A traditional Chinese medicine composition for treating Parkinson's disease, a traditional Chinese medicine preparation and application thereof

Abstract

The application discloses a traditional Chinese medicine composition for treating Parkinson's disease and a traditional Chinese medicine preparation and application thereof. The traditional Chinese medicine composition comprises, in terms of weight parts, 9-20 parts of Schisandra chinensis, 10-30 parts of Ophiopogon japonicus, 3-15 parts of Radix ginseng rubra, 10-30 parts of Uncaria, 10-30 parts of Cuscuta chinensis, 10-30 parts of Semen ecliptae prostratae, 3-5 parts of ginger and 5-15 parts of Jujube. The application further discloses a preparation of the traditional Chinese medicine composition. The traditional Chinese medicine composition and the preparation thereof can effectively improve the motor function of Parkinson's disease patients, reduce the dosage of Madopa, and thus reduce the related side effects caused by dopamine replacement therapy.

Description

A traditional Chinese medicine composition for treating Parkinson's disease, its preparation and application Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology. More specifically, it relates to a traditional Chinese medicine composition for treating Parkinson's disease, its preparation, and its application. Background Technology

[0002] Parkinson's disease is a common disease among middle-aged and elderly people, with a global incidence rate exceeding 6 million. With the aging population, the incidence of Parkinson's disease continues to rise. Clinically, it is mainly characterized by resting tremor, muscle rigidity, bradykinesia, and postural and gait disturbances, affecting patients' daily living abilities and imposing a certain economic burden on patients and their families. Therefore, exploring new treatments for Parkinson's disease, improving patients' quality of life, and slowing the progression of disease symptoms are current research priorities.

[0003] Currently, the main treatment for Parkinson's disease both domestically and internationally is dopamine replacement therapy, which includes drugs that increase dopamine levels and drugs that improve dopamine receptor function. For example, levodopa (L-DOPA) is currently the most widely used and most effective drug for improving Parkinson's motor symptoms. In addition, monoamine oxidase B inhibitors (MAOBIs) and catechol-O-methyltransferase inhibitors (COMTIs) can improve dopamine bioavailability, thereby increasing dopamine levels. However, dopamine replacement therapy suffers from the problem of drug ineffectiveness after the "honeymoon period," and has many adverse reactions such as dyskinesia, motor fluctuations, hypotension, impulse control disorders, and sleep disturbances. Summary of the Invention

[0004] To address the aforementioned technical problems, one objective of this invention is to provide a novel traditional Chinese medicine composition for treating Parkinson's disease. This composition can improve the patient's motor function, reduce the dosage of levodopa, and thus reduce the related side effects of dopamine replacement therapy.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned traditional Chinese medicine composition.

[0006] Another object of the present invention is to provide various traditional Chinese medicine preparations made using the above-mentioned traditional Chinese medicine composition, and their use in the preparation of medicaments for treating Parkinson's disease.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a traditional Chinese medicine composition for treating Parkinson's disease, wherein the raw materials of the traditional Chinese medicine composition include, by weight, 9-20 parts of Schisandra chinensis, 10-30 parts of Ophiopogon japonicus, 3-15 parts of raw ginseng, 10-30 parts of Uncaria rhynchophylla, 10-30 parts of Cistanche deserticola, 10-30 parts of Cuscuta chinensis, 3-5 parts of ginger, and 5-15 parts of jujube.

[0009] Parkinson's disease falls under the category of "tremor syndrome" in Traditional Chinese Medicine (TCM), with its location in the tendons and vessels, and is closely related to the liver, spleen, and kidneys. It is often caused by factors such as old age and physical weakness, emotional distress, improper diet, and inappropriate work-rest balance, leading to liver and kidney yin deficiency, internal wind stirring, and insufficient qi and blood to nourish the tendons and vessels. Over time, this lack of nourishment can cause limb tremors, followed by tendon and vessel obstruction, stiffness, and bradykinesia. Liver and kidney yin deficiency also generates internal wind, which, when excessive, causes movement. Furthermore, insufficient marrow in the brain can also cause head shaking. Based on clinical experience, the inventor believes that insufficient marrow in the brain is the root cause of Parkinson's disease and proposes a TCM composition containing the above-mentioned herbs to replenish essence, marrow, qi, and yin for the treatment of Parkinson's disease. This composition improves motor function, reduces the dosage of levodopa, and thus reduces the side effects associated with dopamine replacement therapy.

[0010] In the prescription of the present invention, Schisandra chinensis is sour in taste and warm in nature. It mainly benefits qi, supplements deficiencies, strengthens yin, and benefits the essence of men. Chen Xiuyuan said, "Schisandra chinensis is warm in qi and sour in taste, obtaining the qi of growth in the east and governing wind. A person is in the wind but doesn't see the wind, just like a fish is in water but doesn't see water. A person's breath, in and out, will die immediately if separated from the wind for a moment. It can be seen that the reason for a person's survival is wind. The wind qi communicates with the liver, which is the wood qi in the human body. Zhuangzi said, 'Wild horses, dust, the breaths of living things blow against each other.' The character 'breath' has two meanings: one is 'vitality', and the other is'rest'. Schisandra chinensis is warm to promote the flourishing of the wood qi, and sour to converge the wood qi back to its root. Vitality and rest are both ways to benefit its continuously growing qi. If the qi is not regulated, the wind wood combines with the fire qi and attacks the metal. In supplementing qi, Schisandra chinensis has the wonderful functions of opening, closing, ascending, and descending. Some scholars believe that it has five flavors and treats the five zang-organs; some believe that its sourness astringes the lungs, its black color enters the kidneys, and its nucleus resembles the kidneys and tonifies the kidneys. The qi of the lungs and kidneys mutually depend on each other. The qi of the lungs must return to the kidneys at night, and the qi of the kidneys must rise to the lungs during the day. Ophiopogon japonicus is sweet in taste, slightly cold in qi, and descending. It can only benefit the lungs and cannot benefit the kidneys. When Ophiopogon japonicus soothes the lungs, the lung qi can communicate with the kidneys, but the kidneys are not supplemented, so the kidneys still obtain nourishment from the mother organ of the lungs, and the lungs are still not at ease. This is why when supplementing the mother organ of the lungs, it is necessary to supplement the son organ of the kidneys. Once the kidney water is sufficient and does not rely on the qi of the lung metal, the lung qi will be at ease by itself, and it can also generate water, and the lungs will be even more at ease. In ancient times, Ophiopogon japonicus was used with Schisandra chinensis, taking its function of astringing the lungs, and also taking its function of tonifying the kidneys. Dried Ginseng (sheng shai shen) is sweet, slightly bitter, warm, and enters the spleen and lung meridians. It can supplement the heart qi and soothe the mind, and benefit the wisdom of the brain marrow. It is one of the commercial varieties of ginseng recorded in 'Shennong's Classic of Materia Medica'. 'Shennong's Classic of Materia Medica' states that it'mainly supplements the five zang-organs, soothes the spirit, stabilizes the soul, stops palpitations, expels pathogenic factors, improves eyesight, makes the mind happy and increases wisdom. Taking it for a long time can lighten the body and prolong life.' Uncaria rhynchophylla is sweet and bitter in taste, slightly cold in qi, and enters the liver meridian. This herb dispels wind very quickly. For those with wind syndromes, it must be used. It is an important herb for the hand少阴 and foot jueyin meridians. The shaoyin meridian governs fire, and the jueyin meridian governs wind. When wind and fire interact and block the limb meridians, but the generation of wind and fire is mostly due to the deficiency of kidney water, resulting in dry wood and flaming fire. Using Uncaria rhynchophylla in yin-nourishing herbs, the wind and fire can be easily dispersed. Cistanche deserticola is sweet, warm, salty, and sour, and enters the kidneys. The flesh of Cistanche deserticola is transformed from the essence of a horse, so it can arouse yang. The essence of a horse originally comes from the kidneys, so it also benefits yin. It specifically tonifies the water and fire in the kidneys. Cuscuta chinensis is pungent, sweet, and warm in qi. It equally possesses the qi of metal. Its pungency obtains the flavor of metal, so it is a lung herb. However, its application is in the kidneys rather than the lungs. Hua Tuo said, 'The kidneys are the root of life. If the kidneys are supplemented, one can prolong life.' Taken internally, it has the function of filling and supplementing, and taken externally, it has the effect of lubrication. 'For the key to yin and yang, yang must be dense to be firm.' Cistanche deserticola and Cuscuta chinensis tonify the kidney yang and benefit the kidney essence, aiming to strengthen the kidneys by tonifying yang, so that yin is firm with yang, and essence is conserved and does not leak. For those with deficiency of the liver and kidneys and a weak spleen and stomach, the combination of ginger and jujube is a commonly used combination for strengthening the spleen and stomach, aiming to supplement qi and tonify the middle, and can also nourish the spleen and promote fluid production. When all the herbs are combined, they not only nourish the kidney yin but also tonify the kidney yang, not only benefit the kidney essence but also astringe sperm. The traditional Chinese medicine composition of the present invention replenishes essence and marrow, supplements qi and nourishes yin.

[0011] In this invention, by way of example, the traditional Chinese medicine composition for treating Parkinson's disease comprises, by weight, the raw materials of the traditional Chinese medicine composition including: 15 parts of Schisandra chinensis, 15 parts of Ophiopogon japonicus, 6 parts of raw ginseng, 20 parts of Uncaria rhynchophylla, 15 parts of Cistanche deserticola, 15 parts of Cuscuta chinensis, 3 parts of ginger, and 10 parts of jujube.

[0012] For example, the traditional Chinese medicine composition for treating Parkinson's disease, by weight, comprises the following raw materials: 9 parts Schisandra chinensis, 10 parts Ophiopogon japonicus, 3 parts raw ginseng, 10 parts Uncaria rhynchophylla, 10 parts Cistanche deserticola, 10 parts Cuscuta chinensis, 3 parts ginger, and 5 parts jujube.

[0013] For example, the traditional Chinese medicine composition for treating Parkinson's disease, by weight, comprises the following raw materials: 20 parts Schisandra chinensis, 30 parts Ophiopogon japonicus, 15 parts raw ginseng, 30 parts Uncaria rhynchophylla, 30 parts Cistanche deserticola, 30 parts Cuscuta chinensis, 5 parts ginger, and 15 parts jujube.

[0014] The basic formula of the above-mentioned traditional Chinese medicine composition of the present invention can be adjusted according to clinical symptoms during specific implementation to improve certain specific clinical symptoms, further enhance the adaptability of the composition of the present invention, and improve the therapeutic effect. These adjustments made based on the basic formula of the present invention are also within the scope of protection of this application.

[0015] Secondly, the present invention provides a method for preparing the above-mentioned traditional Chinese medicine composition for treating Parkinson's disease, the method comprising: taking the following ingredients according to the formula: Schisandra chinensis, Ophiopogon japonicus, raw ginseng, Uncaria rhynchophylla, Cistanche deserticola, Cuscuta chinensis, ginger, and jujube, and extracting them with water to obtain the product;

[0016] Furthermore, the extraction method is selected from water decoction, water heating reflux, or ultrasonic extraction.

[0017] Furthermore, the preparation method further includes the step of concentrating the aqueous extract into an extract; optionally, the extract is dried to form a dry powder.

[0018] Thirdly, the present invention provides a traditional Chinese medicine preparation for treating Parkinson's disease, wherein the raw materials of the traditional Chinese medicine preparation include the above-mentioned traditional Chinese medicine composition.

[0019] Depending on actual needs, the traditional Chinese medicine preparations may also include pharmaceutically acceptable excipients. Various dosage forms, such as decoctions, tablets, granules, pills, powders, capsules, or granules, are prepared according to conventional pharmaceutical processes.

[0020] Furthermore, the present invention also provides the application of the above-mentioned traditional Chinese medicine composition and preparation in the preparation of drugs for treating Parkinson's disease.

[0021] In addition, unless otherwise specified, all raw materials of the traditional Chinese medicine composition of the present invention can be obtained commercially available. Any traditional Chinese medicine composition of any range described in the present invention, including the end value and any value between the end values ​​and any sub-range formed by the end value or any value between the end values, can achieve the purpose of treating Parkinson's disease.

[0022] The beneficial effects of this invention are as follows:

[0023] The herbal composition of this invention can reduce α-synuclein aggregation in the brain of MPTP-induced PD model mice, increase TH expression levels, exert neuroprotective effects, and improve motor function. Furthermore, clinical studies have shown that after treatment with the herbal composition of this invention, the reduction in UPDRS (Unified Parkinson's Disease Rating Scale) scores was significantly greater than in the control group, and some patients experienced a reduction in the dosage of levodopa. This demonstrates that the herbal composition of this invention can effectively improve the symptoms of Parkinson's disease. Attached Figure Description

[0024] Figure 1 shows the identification diagram of compounds in the traditional Chinese medicine composition of Example 1.

[0025] Figure 2 shows the comparison of climbing time for each group. ** indicates P<0.01 compared with the control group; ## indicates P<0.01 compared with the model group; △△ indicates P<0.01 compared with the LY294002 group.

[0026] Figure 3 shows the comparison of rotator times for each group. ** indicates P<0.01 compared with the control group; ## indicates P<0.01 compared with the model group; △△ indicates P<0.01 compared with the LY294002 group; ^^ indicates P<0.01 compared with the LY294002+Schisandra decoction group.

[0027] Figure 4 shows the comparison of open field movement distances for each group. A represents the peripheral movement distance of each group, B represents the central movement distance of each group, and C represents the ratio of central movement distance to peripheral movement distance for each group. ** indicates P < 0.01 when compared with the control group; # indicates P < 0.05, ## indicates P < 0.01 when compared with the model group; △ indicates P < 0.05, △△ indicates P < 0.01 when compared with LY294002.

[0028] Figure 5 shows the comparison of open field dwell time for mice in each group; A represents the peripheral dwell time of mice in each group; B represents the central dwell time of mice in each group; C represents the ratio of central dwell time to peripheral dwell time of mice in each group; ** indicates P<0.01 compared with the control group; ## indicates P<0.01 compared with the model group; △△ indicates P<0.01 compared with the LY294002 group; ^ indicates P<0.05 compared with the Schisandra chinensis decoction group.

[0029] Figure 6 shows the protein expression of α-Syn, the protein expression of TH, and immunohistochemistry in each group; A is the WB electrophoresis of α-Syn protein; B is the relative expression level of α-Syn protein in each group; C is the WB electrophoresis of TH protein; D is the relative expression level of TH protein in each group; E is the immunohistochemical result of TH protein; * indicates P<0.05, ** indicates P<0.01 compared with the control group; # indicates P<0.05 compared with the model group; △ indicates P<0.05, △△ indicates P<0.01 compared with the LY294002 group.

[0030] Figure 7 shows the relative expression of autophagy-related proteins LC3 and p62 / SQSTM in each group; A is the WB electrophoresis image of LC3I / II and p62 protein expression; B and C are the relative expression levels of LC3 II / I and p62 proteins in each group, respectively; D is the relative expression level of LC3 mRNA; * indicates P<0.05, ** indicates P<0.01 compared with the control group; ## indicates P<0.01 compared with the model group; △ indicates P<0.05, △△ indicates P<0.01 compared with the LY294002 group.

[0031] Figure 8 shows the relative expression of PTEN and PI3K in each group; A is the WB protein expression electrophoresis diagram of PTEN and PI3K; B and C are the relative expression levels of PTEN and PI3K proteins in each group; D and E are the relative mRNA expression levels of PTEN and PI3K; * indicates P<0.05, ** indicates P<0.01 compared with the control group; # indicates P<0.05, ## indicates P<0.01 compared with the model group; △ indicates P<0.05, △△ indicates P<0.01 compared with the LY294002 group; ^^ indicates P<0.01 compared with the LY294002 + Schisandra chinensis decoction group.

[0032] Figure 9 shows the protein expression of p-AKT / AKT, p-mTOR / mTOR, and p70s6k in each group; A is the WB electrophoresis image of p-AKT / AKT, p-mTOR / mTOR, and p70s6k; B, C, and D are the relative protein expression levels of p-AKT / AKT, p-mTOR / mTOR, and p70s6k in each group, respectively; * indicates P<0.05, ** indicates P<0.01 compared with the control group; # indicates P<0.05, ## indicates P<0.01 compared with the model group; △ indicates P<0.05, △△ indicates P<0.01 compared with the LY294002 group. Detailed Implementation

[0033] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1

[0035] Raw material formula: Schisandra chinensis 15g, Ophiopogon japonicus 15g, raw ginseng 6g, Uncaria rhynchophylla 20g, Cistanche deserticola 15g, Cuscuta chinensis 15g, ginger 3g, jujube 10g.

[0036] Prepared as a decoction using conventional methods.

[0037] Example 2

[0038] Raw material formula: Schisandra chinensis 9g, Ophiopogon japonicus 10g, raw ginseng 3g, Uncaria rhynchophylla 10g, Cistanche deserticola 10g, Cuscuta chinensis 10g, ginger 3g, jujube 5g.

[0039] Prepared as a decoction using conventional methods.

[0040] Example 3

[0041] Raw material formula: Schisandra chinensis 20g, Ophiopogon japonicus 30g, raw ginseng 15g, Uncaria rhynchophylla 30g, Cistanche deserticola 30g, Cuscuta chinensis 30g, fresh ginger 5g, jujube 15g.

[0042] Prepared as a decoction using conventional methods.

[0043] Example 4: Animal experiment on the treatment of Parkinson's disease with the traditional Chinese medicine composition of the present invention.

[0044] 1. Experimental Methods

[0045] 1.1 Identification of compounds in the traditional Chinese medicine composition of the present invention based on UPLC-Q-TOF / MS technology

[0046] Liquid chromatography conditions: 0.1% formic acid in water and pure acetonitrile, using a large gradient 35-minute method: 0-2, 5% acetonitrile; 2-32, 5-100% acetonitrile; 32-33, 100% acetonitrile; 33.5, 5% acetonitrile; 33.5-35, 5% acetonitrile. Flow rate: 0.3 ml / min; injection volume: 2 μL; column temperature: 35℃; sample chamber temperature: 10℃. Mass spectrometry method: ESI ion source, positive and negative ion modes scanned separately, MSE continue full scan mode, scan time 0.2 s, scan range 50–1200. Collision energies were used in MSE: low collision energy 6 V, high collision energy 15–45 V. Sodium formate was used for mass spectrometry calibration, and leucine enkephalin (positive ion mode m / z 556.2771, negative ion mode m / z 554.2615) was used for real-time mass calibration.

[0047] 1.2 Preparation and grouping of Parkinson's disease (PD) models

[0048] Fifty mice were randomly divided into a control group (n=10) and a model group (n=40). The model group mice were further divided into four subgroups (n=10 each): MPTP group, LY294002 group, Schisandra decoction group (Example 1), and LY294002 + Schisandra decoction group (LY294002 + Schisandra). After grouping, mice in the modeling group were intraperitoneally injected with MPTP 30 mg / kg, diluted with physiological saline to 1 mg / ml, once daily for 7 consecutive days to establish a Parkinson's disease model; mice in the normal group were injected daily with the same volume of physiological saline; the Schisandra chinensis decoction group was administered Schisandra chinensis decoction by gavage at a dose of 0.75 g / 100 g of raw drug once daily for 7 days during modeling; the LY294002 group was administered LY294002 intraperitoneally at a dose of 50 mg / kg of reagent once daily for 7 days during modeling; the LY294002+Schisandra group was administered LY294002 and Schisandra chinensis decoction simultaneously with modeling according to the above administration standards.

[0049] 1.3 Behavioral Indicator Measurement

[0050] During the modeling period, the mice's mental state, posture, coat color, activity level, and other overall condition were observed. Behavioral indicators were tested 48 hours after modeling. All experiments were conducted between 9:00 AM and 12:00 PM. Mice feces and urine were thoroughly cleaned before each test to ensure a clean experimental environment.

[0051] 1.3.1 Pole Climbing Test

[0052] Before the experiment, all mice were trained on a climbing pole for two days. After the experiment began, the mice were placed on the pole, and the time it took for each mouse to turn around and climb from one end of the pole to the other was observed and recorded. Each mouse's pole-climbing experiment was repeated three times, with each measurement spaced 30 minutes apart. The average of the three measurements was the mouse's final pole-climbing time. If any mouse turned around or stopped climbing, the experiment was repeated. A shorter pole-climbing time indicates better motor coordination in the mouse; conversely, a longer time indicates motor coordination dysfunction. (Two days of training, one day of testing).

[0053] 1.3.2 Fatigue Rotating Bar Test

[0054] Before the experiment, mice were pre-trained on a rotating rod at a fixed speed (10 rpm) for 3 days, 3 minutes each day. During the experiment, mice were placed on a stationary rotating rod for 5 minutes to adapt, starting at 5 rpm and gradually increasing to 40 rpm. The start time of the mouse's rotation and the end time of its fall were recorded, and the time spent on the rotating rod was calculated. The rotating rod experiment was repeated 3 times for each mouse, with each measurement 30 minutes apart. The average of the 3 measurements was the mouse's final movement time. The longer the mouse spent on the roller, the better its motor function. (3 days of training, 1 day of testing).

[0055] 1.3.3 Open Field Test

[0056] The open field test is used to assess the autonomous behavior, exploratory behavior, and motor abilities of mice in a new environment. At the start of the experiment, a mouse is placed in the experimental area and allowed to explore freely for 15 minutes. The time and distance of movement around the perimeter and the time and distance of movement in the center are recorded. Normal mice tend to move in the center of the open field; that is, the more time and distance spent in the open area, the better their motor function. (Test for 1 day).

[0057] 1.4 Brain tissue preparation

[0058] After the behavioral tests were completed, the mice were anesthetized with 4% isoflurane and maintained with 2% isoflurane. The mice were euthanized by cervical dislocation, and the brain tissue was collected. The substantia nigra on the coronal section of the brain was quickly separated on ice with a scalpel. The tissue was fixed with 4% paraformaldehyde for 24 hours, dehydrated, and then frozen into sections. The remaining tissue was frozen in liquid nitrogen for subsequent RT-qPCR analysis, and a portion was used for WB protein sample analysis.

[0059] 1.5 RT-qPCR detection of PTEN, PI3K, and LC3 transcription levels in brain tissue

[0060] (1) Total RNA extraction: Total RNA was extracted from mouse brain tissue using Trizol lysis buffer, and the RNA concentration of the sample was detected using a UV spectrophotometer. (2) Reverse transcription of RNA: The reverse transcription kit was followed according to the instructions. After analysis, the RNA in the sample was reverse transcribed into cDNA for quantitative fluorescence analysis. (3) Real-time quantitative PCR: The tubes after instantaneous centrifugation were placed in a real-time quantitative PCR instrument for amplification. RT-qPCR was performed using the Biosystems 7500 real-time PCR detection system, with the β-actin gene as an internal control, to detect mRNA levels. (4) Result analysis: The reliability of the RT-qPCR results was judged by the amplification curve and melting curve, and the relative expression level of the target gene in each sample was calculated.

[0061] 1.6 Immunoblotting was used to determine the expression of LC3I / II, Akt, p-Akt, PTEN, PI3K, mTOR, p-mTOR, p70s6K, p62 / SQSTM1, α-syn, and TH proteins in brain tissue.

[0062] (1) Homogenization: Take out the brain tissue sample flash-frozen in liquid nitrogen and homogenize it thoroughly with a grinder until completely homogenized. (2) Centrifugation and washing: Transfer the homogenized sample to a tissue homogenizer, wash twice with frozen PBS buffer, and centrifuge at 4°C and 12,000 rpm for 40 min. (3) Protein lysis: Collect the supernatant, add protein lysis buffer and protease inhibitor on ice, and incubate at 4°C for 40 min. (4) Protein quantification: Use the colorimetric method (BCA method) with BSA as the standard to determine the protein concentration in the supernatant. (5) Gel electrophoresis: 20 μg of protein sample was electrophoretically separated on a 10% SDS-polyacrylamide gel using a Biora electrophoresis apparatus at a constant voltage of 130V for 1.5 h. (6) Blocking: After transferring to a PVDF membrane at 100V for 1 h, the membrane was blocked at 37℃ with 5% skim milk powder blocking solution for 1 h. (7) Primary antibody incubation: Protein bands were sliced ​​and incubated with primary antibody at 4℃ overnight. (8) Secondary antibody incubation: The membrane was washed 3 times with PBS buffer at room temperature, and then labeled with ALP secondary antibody and gently shaken for 1 hour. (9) Color development and quantification: The membrane was washed 3 times with PBS buffer and the absorbance (A) value of each segment was displayed by Western blot. The absorbance (A) value of each segment was quantitatively analyzed using image analysis software.

[0063] 1.7 Immunohistochemical staining

[0064] Mouse brain tissue was embedded in Shandon Histocentre 2 and sectioned into 4 μm thick sections using Finesse 32. The sections were incubated in neutral resin wash buffer for 5 min × 3 times, then incubated in 100% ethanol for 10 min × 2 times, and incubated in 95% ethanol for 10 min × 2 times. The sections were then immersed in 1× sodium citrate antigen retrieval solution and kept at boiling temperature for 10 min. After cooling, the sections were washed with distilled water (dH2O) for 5 min × 3 times. The sections were incubated in 3% hydrogen peroxide solution for 10 min, then washed with distilled water (dH2O) for 5 min × 2 times, followed by washing in 1× PBS for 5 min. The sections were blocked with 200 μl of blocking buffer at room temperature for 1 hour, and then incubated overnight at 4°C with a 1:500 diluted primary antibody. Boost Detection Reagent was brought to room temperature, washed with 1×PBS for 5 minutes × 3 times, and 200 μl of [unspecified ingredient] was added. Stain with DAB. Wash with distilled water (dH2O), dehydrate sections, incubate in neutral resin and cover with coverslips for photography.

[0065] 1.8 Statistical Analysis

[0066] Data were analyzed using GraphPad Prism 9.4.1 statistical software. Normally distributed data are expressed as mean ± standard deviation. Tukey's test was used for comparisons between two groups. P < 0.05 was considered statistically significant.

[0067] 2. Test Results

[0068] 2.1 Identification of compounds in Schisandra chinensis decoction

[0069] Take the test medicinal material solution (Example 1), and according to the above chromatographic and mass spectrometric conditions, combined with the literature, obtain the total ion chromatogram and composition in positive and negative ion modes, as shown in Figure 1.

[0070] 2.2 Effects on the behavior of PD model mice

[0071] 2.2.1 Results of the pole climbing test

[0072] As shown in Figure 2, compared with the control group, in the pole climbing test, except for the Schisandra chinensis decoction group, the turning time and the total time from the top of the pole to the bottom platform of the other three groups were significantly increased (P<0.01). The turning time and total time of mice in the LY294002 group were significantly increased compared with the model group (P<0.01). The turning time and total time of mice in the Schisandra chinensis decoction group were both reduced compared with the model group and the LY294002 group (P<0.01). After treatment with LY294002 + Schisandra chinensis decoction, the turning time and total time of mice were significantly reduced compared with the LY294002 group, and dropped to the level of the model group (P<0.01).

[0073] 2.2.2 Results of the Rotating Bar Test

[0074] As shown in Figure 3, in the rotarod test, the residence time of mice in the other four groups on the rotarod was significantly shorter than that in the control group (P<0.01). The residence time of mice in the LY294002 group on the rotarod was less than that in the model group (P<0.01). The residence time of mice in the Schisandra chinensis decoction group was significantly increased compared with both the model group and the LY294002 group (P<0.01). After treatment with LY294002 + Schisandra chinensis decoction, the residence time of mice on the rotarod was significantly less than that in the Schisandra chinensis decoction group (P<0.01), decreasing to the level of the model group, but significantly increased compared with the LY294002 group (P<0.01).

[0075] 2.2.3 Results of open field test

[0076] The results are shown in Figure 4. In the open field test, except for the Schisandra chinensis decoction group, compared with the control group, the peripheral and central movement distances and the central movement distance / peripheral movement distance of mice in the other three groups were significantly reduced (P<0.01). Compared with the control group, the peripheral and central movement distances of mice in the Schisandra chinensis decoction group were also reduced (P<0.01). Compared with the model group, the central movement distance and the central movement distance / peripheral movement distance of mice in the Schisandra chinensis decoction group increased, while those in the LY294002 group decreased (P<0.05 or P<0.01). The movement distance and the central movement distance / peripheral movement distance of mice in the Schisandra chinensis decoction group were both increased compared with the LY294002 group (P<0.05 or P<0.01). However, the central movement distance and the central movement distance / peripheral movement distance after treatment with both LY294002 and Schisandra chinensis decoction were significantly increased compared with the LY294002 group (P<0.01).

[0077] The results are shown in Figure 5. Compared with the control group, except for the Schisandra chinensis decoction group, the central dwell time and central dwell time / peripheral dwell time of mice in the other groups were significantly reduced, but the peripheral dwell time was significantly increased (P<0.01). Compared with the model group, the central dwell time and central dwell time / peripheral dwell time of mice in the LY294002 group were significantly reduced (P<0.01), but the peripheral dwell time was significantly increased compared with the model group (P<0.01). The central dwell time and central dwell time / peripheral dwell time of mice in the Schisandra chinensis decoction group were significantly increased compared with the model group, and the peripheral dwell time was significantly decreased compared with the model group (P<0.01). The central dwell time and central dwell time / peripheral dwell time of mice in the Schisandra chinensis decoction group and the LY294002+Schisandra chinensis decoction group were significantly increased compared with the LY294002 group (P<0.01), and the peripheral dwell time was significantly decreased compared with the LY294002 group (P<0.01). The central residence time and the ratio of central residence time to peripheral residence time in mice treated with LY294002 and Schisandra chinensis decoction were lower than those in the Schisandra chinensis decoction group (P<0.05), while the peripheral residence time was higher than that in the Schisandra chinensis decoction group (P<0.05).

[0078] 2.3 Effects on the expression of α-syn and autophagy-related proteins in PD model mice

[0079] 2.3.1 Protein expression of α-Syn and TH in each group and immunohistochemistry

[0080] Western blot results showed that the expression of α-syn protein in the model group, LY294002 group, and LY294002+Schisandra decoction group was significantly higher than that in the control group (P<0.05 or P<0.01); the expression of α-syn protein in the Schisandra decoction group and the Schisandra decoction+LY294002 co-treatment group was significantly reduced compared with the LY294002 group (P<0.05 or P<0.01), as shown in Figure 6.

[0081] 2.3.2 Protein expression of autophagy-related proteins LC3Ⅱ / Ⅰ and p62 / SQSTM and LC3 mRNA expression in each group

[0082] Western blot results showed that LC3 II / I protein expression was significantly higher in the model group, LY294002 group, and LY294002+Schisandra decoction group than in the control group (P<0.01). Compared with the LY294002 group, LC3 II / I protein expression was downregulated in the Schisandra decoction group (P<0.01). Compared with the control group, p62 protein expression was decreased in both the model group and the LY294002 group (P<0.05 or P<0.01). Compared with the LY294002 group, p62 protein expression was increased in both the Schisandra decoction group and the LY294002+Schisandra decoction group (P<0.05 or P<0.01).

[0083] RT-qPCR results showed that the expression level of LC3 mRNA in the other four groups was significantly higher than that in the control group (P<0.01). After treatment with LY294002, the expression level of LC3 mRNA was significantly higher than that in the model group (P<0.01). Compared with LY294002, the expression of LC3 mRNA in the Schisandra chinensis decoction group and the LY294002+Schisandra chinensis decoction group was significantly decreased (P<0.01). The results are shown in Figure 7.

[0084] 2.4 Impact on the PI3K / AKT / mTOR signaling pathway

[0085] 2.4.1 Effects on PTEN and PI3K expression

[0086] Western blot results showed that, compared with the control group, PTEN protein expression was significantly upregulated and PI3K protein expression was significantly downregulated in both the model group and the LY294002 group (P<0.05 or P<0.01). Simultaneously, PI3K protein expression was also downregulated in the LY294002 + Schisandra chinensis decoction group compared with the control group (P<0.01). Compared with the model group, PI3K protein expression was significantly downregulated in the LY294002 group (P<0.05). PTEN protein expression in both the Schisandra chinensis decoction group and the LY294002 + Schisandra chinensis decoction group was downregulated compared with the LY294002 group (P<0.05). PI3K protein expression was significantly upregulated in the Schisandra chinensis decoction group compared with the LY294002 group, while after co-treatment with LY294002 and Schisandra chinensis decoction, PI3K protein expression was downregulated to the level of the LY294002 group (P<0.01).

[0087] In the RT-qPCR experiment, compared with the control group, the expression levels of PI3K mRNA were downregulated in the other four groups, while the expression levels of PTEN mRNA were upregulated (P<0.01). Compared with the model group, the expression level of PTEN mRNA was upregulated in the LY294002 group and downregulated in the Schisandra chinensis decoction group, while the expression level of PI3K mRNA was downregulated in the LY294002 group and the LY294002+Schisandra chinensis decoction group and upregulated in the Schisandra chinensis decoction group (P<0.01). Compared with the LY294002+Schisandra chinensis decoction group, the PTEN mRNA level was significantly decreased and the PI3K mRNA level was significantly increased in the Schisandra chinensis decoction group (P<0.05, P<0.01). Compared with Schisandra chinensis decoction, the PTEN mRNA expression level was significantly increased and the PI3K mRNA expression level was significantly decreased in the LY294002+Schisandra chinensis decoction group (P<0.01). The results are shown in Figure 8.

[0088] 2.4.2 Effects on the expression of p-AKT / AKT, p-mTOR / mTOR, and p70s6k proteins

[0089] Western blot results showed that the expression levels of p-AKT / AKT and p70s6k proteins in the other four groups were significantly lower than those in the control group, while the expression levels of p-mTOR / mTOR proteins in the model group and the LY294002 group were significantly lower than those in the control group (P<0.05 or P<0.01). Compared with the model group, the expression levels of p-AKT / AKT, p-mTOR / mTOR, and p70s6k proteins in the LY294002 group were both lower (P<0.05 or P<0.01). The expression levels of p-AKT / AKT, p-mTOR / mTOR, and p70s6k proteins in the Schisandra chinensis decoction group were higher than those in the LY294002 group (P<0.01). After treatment with both Schisandra chinensis decoction and LY294002, the expression levels of p70s6k and p-mTOR / mTOR proteins were higher than those in the LY294002 group (P<0.05). The results are shown in Figure 9.

[0090] 3. Conclusion

[0091] This study, through animal experiments, shows that the traditional Chinese medicine composition of the present invention can reduce the aggregation of α-syn in the brain of MPTP-induced PD model mice, increase the expression level of TH, exert a neuroprotective effect, and improve their motor function.

[0092] Furthermore, through the exploration of the mechanism of action of the traditional Chinese medicine composition of the present invention in treating MPTP-induced PD model mice, it was found that, according to domestic and foreign literature, α-syn aggregation is the result of impaired autophagic lysosomal degradation. PD-related autophagy involves multiple signaling pathways, among which the PI3K / AKT / mTOR signaling pathway is the main pathway regulating autophagy.

[0093] In summary, the herbal composition of this invention is effective in treating MPTP-induced PD model mice, and its mechanism of action may be related to the possible activation of the mTOR signaling pathway to inhibit excessive autophagy.

[0094] Example 5 Clinical Study

[0095] 1. Materials and Methods

[0096] 1.1 General Information

[0097] This study included 40 Parkinson's disease patients who visited the outpatient or inpatient department of the Department of Neurology at Wenzhou Municipal Hospital of Traditional Chinese Medicine from April 2023 to April 2024. Among them, 23 were male and 17 were female, aged 55–82 years. They were randomly divided into a treatment group and a control group using a random number table. The treatment group consisted of 20 patients (13 males and 7 females, with an average age of (69.20±7.82) years); the control group consisted of 20 patients (10 males and 10 females, with an average age of (69.20±6.20) years). There were no statistically significant differences in gender or age between the two groups (P>0.05), indicating comparability.

[0098] 1.2 Inclusion Criteria

[0099] (1) Age 40-85 years. (2) According to the diagnostic criteria of traditional Chinese medicine and Western medicine, the Western medicine diagnosis is primary Parkinson's disease, and the traditional Chinese medicine diagnosis is tremor syndrome and marrow deficiency syndrome. (3) No obvious cognitive impairment. (4) Stable use of anti-PD drugs for the past 4 weeks.

[0100] 1.3 Exclusion Criteria

[0101] (1) Atypical or secondary Parkinson's syndrome. (2) Individuals with psychiatric symptoms, hearing impairment, visual impairment, comprehension impairment, or severe cognitive impairment. (3) Individuals with serious life-threatening primary diseases, joint diseases, or gastrointestinal diseases. (4) Individuals deemed unsuitable for participation in this study by the researchers. (5) Individuals with severe organ dysfunction or unwillingness to cooperate.

[0102] 2. Methods

[0103] 2.1 Grouping Method

[0104] The patients were randomly divided into a treatment group and a control group using a random number table. Both groups received basic anti-Parkinson's disease medication (those already taking anti-Parkinson's drugs continued their original regimen). The treatment group additionally received a decoction of Schisandra chinensis, with the following prescription: (Schisandra chinensis 15g, Ophiopogon japonicus 15g, Panax ginseng 6g, Uncaria rhynchophylla 20g, Cistanche deserticola 15g, Cuscuta chinensis 15g, Zingiber officinale 3g, Ziziphus jujuba 10g). One dose was taken daily, decocted twice, and the two decoctions were mixed to obtain a total of 300ml, which was taken twice daily, morning and evening, warm. All the herbs were sourced from the pharmacy of Wenzhou Municipal Hospital of Traditional Chinese Medicine. Both groups underwent follow-up examinations after two months of continuous treatment.

[0105] 2.2 Observation Indicators

[0106] Functional assessment: The Unified Parkinson's Disease Rating Scale (UPDRS) was used to assess both groups of patients before and after treatment. The assessments were conducted during the initial phase of treatment.

[0107] Levodopa dosage: Record the dosage of levodopa used before and after treatment in both groups of patients.

[0108] 2.3 Safety Evaluation

[0109] Record any adverse reactions and liver and kidney function during use.

[0110] 2.4 Statistical Methods

[0111] Data analysis was performed using SPSS 25.0 statistical software. Count data were expressed as number of cases (percentage) [n(%)], and comparisons were performed using the chi-square test. Continuous data were expressed as mean ± standard deviation. The independent samples t-test was used for comparisons between groups if the distribution was normal; otherwise, the rank-sum test was used. Paired samples t-tests were used for comparisons within groups before and after treatment. A p-value < 0.05 was considered statistically significant.

[0112] 3. Results

[0113] 3.1 Comparison of gender and age, see Table 1.

[0114] Table 1 Comparison of gender and age between the two groups

[0115]

[0116] The chi-square test showed no statistically significant difference in gender (P > 0.05), indicating comparability. The ages, after normality testing, all followed a normal distribution with homogeneous variances; a two-sample t-test showed no statistically significant difference (P > 0.05), indicating comparability.

[0117] 3.2 Comparison of UPDRS re-examination scores between the two groups of patients before treatment, see Table 2.

[0118] Table 2 Comparison of UPDRS scores between the two groups of patients before treatment.

[0119]

[0120] The t-test showed that t = -0.591, P = 0.558 > 0.05, indicating that there was no statistically significant difference in UPDRS scores between the two groups before treatment, and they were comparable.

[0121] 3.3 Comparison of UPDRS scores between the two groups of patients before and after treatment, see Table 3.

[0122] Table 3 Comparison of UPDRS scores between the two groups before and after treatment.

[0123]

[0124] Note: * indicates that compared with before treatment, P < 0.05.

[0125] 3.3.1 Within-group comparison

[0126] (1) Treatment group: The scores before and after treatment were compared with those before treatment by paired samples t test. The UPDRS score after treatment decreased significantly (t=14.026, P<0.05), and the difference was statistically significant.

[0127] (2) Control group: The scores before and after treatment were compared by paired samples t test. The UPDRS score after treatment was significantly lower than that before treatment (t=14.419, P<0.05), and the difference was statistically significant.

[0128] 3.3.2 Intergroup comparisons

[0129] Independent samples t-test showed that after treatment, the UPDRS score of the treatment group was significantly lower than that of the control group, but the difference was not statistically significant (P>0.05).

[0130] 3.4 Comparison of UPDRS score decrease between the two groups after treatment, see Table 4.

[0131] Table 4 Comparison of UPDRS score decrease between the two groups after treatment.

[0132]

[0133] Note: * indicates that compared with before treatment, P < 0.05.

[0134] The UPDRS scores of both groups after treatment followed a normal distribution with homogeneous variances. According to the t-test, t = 2.869, P = 0.007 < 0.05. The UPDRS score reduction in the treatment group after treatment was significantly greater than that in the control group, and the difference was statistically significant.

[0135] 3.5 Dosage of Levodopa: In the treatment group, 12 patients reduced their daily dosage of Levodopa by an average of 0.5 tablets, while there was no change in the control group.

[0136] 4. Conclusion

[0137] This study found that the UPDRS score decreased after treatment with the herbal composition of this invention compared with the pre-treatment score in both the treatment group and the control group. The decrease in UPDRS score after treatment was significantly greater in the treatment group than in the control group. Some patients also reduced their dosage of Levodopa. Therefore, it can be seen that Schisandra chinensis decoction can effectively improve the symptoms of Parkinson's disease.

[0138] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine composition for treating Parkinson's disease, characterized in that, The traditional Chinese medicine composition is made from the following raw materials in parts by weight: 9-20 parts of Schisandra chinensis, 10-30 parts of Ophiopogon japonicus, 3-15 parts of raw ginseng, 10-30 parts of Uncaria rhynchophylla, 10-30 parts of Cistanche deserticola, 10-30 parts of Cuscuta chinensis, 3-5 parts of ginger, and 5-15 parts of jujube.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition is made from the following raw materials in parts by weight: 15 parts Schisandra chinensis, 15 parts Ophiopogon japonicus, 6 parts raw ginseng, 20 parts Uncaria rhynchophylla, 15 parts Cistanche deserticola, 15 parts Cuscuta chinensis, 3 parts ginger, and 10 parts jujube.

3. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition is made from the following raw materials in parts by weight: 9 parts Schisandra chinensis, 10 parts Ophiopogon japonicus, 3 parts raw ginseng, 10 parts Uncaria rhynchophylla, 10 parts Cistanche deserticola, 10 parts Cuscuta chinensis, 3 parts ginger, and 5 parts jujube.

4. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition is made from the following raw materials in parts by weight: 20 parts Schisandra chinensis, 30 parts Ophiopogon japonicus, 15 parts raw ginseng, 30 parts Uncaria rhynchophylla, 30 parts Cistanche deserticola, 30 parts Cuscuta chinensis, 5 parts fresh ginger, and 15 parts jujube.

5. A method for preparing the traditional Chinese medicine composition for treating Parkinson's disease according to any one of claims 1-4, characterized in that, The method includes: taking the following ingredients according to the formula: Schisandra chinensis, Ophiopogon japonicus, raw ginseng, Uncaria rhynchophylla, Cistanche deserticola, Cuscuta chinensis, ginger, and jujube, and extracting them with water to obtain the final product.

6. The preparation method according to claim 4, characterized in that, The extraction method is selected from water decoction, water heating and reflux, or ultrasonic extraction.

7. The preparation method according to claim 5, characterized in that, The preparation method further includes the step of concentrating the aqueous extract into an extract; optionally, the extract is dried to make a dry powder.

8. A traditional Chinese medicine preparation for treating Parkinson's disease, characterized in that, The traditional Chinese medicine preparation is made from the traditional Chinese medicine composition according to any one of claims 1-4.

9. The traditional Chinese medicine preparation according to claim 8, characterized in that, The raw materials for the traditional Chinese medicine preparations also include pharmaceutically acceptable excipients.

10. The traditional Chinese medicine preparation according to claim 8, characterized in that, The dosage forms of the traditional Chinese medicine preparations include decoctions, tablets, granules, pills, powders, or capsules.

11. The use of the traditional Chinese medicine composition according to any one of claims 1-4 or the traditional Chinese medicine preparation according to any one of claims 8-10 in the preparation of a medicament for treating Parkinson's disease.

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