Application of panoxadiol in preparation of medicine for preventing and / or treating Parkinson's disease

Abstract

The invention discloses an application of panoxadiol in preparation of a product for preventing and / or treating Parkinson's disease. According to the invention, the PD treatment activity and mechanism of panoxadiol are explored in vivo and in vitro. In vitro, the panoxadiol can reduce the activity and cytotoxicity of LPS-induced BV2 cells, and inhibit the generation of proinflammatory factors through a TLR4 / MyD88 / NF-kappa B pathway. In vivo, the panoxadiol can improve movement and intestinal dysfunction of PD mice and repair deletion of dopaminergic neurons. A further research shows that the panoxadiol can be used for relieving the injury of the blood-brain barrier and inhibiting the neuroinflammation of the nigra, so that the injury to dopaminergic neurons is reduced. Subsequently, panoxadiol is found to be capable of relieving peripheral inflammation and remarkably recovering intestinal microflora. A further research shows that the panoxadiol treatment can inhibit a TLR4 / MyD88 / NF-kappa B signal channel in the nigra and a downstream pro-inflammatory product of the TLR4 / MyD88 / NF-kappa B.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to the application of ginsenodiol in the preparation of drugs for preventing and / or treating Parkinson's disease. Background Art

[0002] Parkinson's disease (PD) is considered the second most common neurodegenerative inflammatory disease and poses a major threat to human health and well-being. It is predicted that the global prevalence of PD may increase to between 12 million and 17 million people by 2043. The disease has multiple pathological features, including chronic loss of dopaminergic (DA) neurons in the substantia nigra of the midbrain, abnormal aggregation of α-synuclein, and the presence of neuroinflammation. The underlying etiology of PD remains controversial in the scientific community. There is evidence that an inflammatory response occurs in the substantia nigra of PD patients, characterized by the activation of microglia and upregulation of related cytokines. Therefore, neuroinflammation is considered to be a key factor in the pathogenesis of PD. Levodopa is the main drug treatment for PD; however, it is associated with a series of adverse reactions. Therefore, there is an urgent need to develop safe and effective therapeutic drugs to treat PD and improve the quality of life of patients.

[0003] Ginseng is a traditional Chinese herbal medicine with medicinal and nutritional properties, showing anticancer, antioxidant, anti-inflammatory and cognitive enhancement effects. Panaxadiol, a compound produced by the hydrolysis of active ginsenosides in ginseng, has received increasing attention in recent studies due to its proven antitumor, anti-inflammatory and potential therapeutic effects on diabetes, obesity and Alzheimer's disease. However, research on the relationship between panaxadiol and PD is significantly insufficient. Summary of the invention

[0004] The purpose of the present invention is to provide a new medicinal use of ginsenoside.

[0005] In a first aspect, the present invention provides the use of ginsenodiol in the preparation of a product for preventing and / or treating Parkinson's disease.

[0006] The structural formula of the ginsenoside is shown in Formula I:

[0007]

[0008] Furthermore, the prevention and / or treatment of Parkinson's disease is embodied in at least one of the following aspects:

[0009] 1) Alleviate motor dysfunction caused by Parkinson's disease;

[0010] 2) Alleviate gastrointestinal dysfunction caused by Parkinson's disease;

[0011] 3) Improve the histological features of substantia nigra (SN) caused by Parkinson's disease;

[0012] 4) Improve substantia nigra (SN) inflammation caused by Parkinson's disease;

[0013] 5) Inhibit the activation of microglia and astrocytes;

[0014] 6) Improve blood-brain barrier damage caused by Parkinson's disease;

[0015] 7) Inhibit neuroinflammation caused by Parkinson's disease;

[0016] 8) Alleviate intestinal flora imbalance caused by Parkinson's disease;

[0017] 9) Inhibit the secretion of inflammatory cytokines and the expression of proteins related to the TLR4 / MyD88 / NF-κB signaling pathway in BV2 cells induced by lipopolysaccharide (LPS).

[0018] Furthermore, the alleviation of the motor dysfunction caused by Parkinson's disease may specifically be a significant reduction of the tremor and stiffness caused by Parkinson's disease.

[0019] Furthermore, the alleviation of gastrointestinal dysfunction caused by Parkinson's disease may be significantly inhibiting the reduction of fecal water content and the number of fecal particles caused by Parkinson's disease.

[0020] Furthermore, the improvement of the histological characteristic changes of the substantia nigra (SN) caused by Parkinson's disease can specifically be that the tyrosine hydroxylase (TH) positive cells in the substantia nigra (SN) are arranged in an orderly manner, the number of tyrosine hydroxylase (TH) positive cells is significantly increased, and the expression of tyrosine hydroxylase (TH) is increased.

[0021] Furthermore, the improvement of substantia nigra (SN) inflammation caused by Parkinson's disease can specifically be the inhibition of the release of pro-inflammatory cytokines in the substantia nigra. The pro-inflammatory cytokines include IL-1β, IL-6 and TNF-α.

[0022] Furthermore, the improvement of blood-brain barrier damage caused by Parkinson's disease can specifically be to inhibit the increase in blood-brain barrier permeability caused by Parkinson's disease and restore the expression of three key tight junction proteins ZO-1, occludin and claudin-5.

[0023] Furthermore, the alleviation of intestinal flora imbalance caused by Parkinson's disease can specifically be to regulate the intestinal microbial community structure, normalize the composition of the intestinal microbial community, and restore the homeostasis of the microbial community to a large extent.

[0024] The product includes a medicine, a pharmaceutical composition or a health product.

[0025] The medicine, pharmaceutical composition or health product uses the ginsenoside as an active ingredient.

[0026] Optionally, the drug preparation form includes one or more of a solution, a suspension, a granule, a lyophilized powder, an emulsion, an oil, a syrup, an aerosol, and a nanoformulation.

[0027] In the above applications, when preparing medicines, ginsenodiol can be used as one of the active ingredients or as the only active ingredient.

[0028] In the above applications, carrier materials may also be added when preparing the medicine.

[0029] Carrier materials include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). These materials can be used to make a variety of dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. It can be a common preparation, a sustained-release preparation, a controlled-release preparation, and various microparticle delivery systems. In order to make a unit dosage form into a tablet, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants. , such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, tristearate, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. In order to make the unit dosage form into a pill, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc. In order to prepare the unit dosage form into a suppository, various carriers known in the art can be widely used. Examples of carriers include, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. In order to prepare the unit dosage form into an injectable preparation, such as a solution, emulsion, freeze-dried powder injection and suspension, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol may be added to the injection preparation. In addition, conventional solubilizers, buffers, pH adjusters, etc. may also be added.In addition, if necessary, colorants, preservatives, flavors, flavoring agents, sweeteners or other materials may be added to the pharmaceutical preparations. The above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; cavity administration, such as rectal and vaginal administration; respiratory tract administration, such as nasal cavity; mucosal administration.

[0030] In a second aspect, the present invention provides the use of ginsenodiol in the preparation of a product for alleviating motor dysfunction caused by Parkinson's disease.

[0031] In a third aspect, the present invention provides the use of ginsenodiol in the preparation of a product for alleviating gastrointestinal dysfunction caused by Parkinson's disease.

[0032] In a fourth aspect, the present invention provides the use of ginsenodiol in the preparation of a product for improving blood-brain barrier damage caused by Parkinson's disease.

[0033] In a fifth aspect, the present invention provides the use of ginsenodiol in the preparation of a product for inhibiting neuroinflammation caused by Parkinson's disease.

[0034] In a sixth aspect, the present invention provides the use of ginsenodiol in the preparation of a product for alleviating intestinal flora imbalance caused by Parkinson's disease.

[0035] In a seventh aspect, the present invention also provides a method for preventing and / or treating Parkinson's disease, comprising administering an effective amount of ginsenodiol or a pharmaceutical composition of ginsenodiol to a subject with Parkinson's disease.

[0036] In the present invention, the term "effective amount" refers to a dose that can achieve treatment, prevention, alleviation and / or relief of the diseases or conditions described in the present invention in a subject.

[0037] In the present invention, the term "subject" refers to an individual or animal subject who suffers from or may suffer from heart failure or heart failure-related diseases described in the present invention, and may also refer to an individual or animal subject used for a certain purpose, such as for scientific research purposes. Specifically, the individual is, for example, an animal subject, in particular a mammalian subject, such as a human, pig, dog, cat, cow, sheep, horse, rat, mouse, rabbit, guinea pig, monkey, etc.

[0038] The results of this study suggest that panaxadiol has potential therapeutic effects on Parkinson's disease through multiple mechanisms, including anti-inflammatory properties, enhanced motor function, repair of blood-brain barrier damage, and regulation of intestinal microbiota. These findings lay a scientific foundation for considering panaxadiol as a potential drug for the treatment of Parkinson's disease. Future studies should further investigate the clinical application of panaxadiol and evaluate its long-term therapeutic effects and safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1Effects of ginsenoside on LPS-induced BV2 cells; (A) CCK8 was used to detect cell proliferation; (B) LDH was used to detect cytotoxicity; (C) ELISA was used to detect the expression of inflammatory factors in cell supernatant; (D) Western blot was used to detect the expression of TLR4, MyD88, and p-NF-κB; data are expressed as mean±SEM. # p<0.05, ## p<0.01, ### p<0.001vs control group; * p<0.05, ** p<0.01, *** p<0.001vs model group.

[0040] Figure 2 Treatment with panaxadiol alleviated motor and gastrointestinal dysfunction in PD mice; (AD) motor behavior assessment; (E) pole climbing test; (F) hanging test; (G) colon length; (H) percentage of water in fecal pellets; (I) total number of fecal pellets; data are expressed as mean ± SEM. # p<0.05, ## p<0.01, ### p<0.001vs control group; * p<0.05, ** p<0.01, *** p<0.001vs model group.

[0041] Figure 3 Panaxadiol improves the histological characteristics and inflammation of SN in PD mice. (A) Immunohistochemical staining of TH in the substantia nigra of mice; (B) Western blot detection of TH expression; (C) ELISA detection of inflammatory factor expression; (D) mRNA expression of glial cell markers GFAP and Iba1; data are expressed as mean±SEM. # p<0.05, ## p<0.01, ### p<0.001vs control group; * p<0.05, ** p<0.01, *** p<0.001vs model group.

[0042] Figure 4Panaxadiol can improve blood-brain barrier damage in PD mice. (A) Evans blue test; (B) Representative electron micrographs of BBB tight junction structures in SN; (C) Western blot detection of ZO-1, occludin and claudin-5 expression in SN; (D) mRNA expression of tight junction proteins ZO-1, occludin and claudin-5 in SN; Data are expressed as mean±SEM. # p<0.05, ## p<0.01, ### p<0.001vs control group; * p<0.05, ** p<0.01, *** p<0.001vs model group.

[0043] Figure 5 Panaxadiol alleviates the imbalance of intestinal microbiota in PD mice. (A) Analysis of α diversity of intestinal microbiota; (B) PCoA diagram of β diversity; (CE) Relative abundance of intestinal microbiota at the family level and genus level and the relative abundance of three groups of bacteria; (F) LDA score bar graph; Data are expressed as mean ± SEM. # p<0.05, ## p<0.01, ### p<0.001vs control group; * p<0.05, ** p<0.01, *** p<0.001vs model group. DETAILED DESCRIPTION

[0044] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0045] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0046] Example 1: Pharmacodynamics experiment of ginsenodiol in preventing and / or treating Parkinson's disease

[0047] An experimental method

[0048] Materials and instruments

[0049] Panaxadiol (purity: 98%, C30 H 52 O 3 ) were purchased from Maclean (Shanghai, China).

[0050]

[0051] Cell culture

[0052] BV2 microglial cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM medium containing 10% fetal bovine serum in an atmosphere of 5% CO. 2 and cultured in an incubator at 37°C.

[0053] Animal studies

[0054] Male C57BL / 6 mice, weighing between 18 and 22 g and aged 6 to 8 weeks, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The license number is SCXK (Beijing) 2021-0006. All experimental procedures were in accordance with the Guide for the Care and Use of Laboratory Animals of Shanxi University of Traditional Chinese Medicine (October 19, 2022; AWE202209139). Mice lived in a suitable environment with a constant relative humidity of 50%-60%, a temperature of 22 ± 2 °C, and a light / dark cycle of 12 / 12 h. There were 15 mice in each of the control group, model group, ginsenodiol (25 mg / kg / day) group, ginsenodiol (50 mg / kg / day) group, and L-DOPA (75 mg / kg) group. Mice in the model group, ginsenodiol group, and L-DOPA group were orally administered with rotenone (30 mg / kg / day) for 4 consecutive weeks, followed by oral administration for 2 weeks. The mice were then killed and brain tissue, cell supernatant, blood, etc. were collected.

[0055] CCK-8 and LDH

[0056] In a 96-well plate, BV2 microglia were treated with ginsenodiol (10, 20, and 40 μM) and LPS (1 μg / mL) for 24 h. 10% CCK-8 was added, and the absorbance value was detected at 450 nm using an ELISA reader to calculate cell viability. According to the requirements of the LDH kit, the cell supernatant was taken and the absorbance was measured at 450 nm to calculate the LDH content of each group.

[0057] Fecal Pellet Output and Colon Length

[0058] Mice were orally administered rotenone (30 mg / kg / day) for 4 consecutive weeks. After 2 weeks of oral administration, the feces of the mice were collected after fasting for 2 hours, placed in a sealed test tube and the number was calculated. The feces were dried at 85°C for 24 hours, and the weight of the feces before and after drying was recorded, and the water content was calculated. In addition, the colon length was recorded.

[0059] Behavioral testing

[0060] Motor behavior assessment: The DigiGait system provided by RWD Life Sciences (China) was used to assess the motor status of mice (10 cm / s, recording for 3 seconds). Data were analyzed using DigiGait software. Pole climbing test: A 60 cm long wooden pole with a 5 cm diameter metal ball on the top was used. The mouse was placed on the ball and the time it took to climb to the platform at the bottom of the pole was recorded. Hanging test: First, the front paws of the mouse were hung on a horizontal steel wire and scored based on whether the hind paws were on the wire. 3 points: two hind paws; 2 points: one hind paw; 1 point: zero hind paws; 0 points: the mouse fell. Each experiment was separated by 10 minutes. Each mouse was repeated three times (Zhao Z., NingJ., Bao XQ, Shang M., Ma J., Li G., Zhang D., 2021. Fecal microbiotatransplantation protects rotenone-induced Parkinson's disease mice via suppressing inflammation mediated by the lipopolysaccharide-TLR4 signaling pathway through the microbiota-gut-brain axis.Microbiome,9(1),226.).

[0061] Immunofluorescence staining

[0062] After intracardial perfusion of 0.9% saline and 4% paraformaldehyde, the brain tissue was dissected and fixed in paraformaldehyde for 24 h. After dehydration with different concentrations of sucrose gradient, the brain tissue was embedded in paraffin and cut into 5 μm thick sections. Immunofluorescence staining was performed according to previous studies (Zhao Z., Bao XQ, Zhang Z., Li F., Liu H., Zhang D., 2020. Novel phloroglucinol derivative Compound 21 protects experimental autoimmuneencephalomyelitis rats via inhibiting Th1 / Th17 cell infiltration. Brain Behav Immun. 87, 751-764.). Immunofluorescence intensity was analyzed using Image Pro Plus 6.0 software.

[0063] Evans blue blood-brain barrier permeability test

[0064] Prepare 2% Evans blue dye and sodium chloride solution containing 20U / mL heparin for later use. Anesthetize the mouse with an anesthetic. Inject 2% Evans blue dye from the tail vein or femoral vein. Open the mouse chest cavity, slowly inject 0.9 sodium chloride solution (containing heparin) from the apex of the heart, cut the right atrium until the fluid flowing out of the right atrium is clear, and stop the perfusion. Separate the brain tissue, take the supernatant after homogenization, and measure it with a spectrophotometer at 620nm.

[0065] Transmission electron microscopy

[0066] The ultrastructure of tight junctions in the substantia nigra (SN) was observed using a transmission electron microscope (Hitachi, HT7800, Japan).

[0067] ELISA

[0068] Cell supernatant, blood and brain tissue extracts were collected from each group. The protein content of brain tissue extracts was determined using the BCA method. ELISA kits for detecting mouse inflammatory factor levels were purchased from Shanghai Jianglai Industrial Co., Ltd.

[0069] Quantitative polymerase chain reaction assay

[0070] Take mouse brain tissue samples. Use RNAprep pure Tissue Kit to extract total RNA from the samples and measure the concentration. According to the instructions, use M5 Sprint qPCR RT kit with gDNA remover to transcribe cDNA. Mix cDNA, primers and reagents according to the instructions and use Realtime PCR Super mix for detection on a real-time PCR instrument. Use the 2-ΔΔCt algorithm to calculate the relative quantification of mRNA expression. Primer sequences are shown in Table 1.

[0071] Table 1. Primers used for quantitative PCR

[0072]

[0073] In the above table, the second column is sequences 1-7 from top to bottom, and the third column is sequences 8-14 from top to bottom.

[0074] Western blotting

[0075] Brain tissue and cell samples from different groups were collected. Total protein concentration was determined using the BCA method. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% skim milk at room temperature, the membranes were incubated with anti-TH, anti-ZO-1, anti-Occludin, anti-Claudin-5, anti-TLR4, anti-MyD88, and anti-p-NF-κB antibodies overnight at 4°C. β-actin was used as an internal control. After incubating the membrane with anti-rabbit / anti-mouse secondary antibodies (1:5000), the color development signals were captured by a gel imaging system and analyzed using ImageJ software. DNA extraction, PCR, and Illumina Miseq sequencing of intestinal flora

[0076] The cecal contents of mice were collected, placed in dry ice, and then stored at −80° C. Extraction and determination were performed by Suzhou Panomik Biopharmaceutical Technology Co., Ltd.

[0077] 2. Statistical Analysis

[0078] All data are presented as mean ± standard error. One-way analysis of variance was used for multiple group comparisons. Data were analyzed using GraphPad PRISM 8.0 software (GraphPad Software, Inc., California, USA). P values ​​< 0.05 were considered statistically significant.

[0079] Three results

[0080] Panaxadiol inhibits LPS-induced inflammatory cytokine production in BV2 cells via TLR4 / MyD88 / NF-κB pathway

[0081] The results of CCK-8 assay showed that ginsenoside treatment at 20 and 40 μM concentrations significantly reduced the proliferation capacity of BV2 cells induced by LPS (p<0.05, Figure 1 A). In addition, LDH assay showed that after 24 h of treatment with panaxadiol (20 and 40 μM), LPS-induced LDH release in BV2 cells was significantly reduced, indicating reduced cytotoxicity (p < 0.05, Figure 1 B). Based on these findings, 40 μM ginsenoside was selected for further experiments. ELISA results showed that ginsenoside treatment effectively reversed the increase of inflammatory factors in BV2 cells induced by LPS (p<0.01, Figure 1 C). In addition, Western blot analysis showed that the expression levels of TLR4, MyD88, and p-NF-κB were significantly decreased in the ginsenodiol-treated group (p<0.01, Figure 1 D).

[0082] Panaxadiol alleviates motor and gastrointestinal dysfunction in PD mice

[0083] A series of behavioral experiments were performed to evaluate the protective effect of ginsenosides on PD. In gait analysis, the hind limb foot angle, step length, step frequency, and foot area of ​​the model group mice were significantly changed compared with the control group. In contrast, ginsenosides (50 mg / kg) and L-DOPA treatment significantly alleviated these changes (p<0.05, Figure 2 In addition, panaxadiol (50 mg / kg) and L-DOPA administration resulted in a significant reduction in tremor and rigidity in PD mice in the pole climbing test and hanging test (p < 0.05, Figure 2 E, F).

[0084] Rotenone induced a decrease in colon length in mice. In contrast, panaxadiol (50 mg / kg) and L-DOPA treatment significantly attenuated the decrease in colon length (p < 0.001; Figure 2 G). In addition, the assessment of colonic motility showed that rotenone exposure resulted in a significant decrease in fecal water content (p<0.001) and a significant decrease in fecal particle number (p<0.001). In contrast, panaxadiol (50 mg / kg) and L-DOPA treatment groups showed significant increases in fecal water content (p<0.001) and particle number (p<0.05). Figure 2 H, I).

[0085] Panaxadiol improves histological features and inflammation in the substantia nigra (SN) of PD mice

[0086] To further investigate the protective effect of panaxadiol on motor deficits, we evaluated the histological characteristics and inflammation of the substantia nigra (SN) through a series of experiments. In this study, the number of tyrosine hydroxylase (TH)-positive cells in the SN of mice in the model group was significantly reduced and dispersed (p<0.001). In contrast, the TH-positive cells in the panaxadiol (50 mg / kg) and L-DOPA treatment groups were more orderly arranged, with enhanced cytoplasmic staining, and the number of TH-positive cells was significantly increased (p<0.01, Figure 3 A). Based on these findings, 50 mg / kg of ginsenoside was selected for subsequent experiments. Western blot analysis showed that the expression level in the model group was significantly reduced compared with the control group (p<0.001), while ginsenoside treatment effectively reversed this trend (p<0.001, Figure 3 B). Notably, panaxadiol treatment significantly inhibited the release of pro-inflammatory cytokines (p<0.01, Figure 3C). Activation of microglia and astrocytes during the progression of Parkinson's disease (PD) may lead to neuroinflammation and subsequent dopaminergic neuron death in the SN (Liddelow SA, Guttenplan KA, Clarke LE, Bennett FC, Bohlen CJ, Schirmer L., Bennett ML, Münch AE, Chung WS, Peterson TC, Wilton DK, Frouin A., Napier BA, Panicker N., Kumar M., Buckwalter MS, Rowitch DH, Dawson VL, Dawson TM, Stevens B., Barres BA, 2017. Neurotoxic reactiveastrocytes are induced by activated microglia. Nature. 541(7638), 481-487.). Therefore, we evaluated GFAP and Iba-1, and the results showed that ginsenosides significantly reduced the increase of GFAP and Iba-1 in the SN area of ​​the model group mice (p<0.05; Figure 3 D).

[0087] Panaxadiol improves blood-brain barrier damage in Parkinson's disease mice

[0088] According to existing literature, disruption of the blood-brain barrier (BBB) ​​plays an important role in the pathogenesis of Parkinson's disease (PD) (Gasca-Salas C., Fernández-Rodríguez B., Pineda-Pardo JA, Rodríguez-Rojas R., Obeso I., Hernández-Fernández F., Del M., Mata D., Guida P., Ordás-Bandera C., Montero-Roblas JI, Martínez-Fernández R., Foffani G., Rachmilevitch I., Obeso JA, 2021. Blood-brain barrier opening with focused ultrasound in Parkinson's disease dementia. Nat Commun. 12(1), 779.). To evaluate whether ginsenodiol can reduce neuroinflammation by restoring the integrity of the BBB, we used Evans blue staining and transmission electron microscopy (TEM) analysis. Our results showed that the extravasation of Evans blue dye was significantly increased in Parkinson's mice (p<0.01). Notably, ginsenodiol had a significant inhibitory effect on the extravasation of Evans blue dye (p<0.01; Figure 4 A).

[0089] TEM analysis showed that the tight junctions of the BBB in the model group showed diffuse and disordered structures, accompanied by endothelial cell damage ( Figure 4 B). In addition, Western blot analysis showed that the expression levels of three key tight junction proteins, ZO-1 (p<0.01), occludin (p<0.001), and claudin-5 (p<0.001), were significantly decreased in the model group compared with the control group. In contrast, panaxadiol treatment significantly improved BBB damage and restored the expression of these tight junction proteins ( Figure 4 B, C). In addition, RT-PCR analysis results confirmed that ginsenoside effectively promoted the repair of BBB in Parkinson's disease mice ( Figure 4 D).

[0090] Panaxadiol alleviates intestinal flora imbalance in Parkinson's disease mice

[0091] Parkinson's disease is closely related to intestinal flora. In this study, we evaluated the effect of ginsenodiol on the intestinal flora of Parkinson's disease mice. The results showed that ginsenodiol could regulate the microbial community structure of Parkinson's disease mice. First, we performed α-diversity analysis to evaluate the richness and diversity of bacterial species. As shown in Figure 6A, the Chao1, Simpson, and Shannon indices of the model group increased compared with the control group, while the indices of the ginsenodiol group decreased. In addition, the diversity was evaluated using weighted UniFrac distance to determine the similarity of the composition of the three microbial communities. The microbial community structure of the model group was significantly different from that of the control group, while the ginsenodiol group was significantly different from the model group (Figure 6B). The effects of ginsenodiol on the composition of the intestinal flora at the family and genus levels are shown in Figures 6C and 6D. Significant changes in the relative abundance of intestinal flora were observed. Specifically, the relative abundance of unclassified Clostridiales (p<0.01), unidentified Clostridiales (p<0.001), and Coprococcus (p<0.001) decreased in the model group, while the relative abundance of Ruminococcus (p<0.01) increased. Panaxadiol was found to reverse these changes (Figure 6E). To evaluate the composition and significant changes in the intestinal flora of mice in different groups, we used linear discriminant analysis (LDA) evolutionary branch diagram. The results showed that there were 6 differential bacterial species in the control group, 28 differential species in the model group, and 24 species with LDA scores greater than 2 were significantly enriched in the panaxadiol group (Figure 6F). In summary, the relative abundance of the intestinal flora of Parkinson's disease mice has changed compared with the control group. Panaxadiol has been shown to have the ability to effectively and actively restore the abundance of various microbial communities.

[0092] Discussion

[0093] Panaxadiol is an important sapogenin derived from ginseng by hydrolysis, which exhibits a range of pharmacological activities, including anti-inflammatory, anti-Alzheimer's disease, and anti-tumor effects. Therefore, we hypothesized that panaxadiol may have therapeutic potential for Parkinson's disease. In cell experiments, we observed that panaxadiol effectively inhibited the secretion of proinflammatory cytokines through the TLR4 / MyD88 / NF-κB signaling pathway. Therefore, we further investigated its activity in vivo.

[0094] Rotenone is an insecticide that can penetrate the central nervous system through the blood-brain barrier, causing damage to dopamine neurons. Studies have shown that panaxadiol can improve rotenone-induced motor and intestinal dysfunction in mice. In addition, it has been shown to repair damage to dopamine neurons and inhibit the elevation of inflammatory factors in the substantia nigra. Chronic activation and inflammatory responses of glial cells may exacerbate neurodegenerative processes. In our study, it was surprisingly found that panaxadiol can inhibit the activation of microglia and astrocytes. Therefore, we hypothesized that panaxadiol exerts neuroprotective effects by alleviating chronic activation and inflammatory responses of glial cells.

[0095] The blood-brain barrier plays a key role in maintaining the homeostasis of the brain. An increasing number of studies have shown that the disruption of blood-brain barrier function may be an important factor in the pathogenesis of Parkinson's disease. At the same time, the blood-brain barrier provides a new therapeutic approach for the treatment of Parkinson's disease. This study showed that panaxadiol can reduce the permeability of the blood-brain barrier in Parkinson's disease mice by inhibiting the degradation of tight junction proteins and alleviate the structural damage of the blood-brain barrier. Therefore, panaxadiol reduces the penetration of neurotoxic substances, cells and microbial pathogens from the blood into the brain, while also inhibiting inflammatory responses and neuronal damage. In addition, our results showed that the original intestinal flora of Parkinson's disease mice was significantly damaged. Treatment with panaxadiol was found to normalize the composition of the intestinal microbiota and largely restore the homeostasis of the microbiota.

[0096] In conclusion, our results suggest that panaxadiol has potential therapeutic effects on Parkinson's disease through multiple mechanisms, including anti-inflammatory properties, motor function enhancement, repair of blood-brain barrier damage, and regulation of the gut microbiota. These findings lay a scientific foundation for considering panaxadiol as a potential drug for the treatment of Parkinson's disease. Future studies should further investigate the clinical application of panaxadiol and evaluate its long-term therapeutic effects and safety.

[0097] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Use of ginsenodiol in the preparation of products for preventing and / or treating Parkinson's disease.

2. The use according to claim 1, characterized in that: The prevention and / or treatment of Parkinson's disease is embodied in at least one of the following aspects: 1) Alleviate motor dysfunction caused by Parkinson's disease; 2) Alleviate gastrointestinal dysfunction caused by Parkinson's disease; 3) Improve the histological characteristics of substantia nigra caused by Parkinson's disease; 4) Improve substantia nigra inflammation caused by Parkinson's disease; 5) Inhibit the activation of microglia and astrocytes; 6) Improve blood-brain barrier damage caused by Parkinson's disease; 7) Inhibit neuroinflammation caused by Parkinson's disease; 8) Alleviate intestinal flora imbalance caused by Parkinson's disease; 9) Inhibit the secretion of inflammatory cytokines and the expression of proteins related to the TLR4 / MyD88 / NF-κB signaling pathway in BV2 cells induced by lipopolysaccharide.

3. The use according to claim 2, characterized in that: The said alleviating the motor dysfunction caused by Parkinson's disease specifically means significantly reducing the tremor and stiffness caused by Parkinson's disease; And / or, the alleviating gastrointestinal dysfunction caused by Parkinson's disease is to inhibit the reduction of fecal water content and the number of fecal particles caused by Parkinson's disease; And / or, the improvement of the histological characteristic changes of the substantia nigra caused by Parkinson's disease is that the tyrosine hydroxylase-positive cells in the substantia nigra are arranged in an orderly manner, the number of tyrosine hydroxylase-positive cells is significantly increased, and the expression of tyrosine hydroxylase is increased; And / or, the improving the substantia nigra inflammation caused by Parkinson's disease is to inhibit the release of pro-inflammatory cytokines in the substantia nigra; And / or, the improvement of blood-brain barrier damage caused by Parkinson's disease is to inhibit the increase in blood-brain barrier permeability caused by Parkinson's disease and restore the expression of three key tight junction proteins ZO-1, occludin and claudin-5; And / or, the alleviation of intestinal flora imbalance caused by Parkinson's disease is to regulate the intestinal microbial community structure, normalize the composition of the intestinal microbial community, and restore the homeostasis of the microbial community.

4. Application of ginsenodiol in the preparation of products for alleviating movement dysfunction caused by Parkinson's disease.

5. Application of ginsenodiol in the preparation of products for alleviating gastrointestinal dysfunction caused by Parkinson's disease.

6. Application of ginsenodiol in the preparation of products for improving blood-brain barrier damage caused by Parkinson's disease.

7. Application of ginsenodiol in the preparation of products for inhibiting neuroinflammation caused by Parkinson's disease.

8. Application of ginsenodiol in the preparation of products for alleviating intestinal flora imbalance caused by Parkinson's disease.

9. The use according to any one of claims 1 to 8, characterized in that: The product includes a medicine, a pharmaceutical composition or a health product.

10. A product for preventing and / or treating Parkinson's disease, the active ingredient of which comprises panaxadiol.

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