodia elata extract with γ-aminobutyric acid to treat mice with memory impairment, and has the following effects: (1) upregulates the expression of BDNF and PSD95 proteins in brain tissue and promotes synaptic plasticity repair; (2) balances the levels of 5-HT, NE and GABA neurotransmitters in brain tissue and stabilizes central nervous system function; (3) inhibits excessive hyperactivity of the HPA axis, reduces serum adrenocorticotropic hormone and cortisol levels, and alleviates stress-induced nerve damage; (4) reduces pathological damage to brain and liver tissues and plays an overall protective role in the body; (5) regulates differentially expressed genes and pathways related to neuroplasticity and neurotransmitter metabolism.

[0022] In one embodiment, the mass ratio of Gastrodia elata extract to γ-aminobutyric acid in the product of the present invention is (0.15~0.60):(10~40), and the mass of the Gastrodia elata extract is based on the mass of gastrodin; in another embodiment, the mass ratio of Gastrodia elata extract to γ-aminobutyric acid in the product of the present invention is (0.30~0.60):40. In one embodiment, the concentration of gastrodin in the product of the present invention is 0.15~6.0 mg / mL; in another embodiment, the concentration of gastrodin in the product of the present invention is 0.2~5.0 mg / mL; in another embodiment, the concentration of gastrodin in the product of the present invention is 0.3~4.5 mg / mL; in another embodiment, the concentration of gastrodin in the product of the present invention is 0.5~4.0 mg / mL; in another embodiment, the concentration of gastrodin in the product of the present invention is 0.8~3.0 mg / mL; in another embodiment, the concentration of gastrodin in the product of the present invention is 1.0~2.0 mg / mL; in another embodiment, the concentration of gastrodin in the product of the present invention is 1.5 mg / mL.

[0023] As one implementation method, the products described in this invention include pharmaceuticals, food, and health products.

[0024] In one embodiment, the concentration of gastrodin in the *Gastrodia elata* extract of the present invention is 0.15~0.6 mg / mL; in another embodiment, the concentration of gastrodin in the *Gastrodia elata* extract of the present invention is 0.6 mg / mL. The *Gastrodia elata* extract containing gastrodin prepared by the present invention has the advantage of synergistic effect of multiple active components compared to directly utilizing gastrodin. In addition to gastrodin, p-hydroxybenzyl alcohol, and barisonisides, the extract also contains polysaccharides, phenols, sterols, and other natural active ingredients. The components complement each other and synergistically enhance the core effects of sedation, tranquilization, and memory improvement, and can also regulate the overall nervous state of the body, with a richer range of targets, resulting in better effects than a single gastrodin monomer. In one embodiment, the preparation method of the *Gastrodia elata* extract of the present invention includes: mixing *Gastrodia elata* and water, heating and refluxing for extraction, collecting the extract, and concentrating it to obtain the *Gastrodia elata* extract.

[0025] In one embodiment, the mass ratio of Gastrodia elata to water in this invention is 1:(10~20). In another embodiment, the mass ratio of Gastrodia elata to water in this invention is 1:10. This invention uses water as the extraction solvent, which, compared to other solvents, is green, safe, and leaves no toxic solvent residue, meeting the safety requirements for oral products. Simultaneously, water extraction can efficiently dissolve water-soluble active substances unique to Gastrodin, p-hydroxybenzyl alcohol, barisoniside, polysaccharides, etc., in Gastrodia elata, maximizing the retention of the inherent effective components of the medicinal material. Furthermore, the extraction process is simple, the production cost is low, and it is easy to scale up production.

[0026] In one embodiment, the reflux extraction is performed 1-2 times; in another embodiment, the reflux extraction is performed twice. In one embodiment, the reflux extraction time for each extraction is 1-2 hours; in another embodiment, the reflux extraction time for each extraction is 1.5 hours. Compared to other extraction methods, the reflux extraction method of this invention offers advantages such as thorough dissolution of active ingredients and high extraction efficiency. Simultaneously, the system is heated uniformly, stably retaining water-soluble active components such as gastrodin. The process is highly controllable, has excellent reproducibility, and is suitable for industrial-scale production.

[0027] This invention provides a product for calming the nerves and / or improving memory, wherein the active ingredients of the product include Gastrodia elata extract and γ-aminobutyric acid, and the Gastrodia elata extract includes a water extract of Gastrodia elata; The mass ratio of Gastrodia elata extract to γ-aminobutyric acid in the product is (0.15~0.60):(10~40), and the mass of Gastrodia elata extract is based on the mass of gastrodin.

[0028] In one embodiment, the mass ratio of Gastrodia elata extract to γ-aminobutyric acid in the product of the present invention is (0.30~0.60):40. In one embodiment, the concentration of gastrodin in the product of the present invention is 0.15~6.0 mg / mL; in another embodiment, the concentration of gastrodin in the product of the present invention is 0.2~5.0 mg / mL; in another embodiment, the concentration of gastrodin in the product of the present invention is 0.3~4.5 mg / mL; in another embodiment, the concentration of gastrodin in the product of the present invention is 0.5~4.0 mg / mL; in another embodiment, the concentration of gastrodin in the product of the present invention is 0.8~3.0 mg / mL; in another embodiment, the concentration of gastrodin in the product of the present invention is 1.0~2.0 mg / mL; in another embodiment, the concentration of gastrodin in the product of the present invention is 1.5 mg / mL.

[0029] To further illustrate the present invention, the application of the Gastrodia elata extract combined with γ-aminobutyric acid in sedation, mind-calming and memory improvement provided by the present invention is described in detail below with reference to the accompanying drawings and examples, which should not be construed as limiting the protection scope of the present invention.

[0030] Example 1 1. Materials and Methods (1) Materials and reagents: Gastrodia elata (purchased from Dejiang County, Guizhou Province); γ-aminobutyric acid: food grade, purity ≥99%, from Zaozhuang Jienuo Bio-enzyme Co., Ltd.; PCPA (Sigma, USA, lot number 20230405); Mouse 5-hydroxytryptamine (5-HT), mouse norepinephrine (NE), mouse cortisol, mouse adrenocorticotropic hormone (ACTH) enzyme-linked immunosorbent assay (ELISA) kits (Jiangsu Jingmei Biotechnology Co., Ltd., lot number 202506); GABA content assay kit (Jiangsu Adison Biotechnology Co., Ltd., lot number 202506); Sodium pentobarbital (Sigma, USA, lot number 191201).

[0031] (2) Instruments and equipment: Mouse autonomous activity tester (Chengdu Tailmeng Technology Co., Ltd., model ZZ-6g); Microplate reader (Shenzhen Rayto Life Science Co., Ltd., model RT-6000); Paraffin microtome (Leica, Germany, model RM2245); Inverted fluorescence microscope (Leica, Germany, model DMI8); Imaging system (Nikon, Japan, models Nikon Eclipse C1, Nikon DS-U3); Optical microscope (Olympus, Japan, model IX8I); Slide scanner (Chengdu Zhiyue Technology Co., Ltd., model WS-10).

[0032] (3) Laboratory animals: 204 male SPF-grade ICR mice, weighing 18~22g, purchased from Henan Skebes Biotechnology Co., Ltd., with the license number SCXK (Yu) 2020-0005. The study strictly followed the management and use of laboratory animals, and was approved by the Laboratory Animal Ethics Committee of Anhui University of Chinese Medicine (Ethics No. AHUCM-mouse-2024058). The mice were reared in a 12h light / dark alternating environment, with the temperature controlled at 20~25℃ and the relative humidity at 40%~70%.

[0033] 2. Experimental Methods 2.1. Drug Preparation (1) Preparation of Gastrodia elata extract: Take 500g of Gastrodia elata, add 10 times the amount of purified water, and heat under reflux for extraction (heat at 200℃, boil, then reduce to 80℃ and keep boiling for extraction) twice, each time for 1.5h. Combine the extracts and detect the content of gastrodin by HPLC. The content of gastrodin is 0.17mg / mL. Concentrate under reduced pressure to a content of gastrodin of 0.6mg / mL to obtain concentrated Gastrodia elata extract. Store at 4℃ for later use.

[0034] (2) Preparation of the combined drug: Accurately measure an appropriate amount of Gastrodia elata concentrate, add the corresponding mass of γ-aminobutyric acid, dilute with purified water to prepare three doses of combined drug solution (low, medium, and high). The combined drug solution should be prepared and used immediately. After being magnetically stirred evenly, it should be administered to animals by gavage on the same day; among which, The final concentration of gastrodin in the low-dose combined drug solution was 0.15 mg / mL, and the final concentration of γ-aminobutyric acid was 10 mg / mL. The final concentration of gastrodin in the medium-dose combined drug solution was 0.30 mg / mL, and the final concentration of γ-aminobutyric acid was 20 mg / mL. The final concentration of gastrodin in the high-dose combined drug solution was 0.60 mg / mL, and the final concentration of γ-aminobutyric acid was 40 mg / mL.

[0035] (3) Preparation of diazepam (DZP) solution: Weigh 12mg of diazepam, dissolve it in 10mL of 20% castor oil polyoxyethylene saline solution, and dilute it with 30mL of saline to a concentration of 0.3mg / mL. The dosage of pure diazepam in mice is 3mg / kg.

[0036] (4) Preparation of p-chlorophenylalanine (PCPA) solution: Dissolve p-chlorophenylalanine in 20% castor oil polyoxyethylene saline solution, add saline to make up to a concentration of 40 mg / mL suspension, and administer the pure p-chlorophenylalanine to mice at a dose of 400 mg / kg.

[0037] 2.2. Laboratory Animals and Grouping 2.2.1 Evaluation of the sedative, tranquilizing, and hypnotic effects of Gastrodia elata extract combined with γ-aminobutyric acid (GABA). Forty male SPF-grade ICR mice were acclimatized for 7 days and randomly divided into 5 groups of 8 mice each. The following treatments were performed: Normal control group (NC): The normal control group was given the same volume of physiological saline; Diazepam (DZP) group: mice were administered diazepam solution by gavage at a dose of 3 mg / kg based on the mass of diazepam, once daily for 7 days; Low-dose combined administration (GAS-GABA-L group): mice were administered a low dose of the combined administration solution by gavage at a volume of 10 mL / kg for 7 days, once a day. Medium-dose combined administration (GAS-GABA-M) group: mice were given a medium dose of the combined administration solution by gavage at a volume of 10 mL / kg for 7 days, once a day; High-dose combined administration (GAS-GABA-H group): mice were given a high dose of the combined administration solution by gavage at a volume of 10 mL / kg for 7 days, once a day. The open field test (OFT) is a classic and commonly used method. Generally, a decrease in autonomic nervous activity in mice indicates that the drug has a sedative effect. Therefore, this invention uses normal mice to investigate the sedative and tranquilizing effects of the test drug, with diazepam as a positive control. The open field test was used to observe changes in spontaneous activity and resting time in mice. The open field test was performed on day 7 of drug administration in each treatment group. Mice were placed in the central area of ​​a rectangular box 30 minutes after drug administration, and the test lasted for 6 minutes. The first minute was considered an adaptation period, and the total distance traveled and resting time of the mice within 5 minutes were recorded using a spontaneous activity analysis system.

[0038] The results are as follows Figure 1 and Figure 2 As shown, the normal control group (NC) mice exhibited a typical pattern of alternating exploration and rest behavior, with both total distance traveled and resting time at relatively high levels. Compared to the NC group, mice in the diazepam group (DZP, 3 mg / kg) and the medium- and high-dose groups of Gastrodia elata extract combined with γ-aminobutyric acid (GABA) showed a significantly shorter total distance traveled during spontaneous activity within 5 minutes after administration, suggesting that the test drug and positive control drug could inhibit spontaneous activity in normal mice, exhibiting a certain sedative effect. However, the resting time in the above groups did not show the expected increase, but was significantly lower than that in the normal control group, indicating that the test drug did not exert a sedative effect by prolonging resting time, but rather its mechanism of action was more inclined to inhibit exploratory activity rather than directly inducing a quiet state. The total distance traveled and resting time in the low-dose GAS-GABA group were not significantly different from those in the normal control group, suggesting that this dose had no significant effect on the basic activity of normal mice and did not show an excessive sedative effect.

[0039] 2.2.2. Effects of Gastrodia elata extract combined with γ-aminobutyric acid on a PCPA-induced memory impairment model (1) 112 male SPF-grade ICR mice were acclimatized for 7 days and then divided into 14 groups of 8 mice each. Except for the normal control group (NC), the other mice were intraperitoneally injected with chlorophenylalanine (PCPA) solution. The dosage of PCPA was 400 mg / kg of mice, once a day for 7 consecutive days to establish a memory impairment model. The NC group was injected with an equal volume of physiological saline. From day 8, intraperitoneal injection of PCPA was stopped and a 14-day drug intervention was started. The specific intervention method is as follows: Normal control group (NC): Intraperitoneal injection of an equal volume of physiological saline; Model group (MOD): Intraperitoneal injection of an equal volume of physiological saline; Positive control group (DZP): mice were administered diazepam solution by gavage at a dose of 3 mg / kg of diazepam once a day for 14 days. Low-dose group of Gastrodia elata extract (GAS-L): Gastrodia elata extract was administered to mice by gavage at a dose of 1.5 mg / kg, calculated as gastrodin, for 14 days, once a day. Medium-dose group of Gastrodia elata extract (GAS-M): Gastrodia elata extract was administered to mice by gavage at a dose of 3.0 mg / kg, calculated as gastrodin, for 14 days, once a day. High-dose group of Gastrodia elata extract (GAS-H): mice were administered Gastrodia elata extract by gavage at a dose of 6.0 mg / kg, calculated as gastrodin, for 14 days, once a day. Low-dose γ-aminobutyric acid (GABA-L): γ-aminobutyric acid was administered by gavage at a dose of 100 mg / kg mice for 14 days, once daily. Medium-dose γ-aminobutyric acid group (GABA-M): γ-aminobutyric acid was administered by gavage at a dose of 200 mg / kg mice for 14 days, once daily. High-dose γ-aminobutyric acid (GABA-H) group: γ-aminobutyric acid was administered by gavage to mice at a dose of 400 mg / kg for 14 days, once a day. Low-dose gastrodin combined with low-dose γ-aminobutyric acid (GAS-GABA-L): Gastrodia elata extract and γ-aminobutyric acid were administered by gavage. The dose of gastrodin was 1.5 mg / kg mice and the dose of γ-aminobutyric acid was 100 mg / kg mice, for 14 days, once a day. The medium-dose gastrodin combined with medium-dose γ-aminobutyric acid (GAS-GABA-M) group was administered gastrodin extract and γ-aminobutyric acid by gavage. The dose of gastrodin extract was 3.0 mg / kg mice and the dose of γ-aminobutyric acid was 200 mg / kg mice, for 14 days, once a day. High-dose gastrodin combined with high-dose γ-aminobutyric acid (GAS-GABA-H): Gastrodia elata extract and γ-aminobutyric acid were administered by gavage. The dose of gastrodin was 6.0 mg / kg mice and the dose of γ-aminobutyric acid was 400 mg / kg mice, for 14 days, once a day. The medium-dose gastrodin combined with high-dose γ-aminobutyric acid (GASM-GABA-H) group: Gastrodia elata extract and γ-aminobutyric acid were administered by gavage. The dose of gastrodin was 3.0 mg / kg mice and the dose of γ-aminobutyric acid was 400 mg / kg mice, for 14 days, once a day. High-dose gastrodin combined with medium-dose γ-aminobutyric acid (GASH-GABA-M): Gastrodia elata extract and γ-aminobutyric acid were administered by gavage. The dose of gastrodin extract was 6.0 mg / kg mice and the dose of γ-aminobutyric acid was 200 mg / kg mice, for 14 days, once a day. High-dose gastrodin combined with low-dose γ-aminobutyric acid (GASH-GABA-L): Gastrodia elata extract and γ-aminobutyric acid were administered by gavage. The dose of gastrodin was 6.0 mg / kg mice and the dose of γ-aminobutyric acid was 100 mg / kg mice, once a day for 14 days.

[0040] (2) Water maze experiment Step (1) The Morris water maze test was performed 30 minutes after the last administration of each treatment group. The Morris water maze test was performed on mice in all groups to evaluate their learning and memory abilities. The experiment was divided into two stages: navigation and spatial exploration. The navigation test lasted for 4 days. Each day, the mice were placed in the water from different quadrants. The time it took for the mice to find the platform within 60 seconds (escape latency and swimming speed) was recorded. If the mice did not find the platform, they were guided to stand on the platform for 15 seconds. The latency was recorded as 60 seconds. The spatial exploration test was conducted on the 5th day. The platform was removed, and the mice were placed in the water from the quadrant opposite to the original platform. The percentage of time the mice spent in the target quadrant and the number of times they entered the target quadrant within 60 seconds were recorded.

[0041] The positioning and navigation experiment was conducted continuously for 4 days, and the results were as follows: Figure 3 As shown in Figure A, the escape latency of mice in all groups generally decreased with increasing training days. Compared with the normal control group (NC), the escape latency of mice in the model group (MOD) was significantly prolonged, with a limited decrease, confirming that PCPA can cause impairment of spatial learning function in mice. Compared with the model group, the escape latency of mice in the gastrodin-only group and the GABA-only group was shortened to varying degrees. The combined gastrodin and GABA group showed a better shortening effect on escape latency than the single component, and the effect increased with increasing dosage. The swimming speed results of mice in each group are shown in the figure. Figure 3In the middle B group, during the 4-day training cycle, there was no significant difference in the average swimming speed among the mice in each group, proving that the difference in escape latency among the groups was not affected by the mice's motor ability.

[0042] On the 5th day, a space exploration experiment was conducted, the underwater platform was removed, and the experimental results were as follows: Figure 4 As shown. Compared with the normal control group, the percentage of time spent in the target quadrant in the model group mice ( Figure 4 (A) Number of times entering the target quadrant ( Figure 4 Both B and C decreased; combined with the mouse swimming trajectory ( Figure 4 As shown in Figure C), the model mice exhibited dispersed swimming trajectories and showed less clustering behavior in the quadrant where the original platform was located. Compared to the model group, the gastrodin administration group, the GABA administration group, and the GAS-GABA combination administration group all increased the proportion of time spent in the target quadrant and the number of times mice entered the target quadrant; among them, the high-dose gastrodin and GABA combination group (GAS-GABA-H) showed the best improvement. The above experimental results indicate that gastrodin combined with GABA can improve PCPA-induced spatial learning and memory impairment in mice, and that the combination of gastrodin and GABA has a synergistic effect.

[0043] (3) Serum sample collection After the water maze experiment, mice in each group were fasted for 12 hours, and blood was collected from the orbital cavity. The blood samples were allowed to stand at 4℃ for 24 hours, then centrifuged at 4000 rpm for 15 minutes at 4℃. The supernatant serum was collected and aliquoted into 0.5 mL centrifuge tubes, frozen at -20℃ for 4 hours, and then transferred to -80℃ for later use. Mice were dissected, and tissues such as the heart, liver, kidneys, and spleen were collected. After washing with physiological saline, the weights of each group were recorded. A portion of liver and whole brain tissue was taken from each group and fixed with 4% paraformaldehyde for histopathological observation. The remaining portions were divided into 2 mL cryovials according to group size, frozen at -20℃ for 4 hours, and then transferred to -80℃ for later storage.

[0044] (4) Detection of biochemical indicators in mouse brain tissue The detection was performed using enzyme-linked immunosorbent assay (ELISA). The results of the brain tissue serotonin (5-HT) content detection are as follows: Figure 5 As shown, compared with the normal control group, the level of 5-hydroxytryptamine (5-HT) in the brain tissue of mice in the model group was significantly decreased. P<0.01); indicating that PCPA modeling significantly reduced 5-hydroxytryptamine (5-HT) levels in the brain tissue of mice in the model group, and that this model effectively depletes 5-HT levels in the mouse brain. Compared with the model group, neither the gastrodin (GAS) monotherapy group nor the γ-aminobutyric acid (GABA) monotherapy group significantly upregulated 5-HT levels in brain tissue. However, the high-dose gastrodin combined with high-dose γ-aminobutyric acid (GAS-GABA-H) group and the medium-dose gastrodin combined with high-dose γ-aminobutyric acid (GASM-GABA-H) group significantly increased 5-HT levels in mouse brain tissue. The 5-HT levels in the combined group were close to those in the normal control group, indicating that the combination of gastrodin and GABA has a synergistic effect on the recovery of 5-HT in the brain. The results of the norepinephrine (NE) content detection in brain tissue are as follows: Figure 6 As shown, compared with the normal control group, the concentration of NE in the brain tissue of the model group mice was significantly increased ( P <0.05), indicating that PCPA modeling can lead to NE metabolism disorder in the brain of model mice. Compared with the model group, all treatment groups (single-drug group and combination-drug group) significantly reduced the NE content in mouse brain tissue ( P <0.01), with the high-dose combination therapy group showing better intervention than the diazepam positive control group. The results from the combination therapy group show that when the dose of ginseng (GAS) is medium or high, regardless of the low, medium, or high dose of GABA administered in combination, it effectively reduces the NE concentration in mouse brain tissue, and the intervention effect is relatively stable. Results are as follows... Figure 7 As shown, compared with the normal control group, the GABA content in the brain tissue of the model group mice was significantly reduced ( P <0.001 indicates that PCPA modeling significantly depletes the inhibitory neurotransmitter GABA in the brain, disrupting neurotransmitter homeostasis. Compared with the model group, low-dose gastrodin intervention alone showed no statistical difference, while medium and high-dose gastrodin significantly and extremely significantly increased GABA levels, respectively, showing a dose-dependent improvement trend; only the high-dose group of exogenous GABA monomers significantly upregulated brain GABA content, while the low and medium-dose monomer interventions had weak effects. The combined administration of gastrodin and GABA showed significantly better improvement effects in all groups than the single-drug treatment groups of the same gradient, with the overall difference reaching an extremely significant level ( ). P <0.001), low-dose combination therapy can achieve a regulatory effect close to that of high-dose single-drug therapy, and the GABA recovery of medium- and high-dose combination therapy is comparable to or even exceeds that of the diazepam positive control group; among them, the intervention effect of medium- and high-dose combination therapy with different ratios is stable and outstanding, proving that the combination of the two can synergistically amplify the upregulation of GABA neurotransmitters in the brain, repair the neurotransmitter disorder induced by PCPA from the neurotransmitter level, and the synergistic regulatory advantage is significant.

[0045] comprehensive Figures 5-7It can be seen that gastrodin and GABA alone have no significant regulatory effect on 5-HT levels in the brain, but the combination of the two can significantly restore 5-HT content and significantly reduce abnormally elevated NE in the brain of PCPA model mice. Among them, the NE-reducing effect of medium and high doses of gastrodin combined with GABA is stable, and the intervention effect of the high-dose group is better than that of the positive drug diazepam. This indicates that the combination of gastrodin and GABA can synergistically regulate the imbalance of monoamine neurotransmitters (5-HT and NE) in the brain of PCPA model mice, simultaneously upregulate GABA content, and reshape neurotransmitter homeostasis. This is one of the important mechanisms by which it improves memory impairment in PCPA mice.

[0046] (5) Detection of biochemical indicators in mouse serum The detection was performed using enzyme-linked immunosorbent assay (ELISA). The serum adrenocorticotropic hormone (ACTH) level detection results are as follows: Figure 8 As shown, compared with the normal control group, the serum ACTH content in the model group mice was significantly increased ( P <0.05 indicates successful model construction, with the mouse HPA axis in an abnormally activated state and enhanced stress response, consistent with characteristic manifestations of relevant stress models. After intervention with the positive control drug diazepam (DZP), the serum ACTH concentration in the model mice was significantly reduced ( P <0.05), and the level was close to that of the normal control group. The study found that both high-dose gastrodin alone (GAS-H) and high-dose γ-aminobutyric acid (GABA) alone (GABA-H) significantly reduced serum ACTH concentration in model mice, indicating that gastrodin and GABA, when used alone at high doses, have certain anti-stress effects. They can inhibit excessive activation of the HPA axis by regulating ACTH secretion, providing experimental evidence for their anti-stress activation properties. Combined intervention showed that the overall regulatory effect of the gastrodin and GABA combined intervention group was better than that of the single-dose groups. Specifically, the serum ACTH level in the high-dose combined intervention group was significantly lower than that in the model group (…). P <0.01), and superior to the positive drug DZP group, indicating that the combined use of the two has a synergistic effect; at the same time, the combined intervention of high-dose gastrodin and medium- and low-dose GABA can also significantly reduce ACTH levels ( P The result (<0.01) further illustrates that an appropriate ratio of these two components can fully leverage their synergistic effect. The serum cortisol concentration (CORT) level detection results are as follows... Figure 9 As shown, all treatment groups significantly reduced serum CORT concentration in model mice, further demonstrating that gastrodin, GABA alone, and in combination have an inhibitory effect on excessive activation of the HPA axis. Figures 8-9It can be seen that both gastrodin and GABA, whether used alone or in combination, can bidirectionally regulate the stress response along the HPA axis, from upstream (ACTH secretion) to downstream (CORT secretion). The regulatory effect of the combination therapy is more significant, and the regulatory effects are particularly prominent in the high-dose combination group and the high-dose gastrodin combined with medium and low-dose GABA groups. This is consistent with the mechanism by which the two drugs synergistically inhibit the overactivation of the HPA axis. In addition, the significant downregulation of CORT concentration in each treatment group further illustrates that it can effectively alleviate stress damage in model mice, providing more comprehensive experimental evidence for the use of this combination therapy in anti-stress treatment, and laying the foundation for further in-depth research into its specific molecular mechanism of regulating the HPA axis.

[0047] (6) Pathological section analysis of brain tissue After euthanizing the mice by cervical dislocation, the lower left quarter of the brain and liver tissue were taken and fixed in 10% paraformaldehyde. The tissue was then dehydrated, trimmed, embedded, sectioned, stained, and mounted according to the standard operating procedure (SOP) for pathological examination. The tissue was then examined under a microscope and images were collected.

[0048] The results are as follows Figure 10 and Figure 11As shown, in the control group (NC), no cell degeneration, necrosis, or glial cell proliferation was observed in the brain tissue. No edema, inflammatory cell infiltration, or other lesions were observed in the medulla. Hippocampal pyramidal cells (black arrows) showed normal morphology and structure, with large, round nuclei and tightly packed cells, without obvious pathological changes. In the model group (MOD), pyramidal cells in the CA1 region of the hippocampus were sparsely and disordered, with a reduced number, and occasional pyknosis, deep staining, and necrosis of pyramidal cell nuclei were observed (blue arrows). Occasionally, vacuolar degeneration of neurons was observed in the surrounding area (green arrows), indicating that the model group caused some damage to the brain. In the positive group (DZP), brain tissue returned to normal after administration. Hippocampal pyramidal cells (black arrows) showed normal morphology and structure, with large, round nuclei and tightly packed cells, without obvious pathological changes. In the low-dose gastrodin-only group (GAS-L), occasional pyknosis, deep staining, and necrosis of individual pyramidal cell nuclei were observed in the CA1 region of the hippocampus (blue arrows). Occasionally, vacuolar degeneration of neurons was observed in the surrounding area (green arrows). In the medium-dose gastrodin group (GAS-M), occasional pyknosis, deep staining, and necrosis of pyramidal cell nuclei were observed in the CA2 and CA3 regions of the hippocampus (blue arrows). Vacuole degeneration was also observed in a small number of surrounding neurons (green arrows). In the high-dose gastrodin group (GAS-H), a small number of pyramidal cell nuclei showed pyknosis, deep staining, and necrosis in the CA2 region of the hippocampus (blue arrows). Vacuole degeneration of surrounding neurons was also occasionally observed (green arrows). In the low-dose γ-aminobutyric acid (GABA-L) group, a small number of pyramidal cell nuclei showed pyknosis, deep staining, and necrosis in the CA2 region of the hippocampus (blue arrows). No other pathological changes were observed. In the medium-dose γ-aminobutyric acid (GABA-M) group, numerous pyramidal cell nuclei showed pyknosis, deep staining, and necrosis in the CA1 region of the hippocampus (blue arrows), with disordered arrangement. In the high-dose γ-aminobutyric acid (GABA-H) group, the hippocampal pyramidal cells (black arrows) had normal morphology and structure, large and round nuclei, and were tightly arranged without obvious pathological changes. Vacuole degeneration of surrounding neurons was occasionally observed (green arrows). In the low-dose combined therapy group (GAS-GABA-L), a small number of pyramidal cells in the CA3 region of the hippocampus showed vacuolar degeneration (green arrows). No other significant pathological changes were observed. In the medium / high-dose combined therapy group (GAS-GABA-M / H), brain tissue returned to normal. The hippocampal pyramidal cells (black arrows) had normal morphology and structure, with large, round nuclei and tightly packed cells; the brain tissue condition was similar to that of the control group.

[0049] In summary: The low, medium, and high dose groups of Gastrodia elata extract alone failed to completely reverse brain damage. Pyramidal cell nuclear condensation, necrosis, and neuronal vacuolar degeneration were still observed in different hippocampal regions. Only slight differences in pathological damage manifestations were observed with varying doses, showing some overall damage relief, but the improvement was limited and did not reach the normal levels of the positive control group and the blank control group. The effect of γ-aminobutyric acid (GABA) alone showed a clear dose-dependent effect; low and medium doses had no significant repair effect, while high doses showed significant improvement. The combined treatment showed a significant synergistic repair effect, with overall efficacy superior to either drug alone. In the low-dose combined group, only a small amount of neuronal vacuolar degeneration was observed in the CA3 region of the hippocampus, without serious lesions such as cell necrosis; the degree of damage was far lower than that of the single-drug groups at the same dose. In the medium and high-dose groups, the brain tissue and hippocampal pyramidal cell morphology and arrangement completely returned to normal, pathological changes completely disappeared, and the brain tissue structure was basically consistent with the blank control group, with repair effects comparable to the positive control group.

[0050] Regarding liver tissue: In the blank control group (NC), hepatocytes (black arrows) are arranged radially outward from the central vein (black triangle) of the liver lobules, forming hepatic plates. The connective tissue area between adjacent liver lobules is called the portal tract (green box), which contains interlobular bile ducts, interlobular arteries, and interlobular veins. No degeneration or necrosis of hepatocytes was observed, and no pathological changes such as inflammatory cell infiltration were observed in the sinusoids or portal tract. In the model group (MOD), more inflammatory cell infiltration was observed around the central vein and portal tract (blue arrows), and focal hepatocyte necrosis with surrounding inflammatory cell infiltration was observed in most liver lobules (red arrows). In the positive control group (DZP), the liver tissue structure returned to normal, and hepatocytes (black arrows) within the liver lobules were arranged radially outward to form hepatic plates. No inflammatory cell infiltration was observed around the portal tract and central vein, and no pathological changes such as degeneration or necrosis of hepatocytes were observed. In the low-dose group (GAS-L) of gastrodin alone, a small amount of inflammatory cell infiltration was observed around the central vein and portal area of ​​the liver (blue arrows), and focal hepatocellular necrosis with peripheral inflammatory cell infiltration was observed in some liver lobules (red arrows). In the medium-dose group (GAS-M), a small amount of inflammatory cell infiltration was observed around the portal area of ​​the liver (blue arrows), and focal hepatocellular necrosis with peripheral inflammatory cell infiltration was observed in some liver lobules (red arrows). In the high-dose group (GAS-H), no obvious abnormalities were observed around the portal area and central vein of the liver, and focal hepatocellular necrosis with peripheral inflammatory cell infiltration was observed in a small number of liver lobules (red arrows). After administration of γ-aminobutyric acid (GABA) alone, a large amount of inflammatory cell infiltration was observed around the portal area of ​​the liver in the low-dose group (GABA-L) (blue arrows), and focal hepatocellular necrosis with peripheral inflammatory cell infiltration was observed in some liver lobules (red arrows). In the medium-dose group (GABA-M), no obvious abnormalities were observed in the portal area and around the central vein of the liver, with focal hepatocellular necrosis accompanied by peripheral inflammatory cell infiltration in a small number of liver lobules (red arrows). In the high-dose group (GABA-H), only a small number of liver lobules showed focal hepatocellular necrosis accompanied by peripheral inflammatory cell infiltration (red arrows). After combined administration, a small amount of inflammatory cell infiltration was observed around the portal area of ​​the liver in the low-dose group (GAS-GABA-L) (blue arrows), and no pathological changes such as degeneration or necrosis of hepatocellular cells were observed. In the medium-dose group (GAS-GABA-M) and the high-dose group (GAS-GABA-H), the liver tissue structure was normal, and no degeneration or necrosis of hepatocellular cells (black arrows) was observed. No pathological changes such as inflammatory cell infiltration were observed in the sinusoids, portal area, and around the central vein.

[0051] In summary, gastrodin alone (GAS) showed a dose-dependent protective effect against PCPA-induced liver injury in mice: low and medium dose groups only slightly alleviated inflammation, with a small amount of inflammatory cell infiltration remaining in the portal area and residual focal hepatocyte necrosis; the high dose group essentially eliminated perivascular inflammatory infiltration, leaving only a small area of ​​focal hepatocyte necrosis, and its hepatoprotective effect was significantly better than that of low and medium doses. γ-aminobutyric acid alone (GABA) also showed a dose-dependent effect: the degree of inflammatory infiltration and hepatocyte necrosis in the low dose group was similar to that in the model group, with a weak improvement effect; the medium dose group eliminated perivascular inflammation, leaving only a small amount of focal necrosis in the liver lobules; the high dose group further reduced the extent of necrosis, with only sporadic focal hepatocyte necrosis and no perivascular inflammation. The combined administration (GAS-GABA) showed outstanding synergistic liver protection effect, and the higher the dose, the better the repair effect: the low-dose combined group had only a small amount of inflammatory infiltration in the portal area, and the hepatocytes had no degeneration or necrosis, which significantly reduced the damage compared with the two single-drug low-dose groups; the liver structure of the medium and high-dose combined groups was completely consistent with the blank group and the positive drug group, the hepatocyte morphology was intact, and there was no inflammatory infiltration or cell necrosis in the hepatic sinusoids, central vein and portal area, achieving complete repair of liver pathological damage, and the synergistic effect was clear.

[0052] (7) Immunofluorescence assay was used to detect the expression of neurotrophic factors PSD95 and BDNF in the brain tissue of PCPA-induced insomnia mice.

[0053] The results are as follows Figures 12-14 As shown, compared with the normal control group, the number of BDNF-positive cells, PSD95-positive cells, and BDNF / PSD95 co-localization-positive cells in the hippocampus of the model group mice were significantly reduced. P <0.05) indicates that modeling can cause hippocampal neuronal damage, synaptic structure disruption, and decreased synaptic plasticity. After drug intervention, the levels of the three indicators in the positive drug DZP group significantly recovered. Under monotherapy conditions, gastrodin showed a certain dose-response trend, and the number of BDNF-positive cells in the GAS-H group was significantly higher than that in the GAS-L and GAS-M groups ( P <0.01), and the number of PSD95 positive cells in the GAS-H group was significantly increased ( P <0.05); the effects of each dose of GABA monomer on BDNF, PSD95 positive cells and their co-localization levels were limited, and there were no statistically significant differences in any indicators among the GABA-L, GABA-M, and GABA-H groups. P >0.05). Compared with single administration, gastrodin combined with γ-aminobutyric acid showed more significant improvement, with the number of BDNF-positive cells in the GAS-GABA-M group being significantly higher than that in other combination groups ( P <0.01), and the number of PSD95 positive cells in this group was significantly higher than that in the GAS-GABA-L group ( P<0.01), the number of BDNF / PSD95 co-localized positive cells was significantly higher than that in the GAS-GABA-H group ( P <0.05). There were no significant differences in any indicators among the different combinations of GASM-GABAH, GASH-GABAM, and GASH-GABAL. P >0.05), the overall improvement effect was weaker than that of the fixed-dose compound group. The above results fully demonstrate that the combination of gastrodin and GABA has a synergistic neuroprotective effect; the combination of the two can not only promote the expression of BDNF in hippocampal neurons and maintain the expression level of synaptic marker protein PSD95, but also promote the enrichment of BDNF in the synaptic region, enhance the regulatory ability of BDNF on synaptic plasticity, and thus repair the damaged hippocampal synaptic structure, with the medium-dose compound (GAS-GABA-M) showing the best effect. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative intent, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of Gastrodia elata extract in combination with γ-aminobutyric acid in the preparation of products for sedation, tranquilization and / or memory improvement, wherein the Gastrodia elata extract includes an aqueous extract of Gastrodia elata.

2. The application according to claim 1, characterized in that, The improvement in memory refers to the improvement of memory impairment caused by chlorophenylalanine.

3. The application according to claim 2, characterized in that, The improvement of chlorophenylalanine-induced memory impairment includes one or more of the following: (1) Improves the decrease in 5-hydroxytryptamine content in brain tissue caused by chlorophenylalanine; (2) Improves the increase in norepinephrine levels caused by chlorophenylalanine; (3) Improves the increase in serum adrenocorticotropic hormone caused by chlorophenylalanine; (4) Improves the increase in serum cortisol concentration caused by chlorophenylalanine; (5) It improves the reduced expression levels of BDNF and / or PSD95 proteins in brain tissue caused by chlorophenylalanine, and promotes synaptic plasticity repair; (6) Improves brain tissue damage caused by chlorophenylalanine; (7) Improves liver tissue damage caused by chlorophenylalanine.

4. Application of Gastrodia elata extract combined with γ-aminobutyric acid in the preparation of products that regulate HPA axis function.

5. The application according to any one of claims 1 to 4, characterized in that, The mass ratio of Gastrodia elata extract to γ-aminobutyric acid in the product is (0.15~0.60):(10~40), and the mass of Gastrodia elata extract is based on the mass of gastrodin.

6. The application according to claim 5, characterized in that, The mass ratio of Gastrodia elata extract to γ-aminobutyric acid in the product is (0.30~0.60):

40.

7. The application according to claim 5, characterized in that, The concentration of gastrodin in the product is 0.15~6.0 mg / mL.

8. The application according to claim 5, characterized in that, The products include pharmaceuticals, food, and health supplements.

9. The application according to claim 5, characterized in that, The preparation method of the Gastrodia elata extract includes: mixing Gastrodia elata and water, heating and refluxing for extraction, collecting the extract and concentrating it to obtain the Gastrodia elata extract.

10. A product for calming the nerves and / or improving memory, characterized in that, The active ingredients of the product include Gastrodia elata extract and γ-aminobutyric acid, wherein the Gastrodia elata extract includes Gastrodia elata aqueous extract; The mass ratio of Gastrodia elata extract to γ-aminobutyric acid in the product is (0.15~0.60):(10~40), and the mass of Gastrodia elata extract is based on the mass of gastrodin.

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