Method for identifying effectiveness of gastrodia elata extract in prevention and treatment of Parkinson's disease

Abstract

The invention discloses a method for identifying the effectiveness of gastrodia elata extract in preventing and treating Parkinson's disease. A Parkinson's disease animal model is used for carrying out sports injury and action mechanism research on the gastrodia elata extract, and a potential biomarker is excavated in combination with non-targeted metabonomics analysis. Experimental results show that gastrodia elata can be used for developing ideal candidates of drugs, health care products or functional foods for preventing and treating Parkinson's disease. Besides, a metabonomics technology is combined with behavioral indexes and biochemical indexes, and from the perspective of expression of inflammatory factors, neurotransmitters, proteins and metabolites and metabolic regulation, through objective quantitative and qualitative analysis of symptoms, a new thought is provided for evaluation of effectiveness of the gastrodia elata extract in prevention and treatment of Parkinson's disease. Based thereon, the invention also provides a method of modulating metabolite abnormalities in a cell in vitro.

Description

Technical Field

[0001] The invention relates to the field of effectiveness identification of Gastrodia elata extracts, and in particular 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, progressive neurodegenerative disease that is more common in middle-aged and elderly people. It seriously endangers the physical and mental health of patients. Its main clinical manifestations are a series of extrapyramidal movement disorders such as resting tremor, bradykinesia and abnormal posture and gait, and can also be accompanied by non-motor symptoms such as depression, anxiety, insomnia and autonomic dysfunction. The etiology of PD is complex. The main pathological changes are the degeneration of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) of the midbrain and the deposition of misfolded α-synuclein (α-synuclein, α-Syn) leading to the formation of Lewy bodies (LB). At present, the treatment of PD is mainly based on drug therapy, including levodopa (L-dopa), central anticholinergic drugs, catechol-O-methyltransferase inhibitors, etc. These drugs can effectively relieve PD symptoms, but long-term use can cause many adverse reactions such as gastrointestinal reactions and switching phenomena. Therefore, it is urgent to develop safe, effective, and low-adverse-reaction drugs and related products for the prevention and treatment of PD. Since modern times, many doctors have accumulated a lot of experience in treating PD based on classics and clinical practice. 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 Chinese medicines, include new drugs with active ingredients and new drugs with effective parts. Among them, the screening of candidate drugs is a key step in the development of innovative Chinese medicines. 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 difficulties in screening candidate drugs.

[0004] The identification technology of biological effects of traditional Chinese medicine is the product of the cross-development of quantitative pharmacology and identification of traditional Chinese medicine. Based on biological effects and using biostatistics as a tool, the identification of biological effects of traditional Chinese medicine uses a specific experimental design to compare the specific reactions of organisms to different test products under certain conditions (which can measure changes in quantitative physiological indicators or biological characteristics) to determine the presence and intensity of the biological activity (pharmacological activity or toxicity) of traditional Chinese medicine, and use 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, so the identification of biological effects of traditional Chinese medicine is the best method for identifying the quality of traditional Chinese medicine. At present, there are few reports on the identification methods for the effectiveness of Tianma extract in preventing and treating Parkinson's disease.

[0005] In addition, Chinese medicinal materials contain many secondary metabolites, and each secondary metabolite may have multiple biological activities, so its overall biological activity is complex and difficult to describe accurately. Therefore, the description of the nature and taste of each medicinal material in Chinese medicine classics is also abstract and vague, and clinical application also requires doctors to treat according to experience, thus forming an insurmountable obstacle to the commercialization and industrialization of Chinese medicinal materials with a rich history of clinical application. How to ensure the optimal clinical efficacy of prescriptions is even more difficult in the clinical pharmacy of traditional Chinese medicine. Therefore, there is still a need for a quantitative description and standardized evaluation method for the effectiveness (efficacy) of Chinese medicine for the prevention and treatment of Parkinson's disease in subjects.

[0006] Pharmaceutical science is a scientific system that is closely centered around clinical medicine. The purpose of all pharmaceutical science practices should be derived from and serve the clinic. This is the value of pharmaceutical science. Unfortunately, the quality standards of traditional Chinese medicine are seriously out of touch with clinical needs, which has become a common phenomenon in today's research on the quality of traditional Chinese medicine. It is unreasonable to rely solely on the qualitative or quantitative inspection of one or several chemical components whose content in the medicinal materials is only a few percent, a few thousandths or even lower to show that the clinical efficacy or safety of the medicinal materials is unreasonable. 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 technology is only for illustrating the general background of the present invention and should not be regarded as admitting or suggesting in any form that such information constitutes the prior art known to a person skilled 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 study and analysis of small molecule metabolites in biological samples provide new approaches for early diagnosis of the disease and exploration of its pathogenesis. However, how Gastrodia elata extract affects Parkinson's disease-related metabolites 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 effects of Gastrodia elata extract through quantitative and qualitative evaluation and analysis of metabolites.

[0009] In order to achieve the purpose of the present invention, the first aspect of the present invention provides the use of a Gastrodia elata extract in preparing a product for preventing and treating Parkinson's disease in a subject, wherein the Gastrodia elata extract is prepared by a method comprising the following steps: Take dried Gastrodia elata, cut it into thin slices, soak it in ethanol overnight, extract it repeatedly with boiling ethanol, filter, concentrate, dry and crush it to obtain it.

[0010] In certain embodiments, according to the use of the present invention, the product comprises at least one of a functional food, a health product and a medicine.

[0011] In certain embodiments, according to the use of the present invention, the dosage form of the product includes at least one of tablets, granules, capsules, compressed candies, solid beverages, functional teas, granules and extracts.

[0012] In certain embodiments, according to the use of the present invention, wherein the subject includes an animal model, and the prevention and treatment includes 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.

[0013] The second aspect of the present invention provides a method for identifying the effectiveness of Gastrodia elata extract in preventing and treating Parkinson's disease, comprising: (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 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 index includes the expression level of striatal inflammatory factors, the expression amount of apoptotic proteins in the striatum, the dopamine (DA) and / or dihydroxyphenylacetic acid content (DOPAC) in the striatum, and the expression level of TLR4 and / or NF-κB protein in the striatum. The metabolomics indicators include 4'-hydroxyflurbiprofen, L-prolylglycine, hopkinsin, phosphatidylserine, phenylalanine-phenylalanine-lysine, 3,3'-thiodipropionic acid, 2-carboxy-4-dodecanol, androstenol, anti-tropolamine A, Ent-6,16-quinolinadiene-19-acid, panquinol 2, 3-ethyl-5-methoxyphenol, DL-3,4-dihydroxyphenylethanol, 3-(6-hydroxy-2,3,4-trimethoxyphenyl)prop-2-enoic acid, dihydroxyphenyl Acetic acid, 3,4-dihydroxyphenylethanol, 2-hydroxy-3-(2,4,5-trihydroxyphenyl)propionic acid, 7-aminoclonazepam, serine-leucine-serine-glycine-leucine, 2''-O-trans-p-coumarin, 4-hydroxyphenylacetylglutamate, 1,2-dehydrooctanol, 5-phosphothiophene adenine nucleotide, (S)-C(S)S-methionine sulfoxide, (3E,6Z)-nona-2,4-dien-1-yl acetate, dexpanthenol, 3-o-isoproterenol sulfate.

[0014] In certain embodiments, according to the method for identifying the effectiveness of Gastrodia elata extract in preventing and treating Parkinson's disease of the present invention, the animal model is a Parkinson's disease animal model induced by MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 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.

[0015] In certain embodiments, according to the method for identifying the effectiveness of Gastrodia elata extract in preventing and treating Parkinson's disease as described in the present invention, the inflammatory factors include TNF-α, IL-6 and / or IL-1β, and the apoptotic proteins include Caspase-3 and / or Caspase-9 proteins.

[0016] In certain embodiments, according to the method for identifying the effectiveness of Gastrodia elata extract in preventing and treating Parkinson's disease of the present invention, the effectiveness refers to improving the propulsion index in animal gait analysis, the support phase in gait analysis, shortening the climbing time, improving the animal hanging score, improving the motor injury in the open field experiment, and reducing the expression level of striatal inflammatory factors, 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 the level of callback metabolites.

[0017] In certain embodiments, according to the method for identifying the effectiveness of Gastrodia elata extract for preventing and treating Parkinson's disease of the present invention, the level of the adjusted metabolites includes: up-regulating 4'-hydroxyflurbiprofen, L-prolylglycine, hawkins, 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 yl-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.

[0018] The third aspect of the present invention provides a non-disease diagnosis and treatment method for regulating abnormal metabolites of cells in vitro, which comprises the step of culturing cells in vitro with the Gastrodia elata extract identified by the method according to the present invention, wherein the abnormal metabolites of the cells are regulated, including: up-regulating 4'-hydroxyflurbiprofen, L-prolylglycine, hawkins, phosphatidylserine, 3,3'-thiodipropionic acid, 2-carboxy-4-dodecanol, androstenol, panquinone pentol 2, 3-ethyl-5-methoxyphenol, DL-3,4-dihydroxyphenylethanol, 3-(6-hydroxy-2,3,4-trimethoxyphenyl)prop-2-enoic acid, dihydroxy Phenylacetic 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 phosphate nucleotide.

[0019] The present invention has at least the following advantages and beneficial effects: (1) The present invention provides a method for identifying the effectiveness of Gastrodia elata extract, and provides a theoretical basis for the development and application of Gastrodia elata extract in traditional Chinese medicine for the prevention and treatment of Parkinson's disease, health products or functional foods.

[0020] (2) The present invention uses MPTP-induced subacute Parkinson's disease mice as a model for research, and proves that Gastrodia elata extract can improve the motor function impairment of MPTP-induced subacute Parkinson's disease model mice, specifically by improving gait function and autonomous 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.

[0021] (3) The present invention determined the differential metabolites of Gastrodia elata extract in preventing and treating Parkinson's disease through metabolomics analysis. First, the metabolic data of Parkinson's mice were obtained through non-targeted metabolomics analysis. Then, PCA analysis showed that the effect of Gastrodia elata extract on mouse metabolites was significantly different from that of the model group, and the differential metabolites were further identified. The regulatory effect of the Gastrodia elata intervention group on the differential metabolites was evaluated by comparing the relative abundance of the differential metabolites in different groups. The results showed that after the intervention of Gastrodia elata extract, the contents of 27 differential metabolites were significantly adjusted. Finally, the metabolic pathways most related to Parkinson's disease were further screened out through KEGG enrichment analysis.

[0022] (4) The present invention uses 6-OHDA-induced Parkinson's disease rats as a model for research, proving that Gastrodia elata extract can increase the content of neurotransmitter levels in the striatum of Parkinson's disease model rats, downregulate the expression of apoptotic proteins, and reduce the occurrence of cell apoptosis. This provides a theoretical basis and data support for the treatment of Parkinson's disease with Gastrodia elata.

[0023] The present invention adopts metabolomics technology combined with behavioral and biochemical indicators, starting from the expression of inflammatory factors, neurotransmitters, proteins and metabolites and metabolic regulation, and through objective quantification of symptoms, provides new ideas for evaluating the effectiveness of Gastrodia elata extract in preventing and treating Parkinson's disease, thereby promoting the modernization of traditional Chinese medicine theory. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Effect of Gastrodia elata extract on 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.

[0025] Figure 2 Effect 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.

[0026] 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.

[0027] Figure 4 Effects of Gastrodia elata extract on the suspension 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.

[0028] Figure 5 Effects of Gastrodia elata extract on the open field test in MPTP model mice (mean ± SEM, n=9-10). Compared with the control group, * p<0.05.

[0029] Figure 6Effects of Gastrodia elata extract on the levels of TNF-ɑ, IL-6 and IL-1β in striatal tissue of MPTP model mice. Compared with the control group, ***p<0.001; compared with the model group, #p<0.05, ##p<0.01.

[0030] Figure 7 Effects of Gastrodia elata extract on the expression of Caspase-3 and Caspase-9 proteins 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.

[0031] Figure 8 PCA score plot of metabolite analysis in the striatum of MPTP model mice.

[0032] Fig. 9 OPLS-DA analysis of the effect of Gastrodia elata extract on metabolites in the striatum of MPTP model mice. A: OPLS-DA score of metabolic profile of striatum of MPTP mice; B: OPLS-DA model validation diagram; C: S-plot diagram of striatum of MPTP mice.

[0033] Fig.10 Volcano plot analysis of differential metabolite expression.

[0034] Fig.11 After intervention with Gastrodia elata extract, the contents of 27 differential metabolites were significantly restored.

[0035] Fig.12 Enrichment map of metabolic pathways of different metabolites in the striatum of MPTP model mice.

[0036] Fig.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.

[0037] Fig.14 Effect of Gastrodia elata extract on 6-OHDA model rats in APO-induced rotation test. Compared with the model group, ##p<0.01.

[0038] Fig.15 Effect of Gastrodia elata extract on DA content in striatum of 6-OHDA model rats. Compared with the sham operation group, ***p<0.001; compared with the model group, #p<0.05, ##p<0.01.

[0039] Fig.16Effect of Gastrodia elata extract on DOPAC content in striatum of 6-OHDA model rats. Compared with the sham operation group, ***p<0.001; compared with the model group, #p<0.05.

[0040] Fig.17 Effects 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 DESCRIPTION

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0043] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may 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 the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0044] Example 1 This example shows the identification of components of Gastrodia elata extract by UHPLC-QE-MS.

[0045] 1. Materials and Methods 1.1 Preparation of extracts Gastrodia elata was purchased from Sichuan Chijian Chinese Medicine Technology Co., Ltd. The main steps and methods for preparing Gastrodia elata extract were as follows: Dried Gastrodia elata was cut into thin slices and soaked in 85% ethanol overnight. The extract was repeatedly extracted with boiled 85% ethanol for 3 times. After filtering, the extract was concentrated, dried, and crushed to obtain a powder, which was the Gastrodia elata extract.

[0046] 1.2 Identification of extract components UHPLC-QE-MS was used to identify the chemical components in Gastrodia elata extract. Briefly, an appropriate amount of 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 at 4°C for 10 min, 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 positive ion mode, the mobile phase was 0.1% formic acid acetonitrile (B2) and 0.1% formic acid water (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 negative ion mode, the mobile phases were acetonitrile (B3) and 5 mM ammonium formate 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.

[0047] Thermo Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, USA) was connected to the UPLC system. The electrospray ion source (ESI) was used to collect data in positive and negative ion modes. 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, and the primary full scan was performed at a resolution of 70000. The primary ion scan range was m / z 100-1000, and HCD was used for secondary fragmentation. The collision energy was 30 eV, the secondary resolution was 17500, and the first 3 ions of the acquisition signal were fragmented. At the same time, dynamic exclusion was used to remove unnecessary MS / MS information.

[0048] 2. Results The Gastrodia elata extract was separated and identified by UHPLC-QE-MS in positive and negative ion modes, and a total of 402 components were identified, of which the main components are shown in Table 1.

[0049] Table 1 Main components of Gastrodia elata extract Example 2 The following shows the study on the effect of Gastrodia elata extract on motor impairment in MPTP-induced Parkinson's disease model mice.

[0050] 1. Materials and Methods 1.1 Experimental animals 72 SPF C57BL / 6JNifdc mice, male, 8-10 weeks old, weighing 20-22g, were ordered from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. Feed and pure water were freely provided, and the daily cycle of 12h light (8:30-20:30) and 12h dark (20:30-8:30 the next day) was maintained, the room temperature was 22-25℃, and the humidity was 55±10%. They were adapted for 3 days before the experiment.

[0051] 1.2 Experimental drugs Gastrodia elata was purchased from Sichuan Chijian Chinese Medicine Technology Co., Ltd. The main steps and methods for preparing Gastrodia elata extract were as follows: Dried Gastrodia elata was sliced ​​and soaked in 85% ethanol overnight. The extract was repeatedly extracted with boiled 85% ethanol for 3 times. After filtering, the extract was concentrated, dried, and crushed to obtain a powder, which was the Gastrodia elata extract. Madopar (Shanghai Roche Pharmaceuticals Co., Ltd.), MPTP (Sigma, USA).

[0052] 1.3 Main experimental instruments Real-time detection device for mouse autonomous activities (jointly developed by the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing Kangsen Yiyou Technology Co., Ltd., etc.); computer detection, analysis and processing device for rat and mouse gait (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 Instrument); homemade mouse climbing instrument, etc.

[0053] 1.4 Experimental methods 1.4.1 Grouping 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.

[0054] (1) Control group: gavage with normal saline once a day; (2) Model group: MPTP (30 mg / kg) was intraperitoneally injected at a fixed time every day for 7 consecutive days; (3) Positive drug group: Madopar (112.5 mg / kg) was gavaged once daily for 14 days before modeling and continued until the end of the experiment; (4) Low dose of Gastrodia elata: Gastrodia elata extract (100 mg / kg) was administered orally once daily for 14 days before model establishment and continued until the end of the experiment; (5) Medium dose of Gastrodia elata: Gastrodia elata extract (200 mg / kg) was administered orally once daily for 14 days before model establishment and continued until the end of the experiment; (6) High dose of Gastrodia elata: Gastrodia elata extract (300 mg / kg) was administered orally once daily for 14 days before model establishment and continued until the end of the experiment.

[0055] 1.4.2 Gait analysis experiment The mouse's feet were wetted with water, and the mouse was allowed to continuously pass through the treadmill, and the video was automatically recorded by the camera. After the acquisition was completed, the recorded video was analyzed using computer analysis software.

[0056] 1.4.3 Pole climbing experiment Each mouse was placed head up on a homemade climbing pole. The mouse was considered to have climbed the entire length when both forelimbs touched the platform at the bottom of the pole. The following times were recorded: the time it took the mouse to turn from head up to head down, the time the mouse spent in the upper half of the pole, the time the mouse spent in the lower half of the pole, and the total time taken to climb the entire pole.

[0057] 1.4.4 Suspension experiment The mouse's forelimbs were gently placed on the rope, and the mouse was observed. The scoring method was as follows: if the mouse could grasp the rope with all four paws, it was scored as 3 points; if it grasped the rope with one hind paw, it was scored as 2 points; if neither of the two hind paws could grasp the rope, it was scored as 1 point; if the mouse could not grasp the rope and fell quickly, it was scored as 0 points.

[0058] 1.4.5 Empty field experiment The mouse was gently placed in the test box (30 cm×30 cm×35 cm) with its back facing the wall. The test began after the mouse had adapted to the environment for 3 minutes. The computer automatically monitored and recorded the spontaneous activities of the mouse in the test box within 10 minutes, and the computer analysis system was used to analyze indicators such as the total distance traveled.

[0059] 1.5 Statistical methods The experimental data were statistically analyzed using SPSS 21.0 statistical software, and the data were expressed as mean ± SEM. One-way ANOVA was used to compare the differences among the groups. The significance level was P < 0.01 or P < 0.05.

[0060] 2. Results 2.1 Effect of Gastrodia elata extract on propulsion index in gait analysis of MPTP model mice Parkinson's disease patients often show obvious changes in gait characteristics, such as unstable gait rhythm, narrow stride, unstable posture, etc. Gait analysis experiments can quantify these changes by analyzing gait parameters (such as step length, gait 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. Figure 1), compared with the control group, the model group had significant differences in the left rear, right front, and right rear propulsion index gait indicators (p<0.01, p<0.001), while there was no significant difference in the left front propulsion index (p>0.05). Compared with the model group, the left rear propulsion index and right rear propulsion index of mice in the low, medium, and high dose groups of Gastrodia elata extract and the Madopar group were significantly restored (p<0.001). The right front propulsion index of mice in the high dose group of Gastrodia elata (300 mg / kg) was restored to a certain extent (p<0.05).

[0061] 2.2 Effect of Gastrodia elata extract on the stance phase in gait analysis of MPTP model mice like Figure 2 As shown in the results, compared with the control group, MPTP intervention significantly increased the left posterior, right anterior and right posterior support phases of mice (p<0.01), while there was no significant difference in the left anterior support phase (p>0.05). Compared with the model group, the left posterior support phase of the Madopar group, the low-, medium- and high-dose Gastrodia elata intervention groups significantly decreased (p<0.05, p<0.01); the right anterior support phase of the Madopar group and the high-dose Gastrodia elata group significantly decreased (p<0.05, p<0.01).

[0062] 2.3 Effect of Gastrodia elata extract on the pole climbing test of MPTP model mice The main characteristics of Parkinson's disease are the loss of dopaminergic neurons and the decrease of 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. Figure 3 As shown in the figure, compared with the control group, the time and total time of the mice in the model group increased in the upper half of the climbing pole, while the intervention of Gastrodia elata extract (100, 200, 300 mg / kg) significantly shortened the time of the mice in the upper half of the climbing pole ( Figure 4 B and D, p<0.01, p<0.001). This indicates that Gastrodia elata extract can effectively improve the motor dysfunction of Parkinson's disease.

[0063] 2.4 Effect of Gastrodia elata extract on the hanging score test of MPTP model mice Parkinson's disease patients often have muscle stiffness and difficulty in movement, which leads to significant impairment of their exercise endurance and muscle strength. The suspension test can be used to evaluate the effect of Gastrodia elata extract on the motor function of Parkinson's disease model animals. Figure 4 As shown in the figure, compared with the control group, the hanging score of mice in the model group decreased significantly (p<0.05). Compared with the model group, the scores of the Madopar group and the Gastrodia extract (100, 200, 300 mg / kg) group showed a certain increasing trend but no significant difference (p>0.05).

[0064] 2.5 Effect of Gastrodia elata extract on the open field test of MPTP model mice In Parkinson's disease research, animal models often show motor characteristics such as slow movement and decreased motor ability, which can be observed and evaluated through the animal's exploratory behavior and motor activity level in an open field experiment. Figure 5 As shown, compared with the control group, the total exercise distance index of the model group was significantly decreased (p<0.05); compared with the model group, there was no significant difference between the Madopar group and the Gastrodia elata dose groups (p>0.05).

[0065] Example 3 The following shows the study on the mechanism of action of Gastrodia elata extract on MPTP-induced Parkinson's disease model mice.

[0066] 1. Materials and Methods 1.1 Experimental animals Same as shown in 1.1 in Example 2.

[0067] 1.2 Experimental Reagents Mouse tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β enzyme-linked immunosorbent assay (ELISA) kits were purchased from Shanghai ELISA Biotechnology Co., Ltd.; PMSF (100 mM), 5× protein loading buffer, developer and fixer, electrophoresis buffer, SDS-PAGE gel preparation kit, phosphorylation proteinase 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 Prilai; protein marker was purchased from Therm (Fermentas); PVDF membrane was purchased from Millipore; ECL was purchased from Millipore; secondary antibody HRP goat anti-rabbit, Servicebio (GB23303); β-actin, primary antibody Cleaved-Caspase-3, and primary antibody Cleaved-Caspase-9 were all purchased from Cell Signaling Technology.

[0068] 1.3 Main experimental instruments Desktop high-speed refrigerated centrifuge (Hunan Xiangli Scientific Instrument); Milli-Q ultrapure water instrument (Millipore, USA); high-speed homogenizer (Shanghai Jingxin); fully automatic microplate reader (BIOTEK, USA); vertical electrophoresis instrument, transfer electrophoresis instrument (Tanon).

[0069] 1.4 Experimental methods 1.4.1 Animal grouping According to the results of the previous behavioral experiments, in the subsequent mechanism study, only the high-dose (300 mg / kg) group of Gastrodia elata extract was selected for experiment. The groups were as follows: control group, model group, Madopar group (112.5 mg / kg), and Gastrodia elata high-dose (300 mg / kg) group.

[0070] 1.4.2 Collection of materials After the behavioral test, the whole brain was dissected out using 5% chloral hydrate, and the hippocampus, striatum, and cortex tissues were taken and stored at -80°C for later use.

[0071] 1.4.3 Effects of Gastrodia elata extract on TNF-α, IL-6, and IL-1β levels in brain tissue of MPTP model mice Striatum and cortex tissues were weighed and homogenized, centrifuged at 12000 r / min for 20 min, and the supernatant was placed in an EP tube and stored at -80°C. ELISA detection was performed according to the steps in the kit instructions.

[0072] 1.4.4 Effect of Gastrodia elata extract on the levels of Caspase-3 and Caspase-9 proteins in the striatum of MPTP model mice Mouse striatal tissue was homogenized and lysed, centrifuged, and the supernatant was taken to determine the protein concentration according to the BCA protein quantification kit. After boiling in water for 10 min for denaturation, 40 μg of the sample was loaded, separated by 10% SDS-PAGE electrophoresis (separation gel 120 V, stacking gel 75 V), PVDF transfer (300 mA, 60 min), blocked with 5% skim milk powder for 60 min, added with primary antibody (Caspase-3, Caspase-9, 1:1000) and incubated at 4°C overnight, added with secondary antibody (HRP-labeled goat anti-rabbit, 1:1000) and incubated at room temperature for 60 min, washed, and detected the target bands in the dark room using the ECL method, the fluorescent chemiluminescence gel imaging system, and the gray value of the target band was analyzed by the Image-PRO software processing system.

[0073] 1.5 Statistical methods Same as described in 1.5 of Example 2.

[0074] 2. Results 2.1 Effects of Gastrodia elata extract on the levels of inflammatory factors in the striatum of MPTP model mice 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. Figure 6), compared with the control group, the levels of inflammatory factors TNF-α, IL-6, and IL-1β in the striatum of the model group mice were significantly increased (p<0.001). Compared with the model group, Madopar and Gastrodia elata (300 mg / kg) could significantly reduce the levels of inflammatory factors in PD mice (p<0.05, p<0.01).

[0075] 2.2 Effects of Gastrodia elata extract on the expression of Caspase-3 and Caspase-9 proteins in the striatum of MPTP model mice In Parkinson's disease, apoptosis of dopaminergic neurons is one of the main causes of disease progression. Apoptosis is a controlled cell death process, and its regulation involves a series of apoptosis-related proteins, such as the Bcl-2 family and cysteine ​​proteases (caspases). 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 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).

[0076] Example 4 The following shows the effect of Gastrodia elata extract on MPTP-induced metabolic changes in the striatum of Parkinson's disease mice revealed based on non-targeted metabolomics.

[0077] 1. Materials and Methods 1.1 Experimental animals Same as shown in 1.1 in Example 2.

[0078] 1.2 Experimental Reagents Chromatographic grade methanol (Fisher), chromatographic grade acetonitrile (Fisher), chromatographic grade formic acid (Fisher), chromatographic grade water (Fisher), chromatographic grade propanol (Fisher).

[0079] 1.3 Main experimental instruments Ultra-high performance liquid chromatography-tandem Fourier transform mass spectrometry (UHPLC-Q Exactive HF-X) was purchased from Thermo Fisher Scientific; HSST3 chromatographic column (100 mm×2.1 mm, 1.8 µm) was purchased from Waters Corporation; JXDC-20 nitrogen purge instrument was purchased from Shanghai Jingxin Industrial Development Co., Ltd.; LNG-T88 desktop rapid centrifugal concentrator and dryer was purchased from Taicang Huamei Biochemical Instrument Factory; Wonbio-96c high-throughput tissue disruptor was purchased from Shanghai Wanbo Biotechnology Co., Ltd.; SBL-10TD ultrasonic cleaner 300W-10L was purchased from Ningbo Xinzhi Biotechnology Co., Ltd.; Centrifuge 5430R high-speed refrigerated centrifuge was purchased from Eppendorf, Germany; and NewClassic MF MS105DU electronic balance was purchased from Mettler, Switzerland.

[0080] 1.4 Experimental methods 1.4.1 Sample preparation 25 mg of striatal sample was placed in a 2 mL centrifuge tube and a 6 mm diameter grinding bead was added. 400 μL of extract (methanol: water = 4: 1 (v: v)) containing 0.02 mg / mL of internal standard (L-2-chlorophenylalanine) was used for metabolite extraction. The sample solution was ground in a frozen tissue grinder for 6 min (-10°C, 50 Hz) and extracted by low-temperature ultrasound for 30 min (5°C, 40 kHz). The sample was placed at -20°C for 30 min, centrifuged for 15 min (4°C, 13000 g), and the supernatant was taken into a vial with an inner insert for analysis.

[0081] 1.4.2 LC-MS / MS analysis Chromatographic conditions: 2 μL of sample was separated by HSS T3 column (100 mm×2.1 mm, 1.8 μm) and then entered mass spectrometry detection. Mobile phase A was 95% water + 5% acetonitrile (containing 0.1% formic acid), and mobile phase B was 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid). Separation gradient: 0-3.5 min, mobile phase B increased from 0% to 24.5%, flow rate 0.40 mL / min; 3.5-5 min, mobile phase B increased from 24.5% to 65%, flow rate 0.4 mL / min; 5-5.5 min, mobile phase B increased from 65% to 100%, flow rate 0.4 mL / min; 5.5-7.4 min, mobile phase B maintained at 100%, flow rate increased from 0.4 mL / min to 0.6 mL / min; 7.4-7.6 min, mobile phase B decreased from 100% to 51.5%, flow rate 0.6 mL / min; 7.6-7.8 min, mobile phase B decreased from 51.5% to 0%, flow rate from 0.6 mL / min to 0.5 mL / min; 7.8-9 min, mobile phase B maintained at 0%, flow rate decreased from 0.5 mL / min to 0.4 mL / min; 9-10 min, mobile phase B was maintained at 0%, flow rate was 0.4 mL / min, and column temperature was 40°C.

[0082] Mass spectrometry conditions: The sample mass spectrometry signal was collected in positive and negative ion scanning mode, 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 positive ion mode spray voltage was set to 3500 V, the negative ion mode spray voltage was set to -3500 V, the ion transfer tube temperature was 325°C, and the normalized collision energy was 20-40-60 V cycle collision energy. The primary mass spectrometry resolution was 60000, the secondary mass spectrometry resolution was 7500, and the data were collected in DDA mode.

[0083] 1.4.3 Data Processing 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, and 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 public metabolic database HMDB (http: / / www.hmdb.ca / ) and Metlin (https: / / metlin.scripps.edu / ) to obtain metabolite information. In order to reduce the errors caused by sample preparation and instrument instability, the response intensity of the sample mass spectrometry peak was normalized by the total normalization method to obtain the normalized data matrix. At the same time, the variables with a relative standard deviation (RSD) of QC samples > 30% were deleted, and log10 logarithmic processing was performed to obtain the final data matrix for subsequent analysis.

[0084] The preprocessed matrix files were subjected to differential analysis. The R software package ropls (Version 1.6.2) was used to perform principal component analysis (PCA) and orthogonal least partial squares discriminant analysis (OPLS-DA), and 7 cycles of interactive validation were used to evaluate the stability of the model. In addition, student's t test and differential fold analysis were performed. The selection of differential metabolites was determined based on the variable weight value (VIP) obtained by the OPLS-DA model and the student's t test p value. Metabolites with VIP>1 and p<0.05 were differential metabolites. A total of 594 differential metabolites were screened.

[0085] The differential metabolites were annotated with metabolic pathways using the KEGG database (https: / / www.kegg.jp / kegg / pathway.html) to obtain the pathways that the differential metabolites participated in. The Python package scipy.stats was used for pathway enrichment analysis, and the most relevant biological pathways to the experimental treatments were obtained using Fisher’s exact test.

[0086] 2. Results 2.1 PCA analysis of the effects of Gastrodia elata extract on metabolites in the striatum of MPTP model mice like Figure 8 As shown in A, there is a clear 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 between the groups. It is worth noting that there is some overlap between the GEB and Control groups, indicating that after Gastrodia elata intervened in MPTP-induced Parkinson's disease model mice, the metabolic state in the body tended to be close to that of the normal group ( Figure 8A). There was a clear separation between the Control group and the MPTP group ( Figure 8 B), indicating that the metabolic activity of MPTP-induced mice was systematically dysregulated compared with the Control group. The GEB and Mardopar groups were divided into different groups 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 aggregation of QC samples was good, and the results showed that the entire analysis system has good stability and repeatability, which meets the analysis requirements of metabolomics.

[0087] 2.2 OPLS-DA analysis of the effects of Gastrodia elata extract on metabolites in the striatum of MPTP model mice like Fig. 9 As shown in A1-A3 of Figure 1, each point in the OPLS-DA score graph 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 from left to right are lower than the original blue Q2 point on the far right ( Fig. 9 B1-B3), the OPLS-DA model is effective and not overfitted, so the verified variable importance prediction (VIP) value was 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 inter-group difference variables, the covariance and correlation between metabolites and model classes were analyzed using S-plot. Fig. 9 As shown in C1-C3 of Figure 3, the closer the metabolites are to the two corners, the stronger their importance. Red points indicate that the VIP of these metabolites is ≥ 1, and green points indicate that the VIP of these metabolites is < 1.

[0088] 2.3 Identification of differential metabolites in the striatum of MPTP model mice after intervention with Gastrodia elata extract Using non-targeted metabolomics technology, a total of 594 differential metabolites were identified in the mouse striatum tissue. Compared with the Control group, the MPTP group had a total of 240 differential metabolites, of which 147 metabolites were down-regulated and 93 metabolites were up-regulated ( Fig.10 A); GEB compared with MPTP group, there were 309 differential metabolites, of which 139 were down-regulated and 170 were up-regulated ( Fig.10 B). Each dot in the figure represents a specific metabolite. The size of the dot indicates the VIP value. Significantly upregulated metabolites are shown in red, while significantly downregulated metabolites are shown in blue. Metabolites with no significant difference are shown in gray. The closer the dot is to the sides and the top, the more significant the difference.

[0089] 2.4 Analysis of potential biomarkers in the striatum of MPTP model mice after intervention with Gastrodia elata extract According to VIP value>1.5 and student's t test (p<0.001), 36 differential metabolites were screened between the control group and the model group, as shown in Table 2. Compared with the control group, the content of 29 metabolites in the model group was significantly decreased: 4'-Hydroxyflurbiprofen, Pro Pro Gly, 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(3-isoproterenol sulfate), 2-Carboxy-4-dodecanolide(2-carboxy-4-dodecanol), Androstenol(androstenol), (3E,6Z)-Nonadien-1-ylacetate((3E,6Z)-nonadien-2,4-dien-1-yl acetate), 6-[3-(carboxymethyl)phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid(6-[3-(carboxymethyl)phenoxy]-3,4,5-trihydroxyoxane-2-carboxylic acid), Dexpanthenol(dexpanthenol), Panaquinquecol 2(panaquinquecol 2), Gly Gly Glu(glycyl-glycyl-glutamic acid), 3-ethyl-5-methoxyphenol(3-ethyl-5-methoxyphenol), Dihydroxyphenylacetic acid(dihydroxyphenylacetic acid), DL-3,4-Dihydroxyphenyl glycol(DL-3,4-dihydroxyphenylethanol), 3-(6-hydroxy-2,3,4-trimethoxyphenyl)prop-2-enoic acid(3-(6-hydroxy-2,3,4-trimethoxyphenyl)prop-2-enoic acid), 3,4-Dihydroxyphenyl ethanol(3,4-dihydroxyphenylethanol), 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)SS-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 acid (4-pyridoxalic acid), Ser Leu Ser Gly Leu (serine-leucine-serine-glycine-leucine), 2''-O-trans-p-Coumaroylastragalin (2''-O-trans-p-coumarin), SAICAR (5-phosphothiophene adenine nucleotide). The regulatory effect of each Gastrodia elata intervention group on the differential metabolites was evaluated by comparing the relative abundance of the differential metabolites in different groups. After intervention with Gastrodia elata extract, the contents of 27 differential metabolites were significantly adjusted (, Fig.11 ).

[0090] Table 2 LC-MS identification of significant changes in potential metabolites in the striatum of MPTP-induced Parkinson's disease model mice 2.5 Effects of Gastrodia elata extract on metabolic pathways in the striatum of MPTP model mice like Fig.12As shown in A, KEGG enrichment analysis of differential metabolites in mouse striatum was performed to explore the mechanism of Gastrodia extract in preventing and treating Parkinson's disease. According to Pathway impact>0.1, the five metabolic pathways most related 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 five metabolic pathways: glycerophospholipid metabolism, sphingomyelin metabolism, tyrosine metabolism, arginine and proline metabolism, and arachidonic acid metabolism; compared with the MPTP group, Mardopar regulated seven 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 regulate four differential metabolic pathways: glycerophospholipid metabolism, sphingomyelin metabolism, arachidonic acid metabolism, arginine and proline, which may be the key to anti-PD. Further comprehensive molecular pathway analysis showed that glycerophospholipid metabolism and arachidonic acid metabolism may be important pathway mechanisms for Gastrodia elata extract and Mardopar to prevent and treat Parkinson's disease.

[0091] Among these metabolic pathways, the levels of spermidine, spermine, L-proline, L-arginine, 4-aminobutyraldehyde, and normetanephrine were significantly regulated after GEB treatment ( Fig.12 B). In this example, the relative abundance of spermidine and spermine in PD mice was significantly reduced compared with the control group. However, GEB can 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.

[0092] The relative abundance of L-proline was significantly decreased in MPTP-induced PD mice, indicating that their antioxidant capacity was reduced and accelerated the progression of PD. However, after GEB administration, the relative abundance of L-proline was significantly upregulated, indicating that they alleviated PD disease through an antioxidant mechanism.

[0093] GEB significantly upregulated the level of 4-aminobutyraldehyde in MPTP-induced PD mice, indicating that GEB exerts a protective effect on PD by promoting the conversion of 4-aminobutyraldehyde into GABA substances.

[0094] In this example, a significant decrease in the relative abundance of arginine was observed in MPTP-induced PD mice. After GEB administration, the relative abundance of arginine increased significantly, indicating that it promotes neuroprotection as an anti-PD drug.

[0095] Compared with the control group, this example revealed a significant downregulation of NE in the MPTP group, and after GEB intervention, the relative abundance of NE showed a significant upregulation.

[0096] Example 5 The following is a study on the effect of Gastrodia elata extract on motor impairment in Parkinson's disease model rats induced by 6-OHDA.

[0097] 1. Materials and Methods 1.1 Experimental animals 100 SPF grade SD rats, male, weighing about 300 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Feed and pure water were freely provided, and the rats were kept in a daily cycle of 12 h light (8:30-20:30) and 12 h dark (20:30-8:30 the next day), with room temperature of 22-25 °C and humidity of 55 ± 10%. They were allowed to adapt to the environment for 3 days before the experiment.

[0098] 1.2 Experimental Reagents The steps and method for preparing the Gastrodia elata extract are the same as those shown in Example 1.

[0099] 6-OHDA (Sigma, USA); apomorphine hydrochloride standard (APO, Gaojiaoyan Technology Co., Ltd., catalog number: 100839); ascorbic acid (Shanghai Yuanye Biotechnology Co., Ltd., catalog number: B21293).

[0100] 1.3 Main experimental instruments Computer detection, analysis and processing device for gait of mice and rats, real-time monitoring system for autonomous activities of rats (jointly developed by 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, South Korea), 462901 microinjection pump (Benchmark, USA), microinjection needle (Hamilton, Switzerland), Mili-Q ultrapure water device (Millipore, USA).

[0101] 1.4 Experimental methods 1.4.1 Surgical modeling The right medial forebrain bundle was selected for modeling, and rats were fasted and deprived of water for 8 h before surgery. On the day of surgery, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate. The skin of the rat's skull was incised along the midline of the skull, the periosteum was separated, and the skull surface was cleaned with a 3% hydrogen peroxide cotton ball until the bregma was fully exposed. The coordinates were determined with the bregma as the origin. The injection coordinates were determined according to the SD rat stereotactic brain atlas: 1.8 mm behind the bregma, 2.0 mm beside the sagittal suture, and 8.6 mm below the dura. After the coordinates were determined, disinfection was performed, and 5 μL of 6-OHDA solution was injected into the coordinate point at an injection speed of 1 μL / min. After the injection, the needle was retained for 5 min and the needle withdrawal speed was 1 mm / min. The surgical incision was sutured, and penicillin powder was applied to the epidermis to prevent surgical wound infection. The sham operation group was given an equal amount of saline containing 0.2% ascorbic acid in the same way, and the rats were raised in the same environment after surgery.

[0102] 1.4.2 Grouping and Dosing Rats were randomly divided into 3 groups, namely: ① control group, ② model group, and ③ sham operation group. There were 80 rats in the model group, 10 rats in the control group and 10 rats in the sham operation group. The rats in the control group were group-housed, and the rats in the other groups were housed in single cages. The model was identified by intraperitoneal injection of 0.5 mg / kg APO solution at week 3 after the modeling. The rats with successful modeling in the model group were randomly divided into: model group, Madopar group (7.8 mg / kg), Gastrodia elata low-dose group (100 mg / kg), and Gastrodia elata high-dose group (300 mg / kg). After grouping, Madopar or Gastrodia elata extract was continuously gavaged daily until the end of sampling. The control group, sham operation group, and model group were gavaged with the same volume of pure water.

[0103] 1.4.3 Empty field experiment The empty field experiment was the same as before.

[0104] 1.4.4 Apomorphine-induced rotation test At 1, 2, and 3 weeks after surgery, rats were intraperitoneally injected with apomorphine (APO) and their motor behavior was recorded within 30 minutes. The criteria for the success of the 6-OHDA model are: the rats use the hind limbs on the opposite side of the surgery as a support point, connect the head and tail, and form a ring with their bodies, and quickly rotate toward the opposite side of the surgery, with the number of rotations ≥180 times / 30 minutes or an average of 7 times / minute.

[0105] 1.5 Statistical methods Same as described in 1.5 of Example 2.

[0106] 2 Results 2.1 Empty field experiment In the open-field experiment ( Fig.13), compared with the control group, the total movement distance, average speed and movement time of the 6-OHDA model group decreased significantly (p<0.05, p<0.01), and the freezing time increased significantly (p<0.01); compared with the model group, Gastrodia elata (100 mg / kg) and (300 mg / kg) significantly restored the abnormalities of total movement distance, average speed, movement time and freezing time (p<0.05, p<0.01); there was no significant difference between Madopar and the model group (p>0.05).

[0107] 2.2 Apomorphine-induced rotation test Apomorphine is a typical dopamine receptor agonist, mainly used to treat the motor symptoms of Parkinson's disease. In experiments, if animals show frequent or continuous rotation behavior under certain conditions, this usually implies that the drug activates the dopamine system. In this experiment, neither the blank control group nor the sham operation group induced rotation behavior to the contralateral side, so only the data of the model group and the Tianma intervention group were statistically analyzed. The results are shown in Figure 2. Fig.14 As shown: In the APO-induced rotation experiment, compared with the model group, all the Tianma dose groups showed a certain trend of reducing the number of rotations, but there was no significant difference (p>0.05). Compared with the model group, the number of rotations in the Madopar group was significantly increased (p<0.01).

[0108] Example 6 The following is a study on the mechanism of action of Gastrodia elata extract on 6-OHDA-induced Parkinson's disease model rats.

[0109] 1. Materials and Methods 1.1 Experimental animals Same as described in 1.1 of Example 5.

[0110] 1.2 Experimental Reagents Rat dopamine (DA) ELISA kit and rat 3,4-dihydroxyphenylacetic acid (DOPAC) ELISA kit were purchased from Shanghai ELISA Biological Co., Ltd. Primary antibodies TLR4, NF-κB, p65, and Cleaved-Caspase-3 were purchased from CellSignaling Technology; Bax and Bcl-2 were purchased from Abcam.

[0111] 1.3 Main experimental instruments Same as described in 1.3 of Example 3.

[0112] 1.4 Experimental methods 1.4.1 Grouping Sham operation group, model group, Madopar group (7.8 mg / kg), Gastrodia elata low-dose group (100 mg / kg), Gastrodia elata high-dose group (300 mg / kg).

[0113] 1.5 Statistical methods Same as described in 1.5 of Example 2.

[0114] 2. Results 2.1 Effect of Gastrodia elata extract on DA content in the striatum of 6-OHDA rats One of the main characteristics of Parkinson's disease is the progressive loss of dopamine neurons in the substantia nigra. This neuronal loss results in a decrease in the amount of dopamine in the brain. Fig.15 As shown, compared with the sham operation group, the DA level of rats in the model group was significantly decreased (p<0.001); the DA level of 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 higher than that in the model group, and the difference was significant (p<0.05).

[0115] 2.2 Effect of Gastrodia elata extract on DOPAC content in the striatum of 6-OHDA rats As one of the main metabolites of dopamine, the content of DOPAC reflects the metabolic rate of dopamine and the activity state of the dopamine system to a certain extent. Therefore, by measuring the changes in DOPAC, the functional state of the dopamine system can be indirectly evaluated. Fig.16 As shown, compared with the sham operation group, the DOPAC content of the model group rats was significantly decreased (p<0.001); compared with the model group, the positive drug Madopar significantly restored the DOPAC level of the model group rats (p<0.05); the content of the Gastrodia elata (300 mg / kg) group tended to be higher than that of the model group, but the difference was not significant (p>0.05).

[0116] 2.3 Effect of Gastrodia elata extract on the expression levels of TLR4 and NF-κB proteins in the striatum of 6-OHDA-induced Parkinson's disease model rats Neuronal loss and inflammatory response are important features of the pathological process of Parkinson's disease. TLR4 can trigger neuroinflammatory response 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. Fig.17 As shown in the data, compared with the control group, the expression of TLR4 protein in the model group tended to be upregulated after 6-OHDA induction, and the expression of TLR4 protein tended to be downregulated after intervention with Madopar and Gastrodia elata, but there was no significant difference (p>0.05).

[0117] In Parkinson's disease, NF-κB is usually activated and participates in regulating the occurrence and progression of neuroinflammatory reactions. Activation of NF-κB may further aggravate the damage of dopamine neurons by regulating the expression of inflammatory factors (such as TNF-α, IL-1β, etc.). The expression of NF-κB protein in the model group was significantly increased after 6-OHDA induction (p<0.01); after treatment with Madopar and Gastrodia elata (300 mg / kg), the expression of NF-κB protein was significantly reduced (p<0.05, p<0.01).

[0118] In conclusion, the effects of Gastrodia extract on motor impairment and its mechanism of action in MPTP-induced subacute Parkinson's disease mice and 6-OHDA-induced Parkinson's disease rats were studied, and potential biomarkers were explored by combining non-targeted metabolomics analysis. The results showed that Gastrodia extract could improve motor impairment in MPTP-induced subacute Parkinson's disease mice, which was manifested by improving gait function and autonomous activity ability, reducing the expression of inflammatory factors TNF-α, IL-6, IL-1β and the expression of Caspase-3 and Caspase-9 apoptotic proteins in the striatum, and playing a protective role against MPTP-induced neuroinflammatory response. A total of 594 differential metabolites were found by non-targeted metabolomics analysis of the striatum. Among them, there were 36 significantly different metabolites between the control group and the model group. Compared with the model group, Gastrodia extract could regulate 27 differential metabolites to normal levels. Gastrodia administration intervention can improve metabolic pathways such as glycerophospholipid metabolism, sphingolipid metabolism, tyrosine metabolism, arachidonic acid metabolism, arginine and proline metabolism. In addition, Gastrodia elata extract has a certain improvement effect on the motor function impairment of 6-OHDA-induced PD rats. It can increase the content of dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) neurotransmitter levels in the striatum of 6-OHDA-induced PD rats. It down-regulates the expression of NF-κB, Caspase-3 protein and Bax / Bcl-2 ratio in the striatum of 6-OHDA-induced PD rats, and reduces the occurrence of cell apoptosis. This shows that Gastrodia elata is expected to become an ideal candidate for the development of drugs to treat Parkinson's disease. It provides data support for the study of the mechanism of Gastrodia elata in preventing and improving Parkinson's disease.

[0119] 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 adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation 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 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 / or dihydroxyphenylacetic acid in the striatum, and the expression level of TLR4 and / or NF-κB proteins in the striatum; The metabolomics indicators include 4'-hydroxyflurbiprofen, L-prolylglycine, hopkinsin, phosphatidylserine, phenylalanine-phenylalanine-lysine, 3,3'-thiodipropionic acid, 2-carboxy-4-dodecanol, androstenol, anti-tropolamine A, Ent-6,16-quinolinadiene-19-acid, panquinol 2, 3-ethyl-5-methoxyphenol, DL-3,4-dihydroxyphenylethanol, 3-(6-hydroxy-2,3,4-trimethoxyphenyl)prop-2-enoic acid, dihydroxyphenyl Acetic acid, 3,4-dihydroxyphenylethanol, 2-hydroxy-3-(2,4,5-trihydroxyphenyl)propionic acid, 7-aminoclonazepam, serine-leucine-serine-glycine-leucine, 2''-O-trans-p-coumarin, 4-hydroxyphenylacetylglutamate, 1,2-dehydrooctanol, 5-phosphothiophene adenine nucleotide, (S)-C(S)S-methionine sulfoxide, (3E,6Z)-nona-2,4-dien-1-yl acetate, dexpanthenol, 3-o-isoproterenol sulfate.

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 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 dopamine and / or dihydroxyphenylacetic acid content in the striatum, reducing the expression level of TLR4 and / or NF-κB proteins in the striatum and the level of callback metabolites.

5. The method for identifying the effectiveness of Gastrodia elata extract in preventing and treating Parkinson's disease according to claim 4, characterized in that: 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.

6. 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.

7. A method for regulating abnormal metabolites of cells in vitro, characterized in that: The method comprises the step of contacting the Gastrodia elata extract identified by the method according to any one of claims 1 to 6 with cells in vitro, wherein the abnormal metabolites of the cells are regulated, comprising: upregulating 4'-hydroxyflurbiprofen, L-prolylglycine, hawkins, 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.

Images