Application of a Traditional Chinese Medicine Composition in the Preparation of Drugs for Treating Parkinson's Disease

Abstract

This invention belongs to the field of traditional Chinese medicine and provides a new application of a traditional Chinese medicine composition, specifically its application in the preparation of anti-Parkinson's disease drugs. The traditional Chinese medicine composition of this invention comprises the following raw materials in the following weight ratios: 8 parts of *Cynanchum paniculatum*, 1 part of *Schisandra chinensis*, 4 parts of *Acorus tatarinowii*, and 2 parts of *Scutellaria baicalensis*. Using *C. elegans* as a drug screening model, in vivo experimental results show that this traditional Chinese medicine composition can significantly inhibit the expression of α-synuclein and promote the recovery of neurodegenerative dopamine (DA) neurons, suggesting that this traditional Chinese medicine composition is an effective candidate for anti-Parkinson's disease drugs and can be used in the preparation of such drugs.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine and provides the application of a traditional Chinese medicine composition in the preparation of drugs for treating Parkinson's disease. The traditional Chinese medicine composition of this invention comprises the following raw materials in the following weight ratios: 8 parts of *Corydalis yanhusuo*, 1 part of *Schisandra chinensis*, 4 parts of *Acorus tatarinowii*, and 2 parts of *Scutellaria baicalensis*. In vivo experiments using the model organism *C. elegans* showed that this traditional Chinese medicine composition can significantly inhibit the expression of α-synuclein and promote the recovery of neurodegenerative dopamine neurons, indicating that this traditional Chinese medicine composition has the potential to treat Parkinson's disease and can be used in the preparation of anti-Parkinson's disease drugs. Background Technology

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease, characterized by a decline in motor and cognitive function. Motorly, it manifests as muscle rigidity, bradykinesia, and tremors; behaviorally and cognitively, it presents as dementia, depression, anxiety, and sleep disturbances. The incidence of this disease increases rapidly in people over 60 years of age. Patients' quality of life declines as the disease progresses, eventually burdening their families and placing a heavy burden on society and their households.

[0003] The main pathological features of Parkinson's disease are the accumulation of α-synuclein in brain tissue and the death of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNPc) of the brain. To date, there are no effective treatments.

[0004] Screening for anti-Parkinson's drugs using traditional mammalian Parkinson's disease models is time-consuming and costly. However, using *Caenorhabditis elegans* (C. elegans) can reduce the screening cycle from months to days, making it a highly useful drug screening model. *C. elegans* is inexpensive and easy to culture; it has a short generation cycle, produces a large number of offspring, and yields a large number of individuals with consistent backgrounds, ensuring experimental reproducibility and allowing for large sample sizes to eliminate the influence of individual differences. Furthermore, *C. elegans* shares high conservation with higher organisms in terms of genes and molecular pathways. Therefore, *C. elegans* is increasingly favored by pharmacologists. DevGen, a US company, has had an antiarrhythmic drug screened using humanized nematodes approved by the FDA for clinical trials.

[0005] Meanwhile, after years of development, many traditional Chinese medicine (TCM) treatments for Parkinson's disease have become available for patients. For example, Chinese patent CN104352725A discloses a TCM composition for treating Parkinson's disease, composed of the following TCM raw materials in the indicated weight ratios: calcined dragon bone 20-40g, calcined oyster shell 20-40g, turtle shell 20-40g, white peony root 10-20g, Sichuan lovage rhizome 5-20g, angelica root 5-20g, earthworm 5-20g, albizia flower 10-20g, daylily bud 10-20g, salvia root 25-40g, and prepared licorice root 5-10g. The TCM composition of this invention uses all natural TCM raw materials and has the effects of promoting blood circulation and nourishing blood, tonifying liver deficiency, calming the liver and extinguishing wind, nourishing the kidneys and brain, strengthening the spleen and replenishing qi, and drying dampness and resolving phlegm. It is easy to prepare, has a wide range of medicinal sources, low cost, significant efficacy, mild medicinal properties, an effective rate of 98.21%, and a cure rate of 75%. In addition, Chinese patent CN105267410A discloses a traditional Chinese medicine formula for treating Parkinson's disease, which is prepared by Bupleurum, Rehmannia glutinosa, Paeonia lactiflora and Glycyrrhiza uralensis in a certain weight ratio. It has been shown to improve the symptoms of Parkinson's disease in mice by inducing a subacute Parkinson's disease model by MPTP.

[0006] This invention discloses a traditional Chinese medicine composition that can significantly reduce the fluorescence intensity of yellow fluorescent protein fused with human α-synuclein in the muscle tissue of *C. elegans*, and can also promote the recovery of neurodegenerative dopamine (DA) neurons. This indicates that the traditional Chinese medicine composition of this invention has the potential to treat Parkinson's disease and can be used in the preparation of anti-Parkinson's drugs. Summary of the Invention

[0007] The purpose of this invention is to provide a traditional Chinese medicine composition for treating Parkinson's disease, specifically relating to the application of such a composition in the preparation of anti-Parkinson's drugs. The traditional Chinese medicine composition of this invention comprises the following raw materials in the following weight ratios: 8 parts of *Schisandra chinensis*, 1 part of *Schisandra chinensis*, 4 parts of *Acorus tatarinowii*, and 2 parts of *Scutellaria baicalensis*.

[0008] Preferably, the traditional Chinese medicine composition comprises the following raw materials in the following weight ratio: 200mg of spider lily, 25mg of schisandra fruit, 100mg of acorus tatarinowii, and 50mg of scutellaria baicalensis.

[0009] The preparation method of the above-mentioned traditional Chinese medicine composition is as follows: weigh spider incense, schisandra fruit, acorus tatarinowii and scutellaria baicalensis according to the weight ratio, soak in distilled water, heat to boiling, filter to remove the dregs, collect the liquid for later use, soak the dregs in distilled water again, heat to boiling, filter again to obtain the supernatant, combine the liquids, concentrate and make up to volume, centrifuge, take the supernatant, filter to remove bacteria, dispense and store frozen.

[0010] The soaking time is 30 minutes.

[0011] The heating and boiling time is 30 minutes each time.

[0012] The centrifugation speed was 12,000 rpm.

[0013] The centrifugation time was 10 minutes.

[0014] The application of the traditional Chinese medicine composition described in this invention in the preparation of drugs for treating Parkinson's disease can enable the traditional Chinese medicine composition to be prepared into injections, powders, granules, pills, oral liquids, and tablets. Attached Figure Description

[0015] Figure 1 The therapeutic effect of decoction of traditional Chinese medicine composition on PD nematodes

[0016] Figure 2 A decoction of traditional Chinese medicine composition promotes the recovery of neurodegenerative dopamine neurons. Detailed Implementation

[0017] Example 1: Preparation of the decoction of the traditional Chinese medicine composition

[0018] Weigh 200mg of spider lily, 25mg of schisandra fruit, 100mg of acorus tatarinowii, and 50mg of scutellaria baicalensis. Add 200mL of distilled water and soak for 30 minutes. Heat to a boil and maintain for 30 minutes. Filter the residue through gauze and collect the liquid for later use. Soak the residue again in 150ml of distilled water for 5-10 minutes. Heat to a boil and maintain for 30 minutes. Filter the residue again through gauze to obtain the supernatant. Combine the two liquids, concentrate and adjust the volume to 50ml. Centrifuge twice at 12000rpm×10min, discard the insoluble precipitate, filter sterilize using a 0.22μm aqueous filter, dispense and store at -20℃.

[0019] Example 2: Therapeutic effect of the decoction of the traditional Chinese medicine composition on *C. elegans*, a nematode of Parkinson's disease.

[0020] 1. Biomaterials

[0021] (1) Caenorhabditis elegans OW13 was purchased from Caenorhabditis Genetics Center (CGC). It is a transgenic strain in which human α-synuclein is fused with YFP yellow fluorescent protein and inserted below a muscle-specific promoter. Therefore, this strain of nematodes specifically expresses human α-synuclein in muscle tissue. Fluorescence microscopy shows that the stronger the fluorescence, the higher the expression of α-synuclein. Drugs that have therapeutic effects on Parkinson's disease (PD) can significantly inhibit the fluorescence produced by α-synuclein expression; the stronger the fluorescence inhibition ability of the tested drug, the better its anti-PD activity.

[0022] (2) Escherichia coli OP50 (uracil leak mutant), purchased from Caenorhabditis Genetics Center (CGC), was used as food for Caenorhabditis elegans.

[0023] 2. Reagents

[0024] (1) Composition and preparation of solid NGM (Nematode Growth Mediμm) culture medium (taking 1 liter as an example):

[0025] Element content NaCl 3.00g K2HPO4 2.60g KH2PO4 17.00g peptone 2.80g Agar 17.00g <![CDATA[Add H2O to]]> 1000mL

[0026] After the solid NGM medium is prepared, it is autoclaved at 121℃ for 20 minutes. Under aseptic conditions, add 1 mL of 5 mg / mL cholesterol, 1 mL of 1 M MgSO4, and 1 mL of 1 M CaCl2 and shake well. While still hot, pour the mixture into a sterilized 9 cm culture plate, about 20 mL per plate, and let it stand until the medium solidifies.

[0027] (2) M9 liquid formulation

[0028] Element content <![CDATA[Na2HPO4]]> 6.00g <![CDATA[KH2PO4]]> 3.00g NaCl 5.00g <![CDATA[1M MgSO4]]> 1.00mL <![CDATA[Add H2O to]]> 1000mL

[0029] (3) Preparation of lysis buffer: 6.4% NaClO solution and 1M NaOH solution are mixed at a volume ratio of 1:1.

[0030] 3. Configuring NGM flat panel

[0031] Prepare NGM agar plates containing single herbs: Add *Symplocos edulis* to NGM medium to achieve final concentrations of 0, 25, 50, and 100 mg / mL. Prepare NGM agar plates containing *Schisandra chinensis*, *Acorus tatarinowii*, and *Scutellaria baicalensis* in the same manner as for *Symplocos edulis*.

[0032] Prepare NGM plates containing the traditional Chinese medicine composition: Add the extract of the traditional Chinese medicine composition (Spider Fragrance: Schisandra: Acorus: Scutellaria = 8:1:4:2) to NGM medium to make the final concentration of the medium containing the traditional Chinese medicine composition 375 mg / mL.

[0033] Prepare NGM plates containing n-butenylphthalide: Add n-butenylphthalide to NGM medium to make the final concentration of the medium containing n-butenylphthalide 60 μmol / L, as a positive control.

[0034] Pour the NGM above into each plate and let stand until the culture medium solidifies. Spread E. coli OP50 evenly on the culture medium as food for the nematodes.

[0035] 4. Implementation Steps

[0036] (1) Cultivation of nematodes:

[0037] Nematodes were inoculated onto solid NGM plates coated with Escherichia coli OP50 and then cultured in an incubator at 20°C. When the nematodes grew to adulthood, they were synchronized.

[0038] (2) Nematode synchronization:

[0039] Select NGM medium containing a large number of adult nematodes and some hatched nematode eggs. Rinse the nematodes off the medium with M9 solution and transfer them to centrifuge tubes. Allow the nematodes to settle freely to the bottom of the tubes, and discard the supernatant. Depending on the amount of nematodes, add lysis buffer to the centrifuge tubes and vortex for 5-7 minutes until all nematodes break down. Stop vortexing and centrifuge at 4000 rpm for 3 minutes. Carefully discard the supernatant, add 10 mL of M9 solution, and centrifuge at 4000 rpm for 3 minutes. Repeat this process three more times, centrifuging at 4000 rpm for 3 minutes each time, discarding the supernatant, leaving 2 mL of liquid. Aliquot the collected 2 mL liquid containing nematode eggs into 1.5 mL centrifuge tubes and place them in a 20°C biochemical incubator for incubation.

[0040] (3) The extract of the traditional Chinese medicine composition inhibited the expression of human α-synuclein in the muscle tissue of Caenorhabditis elegans OW13.

[0041] After synchronization, the nematodes were aliquoted into centrifuge tubes and incubated in M9 buffer for 48 hours to become synchronized L1-stage larvae. Excess M9 was removed by centrifugation at 4000 rpm for 5 minutes, and the nematodes were transferred to NGM-containing culture dishes. Each dish contained 60 nematodes, with three dishes per drug concentration serving as parallels. The nematodes were incubated at 20°C for another 72 hours. The mixture was then rinsed with M9, centrifuged at 4000 rpm for 5 minutes to remove excess M9, and 20 mM NaN3 was added to anesthetize the nematodes. The nematodes were observed and photographed under a fluorescence microscope. Twenty-five nematodes were randomly selected from each group, and the fluorescence intensity of all images was quantitatively analyzed using ImageJ software. Results are shown below. Figure 1 .

[0042] Experimental results show that, in this embodiment, compared with the administration of single herbs Spider Fragrance, Schisandra Chinensis, Acorus tatarinowii, and Scutellaria baicalensis at concentrations of 25 mg / mL, 50 mg / mL, and 100 mg / mL, and the positive control group n-butenylphthalide, this traditional Chinese medicine composition significantly inhibited the expression of human α-synuclein in Nematode muscle tissue, suggesting that it has a significant therapeutic effect on PD.

[0043] Example 3: A decoction of the traditional Chinese medicine composition promotes the recovery of neurodegenerative dopamine neurons.

[0044] 1. Biomaterials

[0045] (1) Caenorhabditis elegans BZ555, purchased from Caenorhabditis Genetics Center (CGC), is a transgenic strain. BZ555 transgenic nematodes were treated with 6-OHDA (Sigma, St. Louis, MI) to induce selective degeneration of DA neurons.

[0046] (2) Escherichia coli OP50 (uracil leak mutant), purchased from Caenorhabditis Genetics Center (CGC), was used as food for Caenorhabditis elegans.

[0047] 2. Reagents

[0048] (1) Composition and preparation of solid NGM (Nematode Growth Mediμm) culture medium (taking 1 liter as an example):

[0049] Element content NaCl 3.00g K2HPO4 2.60g KH2PO4 17.00g peptone 2.80g Agar 17.00g <![CDATA[Add H2O to]]> 1000mL

[0050] After the solid NGM medium is prepared, it is autoclaved at 121℃ for 20 minutes. Under aseptic conditions, add 1 mL of 5 mg / mL cholesterol, 1 mL of 1 M MgSO4, and 1 mL of 1 M CaCl2 and shake well. While still hot, pour the mixture into a sterilized 9 cm culture plate, about 20 mL per plate, and let it stand until the medium solidifies.

[0051] (2) M9 liquid formulation

[0052] Element content <![CDATA[Na2HPO4]]> 6.00g <![CDATA[KH2PO4]]> 3.00g NaCl 5.00g <![CDATA[1M MgSO4]]> 1.00mL <![CDATA[Add H2O to]]> 1000mL

[0053] (3) Preparation of lysis buffer: 6.4% NaClO solution and 1M NaOH solution are mixed at a volume ratio of 1:1.

[0054] 3. Configuring NGM flat panel

[0055] Prepare untreated NGM plates according to the solid NGM culture medium formula as a negative control.

[0056] Prepare NGM plates containing 6-OHDA: Add 6-OHDA to NGM medium to make the final concentration of 6-OHDA in the medium 50 mmol / L.

[0057] Prepare NGM agar plates containing 6-OHDA and individual herbs: Add 6-OHDA to NGM medium to a final concentration of 50 mmol / L. Add *Syzygium aromaticum* (a single herb) to NGM medium to final concentrations of 0, 25, 50, and 100 mg / mL. Prepare NGM agar plates containing *Schisandra chinensis*, *Acorus tatarinowii*, and *Scutellaria baicalensis* in the same manner as for *Syzygium aromaticum*.

[0058] Prepare NGM plates containing 6-OHDA and the traditional Chinese medicine composition: Add 6-OHDA to NGM medium to a final concentration of 50 mmol / L. Add the extract of the traditional Chinese medicine composition (Spider Fragrance: Schisandra: Acorus: Scutellaria = 8:1:4:2) to NGM medium to a final concentration of 375 mg / mL.

[0059] Prepare NGM plates containing 6-OHDA and n-butenylphthalide: Add 6-OHDA to NGM medium to a final concentration of 50 mmol / L. Add n-butenylphthalide to NGM medium to a final concentration of 60 μmol / L as a positive control.

[0060] Pour the NGM above into each plate and let stand until the culture medium solidifies. Spread E. coli OP50 evenly on the culture medium as food for the nematodes.

[0061] 4. Implementation Steps

[0062] (1) Cultivation of nematodes:

[0063] Nematodes were inoculated onto solid NGM plates coated with Escherichia coli OP50 and then cultured in an incubator at 20°C. When the nematodes grew to adulthood, they were synchronized.

[0064] (2) Nematode synchronization:

[0065] Select NGM medium containing a large number of adult nematodes and some hatched nematode eggs. Rinse the nematodes off the medium with M9 solution and transfer them to centrifuge tubes. Allow the nematodes to settle freely to the bottom of the tubes, and discard the supernatant. Depending on the amount of nematodes, add lysis buffer to the centrifuge tubes and vortex for 5-7 minutes until all nematodes break down. Stop vortexing and centrifuge at 4000 rpm for 3 minutes. Carefully discard the supernatant, add 10 mL of M9 solution, and centrifuge at 4000 rpm for 3 minutes. Repeat this process three more times, centrifuging at 4000 rpm for 3 minutes each time, discarding the supernatant, leaving 2 mL of liquid. Aliquot the collected 2 mL liquid containing nematode eggs into 1.5 mL centrifuge tubes and place them in a 20°C biochemical incubator for incubation.

[0066] (3) Extracts from traditional Chinese medicine compositions promote the recovery of neurodegenerative dopamine neurons.

[0067] After synchronization, the nematodes were aliquoted into centrifuge tubes and incubated in M9 buffer for 48 hours to become synchronized L1-stage larvae. Excess M9 was removed by centrifugation at 4000 rpm for 5 minutes, and the nematodes were transferred to NGM-containing culture dishes. Each dish contained 60 nematodes, with three dishes per drug concentration serving as parallels. The nematodes were incubated at 20°C for another 72 hours. The mixture was then rinsed with M9, centrifuged at 4000 rpm for 5 minutes to remove excess M9, and 20 mM NaN3 was added to anesthetize the nematodes. The nematodes were observed and photographed under a fluorescence microscope. Twenty-five nematodes were randomly selected from each group, and the fluorescence intensity of all images was quantitatively analyzed using ImageJ software. Results are shown below. Figure 2 .

[0068] Experimental results show that, in this embodiment, compared with the administration of single herbs Spider Fragrance, Schisandra Chinensis, Acorus tatarinowii, and Scutellaria baicalensis at concentrations of 25 mg / mL, 50 mg / mL, and 100 mg / mL, and the positive control group n-butenylphthalide, this traditional Chinese medicine composition significantly promoted the recovery of neurodegenerative dopamine neurons, suggesting that it has a significant therapeutic effect on PD.

[0069] The above examples demonstrate that the traditional Chinese medicine composition provided by this invention has a significant therapeutic effect on PD-positive Caenorhabditis elegans, suggesting that the traditional Chinese medicine composition provided by this invention has the potential to combat PD and can be used in the preparation of drugs for the prevention and treatment of PD.

Claims

1. The application of a traditional Chinese medicine composition in the preparation of an anti-Parkinson's disease drug, characterized in that, The traditional Chinese medicine composition consists of the following raw materials in the following weight ratio: 8 parts of spider lily, 1 part of schisandra fruit, 4 parts of acorus tatarinowii, and 2 parts of scutellaria baicalensis.

2. The application according to claim 1, characterized in that, The traditional Chinese medicine composition consists of the following raw materials: 200mg of spider filament, 25mg of schisandra fruit, 100mg of acorus tatarinowii, and 50mg of scutellaria baicalensis.

3. The application according to claim 2, characterized in that, The preparation method of the traditional Chinese medicine composition is as follows: weigh spider incense, schisandra fruit, acorus tatarinowii and scutellaria baicalensis according to the weight ratio, soak in distilled water, heat to boiling, filter to remove the dregs, collect the liquid for later use, soak the dregs in distilled water again, heat to boiling, filter again to obtain the supernatant, combine the liquids, concentrate and make up to volume, centrifuge, take the supernatant, filter to remove bacteria, dispense and store frozen.

4. The application according to any one of claims 1-3, characterized in that, The dosage form of the traditional Chinese medicine composition is injection, powder, granule, pill, oral liquid or tablet.

Images