Application of ephedra and apricot kernel cough relieving traditional Chinese medicine composition in preparation of medicine for treating Parkinson's disease

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to application of an ephedra-apricot kernel cough-relieving traditional Chinese medicine composition in preparation of a medicine for treating Parkinson's disease, the ephedra-apricot kernel cough-relieving traditional Chinese medicine composition provided by the invention shows a remarkable treatment effect on Parkinson's disease in a caenorhabditis elegans Parkinson's disease pathological model, and the medicine composition can be used for remarkably treating the Parkinson's disease. The polypeptide has the potential of treating the Parkinson's disease and can be applied to preparation of drugs for treating the Parkinson's disease.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine, and particularly relates to application of a traditional Chinese medicine composition of Mahuang and Maxing in preparation of a medicine for treating Parkinson's disease. BACKGROUND

[0002] Parkinson's disease (PD) is a common neurodegenerative disease in the elderly, which is mainly characterized by pathological changes of progressive degeneration of dopaminergic neurons in the substantia nigra and formation of Lewy bodies, biochemical changes of dopamine neurotransmitter reduction in the striatum and imbalance between dopamine and acetylcholine neurotransmitters, and clinical manifestations of motor symptoms such as tremor, muscle rigidity, bradykinesia, postural imbalance, and non-motor symptoms such as sleep disorders, olfactory disorders, autonomic nervous dysfunction, cognitive and mental disorders. [1] Epidemiological investigations show that the prevalence of Parkinson's disease in people over 60 years old in European and American countries reaches 1%, and that in people over 80 years old exceeds 4% [2] The prevalence of Parkinson's disease in people over 65 years old in China is 1.7% [3] China is the country with the largest population in the world, and the number of people with Parkinson's disease in China will increase from 1.99 million in 2005 to 5 million in 2030, which accounts for almost half of the global number of people with Parkinson's disease [4] With the progression of the disease, the motor and non-motor symptoms of Parkinson's disease will gradually worsen, which will not only impair the daily activities of the patients themselves, but also bring huge social and medical burdens.

[0003] Currently, the drugs for improving Parkinson's disease on the market in China include compound levodopa (dopaminergic agents, carbidopa, cabergoline), dopamine receptor agonists non-ergot Das (pramipexole, ropinirole, piribedil, rotigotine), second-generation MAO BI rasagiline, catechol O methyltransferase inhibitors (entacapone, talipexole, opicapone, entacapone biberid), anticholinergic drugs (benztropine), and amantadine. These drugs mainly have the efficacy of improving the symptoms of the patients, and some also have the efficacy of delaying the progression of the disease. However, the progression of the disease and the long-term use of the drugs will produce adverse reactions and motor complications, for example, the long-term use of levodopa will produce motor complications, and dopamine receptor agonists have the risk of drowsiness and mental adverse reactions. Some drugs will also have drug resistance and reduce the efficacy of the drugs if used for too long [1] However, traditional Chinese medicine compositions have the characteristics of multiple active components and multiple targets, so it is promising to screen drug candidates for treating PD from the treasure house of traditional Chinese medicine compositions.

[0004] Alpha-synuclein is a hallmark pathological product of Parkinson's disease. If the drug to be tested can reduce the content of alpha-synuclein in the animal model, it indicates that the drug has the effect of treating Parkinson's disease. The establishment of the alpha-synuclein overexpression Caenorhabditis elegans Parkinson's disease model is to express human alpha-synuclein fused with yellow fluorescent protein (YFP) in Caenorhabditis elegans by using a promoter. The promoter can drive expression to the body wall muscle cells, and visual observation of alpha-synuclein can be achieved. YFP fused with human alpha-synuclein can migrate to inclusion bodies, which can be observed on the second day after hatching, and the number and volume of the inclusion bodies continue to increase in the late adult stage of the worm [5] .

[0005] Dopaminergic neuron damage is a typical pathological manifestation of Parkinson's disease. If the drug to be tested can restore the dopaminergic neurons of the dopaminergic neuron damage animal model, it indicates that the drug has the potential to treat Parkinson's disease. Caenorhabditis elegans contains eight dopamine neurons: four symmetrically arranged head cells (CEPs, two dorsal and two ventral), two bilateral anterior ear bodies (ADEs) located in the head, and two bilateral posterior ear bodies (PDEs) near the vulva. The synaptic extensions of dopamine neurons are distributed throughout the trunk and tail. By combining green fluorescent protein (GFP) with the dopamine transporter gene promoter (Pdat-1), visual observation of dopamine neuron cell bodies and synapses can be achieved to study the mechanism of dopamine neuron degeneration [6] . 6-OHDA can cause dopamine neuron death, and a dopaminergic neuron damage Caenorhabditis elegans Parkinson's disease model can be constructed by adding 6-OHDA.

[0006] Mahuang Xingji Capsules is a compound traditional Chinese medicine composed of Ephedra, bitter apricot kernel, fried licorice and gypsum, mainly used for relieving cough, eliminating phlegm, and relieving asthma, suitable for acute and chronic bronchitis and wheezing, etc. In previous studies, it has been found to have antiviral and anti-inflammatory effects [7] . However, no efficacy in treating Parkinson's disease has been found.

[0007] However, the present application unexpectedly found that the Mahuang Xingji traditional Chinese medicine composition has an application in treating Parkinson's disease. The Mahuang Xingji traditional Chinese medicine composition showed a significant therapeutic effect in the Caenorhabditis elegans Parkinson's disease pathological model, can significantly reduce the fluorescence intensity of the alpha-synuclein overexpression Caenorhabditis elegans Parkinson's disease model, and enhance the fluorescence intensity of the Parkinson's disease damaged dopamine neuron Caenorhabditis elegans model, has the potential to treat Parkinson's disease, and can be applied in the preparation of drugs for treating Parkinson's disease.

[0008] References [1] Parkinson's disease and movement disorders group of Chinese Society of Neurology, Parkinson's disease and movement disorders group of Chinese Medical Doctor Association. Chinese guideline for Parkinson's disease (4th edition) [J]. Chinese Journal of Neurology, 2020, 53(12): 973-86. [2] SAMII A, NUTT J G, RANSOM B R. Parkinson's disease [J]. Lancet, 2004, 363(9423): 1783-93. [3] ZHANG Z X, ROMAN G C, HONG Z, et al. Parkinson's disease in China: prevalence in Beijing, Xian, and Shanghai [J]. Lancet, 2005, 365(9459): 595-7. [4] LI G, MA J, CUI S, et al. Parkinson's disease in China: a forty-year growing track of bedside work [J]. Transl Neurodegener, 2019, 8: 22. [5] KIM S K, VAN HAM T J, THIJSSEN K L, et al. C. elegans Model Identifies Genetic Modifiers of α-Synuclein Inclusion Formation During Aging [J]. PLoS Genetics, 2008, 4(3). [6] PU P, LE W. Dopamine neuron degeneration induced by MPP+ is independent of CED-4 pathway in Caenorhabditis elegans [J]. Cell Research, 2008, 18(9): 978-81. [7] JING Yaoya, XING Lingyi, LI Xiang, et al. Overview of Modern Research on Maxingshigan Decoction [J]. Journal of Tianjin University of Traditional Chinese Medicine, 2023, 42(02): 264-72. SUMMARY The application provides an application of a traditional Chinese medicine composition in preparation of a medicine for treating Parkinson's disease.

[0009] Preferably, the traditional Chinese medicine composition is a water extract of the traditional Chinese medicine composition or an alcohol extract of the traditional Chinese medicine composition.

[0010] Preferably, the water extract of the traditional Chinese medicine composition has the following preparation process: S1: weighing ephedra, bitter almond, gypsum and fried licorice according to the weight parts; S2: using purified water as a solvent, adding 8 times the amount of the volume ratio of purified water to the drug amount, heating and refluxing, centrifuging to collect supernatant, and reserving the residue for use; S3: continuing to add 8 times the amount of water to the residue after centrifuging in S2, heating and refluxing, and centrifuging again to collect supernatant; S4: combining the supernatants in S2 and S3, concentrating, freeze-drying the concentrated liquid, and collecting the freeze-dried powder.

[0011] Preferably, the alcohol extract of the traditional Chinese medicine composition has the following preparation process: S1: weighing ephedra, bitter almond, gypsum and fried licorice according to the weight parts; S2: using 75% ethanol as a solvent, adding 8 times the amount of the volume ratio of 75% ethanol to the drug amount, heating and refluxing, centrifuging to collect supernatant, and reserving the residue for use; S3: continuing to add 8 times the amount of 75% ethanol to the residue after centrifuging in S2, heating and refluxing, and centrifuging again to collect supernatant; S4: combining the supernatants in S2 and S3, concentrating, freeze-drying the concentrated liquid, and collecting the freeze-dried powder.

[0012] Further, the traditional Chinese medicine composition is added with pharmaceutically acceptable adjuvants to form any pharmaceutically acceptable dosage form.

[0013] Preferably, the dosage form includes powders, granules, powders, pills, tablets, capsules, pills and oral liquids.

[0014] The application has the following beneficial effects: The present application provides a kind of treating Parkinson's disease Ma Xing Zhike water extract or alcohol extract composition, and with Caenorhabditis elegans Parkinson's disease pathological model is carried out experiment, prove the composition described in the application to Caenorhabditis elegans Parkinson's disease pathological model has significant therapeutic effect: (1) Ma Xing Zhike traditional Chinese medicine composition water, alcohol extract has significant therapeutic effect to Parkinson's disease pathological product alpha-synuclein high expression Caenorhabditis elegans Parkinson's disease model;(2) Ma Xing Zhike traditional Chinese medicine composition water extract has significant therapeutic effect to Parkinson's disease damaged dopaminergic neuron Caenorhabditis elegans model;(3) Ma Xing Zhike traditional Chinese medicine composition compared with neurotrophic factor synthesis promoter has significant therapeutic effect to Parkinson's disease pathological product alpha-synuclein high expression Caenorhabditis elegans Parkinson's disease model. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Ma Xing Zhike traditional Chinese medicine composition water extract has significant therapeutic effect to Parkinson's disease pathological product alpha-synuclein high expression Caenorhabditis elegans Parkinson's disease model;Wherein, Ctrl is blank control group;P is positive drug levodopa (0.1 mg / mL) group;0.1 is 0.1 mg / mL Ma Xing Zhike traditional Chinese medicine composition water extract group;1 is 1 mg / mL Ma Xing Zhike traditional Chinese medicine composition water extract group;10 is 10 mg / mL Ma Xing Zhike traditional Chinese medicine composition water extract group.

[0016] Figure 2 Ma Xing Zhike traditional Chinese medicine composition alcohol extract has significant therapeutic effect to Parkinson's disease pathological product alpha-synuclein high expression Caenorhabditis elegans Parkinson's disease model;Wherein, Ctrl is blank control group;P is positive drug levodopa (0.1 mg / mL) group;0.1 is 0.1 mg / mL Ma Xing Zhike traditional Chinese medicine composition alcohol extract group;1 is 1 mg / mL Ma Xing Zhike traditional Chinese medicine composition alcohol extract group;10 is 10 mg / mL Ma Xing Zhike traditional Chinese medicine composition alcohol extract group.

[0017] Figure 3 Ma Xing Zhike traditional Chinese medicine composition water extract has significant therapeutic effect to Parkinson's disease damaged dopaminergic neuron Caenorhabditis elegans model;Wherein, Ctrl is blank control group;M is 6-OHDA (50 mM) modeling group;P is positive drug levodopa (0.1 mg / mL) group;0.1 is 0.1 mg / mL Ma Xing Zhike traditional Chinese medicine composition water extract group;1 is 1 mg / mL Ma Xing Zhike traditional Chinese medicine composition water extract group;10 is 10 mg / mL Ma Xing Zhike traditional Chinese medicine composition water extract group.

[0018] Figure 4Therapeutic effect of alcohol extract of traditional Chinese medicine composition of Mahuang Xixin Zhike on Caenorhabditis elegans model of impaired dopaminergic neurons of Parkinson's disease; wherein Ctrl is a blank control group; M is a 6-OHDA (50 mM) modeling group; P is a positive drug levodopa (0.1 mg / mL) group; 0.1 is a 0.1 mg / mL alcohol extract of traditional Chinese medicine composition of Mahuang Xixin Zhike group; 1 is a 1 mg / mL alcohol extract of traditional Chinese medicine composition of Mahuang Xixin Zhike group; and 10 is a 10 mg / mL alcohol extract of traditional Chinese medicine composition of Mahuang Xixin Zhike group.

[0019] Figure 5 Comparison of therapeutic effects of water extract of traditional Chinese medicine composition of Mahuang Xixin Zhike and neurotrophic factor synthesis promoter on Caenorhabditis elegans Parkinson's disease model with high expression of pathological product α-synuclein; wherein Ctrl is a blank control group; P is a positive drug levodopa (0.1 mg / mL) group; 0.1 is a 0.1 mg / mL water extract of traditional Chinese medicine composition of Mahuang Xixin Zhike group; 10 is a 10 mg / mL water extract of traditional Chinese medicine composition of Mahuang Xixin Zhike group; and XMT is a 10 mg / mL water extract of neurotrophic factor synthesis promoter (Xudeng Decoction) group. DETAILED DESCRIPTION

[0020] The following specific examples are provided to achieve the technical solutions described in the present application, but the protection scope of the present application is not limited to the following.

[0021] Example 1 Preparation method of water and alcohol extract of traditional Chinese medicine composition of Mahuang Xixin Zhike Mahuang 6 parts, bitter apricot kernel 4 parts, gypsum 12 parts, and fried licorice 3 parts were weighed according to the weight ratio, crushed into coarse powder, and added with 8 times the amount of distilled water. The condensation reflux device was heated to reflux for 2 h, repeated twice, and the supernatant was filtered and collected for standby use. The supernatant of the two times was combined, and the rotary evaporator was used to concentrate the liquid. Then the concentrated liquid was frozen in a-80℃ refrigerator for 24 h, and then placed in a freeze dryer for freeze-drying. The freeze-dried powder was collected and stored at-20℃ for standby use.

[0022] Mahuang 6 parts, bitter apricot kernel 4 parts, gypsum 12 parts, and fried licorice 3 parts were weighed according to the weight ratio, crushed into coarse powder, and added with 8 times the amount of 75% ethanol. The condensation reflux device was heated to reflux for 2 h, repeated twice, and the supernatant was filtered and collected for standby use. The supernatant of the two times was combined, and the rotary evaporator was used to concentrate the liquid. Then the concentrated liquid was frozen in a-80℃ refrigerator for 24 h, and then placed in a freeze dryer for freeze-drying. The freeze-dried powder was collected and stored at-20℃ for standby use.

[0023] Example 2 Therapeutic effect of water and alcohol extract of traditional Chinese medicine composition of Mahuang Xixin Zhike on Caenorhabditis elegans Parkinson's disease model with high expression of pathological product α-synuclein 1. Biological materials: (1) C. elegans strain NL5901 was purchased from Caenorhabditis Genetics Center (CGC); it is a transgenic line. NL5901 nematodes express human α-synuclein fused to yellow fluorescent protein (YFP) under the control of a muscle-specific unc-54 promoter. (2) E. coli OP50 (uracil leaky mutant) was purchased from Caenorhabditis Genetics Center (CGC) and used as food for C. elegans. 2. Reagents (1) The composition and preparation of solid NGM (Nematode Growth Medium) are shown in Table 1 (using 1000 mL as an example):

[0024] After the solid NGM medium is prepared, it is autoclaved at 121°C for 20 minutes, 1 mL of 5 mg / mL cholesterolethanol solution, 1 mL of 1M MgSO4, and 1 mL of 1M CaCl2 are added under a sterile operating table, and shaken well. Pour about 20 mL / plate into a sterilized 9 cm culture plate while hot. Wait for the medium to solidify, and prepare for use.

[0025] (2) The formula of M9 liquid is shown in Table 2:

[0026] (3) Preparation of lysis solution: mix 6.4% NaClO solution and 1M NaOH solution at a volume ratio of 1:1.

[0027] 3. Preparation of NGM plates containing water and alcohol extracts of Maxing Cough-Relieving Capsule composition Add water to 10 mL to the freeze-dried powder of water extract of Maxing Cough-Relieving Traditional Chinese Medicine Composition, filter and sterilize, and prepare for use.

[0028] Add 25% ethanol to 10 mL to the freeze-dried powder of alcohol extract of Maxing Cough-Relieving Traditional Chinese Medicine Composition, filter and sterilize, and prepare for use.

[0029] Prepare NGM plates containing water and alcohol extracts of Maxing Cough-Relieving Traditional Chinese Medicine Composition: take the appropriate dilution of water and alcohol extracts of Maxing Cough-Relieving Traditional Chinese Medicine Composition, add to NGM medium, and pour the plate.

[0030] The final concentration of water and alcohol extracts of Maxing Cough-Relieving Traditional Chinese Medicine Composition is 10-fold dilution, 100-fold dilution, and 1000-fold dilution of the original prepared liquid. Wait for the medium to solidify. Spread E. coli OP50 evenly on the medium as food for nematodes.

[0031] 4. Implementation steps (1) Culture of nematodes: The nematodes were inoculated on solid NGM plates coated with E. coli OP50 and then incubated in an incubator at 20°C. When the nematodes grew into adults, they were synchronized.

[0032] (2) Synchronization of nematodes: The NGM medium containing a large number of adults and some hatched nematode eggs was selected, the nematodes were washed off the medium with M9 solution and transferred to a centrifuge tube, and the nematodes were allowed to settle freely at the bottom of the tube. The supernatant was discarded. Depending on the amount of nematodes, lysis solution was added to the centrifuge tube, and the nematodes were shaken in a vortex shaker for 5-7 min until the nematodes were completely broken. Then the nematodes were aliquoted into 1.5 mL centrifuge tubes, and the nematode eggs were washed three times with M9 solution.

[0033] (3) Experimental group setting: Control group (Ctrl): nematodes were cultured using ordinary NGM medium; Positive drug levodopa (0.1 mg / mL) group (P): nematodes were cultured using NGM medium containing 0.1 mg / mL levodopa; 0.1, 1, 10 mg / mL water extract of Maxing cough traditional Chinese medicine composition group: nematodes were cultured using NGM medium containing 0.1, 1, 10 mg / mL water extract of Maxing cough traditional Chinese medicine composition; 0.1, 1, 10 mg / mL alcohol extract of Maxing cough traditional Chinese medicine composition group: nematodes were cultured using NGM medium containing 0.1, 1, 10 mg / mL alcohol extract of Maxing cough traditional Chinese medicine composition; (4) Therapeutic effect of water and alcohol extract of Maxing cough traditional Chinese medicine composition on Parkinson's disease pathological product α-synuclein overexpressing C. elegans Parkinson's disease model NL5901 NL5901 nematode drug treatment: the hatched L1 nematodes were transferred to NGM plates coated with OP50 and mixed with different concentrations of water and alcohol extract of Maxing cough traditional Chinese medicine composition, 60 nematodes were inoculated on each plate, and incubated at 20°C for 5 days. After treatment, the nematodes were washed 3 times with M9 buffer to remove residual bacteria. The washed nematodes were placed on a 2% agarose pad, 20 mM NaN3 was added to fix the nematodes, and a fluorescence microscope was used to check the presence of α-synuclein aggregates. At least 30 images of nematodes were taken for each group. ImageJ software was used to quantify α-synuclein aggregation. The fluorescence intensity of each nematode was used as an indicator of α-synuclein aggregation.

[0034] Data processing and statistical analysis: data processing was performed using statistical software SPSS 23.0, data statistics were expressed as mean ± standard deviation (Mean ± SD), data were analyzed by ANOVA single factor analysis of variance, and Tukey test was used for significant difference, P<0.05 represented significant difference, which had statistical significance. Statistical graphs were drawn using GraphPad Prism 9.5.0 software.

[0035] (5) Experimental results The water and alcohol extracts of the Mahxing Jichu traditional Chinese medicine composition had a significant therapeutic effect on the Parkinson's disease pathological product alpha-synuclein overexpressing C. elegans Parkinson's disease model (Figure 1, Figure 2 Data are expressed as mean ± standard deviation. Single factor analysis of variance was used for comparison between groups, ***p<0.001, indicating extremely significant difference.

[0036] Example 3 Therapeutic effect of water and alcohol extracts of Mahxing Jichu traditional Chinese medicine composition on dopamine neuron impaired C. elegans model of Parkinson's disease 1. Biological materials: (1) C. elegans strain BZ555 was purchased from Caenorhabditis Genetics Center (CGC); it is a transgenic strain. BZ555 expresses green fluorescent protein under the control of dopamine transporter (dat-1) promoter, which can visualize dopamine neurons, and dopamine neurons will be damaged after 6-OHDA treatment.

[0037] (2) Escherichia coli OP50 (uracil leak mutant) was purchased from Caenorhabditis Genetics Center (CGC) and used as food for C. elegans. 2. Reagents (1) The composition and preparation of solid NGM (Nematode Growth Medium) are shown in Table 1 (for 1000 mL):

[0038] After the preparation of solid NGM medium, it was autoclaved at 121°C for 20 min, 1 mL of 5 mg / mL cholesterol ethanol solution, 1 mL of 1M MgSO4, and 1 mL of 1M CaCl2 were added under a sterile operation table, and then shaken, and about 20 mL / plate was poured into the sterilized 9 cm culture plate while hot. Wait for the medium to solidify, and prepare for use.

[0039] (2) The formula of M9 liquid is shown in Table 2:

[0040] (3) S base liquid formula as shown in Table 3:

[0041] (4) Citric acid buffer formula as shown in Table 4:

[0042] (5) Trace liquid formula as shown in Table 5:

[0043] (6) S liquid medium as shown in Table 6:

[0044] (7) Preparation of lysis solution: 6.4% NaClO solution and 1M NaOH solution are mixed at a volume ratio of 1:1.

[0045] 3. Preparation of NGM plates of water and alcohol extracts of Maxing Cough Capsules composition NGM Prepared as shown in Example 2 4. Implementation steps (1) Culture of nematodes: The nematodes are inoculated on solid NGM plates coated with E. coli OP50, and then placed in an incubator at 20°C for culture. When the nematodes grow into adults, they are synchronized.

[0046] (2) Nematode synchronization: Select NGM medium containing a large number of adult worms and some nematode eggs that have hatched, wash the nematodes from the medium with M9 solution, transfer them to a centrifuge tube, let them settle freely at the bottom of the tube, and discard the supernatant. Add lysis solution to the centrifuge tube according to the amount of nematodes, shake on a vortex mixer for 5-7 min, stop the vortex when the nematodes are completely broken, and divide into 1.5 mL centrifuge tubes. Wash the nematode eggs with M9 solution three times.

[0047] (3) Experimental group setting: Control group (Ctrl): Use ordinary NGM medium to culture unmodeled nematodes; 6-OHDA modeling group (M): Use ordinary NGM medium to culture modeled nematodes; Positive drug levodopa (0.1 mg / mL) group (P): Use NGM medium containing 0.1 mg / mL levodopa to culture modeled nematodes; 0.1, 1, 10 mg / mL water extract of Maxing Cough Traditional Chinese Medicine Composition group: Use NGM medium containing 0.1, 1, 10 mg / mL water extract of Maxing Cough Traditional Chinese Medicine Composition to culture modeled nematodes; 0.1, 1, 10 mg / mL MA XIN ZHI KE Chinese medicine composition alcohol extract group: use NGM medium containing 0.1, 1, 10 mg / mL MA XIN ZHI KE Chinese medicine composition alcohol extract to culture the model nematodes; (4) Therapeutic effect of MA XIN ZHI KE Chinese medicine composition water and alcohol extract on BZ555, a Parkinson's disease damaged dopaminergic neuron Caenorhabditis elegans model BZ555 nematode drug treatment: the hatched L1 nematodes were transferred to a normal NGM plate coated with OP50 and cultured to L3. The L3 nematodes were washed with M9 buffer for 3 times to remove residual bacteria. Then they were added to S liquid medium containing 10 mM ascorbic acid and 50 mM 6-OHDA, and incubated at 20°C for 1 hour, with gentle shaking every 10 minutes. After treatment, the nematodes were washed with M9 buffer for 3 times, and then transferred to a NGM plate containing MA XIN ZHI KE Chinese medicine composition water and alcohol extract, and incubated at 20°C for 3 days. After treatment, the nematodes were washed with M9 buffer for 3 times to remove residual bacteria. The washed nematodes were placed on a 2% agarose pad, and 20 mM NaN3 was added to fix the nematodes. The fluorescence was examined using an upright fluorescence microscope, and the dopaminergic neurons were observed by GFP fluorescence. At least 30 images of nematodes were collected for each group. ImageJ software was used to quantify the GFP intensity in the head region of each Caenorhabditis elegans to determine the fluorescence intensity of dopaminergic neurons.

[0048] Data processing and statistical analysis: statistical software SPSS 23.0 was used for data processing. The data was represented as mean ± standard deviation (Mean ± SD). ANOVA single factor variance analysis was used, and Tukey test was used to determine significant differences. P<0.05 represents significant difference, which is statistically significant. Statistical graphs were drawn using GraphPad Prism 9.5.0 software.

[0049] (5) Experimental results MA XIN ZHI KE Chinese medicine composition water extract had a significant therapeutic effect on BZ555, a Parkinson's disease damaged dopaminergic neuron Caenorhabditis elegans model, and 10 mg / mL alcohol extract had a significant therapeutic effect (Figure 3, Figure 4 ). The data is represented as mean ± standard deviation. Single factor variance analysis was used for comparison between groups. ***p<0.001 indicates that the difference is extremely significant. *p<0.05 indicates that the difference is significant. ns indicates no significant difference (p>0.05).

[0050] Example 4 Comparison of the therapeutic effect of MA XIN ZHI KE Chinese medicine composition water extract and neurotrophic factor synthesis promoter on BZ555, a Parkinson's disease with high expression of pathological product alpha-synuclein Caenorhabditis elegans Parkinson's disease 1. Preparation of water extract of Mahxing Zhike traditional Chinese medicine composition and neurotrophic factor synthesis promoter The water extract of the Mahxing Zhike traditional Chinese medicine composition group was prepared as shown in Example 2.

[0051] The neurotrophic factor synthesis promoter: 16 g of almond, 12 g of ephedra, 12 g of cassia twig, 12 g of ginseng, 12 g of angelica, 8 g of chuanxiong, 8 g of dried ginger, 8 g of licorice and 24 g of gypsum (Chunming Decoction: 112 g) were mixed, 8 times the amount of distilled water was added, and the mixture was heated and refluxed for 2 h using a condensation reflux device, which was repeated twice. The supernatant was collected by filtration and was ready for use. The supernatant from the two times was combined and the liquid was concentrated using a rotary evaporator. The concentrated liquid was then frozen at -80°C for 24 h, and was then freeze-dried in a freeze-drying machine. The freeze-dried powder was collected and stored at -20°C for later use. When used, a 100 mg / mL stock solution was prepared and was filtered to remove bacteria. When preparing the NGM medium containing the drug, it was diluted 10 times.

[0052] 2. Experimental procedure The specific implementation steps are the same as in Example 2.

[0053] 3. Experimental results The experimental results showed that the Mahxing Zhike traditional Chinese medicine composition and the neurotrophic factor synthesis promoter had a significant therapeutic effect on the Parkinson's disease model NL5901 of Caenorhabditis elegans with high expression of α-synuclein, a pathological product of Parkinson's disease. The Mahxing Zhike traditional Chinese medicine composition was more effective than the neurotrophic factor synthesis promoter in reducing the fluorescence intensity of α-synuclein in Caenorhabditis elegans NL5901 (as shown in FIG. 1). Figure 5 The data are expressed as mean ± standard deviation. Single factor analysis of variance was used for comparison between groups, and ***p<0.001 indicates that the difference is extremely significant.

[0054] The results of the above examples showed that the water and alcohol extracts of the Mahxing Zhike traditional Chinese medicine composition could significantly reduce the fluorescence intensity of Caenorhabditis elegans expressing human α-synuclein labeled with fluorescence. The water extract and 10 mg / mL alcohol extract of the Mahxing Zhike traditional Chinese medicine composition could significantly enhance the fluorescence intensity of dopamine neurons of Caenorhabditis elegans damaged by 6-OHDA. The Mahxing Zhike traditional Chinese medicine composition and the neurotrophic factor synthesis promoter had a significant therapeutic effect on the Parkinson's disease model NL5901 of Caenorhabditis elegans with high expression of α-synuclein, a pathological product of Parkinson's disease. The Mahxing Zhike traditional Chinese medicine composition was more effective than the neurotrophic factor synthesis promoter in reducing the fluorescence intensity of α-synuclein in Caenorhabditis elegans NL5901.

[0055] In summary, the water and alcohol extracts of the Mahaxin Jichu Chinese medicine composition have a significant therapeutic effect on the Parkinson's disease pathological model of C. elegans, indicating that the water and alcohol extracts of the Mahaxin Jichu Chinese medicine composition have the potential to treat Parkinson's disease and can be applied in the preparation of drugs for treating Parkinson's disease.

[0056] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement to part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. The use of the traditional Chinese medicine composition of Ma Xing Ji Ke in the preparation of a medicine for treating Parkinson's disease, characterized in that, The traditional Chinese medicine composition is composed of the following raw materials in parts by weight: Ephedra 6 parts, bitter almond 4 parts, gypsum 12 parts, and fried licorice 3 parts.

2. Use according to claim 1, wherein The traditional Chinese medicine composition is a water extract of the traditional Chinese medicine composition or an alcohol extract of the traditional Chinese medicine composition.

3. The use according to claim 2, wherein the compound is ###0002### The water extract of the traditional Chinese medicine composition has the following preparation process The process comprises the following steps: S1: weigh the ephedra, bitter almond, gypsum, and fried licorice according to the parts by weight; S2: soak with purified water as the solvent, 8 times the amount of the drug by volume ratio, heat reflux, centrifugal collect supernatant, and reserve the residue for use; S3: continue to add 8 times the amount of water to the residue after centrifugation in S2, heat reflux, and centrifugal collect supernatant again; S4: combine the supernatants in S2 and S3, concentrate, freeze-dry the concentrated liquid, and collect the freeze-dried powder.

4. The use according to claim 2, wherein the compound is ###0002### The alcohol extract of the traditional Chinese medicine composition has the following preparation process The process comprises the following steps: S1: weigh the ephedra, bitter almond, gypsum, and fried licorice according to the parts by weight; S2: soak with 75% ethanol as the solvent, 8 times the amount of the drug by volume ratio, heat reflux, centrifugal collect supernatant, and reserve the residue for use; S3: continue to add 8 times the amount of 75% ethanol to the residue after centrifugation in S2, heat reflux, and centrifugal collect supernatant again; S4: combine the supernatants in S2 and S3, concentrate, freeze-dry the concentrated liquid, and collect the freeze-dried powder.

5. The application as described in any one of claims 1-4, characterized in that, The traditional Chinese medicine composition is added with pharmaceutically acceptable excipients to form any pharmaceutically acceptable dosage form.

6. The use according to claim 5, wherein the compound is ###0002### The dosage form includes powder, granules, powder, pills, tablets, capsules, pills, and oral liquid.

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