A method for identifying the effectiveness of Gastrodia elata extract in preventing and treating Parkinson's disease

Abstract

The present invention discloses a method for identifying the effectiveness of Gastrodia elata extract in preventing and treating Parkinson's disease. The present invention uses an animal model of Parkinson's disease to study the effect of Gastrodia elata extract on motor impairment and its mechanism of action, and combines non-targeted metabolomics analysis to explore potential biomarkers. The experimental results show that Gastrodia elata can be an ideal candidate for developing drugs, health products or functional foods for preventing and treating Parkinson's disease. In addition, the present invention uses metabolomics technology in combination with behavioral indicators and biochemical indicators, starting from the expression of inflammatory factors, neurotransmitters, proteins and metabolites and metabolic regulation, and through objective quantitative and qualitative analysis of symptoms, provides a new idea for evaluating the effectiveness of Gastrodia elata extract in preventing and treating Parkinson's disease. Based on this, the present invention also provides a method for regulating abnormal metabolites of cells in vitro.

Description

Technical Field

[0001] The present invention relates to the field of effectiveness identification of Gastrodia elata extracts, and particularly to a method for identifying the effectiveness of Gastrodia elata extracts in preventing and treating Parkinson's disease. Background Art

[0002] Parkinson's disease (PD) is a chronic and progressive neurodegenerative disease, which is more common in middle-aged and elderly people and seriously endangers the physical and mental health of patients. Its main clinical manifestations include a series of extrapyramidal movement disorders such as resting tremor, bradykinesia, and abnormal postural gait, and non-motor symptoms such as depression, anxiety, insomnia, and autonomic dysfunction can also occur. The etiology of PD is complex, and the main pathological changes are the degeneration of dopamine (DA)-ergic neurons in the substantia nigra pars compacta (SNpc) of the midbrain and the deposition of misfolded α-synuclein (α-Syn), which leads to the formation of Lewy bodies (LB). At present, the treatment of PD mainly relies on drug therapy, including levodopa (L-dopa), central anticholinergics, catechol-O-methyltransferase inhibitors, etc. These drugs can effectively relieve the symptoms of PD, but long-term use will cause many adverse reactions such as gastrointestinal reactions and on-off phenomena. Therefore, there is an urgent need to develop safe, effective, and less adverse reaction drugs and related products for the prevention and treatment of PD. Since modern times, many medical practitioners have based on classics and rooted in clinical practice, accumulating a lot of experience in the treatment of PD. Traditional Chinese medicine has a long history in the treatment of PD, and traditional Chinese medicine has the pharmacological characteristics of multiple components, multiple pathways, and multiple targets, which can provide new strategies and ideas for the treatment of PD.

[0003] On the other hand, innovative drugs, especially innovative traditional Chinese medicine drugs, include new drugs of active ingredients and new drugs of effective parts. Among them, the screening of candidate drugs is a key step in the development of innovative traditional Chinese medicine drugs. However, in the process of screening candidate drugs using experimental animals, there is still a lack of effective detection indicators. How to effectively detect or identify the effectiveness or efficacy of candidate drugs in preventing and treating Parkinson's disease is still one of the problems in the difficult screening of candidate drugs.

[0004] The identification technology of biological effects of traditional Chinese medicine is the product of the cross-development of quantitative pharmacology and traditional Chinese medicine identification. The identification of biological effects of traditional Chinese medicine is based on biological effects, with biostatistics as a tool. Using specific experimental designs, it compares the specific responses (changes in measurable and quantifiable physiological indicators or biological characteristics) of organisms to different test samples under certain conditions to determine the presence or absence and intensity differences of the biological activities (pharmacodynamic activities or toxicities) of traditional Chinese medicine, and uses this as one of the bases for identifying traditional Chinese medicine. The identification of biological effects of traditional Chinese medicine is based on the effectiveness of traditional Chinese medicine. Therefore, the identification of biological effects of traditional Chinese medicine is an optimal method for evaluating the quality of traditional Chinese medicine. Currently, there are few reports on the identification methods for the effectiveness of Gastrodia elata extract in preventing and treating Parkinson's disease.

[0005] In addition, traditional Chinese medicinal materials contain numerous secondary metabolites, and each secondary metabolite may also have multiple biological activities, making its overall biological activity complex and difficult to accurately describe. Therefore, the descriptions of the properties and flavors of each medicinal material in traditional Chinese medicine classics are abstract and vague. In clinical applications, doctors often need to make differential diagnoses based on experience, which poses an insurmountable obstacle to the commercialization and industrialization of traditional Chinese medicinal materials with a long history of clinical application. How to ensure the optimal clinical efficacy of prescriptions is an even more difficult problem in the clinical pharmacy of traditional Chinese medicine. Therefore, there is still a need for a method for quantitatively describing and standardizing the evaluation of the effectiveness (pharmacodynamic effects) of traditional Chinese medicine for preventing and treating Parkinson's disease in subjects.

[0006] Pharmaceutical science is a scientific system closely centered around clinical medicine. The purpose of all pharmaceutical science practices should be derived from clinical needs and serve clinical practice. This is where the value of pharmaceutical science lies. Unfortunately, the serious disconnection between the quality standards of traditional Chinese medicine and clinical needs has become a common phenomenon in the current quality research of traditional Chinese medicine. It is unreasonable to explain the clinical pharmacodynamic effects or safety of medicinal materials merely by relying on the qualitative or quantitative inspection of one or a few chemical components that account for only a few percent, a few per thousand, or even less in the medicinal materials. Therefore, a quality control system that can be closely linked to the effectiveness of clinical medication is an urgent task for the modernization of traditional Chinese medicine.

[0007] The information in the background art is only for explaining the general background of the present invention and should not be regarded as an admission or an indication in any form that this information constitutes the prior art known to those of ordinary skill in the art. Summary of the Invention

[0008] The pathogenesis of Parkinson's disease is complex. With the emergence and development of metabolomics technology, the systematic research and analysis of small molecule metabolites in biological samples provide new ways for the early diagnosis of diseases and the exploration of pathogenesis. However, how Gastrodia elata extract affects metabolites related to Parkinson's disease is still largely unknown. The purpose of the present invention is to provide a method for identifying the effectiveness of Gastrodia elata extract, and to determine the relationship between metabolites and the therapeutic effect of Gastrodia elata extract through quantitative and qualitative evaluation and analysis of metabolites.

[0009] To achieve the object of the present invention, in a first aspect of the present invention, there is provided an application of gastrodia extract in the preparation of a product for preventing and treating Parkinson's disease in a subject, wherein the gastrodia extract is prepared by a method comprising the following steps:

[0010] Take dry gastrodia, cut it into thin slices, soak it in ethanol overnight, repeatedly extract it with boiling ethanol, and obtain it through filtration, concentration, drying, and pulverization.

[0011] In certain embodiments, according to the application of the present invention, wherein the product comprises at least one of functional foods, health products, and pharmaceuticals.

[0012] In certain embodiments, according to the application of the present invention, wherein the dosage form of the product comprises at least one of the types such as tablets, granules, capsules, compressed tablets, solid beverages, functional teas, extracts, and infusions.

[0013] In certain embodiments, according to the application of the present invention, wherein the subject comprises an animal model, and the prevention and treatment comprise regulating one of the following:

[0014] Improve the propulsion index in gait analysis of MPTP model mice;

[0015] Improve the stance phase in gait analysis of MPTP model mice;

[0016] Shorten the pole climbing time of MPTP model mice;

[0017] Improve the hanging score of MPTP model mice;

[0018] Reduce the expression level of inflammatory factors in the striatum of MPTP model mice, and the inflammatory factors include TNF-α, IL-6, and IL-1β;

[0019] Reduce the expression levels of apoptotic proteins in the striatum of MPTP model mice, including Caspase-3 and Caspase-9 proteins;

[0020] Improve the metabolic disorders in the striatum of MPTP model mice;

[0021] Improve the glycerophospholipid metabolism, sphingolipid metabolism, tyrosine metabolism, arginine and proline metabolism, and arachidonic acid metabolism pathways of MPTP model mice;

[0022] Improve the motor injury of 6-OHDA model rats in the open field test;

[0023] Improve the contents of DA and DOPAC in the striatum of 6-OHDA model rats;

[0024] Reduce the protein expression levels of TLR4 and NF-κB in the striatum of 6-OHDA model rats.

[0025] In a second aspect of the present invention, there is provided a method for identifying the effectiveness of gastrodia extract in preventing and treating Parkinson's disease, which comprises:

[0026] (1) Construct an animal model of Parkinson's disease;

[0027] (2) Measure the behavioral indices, biochemical indices and metabolomics indices of the said model;

[0028] (3) Measure the behavioral indices, biochemical indices and metabolomics indices of the said model after administration of gastrodia extract;

[0029] Among them, the said behavioral indices include at least one of the propulsion index in gait analysis of model animals, the stance phase in gait analysis, the pole climbing time, the hanging score, and the movement impairment in the open field experiment. The said biochemical indices include the expression levels of striatal inflammatory factors, the expression levels of apoptotic proteins in the striatum, the content of dopamine (DA) and / or dihydroxyphenylacetic acid (DOPAC) in the striatum, and the protein expression levels of TLR4 and / or NF-κB in the striatum. The said metabolomics indices include 4'-hydroxyflurbiprofen, L-prolylglycine, hawkinsin, phosphatidylserine, phenylalanine-phenylalanine-lysine, 3,3'-thiodipropionic acid, 2-carboxy-4-dodecanol, androstenol, retamide A, Ent-6,16-quinolinadiene-19-oic acid, pantethol 2, 3-ethyl-5-methoxyphenol, DL-3,4-dihydroxyphenethyl alcohol, 3-(6-hydroxy-2,3,4-trimethoxyphenyl)prop-2-enoic acid, dihydroxyphenylacetic acid, 3,4-dihydroxyphenethyl alcohol, 2-hydroxy-3-(2,4,5-trihydroxyphenyl)propanoic acid, 7-aminonitrazepam, serine-leucine-serine-glycine-leucine, 2''-O-trans-p-coumarate, 4-hydroxyphenylacetylglutamic acid, 1,2-dehydrosalt octanol, 5-phosphothiophene adenosine monophosphate, (S)-C(S)S-methionine sulfoxide, (3E,6Z)-non-2,4-dien-1-yl acetate, dexapanthenol, 3-O-isopropylsulfate isoprenaline.

[0030] In certain embodiments, the method for identifying the effectiveness of gastrodia extract in preventing and treating Parkinson's disease according to the present invention, wherein the animal model is a Parkinson's disease animal model induced by MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine) or 6-OHDA. In a specific embodiment, the Parkinson's disease model is a mouse Parkinson's disease model induced by MPTP. In a specific embodiment, the Parkinson's disease model is a rat Parkinson's disease model induced by 6-OHDA.

[0031] In certain embodiments, the method for identifying the effectiveness of gastrodia extract in preventing and treating Parkinson's disease according to the present invention, wherein the inflammatory factors include TNF-α, IL-6 and / or IL-1β, and the apoptotic proteins include Caspase-3 and / or Caspase-9 proteins.

[0032] In certain embodiments, the method for identifying the effectiveness of gastrodia extract in preventing and treating Parkinson's disease according to the present invention, wherein the effectiveness refers to improving the propulsion index in animal gait analysis, the stance phase in gait analysis, shortening the pole climbing time, improving the animal hanging score, improving the motor impairment in the open field test, and reducing the expression level of inflammatory factors in the striatum, the expression level of apoptotic proteins, improving the content of dopamine and / or dihydroxyphenylacetic acid in the striatum, reducing the expression level of TLR4 and / or NF-κB proteins in the striatum, and restoring the level of metabolites.

[0033] In certain embodiments, the method for identifying the effectiveness of gastrodia extract in preventing and treating Parkinson's disease according to the present invention, wherein the restoration of the metabolite level includes: upregulating 4'-hydroxyflurbiprofen, L-prolylglycine, hawkinsin, phosphatidylserine, 3,3'-thiodipropionic acid, 2-carboxy-4-dodecanol, androstenol, pantethine 2, 3-ethyl-5-methoxyphenol, DL-3,4-dihydroxyphenethyl alcohol, 3-(6-hydroxy-2,3,4-trimethoxyphenyl)prop-2-enoic acid, dihydroxyphenylacetic acid, 3,4-dihydroxyphenethyl alcohol, 2-hydroxy-3-(2,4,5-trihydroxyphenyl)propionic acid, 7-aminonitrazepam, 4-hydroxybenzoylglutamic acid, 1,2-dehydrosalt octanol, (S)-C(S)S-methionine sulfoxide, (3E,6Z)-nona-2,4-dien-1-yl acetate, dexpanthenol, 3-O-isopropylsulfate isoprenaline, and downregulating phenylalanine-phenylalanine-lysine, curvulamine A, Ent-6,16-quinoline diene-19-oic acid, serine-leucine-serine-glycine-leucine, 2''-O-trans-p-coumarin, 5-phosphothienyladenosine monophosphate.

[0034] In a third aspect of the present invention, there is provided a non-disease diagnosis and treatment method for in vitro regulating abnormal metabolites of cells, which includes the step of culturing gastrodia extract identified by the method according to the present invention with cells in vitro. The regulation of abnormal metabolites of cells includes: up-regulating 4'-hydroxyflurbiprofen, L-prolylglycine, hodgkinsin, phosphatidylserine, 3,3'-thiodipropionic acid, 2-carboxy-4-dodecanol, androsteneol, pantethine 2, 3-ethyl-5-methoxyphenol, DL-3,4-dihydroxyphenethyl alcohol, 3-(6-hydroxy-2,3,4-trimethoxyphenyl)prop-2-enoic acid, dihydroxybenzeneacetic acid, 3,4-dihydroxyphenethyl alcohol, 2-hydroxy-3-(2,4,5-trihydroxyphenyl)propionic acid, 7-aminonitrazepam, 4-hydroxybenzoylglutamic acid, 1,2-dehydrosalt octanol, (S)-C(S)S-methionine sulfoxide, (3E,6Z)-nona-2,4-dien-1-yl acetate, dexapanthenol, 3-O-sulfate isoprenaline, and down-regulating phenylalanine-phenylalanine-lysine, curvulamine A, Ent-6,16-quinoline diene-19-oic acid, serine-leucine-serine-glycine-leucine, 2''-O-trans-p-coumarin, 5-phosphothienyladenosine monophosphate.

[0035] The present invention has at least the following advantages and beneficial effects:

[0036] (1) The present invention provides a method for identifying the effectiveness of gastrodia extract, providing a theoretical basis for the development and application of gastrodia extract in traditional Chinese medicine for the prevention and treatment of Parkinson's disease, health products or functional foods.

[0037] (2) The present invention uses MPTP-induced subacute Parkinson's disease mice as a model for research, demonstrating that gastrodia extract can improve the motor function impairment of MPTP-induced subacute Parkinson's disease model mice, specifically manifested as improving gait function and spontaneous activity ability, reducing the expression of inflammatory factors and apoptotic proteins in the striatum, and having a protective effect on MPTP-induced neuroinflammatory response, providing a theoretical basis for the treatment of Parkinson's disease with traditional Chinese medicine.

[0038] (3) The present invention determines the differential metabolites of gastrodia extract for the prevention and treatment of Parkinson's disease through metabolomics analysis. First, the metabolic data of Parkinson's mice are obtained through non-targeted metabolomics analysis. Subsequently, through PCA analysis, it is found that the effect of gastrodia extract on mouse metabolites is significantly different from that of the model group, and the differential metabolites are further identified. The regulatory effect of the gastrodia intervention group on the differential metabolites is evaluated by comparing the relative abundances of the differential metabolites in different groups. The results show that after the intervention of gastrodia extract, the contents of 27 differential metabolites are significantly reversed. Finally, through KEGG enrichment analysis, the metabolic pathways most relevant to Parkinson's disease are further screened out.

[0039] (4) The present invention uses 6-OHDA-induced Parkinson's disease rats as a model for research, and proves that Gastrodia elata extract can increase the content of neurotransmitter levels in the striatum of Parkinson's disease model rats, down-regulate the expression of apoptotic proteins, and reduce the occurrence of apoptosis, providing a theoretical basis and data support for the treatment of Parkinson's disease with Gastrodia elata.

[0040] The present invention combines metabolomics technology with behavioral and biochemical indexes, starting from the perspectives of the expression of inflammatory factors, neurotransmitters, proteins and metabolites, as well as metabolic regulation, and provides new ideas for the evaluation of the effectiveness of Gastrodia elata extract in preventing and treating Parkinson's disease through the objective quantification of symptoms, thereby promoting the modernization process of traditional Chinese medicine theory. Brief Description of the Drawings

[0041] Figure 1 Effects of Gastrodia elata extract on the propulsion index in gait analysis of MPTP model mice (mean ± SEM, n = 9 - 10). Compared with the control group, ** p < 0.01, *** p < 0.001; compared with the model group, # p < 0.05, ## p < 0.01, p < 0.001.

[0042] Figure 2 Effects of Gastrodia elata extract on the stance phase in gait analysis of MPTP model mice (mean ± SEM, n = 9 - 10). Compared with the control group, ** p < 0.01, *** p < 0.001; compared with the model group, # p < 0.05, ## p < 0.01, p < 0.001.

[0043] Figure 3 Effects of Gastrodia elata extract on the pole climbing test of MPTP model mice (mean ± SEM, n = 9 - 10). Compared with the control group, ** p < 0.01, *** p < 0.001; compared with the model group, # p < 0.05, ## p < 0.01, p < 0.001.

[0044] Figure 4 Effects of Gastrodia elata extract on the hanging test of MPTP model mice. (mean ± SEM, n = 9 - 10). Compared with the control group, ** p < 0.01, *** p < 0.001; compared with the model group, # p < 0.05, ## p < 0.01, p < 0.001.

[0045] Figure 5 Effects of Gastrodia elata extract on the open field test of MPTP model mice (mean ± SEM, n = 9 - 10). Compared with the control group, * p < 0.05.

[0046] Figure 6Effects of Gastrodia elata extract on the contents of TNF-α, IL-6 and IL-1β in the striatum of MPTP model mice. Compared with the control group, ***p<0.001; compared with the model group, #p<0.05, ##p<0.01.

[0047] Figure 7 Effects of Gastrodia elata extract on the protein expressions of Caspase-3 and Caspase-9 in the striatum of MPTP model mice (mean ± SEM, n=9-10). Compared with the control group, ** p<0.01, *** p<0.001; compared with the model group, # p<0.05, ## p<0.01, p<0.001.

[0048] Figure 8 PCA score plot of metabolite analysis in the striatum of MPTP model mice.

[0049] Figure 9 Effects of Gastrodia elata extract on the metabolites in the striatum of MPTP model mice analyzed by OPLS-DA. A: OPLS-DA score plot of the metabolic profile of the striatum in MPTP mice; B: Validation plot of the OPLS-DA model; C: S-plot of the striatum in MPTP mice.

[0050] Figure 10 Volcano plot analysis of differential metabolite expression.

[0051] Figure 11 After the intervention of Gastrodia elata extract, the contents of 27 differential metabolites were significantly reversed.

[0052] Figure 12 Enrichment map of different metabolite metabolic pathways in the striatum of MPTP model mice.

[0053] Figure 13 Effects of Gastrodia elata extract on 6-OHDA model rats in the open field test. Compared with the control group, *p<0.05, **p<0.01; compared with the model group, #p<0.05, ##p<0.01.

[0054] Figure 14 Effects of Gastrodia elata extract on 6-OHDA model rats in the APO-induced rotation test. Compared with the model group, ##p<0.01.

[0055] Figure 15 Effects of Gastrodia elata extract on the DA content in the striatum of 6-OHDA model rats. Compared with the sham operation control group, ***p<0.001; compared with the model group, #p<0.05, ##p<0.01.

[0056] Figure 16Effect of Gastrodia elata extract on the content of DOPAC in the striatum of 6-OHDA model rats. Compared with the sham operation group and the control group, ***p < 0.001; compared with the model group, #p < 0.05.

[0057] Figure 17 Effect of Gastrodia elata extract on the expression of TLR4 and NF-κB in the striatum of 6-OHDA rats. Compared with the control group, **p < 0.01; compared with the model group, #p < 0.05; ##p < 0.01. Detailed implementation manners

[0058] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0059] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that the upper and lower limits of the range and each intermediate value therebetween are specifically disclosed. Each intermediate value within any stated value or stated range and each smaller range between any other stated value or intermediate value within the stated range are also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0060] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0061] Example 1

[0062] This example shows the identification of the components of Gastrodia elata extract by UHPLC-QE-MS.

[0063] 1. Materials and methods

[0064] 1.1 Preparation of the extract

[0065] The Gastrodia elata medicinal materials were purchased from Sichuan Chijian Traditional Chinese Medicine Technology Co., Ltd. The main steps and methods for preparing the Gastrodia elata extract: Cut the dried Gastrodia elata into thin slices and soak them in 85% ethanol overnight. Repeatedly extract with boiling 85% ethanol three times. After filtration, concentrate, dry and pulverize to obtain a powder, which is the Gastrodia elata extract.

[0066] 1.2 Identification of the components of the extract

[0067] The chemical constituents in Gastrodia elata extract were identified by UHPLC-QE-MS. Briefly, an appropriate amount of the sample was accurately weighed into a 2 mL centrifuge tube, 600 μL of methanol containing 2-chloro-L-phenylalanine (4 ppm) was added, vortexed for 30 s, centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was filtered through a 0.22 μm membrane for LC-MS detection. The column temperature was 40°C. The injection volume was 2 μL. In the positive ion mode, the mobile phase was acetonitrile with 0.1% formic acid (B2) and water with 0.1% formic acid (A2), and the gradient elution program was: 0 - 1 min, 8% B2; 1 - 8 min, 8% - 98% B2; 8 - 10 min, 98% B2; 10 - 10.1 min, 98% - 8% B2; 10.1 - 12 min, 8% B2. In the negative ion mode, the mobile phase was acetonitrile (B3) and 5 mM ammonium formate in water (A3), and the gradient elution program was: 0 - 1 min, 8% B3; 1 - 8 min, 8% - 98% B3; 8 - 10 min, 98% B3; 10 - 10.1 min, 98% - 8% B3; 10.1 - 12 min, 8% B3.

[0068] The Thermo Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, USA) was connected to the UPLC system. The electrospray ionization source (ESI) was used to collect data in positive and negative ion modes respectively. The positive ion spray voltage was 3.50 kV, the negative ion spray voltage was -2.50 kV, the sheath gas was 40 arb, and the auxiliary gas was 10 arb. The capillary temperature was 325°C. The first-level full scan was performed at a resolution of 70000, the first-level ion scan range was m / z 100 - 1000, and HCD was used for second-level fragmentation with a collision energy of 30 eV and a second-level resolution of 17500. The first 3 ions of the collected signals were fragmented, and at the same time, dynamic exclusion was used to remove unnecessary MS / MS information.

[0069] 2. Results

[0070] The Gastrodia elata extract was separated and identified by UHPLC-QE-MS. A total of 402 components were identified in positive and negative ion modes, and the main components are shown in Table 1.

[0071] Table 1 Main components in Gastrodia elata extract

[0072]

[0073] Example 2

[0074] The following shows the study on the effect of Gastrodia elata extract on the motor impairment of mice in a Parkinson's disease model induced by MPTP.

[0075] 1. Materials and Methods

[0076] 1.1 Experimental Animals

[0077] 72 SPF-grade C57BL / 6JNifdc mice, male, 8 - 10 weeks old, weighing 20 - 22 g, were ordered from Beijing Vital River Laboratory Animal Technology Co., Ltd. Feed and pure water were freely provided, and they were maintained under an alternating cycle of 12 h light (8:30 - 20:30) and 12 h darkness (20:30 - 8:30 the next day). The room temperature was 22 - 25 °C, and the humidity was 55 ± 10%. They were acclimatized for 3 days before the experiment.

[0078] 1.2 Experimental Drugs

[0079] The Gastrodia elata medicinal materials were purchased from Sichuan Chijian Traditional Chinese Medicine Technology Co., Ltd. The main steps and methods for preparing the Gastrodia elata extract: The dried Gastrodia elata was cut into thin slices and soaked in 85% ethanol overnight. It was repeatedly extracted 3 times with boiling 85% ethanol. After filtration, it was concentrated, dried, and pulverized to obtain the powder, which was the Gastrodia elata extract. Madopar (Shanghai Roche Pharmaceuticals Ltd.), MPTP (Sigma-Aldrich, USA).

[0080] 1.3 Main Experimental Instruments

[0081] Real-time detection device for the spontaneous activity of mice (jointly developed by the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing Kangsen Yiyou Technology Co., Ltd., etc.); Computer detection and analysis device for the gait of mice and rats (jointly developed by the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing Xinhai Instrument Technology Co., Ltd., etc.); AL104 electronic analytical balance (Shanghai Mettler-Toledo Instruments); Self-made mouse pole climbing instrument, etc.

[0082] 1.4 Experimental Methods

[0083] 1.4.1 Grouping

[0084] The mice were randomly divided into 6 groups: control group, model group, Madopar group (112.5 mg / kg), low-dose Gastrodia elata group (100 mg / kg), medium-dose Gastrodia elata group (200 mg / kg), and high-dose Gastrodia elata group (300 mg / kg), with 12 mice in each group.

[0085] (1) Control group: Gastric gavage with normal saline once a day;

[0086] (2) Model group: Intraperitoneal injection of MPTP (30 mg / kg) at a fixed time every day for 7 consecutive days;

[0087] (3) Positive drug group: Administer Madopar (112.5 mg / kg) by gavage once a day for 14 days before modeling, continuing until the end of the experiment;

[0088] (4) Low-dose Gastrodia elata: Administer Gastrodia elata extract (100 mg / kg) by gavage once a day for 14 days before modeling, continuing until the end of the experiment;

[0089] (5) Medium-dose Gastrodia elata: Administer Gastrodia elata extract (200 mg / kg) by gavage once a day for 14 days before modeling, continuing until the end of the experiment;

[0090] (6) High-dose Gastrodia elata: Administer Gastrodia elata extract (300 mg / kg) by gavage once a day for 14 days before modeling, continuing until the end of the experiment.

[0091] 1.4.2 Gait analysis experiment

[0092] Dip the mouse's feet in water, let the mouse continuously pass through the treadmill, and automatically record the video with a camera. After the collection is completed, use computer analysis software to analyze the recorded video.

[0093] 1.4.3 Pole climbing experiment

[0094] Place each mouse head-up on the self-made pole for the experiment. When the mouse's two front limbs touch the bottom platform of the pole, it is considered to have climbed the full length. Record the following times: the turning time of the mouse from head-up to head-down, the time the mouse spends in the upper half of the pole, the time the mouse spends in the lower half of the pole, and the total time to climb the entire pole.

[0095] 1.4.4 Hanging experiment

[0096] Gently place the mouse's front limbs on the rope and observe the mouse. The scoring method is as follows: If the mouse can grasp the hanging rope with all four paws, it is recorded as 3 points; if it grasps the hanging rope with one hind paw, it is recorded as 2 points; if both hind paws cannot grasp the hanging rope, it is 1 point; if the mouse cannot grasp the hanging rope and quickly falls, it is recorded as 0 points.

[0097] 1.4.5 Open field experiment

[0098] Gently place the mouse with its back to the wall of the test box into the test box (30 cm × 30 cm × 35 cm). After the mouse adapts to the environment for 3 minutes, start the detection. The computer automatically monitors and records the spontaneous activity of the mouse in the test box for 10 minutes, and uses a computer analysis system to analyze indicators such as the total distance.

[0099] 1.5 Statistical methods

[0100] The obtained experimental data were statistically analyzed using the statistical software SPSS 21.0, and the data were expressed as mean ± standard error of the mean (mean ± SEM). One-way analysis of variance (one-way ANOVA) was used to compare the differences between groups. The significance level was P < 0.01 or P < 0.05.

[0101] 2. Results

[0102] 2.1 Effect of Gastrodia elata extract on propulsion index in gait analysis of MPTP model mice

[0103] Parkinson's disease patients often show obvious changes in gait characteristics, such as unstable gait rhythm, narrowed stride length, and unstable posture. Gait analysis experiments can quantify these changes by analyzing gait parameters (such as step length, walking speed, gait cycle, etc.). By quantitatively analyzing the changes in gait characteristics, it provides important tools and methods for the early diagnosis, treatment effect evaluation, and disease monitoring of Parkinson's disease. In gait analysis ( Figure 1 ), compared with the control group, there were significant differences in the left posterior, right anterior, and right posterior propulsion index gait indicators in the model group (p < 0.01, p < 0.001), while there was no significant difference in the left anterior propulsion index (p > 0.05). Compared with the model group, the left posterior and right posterior propulsion indices of the low, medium, and high dose groups of Gastrodia elata extract and the Madopar group in mice were significantly restored (p < 0.001). In the high dose group of Gastrodia elata (300 mg / kg) in mice, there was a certain degree of restoration in the right anterior propulsion index (p < 0.05).

[0104] 2.2 Effect of Gastrodia elata extract on stance phase in gait analysis of MPTP model mice

[0105] As Figure 2 shown, compared with the control group, MPTP intervention could significantly increase the left posterior, right anterior, and right posterior stance phases in mice (p < 0.01), while there was no significant difference in the left anterior stance phase (p > 0.05). Compared with the model group, the left posterior stance phase of the Madopar group and the low, medium, and high dose intervention groups of Gastrodia elata was significantly reduced (p < 0.05, p < 0.01); the right anterior stance phase of the Madopar group and the high dose group of Gastrodia elata was significantly reduced (p < 0.05, p < 0.01).

[0106] 2.3 Effect of Gastrodia elata extract on pole climbing experiment in MPTP model mice

[0107] The main characteristics of Parkinson's disease are the loss of dopaminergic neurons and the decrease in dopamine levels, which affect motor control. The pole climbing experiment can help evaluate the effect of Gastrodia elata extract on the motor function of Parkinson's disease model animals. As Figure 3As shown, compared with the control group, the time of the model group mice in the upper half of the pole climbing and the total time both increased, while after the intervention of gastrodia extract (100, 200, 300 mg / kg), the time of mice in the upper half of the pole climbing could be significantly shortened ( Figure 4 B and D, p < 0.01, p < 0.001). It indicates that gastrodia extract can effectively improve the motor dysfunction in Parkinson's disease.

[0108] 2.4 Effect of gastrodia extract on the hanging score experiment of MPTP model mice

[0109] Parkinson's disease patients often suffer from muscle stiffness and movement difficulties, which lead to obvious impairment of their exercise endurance and muscle strength. The hanging experiment can be used to evaluate the effect of gastrodia extract on the motor function of Parkinson's disease model animals. As Figure 4 shown, compared with the control group, the hanging score of the model group mice decreased significantly (p < 0.05). Compared with the model group, there was a certain increasing trend in the scores of the Madopar group and the gastrodia extract (100, 200, 300 mg / kg) groups, but there was no significant difference (p > 0.05).

[0110] 2.5 Effect of gastrodia extract on the open field experiment of MPTP model mice

[0111] In the study of Parkinson's disease, animal models often show motor characteristics such as slow movement and decreased motor ability, and these characteristics can be observed and evaluated through the exploratory behavior and motor activity level of animals in the open field experiment. As Figure 5 shown, compared with the control group, the total movement distance index of the model group decreased significantly (p < 0.05); compared with the model group, there was no significant difference among the Madopar group and each dose group of gastrodia (p > 0.05).

[0112] Example 3

[0113] The following shows the research on the action mechanism of gastrodia extract on MPTP-induced Parkinson's disease model mice.

[0114] 1. Materials and methods

[0115] 1.1 Experimental animals

[0116] Same as that shown in 1.1 of Example 2.

[0117] 1.2 Experimental reagents

[0118] Mouse Tumor necrosis factor (TNF)-α, Interleukin (IL)-6, and IL-1β enzyme-linked immunosorbent assay (ELISA) kits were all purchased from Shanghai Enzyme-linked Biotechnology Company; PMSF (100 mM), 5× protein loading buffer, developing and fixing reagents, electrophoresis buffer, SDS-PAGE gel preparation kit, phosphorylated protease inhibitor, RIPA lysis buffer, and TBS buffer were all purchased from Servicebio; skim milk powder was purchased from Erie; TWEEN20 was purchased from Solarbio; BCA protein quantification detection kit was purchased from Pulilai; protein Marker was purchased from Therm (Fermentas); PVDF membrane was purchased from Millipore; ECL was purchased from Millipore; secondary antibody HRP goat anti-rabbit was from Servicebio (GB23303); β-actin, primary antibody Cleaved-Caspase-3, and primary antibody Cleaved-Caspase-9 were all purchased from Cell Signaling Technology.

[0119] 1.3 Main experimental instruments

[0120] Tabletop high-speed refrigerated centrifuge (Hunan Xiangli Scientific Instruments); Milli-Q ultrapure water instrument (Millipore, USA); high-speed homogenizer (Shanghai Jingxin); full-automatic microplate reader (BIOTEK Co., Ltd., USA); vertical electrophoresis instrument, transfer electrophoresis instrument (Tanon).

[0121] 1.4 Experimental methods

[0122] 1.4.1 Animal grouping

[0123] According to the results of the previous behavioral experiments, in the subsequent mechanism study, only the high-dose (300 mg / kg) group of gastrodia extract was selected for the experiment. The grouping was as follows: control group, model group, Madopar group (112.5 mg / kg), and high-dose gastrodia (300 mg / kg) group.

[0124] 1.4.2 Sampling

[0125] After the behavioral tests were completed, the whole brain was dissected using 5% chloral hydrate, and hippocampus, striatum, and cortical tissues were collected and stored at -80 °C for later use.

[0126] 1.4.3 Effects of gastrodia extract on the levels of TNF-α, IL-6, and IL-1β in the brain tissues of MPTP model mice

[0127] The striatum and cortical tissues were weighed and homogenized, centrifuged at 12,000 r / min for 20 min, and the supernatant was taken and placed in an EP tube for storage at -80°C. ELISA detection was carried out according to the steps in the kit instruction manual.

[0128] 1.4.4 Effects of Gastrodia elata extract on the protein levels of Caspase-3 and Caspase-9 in the striatum of MPTP model mice

[0129] The striatum tissues of mice were homogenized and lysed, centrifuged, and the supernatant was taken. The protein concentration was determined according to the BCA protein quantification kit. It was denatured by boiling in water for 10 min, 40 μg was loaded, and separated by 10% SDS-PAGE electrophoresis (separating gel at 120 V, stacking gel at 75 V), transferred to PVDF membrane (300 mA, 60 min), blocked with 5% skim milk for 60 min, incubated with primary antibodies (Caspase-3, Caspase-9, 1:1000) at 4°C overnight, incubated with secondary antibody (HRP-labeled goat anti-rabbit, 1:1000) at room temperature for 60 min, washed, and the target bands were detected by ECL method in the darkroom using a fluorescence chemiluminescence gel imaging system, and the gray value of the target band was analyzed by the Image-PRO software processing system.

[0130] 1.5 Statistical methods

[0131] Same as described in 1.5 of Example 2.

[0132] 2. Results

[0133] 2.1 Effects of Gastrodia elata extract on the levels of inflammatory factors in the striatum of MPTP model mice

[0134] The overexpression of inflammatory factors such as TNF-α, IL-6, and IL-1β can promote the damage and death of dopaminergic neurons and accelerate the progression of Parkinson's disease. The ELISA detection results showed ( Figure 6 ), compared with the control group, the levels of inflammatory factors TNF-α, IL-6, and IL-1β in the striatum of model group mice were significantly increased (p < 0.001). Compared with the model group, both Madopar and Gastrodia elata (300 mg / kg) could significantly reduce the levels of inflammatory factors in PD mice (p < 0.05, p < 0.01).

[0135] 2.2 Effects of Gastrodia elata extract on the expression levels of Caspase-3 and Caspase-9 proteins in the striatum of MPTP model mice

[0136] In Parkinson's disease, the apoptosis of dopaminergic neurons is one of the main reasons for disease progression. Apoptosis is a controlled process of cell death, and its regulation involves a series of apoptosis-related proteins, such as the Bcl-2 family, caspases, etc. The results of Western blot showed ( Figure 7 ), compared with the control group, the expression levels of Caspase-3 and Caspase-9 proteins in the model group were significantly increased (p < 0.001); the Madopar group and the Gastrodia elata (300 mg / kg) group could significantly reduce the expression levels of Caspase-3 and Caspase-9 proteins in the striatum of PD mice (p < 0.05, p < 0.01, p < 0.001).

[0137] Example 4

[0138] The following shows the effects of Gastrodia elata extract on the metabolic changes in the striatum of MPTP-induced Parkinson's disease mice revealed by non-targeted metabolomics.

[0139] 1. Materials and Methods

[0140] 1.1 Experimental Animals

[0141] Same as shown in 1.1 of Example 2.

[0142] 1.2 Experimental Reagents

[0143] Chromatographically pure methanol (Fisher), chromatographically pure acetonitrile (Fisher), chromatographically pure formic acid (Fisher), chromatographically pure water (Fisher), chromatographically pure propanol (Fisher).

[0144] 1.3 Main Experimental Instruments

[0145] Ultra-high performance liquid chromatography tandem Fourier transform mass spectrometry UHPLC-Q Exactive HF-X, Thermo Fisher Scientific; HSST3 chromatographic column (100 mm × 2.1 mm, 1.8 µm), Waters, USA; JXDC-20 type nitrogen purging instrument, Shanghai Jingxin Industrial Development Co., Ltd.; LNG-T88 type desktop rapid centrifugal concentrator and dryer, Taicang Huamei Biochemical Instrument Factory; Wonbio-96c type high-throughput tissue homogenizer, Shanghai Wanbai Biotechnology Co., Ltd.; SBL-10TD type ultrasonic cleaner 300W-10L, Ningbo Xinzhi Biotechnology Co., Ltd.; Centrifuge 5430R type high-speed refrigerated centrifuge, Eppendorf, Germany; NewClassic MF MS105DU type electronic balance, Mettler, Switzerland.

[0146] 1.4 Experimental Methods

[0147] 1.4.1 Sample Preparation

[0148] Take 25 mg of striatum samples into a 2 mL centrifuge tube and add a grinding bead with a diameter of 6 mm. Add 400 μL of extraction solution (methanol: water = 4:1 (v:v)) containing 0.02 mg / mL of internal standard (L-2-chlorophenylalanine) for metabolite extraction. Grind the sample solution in a cryogenic tissue grinder for 6 min (-10°C, 50 Hz), and perform low-temperature ultrasonic extraction for 30 min (5°C, 40 kHz). Let the sample stand at -20°C for 30 min, centrifuge for 15 min (4°C, 13000 g), and take the supernatant into an injection vial with an inner cannula for on-machine analysis.

[0149] 1.4.2 LC-MS / MS Analysis

[0150] Chromatographic conditions:

[0151] 2 μL of the sample is separated by an HSS T3 chromatographic column (100 mm × 2.1 mm, 1.8 µm) and then enters the mass spectrometry detection. Mobile phase A is 95% water + 5% acetonitrile (containing 0.1% formic acid), and mobile phase B is 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid). Separation gradient: 0 - 3.5 min, mobile phase B rises from 0% to 24.5% at a flow rate of 0.40 mL / min; 3.5 - 5 min, mobile phase B rises from 24.5% to 65% at a flow rate of 0.4 mL / min; 5 - 5.5 min, mobile phase B rises from 65% to 100% at a flow rate of 0.4 mL / min; 5.5 - 7.4 min, mobile phase B maintains 100% and the flow rate rises from 0.4 mL / min to 0.6 mL / min; 7.4 - 7.6 min, mobile phase B drops from 100% to 51.5% at a flow rate of 0.6 mL / min; 7.6 - 7.8 min, mobile phase B drops from 51.5% to 0% and the flow rate drops from 0.6 mL / min to 0.5 mL / min; 7.8 - 9 min, mobile phase B maintains 0% and the flow rate drops from 0.5 mL / min to 0.4 mL / min; 9 - 10 min, mobile phase B maintains 0% and the flow rate is 0.4 mL / min. The column temperature is 40°C.

[0152] Mass spectrometry conditions:

[0153] The quality spectrum signals of the samples were collected in both positive and negative ion scanning modes, with a mass scanning range of 70 - 1050 m / z. The sheath gas flow rate was 50 psi, the auxiliary gas flow rate was 13 psi, the auxiliary gas heating temperature was 425 °C, the spray voltage in positive ion mode was set at 3500 V, the spray voltage in negative ion mode was set at -3500 V, the ion transfer tube temperature was 325 °C, and the normalized collision energy was 20 - 40 - 60 V cyclic collision energy. The resolution of the first - stage mass spectrum was 60000, and the resolution of the second - stage mass spectrum was 7500. Data were collected using the DDA mode.

[0154] 1.4.3 Data processing

[0155] The LC - MS raw data were imported into the metabolomics processing software Progenesis QI (Waters Corporation, Milford, USA) for baseline filtering, peak identification, integration, retention time correction, and peak alignment. Finally, a data matrix of retention time, mass - to - charge ratio, and peak intensity was obtained. At the same time, the mass spectrometry information was compared with the metabolic public databases HMDB (http: / / www.hmdb.ca / ) and Metlin (https: / / metlin.scripps.edu / ) to obtain metabolite information. To reduce the errors caused by sample preparation and instrument instability, the response intensity of the sample mass spectrum peaks was normalized using the total sum normalization method to obtain a normalized data matrix. At the same time, variables with a relative standard deviation (RSD) > 30% in QC samples were deleted, and log10 logarithmic transformation was performed to obtain the final data matrix for subsequent analysis.

[0156] Differential analysis was performed on the pre - processed matrix file. The R software package ropls (Version1.6.2) was used for principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS - DA), and 7 - cycle cross - validation was used to evaluate the stability of the model. In addition, student’s t - test and fold - change analysis were performed. The selection of differential metabolites was determined based on the variable importance in the projection (VIP) values obtained from the OPLS - DA model and the p - values of the student’s t - test. Metabolites with VIP > 1 and p < 0.05 were differential metabolites. A total of 594 differential metabolites were screened.

[0157] The differential metabolites were annotated for metabolic pathways through the KEGG database (https: / / www.kegg.jp / kegg / pathway.html) to obtain the pathways involved in the differential metabolites. The Python software package scipy.stats was used for pathway enrichment analysis, and the biological pathways most relevant to the experimental treatment were obtained through Fisher's exact test.

[0158] 2. Results

[0159] 2.1 PCA analysis of the effects of Gastrodia elata extract on metabolites in the striatum of MPTP model mice

[0160] As Figure 8 shown in A of Figure 8 , there was a significant separation between the model group (MPTP) and the control group (Control), the positive drug group (Mardopar), and the Gastrodia elata group (GEB). The first two principal components (PC1 and PC2) explained 22.70% and 15.70% respectively, and there were significant differences among the groups. It is worth noting that there was partial overlap between the GEB and Control groups, indicating that after Gastrodia elata intervened in the MPTP-induced Parkinson's disease model mice, the in vivo metabolic state tended to be close to that of the normal group ( Figure 8 A). There was a significant separation between the Control group and the MPTP group ( Figure 8 B), indicating that there was a systemic disorder in the metabolic activities of MPTP-induced mice compared with the Control group. The GEB group and the Mardopar group were divided into different clusters from the MPTP group ( Figure 8 C, D), indicating that Gastrodia elata extract and Madopar have a regulatory effect on the metabolism of MPTP-induced Parkinson's mice. The QC samples had good aggregation, and the results showed that the entire analysis system had good stability and repeatability, meeting the analysis requirements of metabolomics.

[0161] 2.2 OPLS-DA analysis of the effects of Gastrodia elata extract on metabolites in the striatum of MPTP model mice

[0162] As Figure 9 shown in A1 - A3 of Figure 9 , each point in the OPLS-DA score plot represents a sample. Samples in the same group are clustered together, and samples in different groups are clearly distinguished. In addition, a permutation test was performed on the OPLS-DA model to further verify the reliability of the model. The results showed that all blue Q2 points were lower than the rightmost original blue Q2 point from left to right ( Figure 9 B1 - B3). The OPLS-DA model was effective and there was no overfitting, so the variable importance in projection (VIP) values that were verified were obtained from this model. In order to identify potential metabolites from a statistical and biochemical perspective under the OPLS-DA model and better screen the differential variables between groups, the covariance and correlation between metabolites and model classes were analyzed using S-plot. As Figure 9 shown in C1 - C3 of

[0163] , the metabolites closer to the two corners had stronger importance. The red points indicated that the VIP of these metabolites ≥1, and the green points indicated that the VIP of these metabolites <1.

[0164] By non-targeted metabolomics technology, a total of 594 differential metabolites were identified in the striatum tissues of mice. Compared with the Control group, there were 240 differential metabolites in the MPTP group, among which 147 metabolites were down-regulated and 93 metabolites were up-regulated( Figure 10 A); compared with the MPTP group, there were 309 differential metabolites in the GEB group, among which 139 differential metabolites were down-regulated and 170 differential metabolites were up-regulated( Figure 10 B). Each point in the figure represents a specific metabolite. The size of the point represents the VIP value. Significantly up-regulated metabolites are shown in red, while significantly down-regulated metabolites are shown in blue, and metabolites with insignificant differences are shown in gray. The points closer to both sides and above indicate more significant differences.

[0165] 2.4 Analysis of potential biomarkers in the striatum of MPTP model mice after intervention with Gastrodia elata extract

[0166] Thirty-six differential metabolites were screened between the control group and the model group according to VIP value > 1.5 and student’s t-test (p < 0.001), as shown in Table 2. Compared with the control group, the contents of 29 metabolites in the model group decreased significantly: 4'-Hydroxyflurbiprofen, Pro Pro Gly (L-prolylglycine), Hawkinsin, PS(18:3(6Z,9Z,12Z) / 15:0) (phosphatidylserine(18:3(6Z,9Z,12Z) / 15:0)), Deoxycytidine, 3,3'-Thiobispropanoic acid, Genipin, Metaproterenol 3-O-sulfate, 2-Carboxy-4-dodecanolide, Androstenol, (3E,6Z)-Nonadien-1-ylacetate, 6-[3-(carboxymethyl)phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid, Dexpanthenol, Panaquinquecol 2, Gly Gly Glu (glycyl-glycyl-glutamic acid), 3-ethyl-5-methoxyphenol, Dihydroxyphenylacetic acid, DL-3,4-Dihydroxyphenyl glycol, 3-(6-hydroxy-2,3,4-trimethoxyphenyl)prop-2-enoic acid, 3,4-Dihydroxyphenyl ethanol, 2-hydroxy-3-(2,4,5-trihydroxyphenyl)propanoic acid, 2-hydroxy-3-(2,4,5-Trihydroxyphenyl)propionic acid), 7-Aminoclonazepam, (+ / -)-Enterolactone, 4-Hydroxyphenylacetylglutamic acid, Cholic acid, Deoxycholic acid, Allocholic acid, 1,2-Dehydrosalsolinol, (S)C(S)S-S-Methylcysteine sulfoxide; the contents of 7 metabolites were significantly increased: Phe Phe Lys, Retrofractamide A, Ent-6,16-Kauradien-19-oic acid, 4-Pyridoxic acid, Ser Leu Ser Gly Leu, 2''-O-trans-p-Coumaroylastragalin, SAICAR. By comparing the relative abundances of differential metabolites in different groups, the regulatory effects of each Gastrodia elata intervention group on differential metabolites were evaluated. After the intervention with Gastrodia elata extract, the contents of 27 differential metabolites were significantly reversed (, Figure 11 ).

[0167] Table 2. Table of significant changes in potential metabolites in the striatum of MPTP-induced Parkinson's disease model mice identified by LC-MS

[0168]

[0169] 2.5 Effects of Gastrodia elata extract on the metabolic pathways in the striatum of MPTP model mice

[0170] Such as Figure 12As shown in A of , KEGG enrichment analysis was performed on the differential metabolites in the mouse striatum to explore the mechanism of gastrodia extract in preventing and treating Parkinson's disease. According to Pathway impact>0.1, 5 metabolic pathways most relevant to Parkinson's disease were screened out: alanine, aspartate and glutamate metabolism, vitamin B6 metabolism, purine metabolism, arachidonic acid metabolism, and tyrosine metabolism. Compared with the MPTP group, GEB regulated 5 metabolic pathways: glycerophospholipid metabolism, sphingomyelin metabolism, tyrosine metabolism, arginine and proline metabolism, and arachidonic acid metabolism; compared with the MPTP group, Mardopar regulated 7 metabolic pathways: sphingomyelin metabolism, arginine and proline metabolism, glycerophospholipid metabolism, arginine biosynthesis, caffeine metabolism, vitamin B6 metabolism, and arachidonic acid metabolism. GEB and Mardopar jointly regulated four differential metabolic pathways of glycerophospholipid metabolism, sphingomyelin metabolism, arachidonic acid metabolism, and arginine and proline, which might be the key to anti-PD. Further comprehensive molecular pathway analysis showed that glycerophospholipid metabolism and arachidonic acid metabolism might be the important pathway mechanisms of gastrodia extract and Madopar in preventing and treating Parkinson's disease.

[0171] In these metabolic pathways, after treatment with GEB, the levels of spermidine, spermine, L-proline, L-arginine, 4-aminobutyraldehyde, and normetanephrine were significantly regulated ( Figure 12 as shown in B of ). In this example, compared with the control group, the relative abundances of spermidine and spermine in PD mice were significantly reduced. However, GEB could significantly restore the levels of spermidine and spermine in the striatum of PD mice. The results of this example suggest that these metabolites may play a neuroprotective role by regulating autophagy.

[0172] The relative abundance of L-proline in MPTP-induced PD mice was significantly reduced, indicating a decrease in its antioxidant capacity and accelerating the progression of PD. However, after administration of GEB, the relative abundance of L-proline was significantly upregulated, indicating that they relieve PD disease through an antioxidant mechanism.

[0173] GEB significantly upregulated the level of 4-aminobutyraldehyde in MPTP-induced PD mice, indicating that GEB plays a PD-protective role by promoting the conversion of 4-aminobutyraldehyde into GABA.

[0174] In this example, a significant decrease in the relative abundance of arginine was observed in MPTP-induced PD mice. After administration of GEB, the relative abundance of arginine increased significantly, indicating that it promotes neuroprotection as an anti-PD drug.

[0175] Compared with the control group, this example revealed a significant downregulation of NE in the MPTP group. After GEB intervention, the relative abundance of NE showed a significant upregulation.

[0176] Example 5

[0177] The following shows the study on the effect of Gastrodia elata extract on the motor impairment in a rat model of Parkinson's disease induced by 6-OHDA.

[0178] 1. Materials and Methods

[0179] 1.1 Experimental Animals

[0180] 100 SPF-grade SD rats, male, weighing about 300 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Feed and pure water were freely provided, and they were maintained under an alternating cycle of 12 h of light (8:30 - 20:30) and 12 h of darkness (20:30 - 8:30 the next day). The room temperature was 22 - 25°C, and the humidity was 55 ± 10%. They were acclimated to the environment for 3 days before the experiment.

[0181] 1.2 Experimental Reagents

[0182] The steps and methods for preparing the Gastrodia elata extract were the same as those shown in Example 1.

[0183] 6-OHDA (Sigma, USA); Apomorphine hydrochloride standard (APO, High Education Research and Technology Co., Ltd., product number: 100839); Ascorbic acid (Shanghai Yuanye Bio-Technology Co., Ltd., product number: B21293).

[0184] 1.3 Main Experimental Instruments

[0185] Gait computer detection and analysis processing device for mice and rats, real-time monitoring system for the spontaneous activity of rats (jointly developed by the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, China Astronaut Center, etc.), Stereotaxic 463701 brain stereotaxic instrument (Benchmark, USA), strong90 + 102 skull drill (Saeshin, Korea), 462901 type micro-injection pump (Benchmark, USA), micro-injection needle (Hamilton, Switzerland), Mili-Q ultrapure water purifier (Millipore, USA).

[0186] 1.4 Experimental Methods

[0187] 1.4.1 Surgical Modeling

[0188] Select the right medial forebrain bundle area for modeling. Fast the rats for 8 hours before the operation, and withhold water for 8 hours. On the day of the operation, anesthetize the rats by intraperitoneal injection of 10% chloral hydrate. Incise the skin on the top of the rat's skull along the midline of the head, separate the periosteum, and clean the surface of the skull with a cotton ball soaked in 3% hydrogen peroxide until the anterior fontanelle is fully exposed. Determine the coordinates with the anterior fontanelle as the origin. According to the stereotaxic brain atlas of SD rats, determine the injection coordinates: 1.8 mm posterior to the anterior fontanelle, 2.0 mm beside the sagittal suture, and 8.6 mm under the dura mater. After determining the coordinates, disinfect, and inject 5 μL of 6-OHDA solution into the coordinate point at a injection speed of 1 μL / min. After injection, keep the needle in place for 5 minutes and withdraw the needle at a speed of 1 mm / min. Suture the surgical incision and apply penicillin powder to the epidermis to prevent surgical wound infection. The sham operation group was given an equal amount of normal saline containing 0.2% ascorbic acid in the same way, and the rats were raised in the same environment after the operation.

[0189] 1.4.2 Grouping and administration

[0190] Randomly divide the rats into 3 groups, namely: ① control group, ② model group, and ③ sham operation group. There are 80 rats in the model group, and 10 rats in each of the control group and the sham operation group. The rats in the control group are raised in groups, and the rats in the other groups are raised individually in cages. Three weeks after the end of modeling, identify the model by intraperitoneal injection of 0.5 mg / kg of APO solution. Randomly divide the rats with successful modeling in the model group into: model group, Madopar group (7.8 mg / kg), low-dose Gastrodia elata group (100 mg / kg), and high-dose Gastrodia elata group (300 mg / kg). After grouping, continuously administer Madopar or Gastrodia elata extract by gavage every day until the end of sampling. The control group, sham operation group, and model group are given the same volume of pure water by gavage.

[0191] 1.4.3 Open field test

[0192] The open field test is the same as shown before.

[0193] 1.4.4 Apomorphine-induced rotation test

[0194] At 1, 2, and 3 weeks after the operation, intraperitoneally inject apomorphine (APO) into the rats, and record the locomotor behavior of the rats within 30 minutes 10 minutes after injection. The standard for a successful 6-OHDA model is: the rat uses the hind limb on the contralateral side of the operation as the support point, the head and tail are connected, the body is in a circular shape, and it quickly rotates to the contralateral side of the operation, with the number of rotation circles ≥ 180 times / 30 min or an average rotation of 7 times / min.

[0195] 1.5 Statistical methods

[0196] Same as described in 1.5 of Example 2.

[0197] 2 Results

[0198] 2.1 Open-field experiment

[0199] In the open-field experiment ( Figure 13 ), compared with the control group, the total movement distance, average speed, and movement time of the 6-OHDA model group showed significant decreases (p < 0.05, p < 0.01), while the immobility time increased significantly (p < 0.01); compared with the model group, Gastrodia elata (100 mg / kg) and (300 mg / kg) significantly restored the abnormalities in total movement distance, average speed, movement time, and immobility time (p < 0.05, p < 0.01); there was no significant difference between the Madopar group and the model group (p > 0.05).

[0200] 2.2 Apomorphine-induced rotation experiment

[0201] Apomorphine is a typical dopamine receptor agonist, mainly used to treat the motor symptoms of Parkinson's disease. In the experiment, if the animals showed frequent or continuous rotation behavior under specific conditions, this usually implied the activation effect of the drug on the dopamine system. In this experiment, neither the blank control group nor the sham operation group induced contralateral rotation behavior. Therefore, only the data of the model group and the Gastrodia elata intervention group were statistically analyzed, and the results are as Figure 14 shown: In the APO-induced rotation experiment, compared with the model group, each dose group of Gastrodia elata showed a certain trend of reducing the number of rotation circles, but there was no significant difference (p > 0.05). Compared with the model group, the number of rotation circles in the Madopar group was significantly increased (p < 0.01).

[0202] Example 6

[0203] The following shows the study on the mechanism of action of Gastrodia elata extract on rats with 6-OHDA-induced Parkinson's disease model.

[0204] 1. Materials and methods

[0205] 1.1 Experimental animals

[0206] Same as described in 1.1 of Example 5.

[0207] 1.2 Experimental reagents

[0208] Rat dopamine (DA) ELISA detection kit and rat 3,4-dihydroxyphenylacetic acid (DOPAC) ELISA detection kit were both purchased from Shanghai Enzyme-linked Biotechnology Company. Primary antibodies TLR4, NF-κB, p65, Cleaved-Caspase-3 were purchased from Cell Signaling Technology; Bax and Bcl-2 were purchased from Abcam.

[0209] 1.3 Main experimental instruments

[0210] Same as described in 1.3 of Example 3.

[0211] 1.4 Experimental methods

[0212] 1.4.1 Grouping

[0213] Sham operation group, model group, Madopar group (7.8 mg / kg), low-dose Gastrodia elata group (100 mg / kg), high-dose Gastrodia elata group (300 mg / kg).

[0214] 1.5 Statistical methods

[0215] Same as described in 1.5 of Example 2.

[0216] 2. Results

[0217] 2.1 Effect of Gastrodia elata extract on DA content in the striatum of 6-OHDA rats

[0218] One of the main characteristics of Parkinson's disease is the progressive loss of dopamine neurons in the substantia nigra. This neuronal loss leads to a decrease in the content of dopamine in the brain. As Figure 15 shown, compared with the sham operation group, the DA level in the model group rats was significantly decreased (p<0.001); the DA level in the positive drug Madopar group was significantly increased (p<0.01); the DA content in the striatum of rats in the Gastrodia elata (300 mg / kg) group was increased compared with the model group, and the difference was significant (p<0.05).

[0219] 2.2 Effect of Gastrodia elata extract on DOPAC content in the striatum of 6-OHDA rats

[0220] As one of the main metabolites of dopamine, the content of DOPAC reflects to a certain extent the metabolic rate of dopamine and the activity state of the dopamine system. Therefore, by measuring the change of DOPAC, the functional state of the dopamine system can be indirectly evaluated. As Figure 16 shown, compared with the sham operation group, the DOPAC content in the model group rats was significantly decreased (p<0.001); compared with the model group, the positive drug Madopar significantly restored the DOPAC level in the model group rats (p<0.05); the content in the Gastrodia elata (300 mg / kg) group had an increasing trend compared with the model group, but the difference was not significant (p>0.05).

[0221] 2.3 Effect of Gastrodia elata extract on the expression levels of TLR4 and NF-κB proteins in the striatum of Parkinson's disease model rats induced by 6-OHDA

[0222] During the pathological process of Parkinson's disease, neuronal loss and inflammatory responses are important features. TLR4 can trigger neuroinflammatory responses by recognizing endogenous and exogenous pathogenic factors (such as α-synuclein and other inflammatory factors), which may promote the damage and death of dopamine neurons. As Figure 17 shown, compared with the control group, the expression of TLR4 protein in the model group tended to increase after induction with 6-OHDA. After treatment with Madopar and Gastrodia elata, the expression of TLR4 protein tended to decrease, but there was no significant difference (p>0.05).

[0223] In Parkinson's disease, NF-κB is usually activated and participates in regulating the occurrence and progression of neuroinflammatory responses. The activation of NF-κB may further exacerbate the damage of dopamine neurons by regulating the expression of inflammatory factors (such as TNF-α, IL-1β, etc.). The expression level of NF-κb protein in the model group increased significantly after induction with 6-OHDA (p<0.01); after treatment with Madopar and Gastrodia elata (300mg / kg), the expression of NF-κB protein decreased significantly (p<0.05, p<0.01).

[0224] In summary, using subacute Parkinson's disease mice induced by MPTP and Parkinson's disease rats induced by 6-OHDA as models respectively, the study on the motor injury and mechanism of action of Gastrodia elata extract on Parkinson's disease model mice and rats was carried out, and combined with non-targeted metabolomics analysis to explore potential biomarkers. The results showed that Gastrodia elata extract could improve the motor function injury of subacute Parkinson's disease model mice induced by MPTP, specifically manifested as improving gait function and spontaneous activity ability, reducing the expression of inflammatory factors TNF-α, IL-6, IL-1β and the expression levels of Caspase-3 and Caspase-9 apoptotic proteins in the striatum, and playing a protective role in the neuroinflammatory response induced by MPTP. Through non-targeted metabolomics analysis of the striatum, a total of 594 differential metabolites were found. Among them, there were 36 significantly differential metabolites between the control group and the model group. Compared with the model group, Gastrodia elata extract could regulate 27 differential metabolites to return to normal levels. After Gastrodia elata administration intervention, it could improve metabolic pathways such as glycerophospholipid metabolism, sphingolipid metabolism, tyrosine metabolism, arachidonic acid metabolism, arginine and proline metabolism. In addition, Gastrodia elata extract had a certain improvement effect on the motor function injury of PD rats induced by 6-OHDA. It could increase the contents of neurotransmitters dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) in the striatum of PD rats induced by 6-OHDA. Down-regulate the expression of NF-κB, Caspase-3 proteins and the Bax / Bcl-2 ratio in the striatum of PD rats induced by 6-OHDA, and reduce the occurrence of apoptosis. It indicates that Gastrodia elata is expected to become an ideal candidate for developing drugs for the treatment of Parkinson's disease. Provide data support for the mechanism study of Gastrodia elata in preventing and improving Parkinson's disease.

[0225] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various modifications or variations can be made to the exemplary embodiments of the present invention specification without departing from the scope or spirit of the present invention. The scope of the claims should be interpreted in the broadest manner to cover all modifications and equivalent structures and functions.

Claims

1. A method for identifying the effectiveness of Gastrodia elata extract in preventing and treating Parkinson's disease, characterized in that: include: (1) Establishing an animal model of Parkinson's disease; (2) measuring behavioral, biochemical, and metabolomic indicators of the model; (3) measuring behavioral, biochemical, and metabolomic indices of the model after administration of Gastrodia elata extract; The effectiveness refers to improving the propulsion index in animal gait analysis, the support phase in gait analysis, shortening the climbing pole time, improving the animal hanging score, improving the sports injury in the open field experiment, and reducing the expression level of inflammatory factors in the striatum, the expression amount of apoptotic proteins, improving the content of dopamine and / or dihydroxyphenylacetic acid in the striatum, reducing the expression level of TLR4 and / or NF-κB proteins in the striatum, and adjusting the level of metabolites in the striatum; The behavioral index includes at least one of the propulsion index in the gait analysis of the model animal, the support phase in the gait analysis, the climbing time, the hanging score, and the motor injury in the open field experiment; The biochemical indices include the expression level of inflammatory factors in the striatum, the expression amount of apoptotic proteins in the striatum, the content of dopamine and dihydroxyphenylacetic acid in the striatum, and the expression level of TLR4 and NF-κB proteins in the striatum; The levels of the callback metabolites include: up-regulating 4'-hydroxyflurbiprofen, L-prolylglycine, hopkinsin, phosphatidylserine, 3,3'-thiodipropionic acid, 2-carboxy-4-dodecanol, androstenol, panquinol 2, 3-ethyl-5-methoxyphenol, DL-3,4-dihydroxyphenylethanol, 3-(6-hydroxy-2,3,4-trimethoxyphenyl)prop-2-enoic acid, dihydroxyphenylacetic acid, 3,4-dihydroxyphenylethanol, 2-hydroxy-3-(2,4,5-trihydroxyphenyl)propionic acid, 7-aminoclonazepam, 4-hydroxyphenylacetylglutamate, 1,2-dehydrogenated salt octanol, (S)-C(S)S-methionine sulfoxide, (3E,6Z)-nona-2,4-dien-1-yl acetate, dexpanthenol, 3-o-isoproterenol sulfate, and down-regulated phenylalanine-phenylalanine-lysine, anti-tropolamine A, Ent-6,16-quinolinadiene-19-acid, serine-leucine-serine-glycine-leucine, 2''-O-trans-p-coumarin, 5-thienyl adenine nucleotide phosphate.

2. The method for identifying the effectiveness of Gastrodia elata extract in preventing and treating Parkinson's disease according to claim 1, characterized in that: The animal model is a Parkinson's disease animal model induced by MPTP or 6-OHDA.

3. The method for identifying the effectiveness of Gastrodia elata extract in preventing and treating Parkinson's disease according to claim 1, characterized in that: The inflammatory factors include TNF-α, IL-6 and / or IL-1β, and the apoptotic proteins include Caspase-3 and / or Caspase-9 proteins.

4. The method for identifying the effectiveness of Gastrodia elata extract in preventing and treating Parkinson's disease according to claim 1, characterized in that: The control comprises regulating one of the following: Improve the propulsion index in gait analysis of MPTP model mice; Improve the stance phase in gait analysis of MPTP model mice; Shorten the climbing time of MPTP model mice; Improve the hanging score of MPTP model mice; Reduce the expression level of inflammatory factors in the striatum of MPTP model mice, including TNF-α, IL-6, and IL-1β; Reduce the expression of apoptotic proteins in the striatum of MPTP model mice, including Caspase-3 and Caspase-9 proteins; Improve metabolic disorders in the striatum of MPTP model mice; Improve the metabolism of glycerophospholipids, sphingomyelin, tyrosine, arginine and proline, and arachidonic acid in MPTP model mice; Improve the motor impairment of 6-OHDA model rats in the open field test; Improved DA and DOPAC content in the striatum of 6-OHDA model rats; Reduce the expression levels of TLR4 and NF-κB proteins in the striatum of 6-OHDA model rats.

5. A non-disease diagnosis and treatment method for regulating abnormal metabolites of the striatum, characterized in that: The method comprises the steps of using a Gastrodia elata extract identified by the method according to any one of claims 1 to 4, wherein the regulation comprises: upregulating 4'-hydroxyflurbiprofen, L-prolylglycine, hopkinsin, phosphatidylserine, 3,3'-thiodipropionic acid, 2-carboxy-4-dodecanol, androstenol, panquinol 2, 3-ethyl-5-methoxyphenol, DL-3,4-dihydroxyphenylethanol, 3-(6-hydroxy-2,3,4-trimethoxyphenyl)prop-2-enoic acid, dihydroxyphenylacetic acid, 3,4-dihydroxyphenylethanol, 2-hydroxy-3-(2,4 ,5-trihydroxyphenyl)propionic acid, 7-aminoclonazepam, 4-hydroxyphenylacetylglutamate, 1,2-dehydrooctanol, (S)-C(S)S-methionine sulfoxide, (3E,6Z)-nona-2,4-dien-1-yl acetate, dexpanthenol, 3-o-isoproterenol sulfate, and down-regulated phenylalanine-phenylalanine-lysine, anti-tropolamine A, Ent-6,16-quinolinadiene-19-acid, serine-leucine-serine-glycine-leucine, 2''-O-trans-p-coumarin, 5-thienyl adenine nucleotide phosphate.

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