A traditional Chinese medicine composition for preventing and / or treating Parkinson's disease

Abstract

The application provides a traditional Chinese medicine composition for preventing and / or treating Parkinson disease, and belongs to the field of traditional Chinese medicines.The traditional Chinese medicine composition provided by the application is composed of Radix Rehmanniae Preparata 130-180 parts, Cortex Cinnamomi 20-40 parts, Rhizoma Alpiniae Oxyphyllae 20-40 parts and Radix Polygalae 20-40 parts, has a simple preparation method, and has no obvious difference in drug efficacy compared with traditional Parkinson treatment drugs, and has practical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition for preventing and / or treating Parkinson's disease. BACKGROUND

[0002] The statements in this section merely provide background information related to the present application and do not necessarily constitute the prior art.

[0003] Parkinson's disease is a common neurodegenerative disease in the elderly, with high heterogeneity, and its clinical symptoms include extensive motor symptoms and non-motor symptoms. Typical motor symptoms are bradykinesia, resting tremor and muscle rigidity; meanwhile, it is accompanied by different degrees of non-motor symptoms such as cognitive impairment, mental disorders, autonomic nervous disorders, sleep disorders, and hyposmia. The typical pathological manifestations are the damage, necrosis and loss of dopaminergic neurons in the substantia nigra of the midbrain, accompanied by the generation of pathological products Lewy bodies. Due to the increasing social and economic burden caused by Parkinson's disease, it has become a national health problem that cannot be ignored.

[0004] At present, there are therapeutic drugs for Parkinson's disease. Western medicines usually use levodopa, dopamine receptor agonists, and anticholinergic drug amantadine, but they can only suppress symptoms and cannot intervene in the delay of neuronal degenerative damage.

[0005] In traditional Chinese medicine, Parkinson's disease belongs to the category of "shaking disease", and its pathogenesis is complex, mainly including two aspects of deficiency in origin and excess in superficiality. In the clinical diagnosis and treatment strategy of traditional Chinese medicine, emphasis is placed on syndrome typing and intervention according to the syndrome of the disease. Among them, Parkinson's disease with kidney essence deficiency syndrome caused by kidney essence deficiency, marrow deficiency and muscle and bone malnutrition is common in syndrome typing. Under the condition that the first-line western medicine has not achieved satisfactory results, some traditional Chinese medicine preparations for intervention according to the syndrome provide a possibility for the treatment of Parkinson's disease due to their neuroprotective effect in delaying disease progression. However, the existing traditional Chinese medicine compound preparations for Parkinson's disease with kidney essence deficiency syndrome have many flavors, and the preparation process is complicated, the active ingredients are not easy to be clear, and there is a probability of side reactions between multiple drugs. SUMMARY

[0006] In order to overcome the shortcomings of the prior art, the present application provides a traditional Chinese medicine composition for preventing and / or treating Parkinson's disease. The traditional Chinese medicine composition provided by the present application has simple drug composition and preparation method, and has no obvious difference in efficacy compared with traditional treatment drugs for Parkinson's disease (especially Parkinson's disease with kidney essence deficiency syndrome), and has practical application value.

[0007] To achieve the above object, one or more embodiments of the present application provide the following technical solutions:

[0008] In a first aspect of the present application, a traditional Chinese medicine composition for preventing and / or treating Parkinson's disease is provided, which is composed of Radix Rehmanniae Preparata 130-180 parts, Cinnamomum Cassia 20-40 parts, Acorus gramineus 20-40 parts, and Polygala 20-40 parts.

[0009] In a specific embodiment of the present application, the traditional Chinese medicine composition is composed of Radix Rehmanniae Preparata 150 parts, Cinnamomum Cassia 30 parts, Acorus gramineus 30 parts, and Polygala 30 parts.

[0010] In a second aspect of the present application, a traditional Chinese medicine extract is provided, which is obtained by soaking the traditional Chinese medicine composition of the first aspect in water and decocting 1-3 times, combining the decocting liquid, and concentrating into a 150-200% suspension, i.e., a suspension with a relative density of 0.027-0.036 g / mL.

[0011] In a specific embodiment of the present application, the traditional Chinese medicine composition is soaked in 10 times the volume of water for 2 hours, and the liquid is filtered after 1 hour of decoction. Then, an equal amount of water is added for 1 hour of decoction, and the liquid is filtered. The two liquids are mixed, heated, and concentrated into a 178% suspension.

[0012] In a third aspect of the present application, a traditional Chinese medicine preparation is provided, which comprises the traditional Chinese medicine extract of the second aspect.

[0013] In a specific embodiment of the present application, the traditional Chinese medicine preparation further comprises a pharmaceutically acceptable carrier.

[0014] In a specific embodiment of the present application, the medicine further comprises other components for treating Parkinson's disease, such as levodopa, dopamine receptor agonists, and anticholinergic drug amantadine.

[0015] In a fourth aspect of the present application, the use of the traditional Chinese medicine composition or the traditional Chinese medicine extract or the traditional Chinese medicine preparation in the preparation of a medicine for preventing and / or treating Parkinson's disease is provided.

[0016] In a specific embodiment of the present application, the Parkinson's disease is Parkinson's kidney essence deficiency syndrome.

[0017] Kidney essence deficiency is also known as kidney essence deficiency, kidney essence deficiency, and kidney essence deficiency. Children's growth and development are delayed, and adults' reproductive function is reduced. Early aging, tinnitus, hair loss, loose teeth, forgetfulness, and other common symptoms are common. Kidney essence deficiency usually manifests as dizziness, tinnitus, soreness of the waist and knees, decreased sexual function, decreased semen in men, early exhaustion of "Tianqi" in women, premature aging, fatigue, forgetfulness, pale tongue, less moss, and other symptoms. In the present application, the Parkinson's kidney essence deficiency syndrome animal model is constructed by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) combined with D-galactose.

[0018] In a fifth aspect of the present application, the application provides use of the above-mentioned traditional Chinese medicine composition or traditional Chinese medicine extract or traditional Chinese medicine preparation in the preparation of a medicament for alleviating movement disorders caused by Parkinson's disease, improving damage to dopaminergic neurons in the substantia nigra compacta, or improving the product of striatal neuron burst firing.

[0019] In the detailed description of the present application, the movement disorders caused by Parkinson's disease are manifested as one or more of the following: a shortened duration of the rotarod test, weakened balance and coordination, an extended time for climbing a pole, an increased duration of gait, an increased step frequency, a shortened step length, or a shortened standing time.

[0020] In the detailed description of the present application, the medicament for preventing and / or treating Parkinson's disease includes a drug for inhibiting a Parkinson's disease model.

[0021] Preferably, the drug administration subject can be a human or a non-human mammal, such as a mouse, a rat, a guinea pig, a rabbit, a dog, a monkey, a chimpanzee, etc.

[0022] The above one or more technical solutions have the following beneficial effects:

[0023] The present application optimizes the existing prescription for treating Parkinson's disease (kidney essence deficiency syndrome) with Dihuang Yinzi, and obtains the traditional Chinese medicine composition (Jingyi Qiangzhi Recipe) of the present application. Through biological experiments, it is verified that the Jingyi Qiangzhi Recipe provided by the present application has no obvious difference in the prevention or treatment effect on Parkinson's disease with kidney essence deficiency syndrome on the basis of the simplified Dihuang Yinzi.

[0024] The traditional Chinese medicine composition prepared by the present application is verified by pharmacodynamic experiments to have a significant pharmacodynamic effect, high bioavailability, and stable and reliable quality.

[0025] The traditional Chinese medicine composition can be prepared into different dosage forms according to the needs of patients, is convenient to carry and easy to take, and has practical application value.

[0026] The advantages of the additional aspects of the present application will be partially given in the following description, partially will become obvious from the following description, or will be understood through the practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0027] The drawings accompanying the specification of the present application serve to provide a further understanding of the present application, and the illustrative embodiments of the present application and their descriptions serve to explain the present application, and do not constitute an improper limitation on the present application.

[0028] Figure 1 Figure 1 is a flowchart of the preparation of a Parkinson's disease kidney essence deficiency syndrome mouse model and the pharmacodynamic evaluation experiment of the Yijing Qiangzhi Recipe according to Embodiment Three of the present application. PD: Parkinson's disease; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; i.p.: intraperitoneal injection; i.g.: oral administration.

[0029] Figure 2 To prolong the duration of the rotating stick experiment of the Parkinson's disease kidney essence deficiency syndrome mouse model by the Yijing Qiangzhi of the present application, ** p<0.01, **** p<0.001). Control: normal mice, Model: Parkinson's disease kidney essence deficiency syndrome mouse model, Model+DHYZ: Parkinson's disease kidney essence deficiency syndrome mouse model treated with Dihuang Yinzi, Model+YJQZ: Parkinson's disease kidney essence deficiency syndrome mouse model treated with Yijing Qiangzhi, Model+L-DOPA: Parkinson's disease kidney essence deficiency syndrome mouse model treated with levodopa.

[0030] Figure 3 To reduce the time of the pole climbing experiment of the Parkinson's disease kidney essence deficiency syndrome mouse model by the Yijing Qiangzhi of the present application, * p<0.05, ** p<0.01). Control: normal mice, Model: Parkinson's disease kidney essence deficiency syndrome mouse model, Model+DHYZ: Parkinson's disease kidney essence deficiency syndrome mouse model treated with Dihuang Yinzi, Model+YJQZ: Parkinson's disease kidney essence deficiency syndrome mouse model treated with Yijing Qiangzhi, Model+L-DOPA: Parkinson's disease kidney essence deficiency syndrome mouse model treated with levodopa.

[0031] Figure 4 To obtain the footprint graph and three-dimensional footprint graph of each group of mice completing the runway in the automatic gait analysis test of the present application. Control: normal mice, Model: Parkinson's disease kidney essence deficiency syndrome mouse model, Model+DHYZ: Parkinson's disease kidney essence deficiency syndrome mouse model treated with Dihuang Yinzi, Model+YJQZ: Parkinson's disease kidney essence deficiency syndrome mouse model treated with Yijing Qiangzhi, Model+L-DOPA: Parkinson's disease kidney essence deficiency syndrome mouse model treated with levodopa.

[0032] Figure 5 To obtain the time and speed of each group of mice passing through the runway in the automatic gait analysis of the present application. A: time of each group of mice passing through the runway in the gait analysis, B: speed of each group of mice passing through the runway in the gait analysis *** p<0.001,

[0033] ****p<0.0001. Control: normal mice, Model: Parkinson's disease kidney essence deficiency syndrome mouse model, Model+DHYZ: Parkinson's disease kidney essence deficiency syndrome mouse model treated with Dihuang Yinzi, Model+YJQZ: Parkinson's disease kidney essence deficiency syndrome mouse model treated with Yijing Qiangzhi recipe, Model+L-DOPA: Parkinson's disease kidney essence deficiency syndrome mouse model treated with levodopa.

[0034] Figure 6 For example three of the present application, automatic gait analysis of each group of mice four limbs step circumference, standing time, A-D automatic gait analysis of each group of mice four limbs standing time E-H automatic gait analysis of each group of mice four limbs step circumference **** p<0.0001. Control: normal mice, Model: Parkinson's disease kidney essence deficiency syndrome mouse model, Model+DHYZ: Parkinson's disease kidney essence deficiency syndrome mouse model treated with Dihuang Yinzi, Model+YJQZ: Parkinson's disease kidney essence deficiency syndrome mouse model treated with Yijing Qiangzhi recipe, Model+L-DOPA: Parkinson's disease kidney essence deficiency syndrome mouse model treated with levodopa.

[0035] Figure 7 For example three of the present application, Yijing Qiangzhi relieves the damage of substantia nigra compacta tyrosine hydroxylase positive neurons in Parkinson's disease kidney essence deficiency syndrome mouse model, A each group of mice substantia nigra compacta tyrosine hydroxylase positive neurons schematic diagram B each group of mice substantia nigra compacta tyrosine hydroxylase positive neurons statistical graph. Control: normal mice, Model: Parkinson's disease kidney essence deficiency syndrome mouse model, Model+YJQZ: Parkinson's disease kidney essence deficiency syndrome mouse model treated with Yijing Qiangzhi recipe, Model+L-DOPA: Parkinson's disease kidney essence deficiency syndrome mouse model treated with levodopa.

[0036] Figure 8 For example three of the present application, Yijing Qiangzhi relieves the abnormal burst discharge of striatal neurons in PD kidney essence deficiency syndrome mouse model, A each group of mice striatal neurons burst discharge times per minute statistical graph B each group of mice striatal neurons burst discharge neuron ratio statistical graph * p<0.05, ** p<0.01, *** p<0.001. Control: normal mice, Model: Parkinson's disease kidney essence deficiency syndrome mouse model, Model+YJQZ: Parkinson's disease kidney essence deficiency syndrome mouse model treated with Yijing Qiangzhi recipe, Model+L-DOPA: Parkinson's disease kidney essence deficiency syndrome mouse model treated with levodopa. DETAILED DESCRIPTION

[0037] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not intended to limit the scope of the application.

[0038] In the present application, the term "treatment" has its general meaning and in particular herein refers to the use of the medicaments of the present application for the management of a mammalian subject (preferably a human) who has already acquired a disease, in order to produce a therapeutic, healing, remission-inducing, palliative or prophylactic effect, to obtain a desired pharmacological and / or physiological effect.

[0039] "Pharmaceutically acceptable carrier" is art-recognized and includes a pharmaceutically-acceptable material, composition or carrier, suitable for administering the compounds of the present application to mammals. The carrier includes a liquid or solid filler, diluent, excipient, solvent or encapsulating material involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; and

[0040] The formulations of the present application can conveniently be presented in unit dosage form and can be prepared by any methods well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.

[0041] It should be noted that the present application is not limited in scope by the specific examples described herein, which are intended as illustrations only and not limiting of the application's scope. Any methods that are functionally equivalent are within the scope of the application. Various modifications of the application in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. All publications, patents and patent applications cited herein, including during the priority period, are hereby incorporated by reference in their entirety for all purposes.

[0042] In the following examples, the materials used, reagents, etc. are of commercial origin unless otherwise specified.

[0043] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not intended to limit the scope of the application.

[0044] Example 1

[0045] The embodiment discloses a traditional Chinese medicine composition for preventing or treating Parkinson's disease and a preparation method thereof, and the traditional Chinese medicine composition comprises 150g of Radix Rehmanniae Preparata., 30g of Cinnamomum cassia Presl, 30g of Acorus tatarinowii Schott and 30g of Polygala tenuifolia Willd.

[0046] Medicine preparation: 10 times the volume of water of the original medicine is added to soak for 2 hours, and the medicine liquid is filtered out after decocting for 1 hour; then the same amount of water is added to decoct for 1 hour, and the medicine liquid is filtered out; the medicine residues are squeezed dry, the medicine liquids taken out twice are mixed, and the mixture is heated and concentrated into a suspension of 178%; that is, each medicine is concentrated into 55.6ml, and each milliliter contains 1.78g of crude medicine; the obtained medicine is named Yijing Qiangzhi Decoction; and after high-pressure sterilization, the Yijing Qiangzhi Decoction is stored in a refrigerator at 4 DEG C for cold storage and standby.

[0047] Example 2

[0048] The embodiment discloses a traditional Chinese medicine composition for preventing or treating Parkinson's disease and a preparation method thereof, and the traditional Chinese medicine composition comprises 170g of Radix Rehmanniae Preparata., 20g of Cinnamomum cassia Presl, 20g of Acorus tatarinowii Schott and 40g of Polygala tenuifolia Willd.

[0049] Medicine preparation: 10 times the volume of water of the original medicine is added to soak for 2 hours, and the medicine liquid is filtered out after decocting for 1.5 hours; then the same amount of water is added to decoct for 1 hour, and the medicine liquid is filtered out; the medicine residues are squeezed dry, the medicine liquids taken out twice are mixed, and the mixture is heated and concentrated into a suspension of 178%; that is, each medicine is concentrated into 55.6ml, and each milliliter contains 1.78g of crude medicine; the obtained medicine is named Yijing Qiangzhi Decoction; and after high-pressure sterilization, the Yijing Qiangzhi Decoction is stored in a refrigerator at 4 DEG C for cold storage and standby.

[0050] Comparative Example 1

[0051] The comparative example adopts Dihuang Yinzi, and the formula of the Dihuang Yinzi is as follows: 12g of Radix Rehmanniae Preparata., 9g of Fructus Corni, 9g of Herba Cistanche, 9g of Radix Morindae Officinalis, 6g of Aconiti Praeparata, 6g of Cinnamomum cassia Presl, 6g of Radix Ophiopogonis, 6g of Schisandra chinensis, 9g of Dendrobium nobile, 6g of Acorus tatarinowii Schott, 6g of Polygala tenuifolia Willd., 6g of White Poria, 3g of Mentha haplocalyx, 3g of ginger and 3g of Jujube.

[0052] The medicine preparation method of the Dihuang Yinzi is as follows: 10 times the volume of water of the original medicine is added to soak for 2 hours, and the medicine liquid is filtered out after decocting for 1 hour; then the same amount of water is added to decoct for 1 hour, and the medicine liquid is filtered out; the medicine residues are squeezed dry, the medicine liquids taken out twice are mixed, and the mixture is heated and concentrated into a suspension of 178%; that is, each medicine is concentrated into 55.6ml, and each milliliter contains 1.78g of crude medicine; and after high-pressure sterilization, the Yijing Qiangzhi Decoction is stored in a refrigerator at 4 DEG C for cold storage and standby.

[0053] Example 3

[0054] The embodiment discloses application of Yijingqiangzhi prescription in preventing or treating Parkinson (especially Parkinson disease with kidney essence deficiency syndrome).

[0055] The main preparation method for the mouse model of Parkinson disease with kidney essence deficiency syndrome is intraperitoneal injection of D-galactose and MPTP. At present, MPTP is commonly used to construct a subacute Parkinson disease model. The D-galactose-induced experimental aging animal model is a recognized ideal modeling method. D-galactose is converted into glycosylation end products in vivo, and the body metabolism is not as good as before, which can accelerate aging. The induced aging can simulate the kidney essence deficiency syndrome. Therefore, the method of combining MPTP with D-galactose is a possible method for constructing the PD kidney essence deficiency syndrome animal model (reference: Sun K, Yang P, Zhao R, et.al. D-Galactose-Induced Aging-Related Behavior in Mice via Inhibition of Cellular Senescence and Oxidative Stress. Oxidative medicine and cellular longevity 2018, 7108604.).

[0056] 1. Experimental materials

[0057] 1.1 Experimental animals

[0058] Healthy male SPF C57BL / 6J mice, weighing 20±2 g, 8 weeks old, purchased from Beijing Vantoll Life Experimental Animal Technology Co., Ltd. [SCXK (Jing) 2016-0006], all animals were bred in the SPF mouse laboratory of Shandong University of Traditional Chinese Medicine [SYXK (Lu) 2022 0009], the mice were randomly numbered using ear tag pliers after arrival, 5 mice were fed in each cage, and the light and dark cycle was maintained at 12 h / 12 h, the day and night were reversed (lights on at 20:00, lights off at 8:00), the humidity was 65%±2%, and the temperature was 21.0±1.0℃. The experimental protocol in this study was approved by the Ethics Committee of Experimental Animals of Shandong University of Traditional Chinese Medicine (No. SDUTCM2022111449), and the animal experiments were conducted in accordance with the 3R principle. After the animals arrived, they were adaptively fed for 1 week, and the parallel operation of grasping was performed daily to eliminate the influence of human operation, and the animals were free to eat and drink during the experiment, and the bedding was replaced every 3 days.

[0059] 1.2 Reagents and drugs

[0060] Normal saline: Chenxin Pharmaceutical Co., Ltd., item number: H37022336

[0061] D-galactose: MACKLIN, item number: D810319

[0062] MACKLIN, Cat. No.: M832929

[0063] Sodium carboxymethylcellulose: Source Leaf Biotech, Cat. No.: S14016

[0064] Yijing Qiangzhi: Self-made by the Pharmacy Department of the Affiliated Hospital of Shandong University of Chinese Medicine

[0065] Dihuang Yinzi: Self-made by the Pharmacy Department of the Affiliated Hospital of Shandong University of Chinese Medicine Levodopa: MACKLIN, Cat. No.: M832929

[0066] D807434

[0067] 1x PBS buffer (pH 7.2-7.4): Biosharp, Cat. No.: BL550A

[0068] 4% paraformaldehyde / Universal tissue fixative: Biosharp, Cat. No.: BL539A

[0069] Sodium pentobarbital: Hubei Hongyunlong Biological Technology Co., Ltd.

[0070] 1.3 Equipment and instruments

[0071] Animal behavior analysis system: Shanghai Xinsuo Information Technology Co., Ltd., XR-SuperMaze

[0072] Open field test system: Shanghai Xinsuo Information Technology Co., Ltd., XR-XZ301

[0073] Rope climbing experiment device: Self-made

[0074] Rotarod fatigue instrument: Ugo Basile, 47650

[0075] Animal gait analysis system: Noldus Information Technology (Beijing) Co., Ltd., CatWalk XT

[0076] Cycloidal shaker: Labyeah, CS-1003K

[0077] Electronic analytical balance of 100,000th: Sartorius, CP225D

[0078] Milli-Q pure water machine

[0079] Manual pipette: Eppendorf

[0080] Pipette tip: axygen

[0081] Centrifuge: Eppendorf

[0082] Surgical instrument kit: Ruivode

[0083] 2. Experimental method

[0084] Preparation of mouse model of Parkinson's disease with kidney essence deficiency and evaluation process of Yijingqiangzhi and Dihuang Yinzi Figure 1 as shown.

[0085] 2.1 Experimental grouping

[0086] After the mice were adaptively fed in SPF environment for 1 week, they were randomly divided into normal control group (Control), Parkinson's disease with kidney essence deficiency model group (Model), Parkinson's disease with kidney essence deficiency Dihuang Yinzi treatment group (Model+DHYZ), Parkinson's disease with kidney essence deficiency Yijingqiangzhi treatment group (Model+YJQZ), and Parkinson's disease with kidney essence deficiency levodopa treatment group (Model+L-DOPA) according to the total distance in the open field and the body weight of the mice using the Rand function.

[0087] 2.2 Preparation of mouse model of Parkinson's disease with kidney essence deficiency

[0088] The animals were injected intraperitoneally with drugs at a dose of 1 ml / 100 g body weight. Subacute aging mouse models with kidney essence deficiency were constructed by intraperitoneal injection of D-galactose (150 mg / kg / d), and the injection was performed at 8:30 every day for 6 weeks. The normal control group was given an equal volume of normal saline (10 ml / kg / d). Parkinson's disease mouse models were constructed by intraperitoneal injection of MPTP (30 mg / kg / d), and the injection was performed at 14:30 every day for 1 week. The normal control group was given an equal volume of normal saline (10 ml / kg / d).

[0089] 2.3 Drug administration method: After intraperitoneal injection of D-galactose for 6 weeks and MPTP for 1 week, the mice were given oral administration. According to the equivalent ratio measurement table for body surface area conversion between mice and humans, the equivalent dose of mice was calculated: the Model+DHYZ group was given Dihuang Yinzi at a dose of 0.5 ml / 100 g by gavage, the Model+YJQZ group was given Yijingqiangzhi at a dose of 0.5 ml / 100 g by gavage, the Model+L-DOPA group was given levodopa at a dose of 50 mg / kg by gavage, and levodopa was dissolved in 0.5% carboxymethylcellulose sodium solution. The other groups of mice were given an equal volume of 0.5% carboxymethylcellulose sodium solution, and the administration was continued for 2 weeks.

[0090] 2.4 Behavioral test

[0091] 2.4.1 Rotarod test

[0092] The rotarod apparatus consists of a 5-channel rotating rod (3.5 cm in diameter) and a trip box, a touch screen, different speed modes and a drive device. Three days before the experiment, the mice were placed in the laboratory one hour in advance to adapt to the experimental environment. The experiment was carried out in a quiet weak red light environment. The mice were placed on the rotating rod of the rotarod apparatus, which rotated at a constant speed (12 r / min) for 60 seconds. If the mouse fell in the middle, it was put back on the rotating rod. In the formal experiment, the acceleration mode (5 r / min to 40 r / min, lasting 10 min) was used for testing. The rotarod fall latency of the mice was recorded. If the mouse did not fall from the rotating rod, the fall time was considered to be 600 seconds. After each mouse test, the rotarod apparatus was cleaned. Each mouse was tested 4 times with an interval of 30 minutes, and the average value was used for statistical analysis.

[0093] 2.4.2 Pole climbing experiment

[0094] The pole climbing apparatus consists of a 50 cm long wooden pole with a diameter of 1 cm, a circular ball with a diameter of 2.5 cm at the top, and a base. The outside of the ball and the pole is wrapped with gauze, and the wooden pole is perpendicular to the base. One hour in advance, the experimental mice were placed in the laboratory to adapt to the experimental environment in advance. The experiment was carried out in a quiet weak red light environment. The mice were placed on the top of the ball with their heads up, and the time from the top to the base was recorded. After each mouse test, the pole climbing apparatus was cleaned. Each mouse was tested 4 times with an interval of 30 minutes, and the average value was used for statistical analysis.

[0095] 2.4.3 Automatic gait analysis

[0096] The automatic gait analysis system includes a glass plate runway, a light plate above the glass plate, and an image automatic acquisition and analysis processing system including a camera, a display, and analysis processing software. Three days before the experiment, uniform gait training is performed. One hour in advance, the experimental mice are placed in the laboratory to adapt to the experimental environment. The experiment is performed in a quiet weak red light environment. During training, the mice are placed at the front end of the CatWalk instrument runway, and they are allowed to freely explore until they walk the entire runway without turning around. Entering the dark box at the end of the instrument is considered as one training. If the mouse turns around in the middle, it is considered as a failed training, and the mouse is taken out and placed at the front end of the runway to start a new training. If the mouse hesitates to move forward, a certain amount of wind is blown from the back of the mouse to stimulate it, or food is placed at the end of the runway to attract it, until it enters the dark box and adapts for 30 seconds. After each mouse training, the residues on the runway are cleaned with 75% alcohol solution to prevent the odor of the previous mouse's movement trail from interfering with the next test mouse. Each mouse is trained for 3 times. In the formal experiment, the mice are placed at the front end of the CatWalk instrument runway, and they walk the entire runway without turning around and enter the dark box at the end of the instrument. This is considered as one successful test. If the mouse hesitates to move forward or turns around during the walking process, the test fails and needs to be retested. Each mouse is tested for 3 times, and the average value is used for evaluation.

[0097] 2.5 Immunohistochemical staining to detect the expression of tyrosine hydroxylase in the substantia nigra compacta

[0098] In this study, the antigen-antibody binding staining technique was used to detect the number of tyrosine hydroxylase positive neurons in the substantia nigra compacta of mice. The specific operation steps are as follows:

[0099] The complete mouse whole brain was placed on the tray of the freezing microtome, and the mouse brain atlas was combined. The substantia nigra compacta was cut into 40 um thick coronal sections at -20℃. The brain sections were placed in a 12-well plate from front to back, soaked in 1x phosphate buffer solution (PBS), and prepared for use.

[0100] Washing: 5 min x 2 times with TBS containing 0.025% Triton X-100, and placed on a shaker

[0101] Blocking: 2h of TBS solution containing 10% normal goat serum at room temperature, after blocking, the waste liquid was discarded

[0102] Primary antibody incubation: goat serum blocking solution was used as diluent, and the primary antibody used was Tyrosine Hydroxylase (1:1000) (E2L6M, Cell Signaling Technology). After adding the primary antibody, it was incubated at 4℃ overnight

[0103] Wash: 5 min x 2 with 0.025% concentration Triton X-100 in TBS on a shaker

[0104] Hydrogen Peroxide Block: 15 min incubation of the slides in Hydrogen Peroxide Block

[0105] Wash: 5 min x 2 with 0.025% concentration Triton X-100 in Tris buffered saline (TBS) on a shaker

[0106] Secondary antibody incubation: 10 min, goat anti-rabbit antibody (ab64261, Rabbit Specific HRP / DAB (ABC) Detection IHC Kit) was used to flood the slides (30ul per drop, 24 well plate, 3-4 drops per well)

[0107] Wash: 5 min x 2 with 0.025% concentration Triton X-100 in TBS on a shaker

[0108] Streptavidin Peroxidase incubation: 10 min incubation at room temperature

[0109] Wash: 5 min x 2 with 0.025% concentration Triton X-100 in TBS on a shaker

[0110] Chromogen: freshly prepared, mix the chromogen and substrate in a ratio of 1:50 (avoid light)

[0111] Example: if 500ul total volume is needed, then 10ul of DAB (Chromogen) and 490ul of DAB substrate are needed

[0112] Wash: distilled water (depending on the degree of color development of the slices in the well plate, it is generally not more than 1 minute, if the time is too long, it will cause the slices to be too dark)

[0113] Mounting: the slices in the well plate are flatly mounted on the adhesive glass slide (as close as possible to the bottom of the glass slide to save reagents when dehydrating)

[0114] Dehydration: 1 min x 4, the glass slide is sequentially immersed in 75%, 95%, 100%, and 100% alcohol for dehydration

[0115] 3 min x 2, the glass slide is immersed in grade I and grade II (second time) xylene for transparency

[0116] Wait for the slide with the brain section to dry before placing it in alcohol dehydration (to prevent the section from falling off)

[0117] Mounting: Drop neutral resin on the brain section, place the cover glass on one side first, try to avoid air bubbles that will affect the observation.

[0118] Observe under the microscope after mounting.

[0119] 2.6 Resting mouse striatal neuron electrophysiology detection

[0120] 2.6.1 Animal electrode implantation surgery

[0121] Anesthetize the mouse with an anesthetic machine (isoflurane) at a pressure of 0.8 nL / min, place the mouse in the chamber, and after it is anesthetized, adjust the anesthetic mode to mask (after entering the anesthetic state, the small valve 0.15 nL / min can maintain anesthesia). After the mouse's tendon reflex and blinking reflex disappear, it is completely anesthetized, the hair between the two ears of the mouse is cut, and the area between the two ears is disinfected with iodophor and alcohol. According to the order of iodophor first and then alcohol (three times), disinfect the area. Take the anterior fontanel as the origin, move the same distance (2 mm) left and right, and lower the needle to contact the surface of the skull. Read the z-axis coordinates of the corresponding points respectively, and judge whether it is flush (<0.05 mm) through the reading of the z-axis coordinates. Then take the anterior fontanel as the origin, and locate the posterior fontanel (<0.05 mm). After the four points of the anterior fontanel and the posterior fontanel are basically maintained on the same plane, first locate the four points according to the range coordinates of the striatum (STR) AP: 0.97-1.30 mm, ML: ±2.00-±2.80 m. Use a syringe needle or other sharp object to dip paint as a marker to mark the location, use a skull drill to drill the marker point, slowly grind the skull to expose the dura mater, and try to avoid bleeding (generally a square skylight). Carefully peel off the dura mater with a fine tweezer, and carefully remove the remaining bone fragments (be careful not to damage the brain tissue). Then test whether there is residual skull with a discarded electrode wire. Then drill two screw holes on the opposite side of the skylight (avoid the skull sutures of the anterior fontanel and the posterior fontanel) to fix the skull screws (use the skull drill just touching the skull surface as the standard, there will be a clear empty feeling). Make the screw touch the brain tissue, but not too much pressure. Connect the ground wire (the screw should just touch the dura mater, and should not be shaken; the ground wire is tightly wrapped around the screw to effectively avoid motion noise). Use the electrode holder to hold the electrode, first locate it above the skylight. The electrode tip is next to the brain tissue surface. Then, use the micro-advance to slowly advance, so that the electrode enters the brain tissue (DV: 2.00-2.80 mm). Pay attention to the speed of the advance, so that the electrode slowly reaches the brain area you want to record, while observing the signal changes (use the positioning data to guide the selection of the position with the best signal-to-noise ratio in the brain area to fix the point). Dissolve the polyethylene glycol, fix the electrode point (use dental cement), and complete the electrode implantation.

[0122] 2.6.2 Animal electrical signal acquisition

[0123] The OmniPlex 16-channel signal acquisition system of Plexon Company is used to synchronously acquire the single cell spike activity of the rat in a clear and static (clear, without any movement, and without attention to the external environment) state. Spike, i.e., the single cell action potential of neurons, its activity can reflect the spontaneous firing of individual neurons.

[0124] The signal reaches the micro preamplifier through the recording electrode, and then reaches the acquisition system through the transmission line. For Spike signal, set its band-pass filter to 300-8000 Hz, sampling rate 40 kHz, and only Spike signal with signal-to-noise ratio > 2:1 is recorded during acquisition.

[0125] 2.6.2 Analysis of electrical signals of animals

[0126] The acquired original signal is imported into Offline Sorter 4.0 software (Plexon, USA), and the threshold line is manually set according to the principle of signal-to-noise ratio > 2:1 in the channel recording Spike signal, and at the same time, the automatic threshold setting function in the software is combined to define the sequence exceeding the threshold line as Spike discharge sequence. In order to eliminate the noise interference contained in the Spike sequence, first, the signal with discharge interval < 2 ms (less than the refractory period of neurons) in each channel is removed by using the self-contained function in the software, and then the neurons discharge and noise are separated by three-dimensional principal component analysis combined with k-means clustering algorithm. Finally, in order to ensure accuracy, the noise interference is manually removed after careful observation of the whole signal. The intercepted signal is imported into Neuroexplorer 5.0 software (Nex, USA), and burst analysis algorithm is used for burst discharge analysis to obtain the burst discharge rate per minute of neurons in each group of mice and the proportion of neuron burst discharge.

[0127] 3. Experimental results

[0128] 3.1 Yijing Qiangzhi Decoction can significantly improve the motor dysfunction of Parkinson's disease mice with kidney essence deficiency

[0129] 3.1.1 Rotarod test results

[0130] Rotarod test is a gold standard for detecting motor symptoms of Parkinson's disease mouse model. In the rotarod test, compared with the control group, the time for the mice in the model group to fall from the rotating rod to the ground was significantly shortened (p<0.01). It shows that the balance and coordination ability is weakened, and the time for the mice to fall from the rotating rod to the ground is increased after the treatment of Dihuang Yinzi and Yijing Qiangzhi (p<0.01). The time for the mice to fall from the rotating rod to the ground is increased after the treatment of positive drug levodopa (p<0.001), and there is no significant difference between the treatment group of Yijing Qiangzhi Decoction and the treatment group of Dihuang Yinzi or levodopa. The results are shown in Figure 2 .

[0131] 3.1.2 Pole climbing test results

[0132] In the pole climbing test, compared with the control group, the time from the top of the pole to the ground in the model group was significantly prolonged (p<0.01), indicating impaired motor coordination and grasping ability. After treatment with Dihuang Yinzi, the above-mentioned motor impairment was alleviated (p<0.05), and after treatment with Yijing Qiangzhi, the above-mentioned motor impairment was also alleviated (p<0.01). The positive drug levodopa significantly improved the pole climbing ability of the PD kidney essence deficiency model mice (p<0.01). There was no significant difference between the Yijing Qiangzhi group and the Dihuang Yinzi or levodopa group. The results are shown in Figure 3 .

[0133] 3.1.3 Automatic gait analysis experimental results

[0134] In the gait experiment, from the gait footprint map, it was observed that compared with normal mice, the model mice had disordered stride, and the speed was not uniform. Yijing Qiangzhi had a therapeutic effect on restoring motor function. The footprint maps of mice in each group completing the runway are shown in Figure 4 , the time and speed are shown in Figure 5 , and the limb step circumference and standing time of mice in each group are shown in Figure 6 .

[0135] Compared with normal mice, the time of model mice through the runway was longer (p<0.0001), and the speed was smaller (p<0.0001). After treatment with Dihuang Yinzi, the time of mice through the runway was shorter (p<0.0001), and the speed was larger (p<0.0001). After treatment with Yijing Qiangzhi, the time of mice through the runway was shorter (p<0.0001), and the speed was larger (p<0.001). After treatment with levodopa, the time of mice through the runway was shorter (p<0.0001), and the speed was larger (p<0.0001). There was no significant difference between the Yijing Qiangzhi group and the Dihuang Yinzi or levodopa group. The results are shown in Figure 5 .

[0136] Compared with normal mice, the limb step circumference of model mice was prolonged (right front limb p<0.0001, left front limb p<0.0001, right rear limb p<0.0001, left rear limb p<0.0001), and the standing time was prolonged (right front limb p<0.0001, right rear limb p<0.0001, left rear limb p<0.0001), indicating that the model mice exhibited typical Parkinson's-like behavior characteristics. After treatment with Dihuang Yinzi, Yijing Qiangzhi, and levodopa, the step circumference was significantly reduced (right front limb p<0.0001, left front limb p<0.0001, right rear limb p<0.0001, left rear limb p<0.0001), and the standing time was reduced (right front limb p<0.0001, left front limb p<0.0001, right rear limb p<0.0001, left rear limb p<0.0001). There was no significant difference between the Yijing Qiangzhi group and the Dihuang Yinzi or levodopa group. The results are shown in Figure 6 .

[0137] The above results indicate that during the treatment of Parkinson's disease, in terms of the changes in the motor skill disorders of mice, the Yijing Qiangzhi formula treatment group has a significant improvement effect compared with the model group, and its efficacy has no obvious difference from that of the commonly used Dihuang Yinzi and levodopa at present.

[0138] 3.2 Yijing Qiangzhi formula can relieve the damage of dopaminergic neurons in the substantia nigra pars compacta

[0139] Through immunohistochemical experiments, the tyrosine hydroxylase (TH) - positive neurons in the substantia nigra pars compacta of mice in each group were stained. Compared with the control group, the number of tyrosine hydroxylase - positive neurons (TH + ) in the substantia nigra pars compacta of the model group was significantly reduced (p < 0.01), and the morphology of the remaining neurons was severely damaged. After treatment with Yijing Qiangzhi, the damage and the reduction in the number of tyrosine hydroxylase - positive neurons in the substantia nigra pars compacta were protected (p < 0.05), thereby protecting dopaminergic neurons. The treatment effect of the positive drug levodopa was also significant (p < 0.05). The results are shown in Figure 7 .

[0140] 3.3 Yijing Qiangzhi formula can improve the abnormal burst firing of striatal neurons

[0141] The results of electrophysiological analysis showed that compared with the control group, the number of burst firing times of striatal neurons in the model group per minute was significantly increased (p < 0.01). After intragastric administration of the Yijing Qiangzhi formula, the number of burst firing times of striatal neurons was improved (p < 0.05). The treatment with the positive drug levodopa significantly reduced the number of burst firing times of striatal neurons in the mouse model of kidney essence deficiency syndrome of PD (p < 0.001). Compared with the control group, the proportion of burst - firing neurons in the striatum of the model group was significantly increased (p < 0.05). After intragastric administration of the Yijing Qiangzhi formula, the proportion of burst - firing neurons in the striatum was reduced (p < 0.05). The treatment with the positive drug levodopa significantly reduced the proportion of burst - firing neurons in the striatum of the mouse model of kidney essence deficiency syndrome of PD (p < 0.001). The results are shown in Figure 8 .

[0142] The above - mentioned are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions recorded in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine composition for treating Parkinson's disease with kidney essence deficiency syndrome, characterized in that, The traditional Chinese medicine composition is made from Radix Rehmanniae Preparata 130-180 parts, Cinnamomum Cassia 20-40 parts, Acorus gramineus 20-40 parts and Polygala 20-40 parts.

2. The traditional Chinese medicine composition of claim 1, wherein, The traditional Chinese medicine composition is made from Radix Rehmanniae Preparata 150 parts, Cinnamomum Cassia 30 parts, Acorus gramineus 30 parts and Polygala 30 parts.

3. A traditional Chinese medicine extract for treating Parkinson's disease with kidney essence deficiency syndrome, characterized in that, The traditional Chinese medicine composition in claim 1 or 2 is soaked with water and decocted for 1-3 times, the decocted liquid is combined and concentrated into a 150-200% suspension.

4. The traditional Chinese medicine extract of claim 3, wherein, The traditional Chinese medicine composition is soaked with 10 times of water for 2 hours, the medicine liquid is filtered out after decoction for 1 hour, and then the same amount of water is added for decoction for 1 hour, the medicine liquid is filtered out, the medicine residue is squeezed dry, and the two times of medicine liquid are mixed and heated to concentrate into a 178% suspension.

5. A traditional Chinese medicine preparation for treating Parkinson's disease with kidney essence deficiency syndrome, characterized in that, The traditional Chinese medicine extract in claim 3.

6. The traditional Chinese medicine preparation of claim 5, characterized in that, Also included is a pharmaceutically acceptable carrier.

7. Use of the traditional Chinese medicine composition in claim 1 or the traditional Chinese medicine extract in claim 3 or the traditional Chinese medicine preparation in claim 5 in the preparation of a medicine for treating Parkinson's kidney essence deficiency syndrome.

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