Application of iridoid compound in medicine with neuroprotective effect on Parkinson's disease

Abstract

The invention belongs to the technical field of medicines, and particularly discloses an application of an iridoid compound in a Parkinson's disease neuroprotective medicine, which is specifically characterized in that an alpha-Syn plasmid is transfected into a mouse dopaminergic neuron MES23.5 cell to establish an alpha-Syn overexpressed PD cell model, and 8-O-acetyl shanzhiside methyl ester is added to intervene the PD cell. Results prove that 8-O-acetyl shanzhiside methyl ester can improve cell viability and reduce cytotoxicity by reducing mitochondrial damage of PD cells, and has a neuroprotective effect on PD damaged neurons; a PD mouse model is established by utilizing MPTP intraperitoneal injection, intervention is carried out through intragastric administration of 8-O-acetyl shanzhiside methyl ester, and a result proves that 8-O-acetyl shanzhiside methyl ester can effectively improve the athletic ability of a PD mouse. The results show that the 8-O-acetyl shanzhiside methyl ester can effectively play a neuroprotective role on PD, and has a good clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to the application of iridoid compounds in drugs for the neuroprotective effect of Parkinson's disease. Background Art

[0002] Parkinson's disease (PD) is one of the most common neurodegenerative movement disorders in humans, second only to Alzheimer's disease. The characteristic pathological feature of PD is the progressive loss of dopaminergic neurons in the substantia nigra (SN) of the midbrain, and the appearance of Lewy body-like structures formed by abnormal aggregation of α-synuclein (α-Syn) in the remaining neurons. The etiology of PD is not yet fully understood. It is of great significance to clarify its specific disease mechanism and develop targeted drugs for the treatment and prevention of PD. Neurotoxins, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), rotenone, etc., can selectively cause damage to dopaminergic neurons. Such animal models can well simulate the damage of dopaminergic neurons in the SN of PD and motor symptoms. At the molecular level, the abnormal increase of α-Syn and the resulting mitochondrial dysfunction have also been proven to be the key factors leading to the progressive loss of dopaminergic neurons in PD patients. Existing studies have confirmed that a part of endogenous or overexpressed α-Syn exists in mitochondria and leads to the down-regulation of complex I activity, resulting in mitochondrial damage. In addition, the morphology of brain mitochondria in α-Syn transgenic mice is damaged. α-Syn may exist in the form of small polymers, inducing mitochondrial fragmentation, leading to subsequent mitochondrial dysfunction and death.

[0003] Mitochondria are the main sites for generating ATP in animal and plant cells. They are important organelles that promote cell energy conversion and participate in cell apoptosis. They are important regulators of cell energy and metabolism and play a crucial role in maintaining cell growth and survival. During the respiratory oxidation process, mitochondria store the generated energy in the form of electrochemical potential energy in the inner mitochondrial membrane, creating an asymmetric distribution of proton and other ion concentrations on both sides of the inner membrane to form the mitochondrial membrane potential (MMP). Normal MMP is a prerequisite for maintaining mitochondrial oxidative phosphorylation and generating adenosine triphosphate. The stability of MMP is beneficial to maintaining the normal physiological functions of cells. A large number of studies have shown that the decrease of mitochondrial membrane potential is related to autophagy, apoptosis or necrosis, etc. Therefore, mitochondrial membrane potential is one of the important indicators for evaluating the normal function of mitochondria.

[0004] 8-O-acetylshanzhiside methyl ester (Barlerin) is a secoiridoid compound isolated from the leaves of Lamiophlomis rotata Kudo, a folk medicinal plant in Tibet. In vitro studies have shown that Barlerin can block the activation of nuclear factor κB (NF-κB) induced by TNF-α in SH-SY5Y cells and reduce the expression of HMGB-1. Barlerin can block the phosphorylation of NF-κB induced by TNF-α in H9C2 cells by reducing the expression of HMGB1. In vivo studies have shown that Barlerin still has a significant neuroprotective effect when administered 4 hours after ischemia-reperfusion, by reducing the histopathological damage of the ischemic brain tissue, reducing brain swelling, inhibiting the activation of NF-κB, reducing the expression of HMGB-1, significantly promoting ischemic cerebral angiogenesis, and improving the functional prognosis after stroke. At the same time, Barlerin also significantly increased vascularization, increased the expression of VEGF, Ang1, and the phosphorylation of Tie2 and Akt, VEGF. Barlerin can significantly shorten the capillary clotting time and reduce blood loss, but has no effect on the activated partial thromboplastin time, prothrombin time, and thrombin time in mice, but significantly prolongs the euglobulin clot lysis time in hyperfibrinolytic mice. In addition, Barlerin has a significant inhibitory effect on NO production in LPS-stimulated BV2 cells. However, so far, there is no relevant research on whether Barlerin has a protective effect on the nerve damage in PD. We hypothesized that Barlerin could reduce the cell damage of PD dopaminergic neurons and relieve the motor dysfunction of PD by inhibiting mitochondrial damage. Summary of the Invention

[0005] The object of the present invention is to provide the application of secoiridoid compounds in drugs for the neuroprotective effect of Parkinson's disease. Specifically, 8-O-acetylshanzhiside methyl ester reduces the cell damage and cytotoxicity of dopaminergic neuron MES23.5 cells induced by overexpressed α-Syn protein, improves the mitochondrial dysfunction caused by overexpressed α-Syn protein in dopaminergic neuron MES23.5, relieves the motor disorder of Parkinson's disease mice, and thus plays a protective role in the nerve damage of Parkinson's disease.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] One of the objects of the present invention is to provide the application of secoiridoid compounds in drugs for the neuroprotective effect of Parkinson's disease.

[0008] Further explanation, the secoiridoid compound is 8-O-acetylshanzhiside methyl ester.

[0009] Further explanation: 8-O-acetylshanzhiside methyl ester plays a protective role in neuronal injury.

[0010] Further explanation: 8-O-acetylshanzhiside methyl ester plays a protective role in Parkinson's disease nerve injury by reducing the damage and cytotoxicity of dopaminergic neuron MES23.5 cells induced by overexpressed α-Syn protein, improving the mitochondrial dysfunction caused by overexpressed α-Syn protein in dopaminergic neuron MES23.5, and alleviating the motor disorders of Parkinson's disease mice.

[0011] The second object of the present invention is to provide a drug with a neuroprotective effect on Parkinson's disease, and the main component of the drug is 8-O-acetylshanzhiside methyl ester.

[0012] Further explanation: The effective concentration of 8-O-acetylshanzhiside methyl ester to reduce mitochondrial damage of Parkinson's cells in vitro, enhance cell viability, and reduce cytotoxicity is 1.25 μM - 5 μM.

[0013] Further explanation: The effective concentration of 8-O-acetylshanzhiside methyl ester to improve the motor ability of PD model mice induced by MPTP in vivo is 1 mg / kg - 8 mg / kg.

[0014] Further explanation: The drug is prepared into a clinically acceptable pharmaceutical preparation with 8-O-acetylshanzhiside methyl ester as the main component, plus pharmaceutically acceptable excipients or auxiliary components.

[0015] Further explanation: The pharmaceutical preparation includes any one of capsules, granules, tablets, pills, syrups, powders, powders for external use, suppositories, drops, emulsions, solutions, and suspensions.

[0016] The present invention also provides a pharmaceutical composition for treating and / or preventing Parkinson's disease. The pharmaceutical composition is prepared into a clinically acceptable pharmaceutical preparation with 8-O-acetylshanzhiside methyl ester as the main component, plus pharmaceutically acceptable excipients or auxiliary components.

[0017] Further explanation: The content of 8-O-acetylshanzhiside methyl ester in the pharmaceutical composition is 1 mg / kg - 8 mg / kg.

[0018] Generally speaking, as a drug, it is usually clinically applied after being prepared into a preparation. The pharmaceutical composition of the present invention can be prepared according to the methods well known in the art. It can be combined with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants to form any dosage form suitable for human or animal use.

[0019] The pharmaceutical composition of the present invention or a pharmaceutical composition containing the same can be administered in unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.

[0020] The dosage form for administration can be a liquid dosage form, a solid dosage form or a semi-solid dosage form. The liquid dosage form can be a solution (including true solution and colloidal solution), an emulsion (including o / w type, w / o type and multiple emulsion), a suspension, an injection (including aqueous injection, powder injection and infusion), an eye drop, a nasal drop, a lotion and a liniment, etc.; the solid dosage form can be a tablet (including ordinary tablet, enteric-coated tablet, buccal tablet, dispersible tablet, chewable tablet, effervescent tablet, orally disintegrating tablet), a capsule (including hard capsule, soft capsule, enteric-coated capsule), a granule, a powder, a pellet, a dropping pill, a suppository, a film, a patch, an aerosol (powder aerosol), a spray, etc.; the semi-solid dosage form can be an ointment, a gel, a paste, etc.

[0021] The pharmaceutical composition of the present invention can be made into a conventional preparation, a sustained-release preparation, a controlled-release preparation, a targeted preparation and various particulate drug delivery systems. In order to make the pharmaceutical composition of the present invention into a tablet, various excipients well-known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, glidants. The diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent can be water, ethanol, isopropanol, etc.; the binder can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia mucilage, gelatin mucilage, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; the disintegrant can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, etc.; the lubricant and glidant can be talc powder, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0022] The tablet can be further made into a coated tablet, such as a sugar-coated tablet, a film-coated tablet, an enteric-coated tablet, or a bilayer tablet and a multilayer tablet.

[0023] To prepare the administration unit in the form of capsules, the active ingredient of the pharmaceutical composition of the present invention can be mixed with a diluent and a glidant, and the mixture can be directly placed into hard capsules or soft capsules. Alternatively, the active ingredient of the pharmaceutical composition of the present invention can be first made into granules or pellets with a diluent, a binder, and a disintegrant, and then placed into hard capsules or soft capsules. The various diluents, binders, wetting agents, disintegrants, and glidants used for preparing tablets of the pharmaceutical composition of the present invention can also be used for preparing capsules of the pharmaceutical composition of the present invention.

[0024] To prepare the pharmaceutical composition of the present invention in the form of an injection, water, ethanol, isopropanol, propylene glycol, or a mixture thereof can be used as a solvent, and appropriate solubilizers, cosolvents, pH adjusters, and osmotic pressure regulators commonly used in the art can be added. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjuster can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. When preparing a freeze-dried powder injection, mannitol, glucose, etc. can also be added as a bulking agent.

[0025] In addition, if necessary, colorants, preservatives, fragrances, flavoring agents, or other additives can also be added to the pharmaceutical preparation. In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are:

[0026] The application of the iridoid compound described in the present invention in drugs for the neuroprotective effect of Parkinson's disease. Specifically, the iridoid compound is 8-O-acetylshanzhiside methyl ester. Through experiments, a PD cell model with overexpression of α-Syn was established by transfecting α-Syn plasmid into mouse dopaminergic neuron MES23.5 cells, and PD cells were intervened by adding 8-O-acetylshanzhiside methyl ester. The cell viability and cytotoxic damage of PD cells were detected by cell viability experiment and LDH release experiment to evaluate the effect of 8-O-acetylshanzhiside methyl ester on the cell viability of PD cells; the mitochondrial membrane potential (MMP) of cells was detected by Mito-Tracker staining experiment to evaluate the effect of 8-O-acetylshanzhiside methyl ester on the mitochondrial function of cells. The experimental results showed that treatment with 5 μM 8-O-acetylshanzhiside methyl ester could effectively increase the cell viability of PD cells, reduce cytotoxicity, and effectively increase the mitochondrial membrane potential of PD cells, saving mitochondrial damage. The above results proved that 8-O-acetylshanzhiside methyl ester could increase cell viability and reduce cytotoxicity by reducing mitochondrial damage in PD cells, playing a neuroprotective role in PD-damaged neurons. A PD mouse model was established by intraperitoneal injection of MPTP, and PD model mice were intervened by gavage with 8-O-acetylshanzhiside methyl ester. Further, the open field experiment and the rotarod test were used to evaluate the effect of 8-O-acetylshanzhiside methyl ester on the motor ability of mice. The results of animal experiments found that 8 mg / kg 8-O-acetylshanzhiside methyl ester could significantly improve the average movement speed of PD model mice in the open field experiment and increase the falling rod latency of PD mice in the rotarod test, indicating that 8-O-acetylshanzhiside methyl ester could effectively improve the motor function of Parkinson's disease mice. Therefore, 8-O-acetylshanzhiside methyl ester has good clinical application prospects for the treatment and / or prevention of Parkinson's disease. Description of the Drawings

[0027] Figure 1 It is a diagram for establishing a PD cell model with overexpression of α-Syn of the present invention. Among them Figure 1 A is the protein electrophoresis result diagram of MES23.5 cells transfected with NC and SNCA plasmids for 48 h. Figure 1 B is the comparison result of the relative expression levels of α-Syn protein in the cell proteins of each group. In the figure, the label Con: blank cell group; NC: group transfected with NC plasmid; SNCA: group transfected with α-Syn plasmid; α-Syn: α-synuclein; β-actin: β-actin. n = 3, ****P < 0.0001.

[0028] Figure 2This is a statistical chart showing that 8-O-acetylshanzhiside methyl ester (Barlerin) of the present invention significantly inhibits the cytotoxic damage of PD model cells. In Figure A of the chart, the results of the cell viability detection experiment show the relative cell viability of each group of cells. In Figure B of the chart, the results of the cytotoxicity detection experiment show the percentage of LDH release of each group of cells. In the figure, Con: blank cell group; NC: group transfected with NC plasmid; SNCA: groups transfected with α-Syn plasmid. n = 3, *P < 0.05, ***P < 0.001, ****P < 0.0001.

[0029] Figure 3 This is a result chart and statistical chart showing that 8-O-acetylshanzhiside methyl ester (Barlerin) of the present invention significantly inhibits the mitochondrial damage of PD model cells. In Figure A of the chart, the staining results of MitoTracker and Hoechst33342 of each group of cells are shown. In Figure B of the chart, the comparison chart of the relative fluorescence intensity of MitoTracker staining of each group of cells is shown. In the figure, Con: blank cell group; NC: group transfected with NC plasmid; SNCA: group transfected with α-Syn plasmid; SNCA+Barlerin: group transfected with α-Syn plasmid and intervened with Barlerin. n = 3, *P < 0.05, **P < 0.01.

[0030] Figure 4 This is a result chart showing that 8-O-acetylshanzhiside methyl ester (Barlerin) of the present invention significantly improves the motor ability of PD model mice. In Figure A of the chart, the open field movement trajectory of each group of mice is shown. In Figure B of the chart, the statistical result chart of the average movement speed of the open field experiment of each group of mice is shown. In Figure C of the chart, the statistical result chart of the falling rod latency of the rotarod test of each group of mice is shown. In the figure, Con: control group; PD: Parkinson's disease group; Barlerin: 8-O-acetylshanzhiside methyl ester. n = 5, **P < 0.01, ***P < 0.001, ****P < 0.0001. Detailed implementation manners

[0031] To make the above objects, features and advantages of the present invention more obvious and understandable, the following will describe the detailed implementation manners of the present invention with reference to the accompanying drawings. Many specific details are set forth in the following description in order to fully understand the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific implementations disclosed below.

[0032] Experiment:

[0033] 1. Materials and methods

[0034] 1.1 Cell culture and treatment

[0035] Establishment of PD cell model: Mouse dopaminergic neurons (MES23.5 cells) were cultured in DMEM / F12 medium (Gibco, New York, USA) containing 10% fetal bovine serum and Sato's. 2 μg of α-Syn plasmid or empty vector (NC) plasmid was transfected into each well of a 6-well plate using Lipofectamine 3000 (Gibco, New York, USA). The cells were divided into 3 groups: Con group, NC group, and SNCA group. The cells were treated for 48 h, and subsequent Western blotting was performed.

[0036] Drug intervention: 100 ng of α-Syn plasmid (SNCA) or empty vector (NC) plasmid was transfected into each well of a 96-well plate using Lipofectamine 3000. 24 h after plasmid transfection, Barlerin dissolved in DMSO (Sigma, MO, USA) was added to the cell culture medium overexpressing α-Syn protein, and the final concentrations were 0 μM, 1.25 μM, 2.5 μM, and 5 μM, respectively. The cells were divided into 6 groups: Con group, NC group, SNCA-Barlerin 0 μM group, SNCA-Barlerin 1.25 μM group, SNCA-Barlerin 2.5 μM group, and SNCA-Barlerin 5 μM group. The cells were treated for 24 h, and subsequent cell viability detection, cytotoxicity detection, and mitochondrial membrane potential detection were performed.

[0037] 1.2 Western blotting experiment

[0038] Cells treated differently were collected and lysed according to the instructions of a cell protein preparation kit (Applygen, Beijing, China). The cells were separated and collected into different tubes. The protein concentration was evaluated using a BCA protein quantification kit (Thermo Fisher Scientific, Massachusetts, USA). Then, 20 μg of protein from each fraction was separated by SDS-PAGE and transferred to a PVDF membrane (Sigma, MO, USA). Then, it was blocked with 5% skim milk powder, and anti-α-Syn and anti-β-actin antibodies were added respectively and incubated overnight. The corresponding fluorescent secondary antibody was combined with the primary antibody at room temperature for 1 h. After four washes, the membrane was scanned and imaged using an ODYSSEY imaging system (LI-COR, Nebraska, USA).

[0039] 1.3 Cell viability detection

[0040] Using CellTiter The cell viability was detected by using the AQueous One Solution Cell Proliferation Assay kit (Promega, WI, USA). The old medium of the cells in the 96-well plate was aspirated, and 100 μL of fresh medium was replaced in each well. 20 μL of the CellTiter AQueous One Solution Reagent was added to the treated group of cells and incubated at 37 °C in a 5% CO 2 environment for 1 h. The absorbance value was read at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0041] 1.4 Cytotoxicity detection

[0042] The LDH release was detected by using the CytoTox Non-Radioactive Cytotoxicity Assay kit (Promega, WI, USA) to evaluate the cytotoxic damage. 50 μL of the old medium was taken from all the test wells and control wells and transferred to a clean 96-well plate. 50 μL of CytoTox Reagent was added, and the mixture was incubated at room temperature in the dark for 30 min. The absorbance value was read at a wavelength of 490 nm using an ELISA reader.

[0043] 1.5 Detection of mitochondrial membrane potential (MMP)

[0044] The cell MMP was detected by using a mitochondrial membrane potential detection kit (Beyotime, Shanghai, China). The old medium of the cells in the 96-well plate was aspirated, and the cells were washed once with PBS. 96.5 μL of binding buffer was added to each well of the cells, followed by the addition of 2 μL of MitoTracker Red CMXRos staining solution and 5 μL of Hoechst33342 staining solution. The mixture was gently mixed and incubated at room temperature in the dark for 20 min. Then, it was observed under a fluorescence microscope. MitoTracker Red CMXRos showed red fluorescence, and Hoechst33342 showed blue fluorescence.

[0045] 1.6 Establishment of the PD model and Barlerin intervention

[0046] Twenty-five 8-week-old male C57 / BL6N mice were purchased from Cyagen Biosciences (Taicang) Co., Ltd. (production license number: SCXK (Jiangsu) 2018-0003), and were housed in the SPF animal room of Guilin Medical University. They had free access to food and water, and the day-night cycle was adjusted to 12 h / 12 h. They were randomly divided into 5 groups, with 5 mice in each group. Among them, four groups were intraperitoneally injected with MPTP for 7 days to establish a PD mouse model. Three of these groups were respectively pre-gastrically administered with 1 mg / kg, 4 mg / kg, and 8 mg / kg of Barlerin for 7 days, and continuously gavage for 7 days during the PD modeling process to establish PD models intervened with different concentrations of Barlerin, namely the PD + 1 mg / kg Barlerin group, the PD + 4 mg / kg Barlerin group, and the PD + 8 mg / kg Barlerin group; the fourth group was pre-gastrically administered with the same volume of normal saline for 7 days and continuously gavage for 7 days during the modeling process, serving as the PD group, and the fifth group was pre-gastrically administered with normal saline for 14 days and intraperitoneally injected with normal saline starting from the 8th day, serving as the Con group. After the modeling, the motor ability of each group of mice was detected by behavioral tests. The open field test was used to evaluate the spontaneous motor ability of mice, that is, the spontaneous movement trajectory and distance of mice within 5 min were recorded, and the average movement speed was calculated according to the distance and time; the rotarod test was used to detect the coordinated motor ability of mice, that is, first trained at a speed of 4 rpm / min for 3 times, 2 min each time, and then the rotarod rotation speed was adjusted to accelerate from 4 rpm / min to 40 rpm / min within 5 min, and the time experienced by each mouse falling off the rotarod was recorded, that is, the fall latency of the rotarod.

[0047] 1.7 Statistical analysis

[0048] Data are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism software 9.0 (GraphPad, California, USA). One-way ANOVA was used to evaluate the differences between groups, and then Tukey's multiple comparison test was used. P < 0.05 was considered statistically significant.

[0049] 2. Results

[0050] 2.1 Establishment of the PD model cell model

[0051] The WB results showed that compared with the Con group (100%) and the NC group (89.90%), the α-Syn expression in the α-Syn transfection group (SNCA group, 1539.25%) was significantly increased (P < 0.0001), indicating that the PD cell model with overexpression of α-Syn was successfully established ( Figure 1 ).

[0052] 2.2 Barlerin significantly inhibits cytotoxic damage in PD model cells

[0053] Cell viability assays and LDH release experiments showed that compared with the Con group (cell viability 100%, LDH release 100%) and the NC group (cell viability 106.96%, LDH release 97.29%), the cell viability (34.57%) of the α-Syn transfection group (SNCA-Barlerin 0 μM group) decreased significantly, and the LDH release (175.14%) increased significantly (P < 0.0001, P < 0.001). Compared with the SNCA-Barlerin 0 μM group, after treatment with 1.25 μM Barlerin, the cell viability increased to 39.28%, and the LDH release decreased to 159.87%; after treatment with 2.5 μM Barlerin, the cell viability increased to 45.79%, and the LDH release decreased to 162.99%; after treatment with 5 μM Barlerin, the cell viability increased to 56.49%, and the LDH release decreased to 121.25%. Among them, the treatment effect of 5 μM Barlerin was the best (P < 0.05, P < 0.05)( Figure 2 A, B).

[0054] 2.3 Barlerin significantly inhibits mitochondrial function damage in PD model cells

[0055] Mitochondrial membrane potential detection results showed that compared with the Con group (100%) and the NC group (97.74%), the mitochondrial membrane potential (MitoTracker fluorescence intensity, 45.44%) of the α-Syn transfection group (SNCA group) decreased significantly (P < 0.01). Compared with the SNCA group, after treatment with 5 μM Barlerin, the MitoTracker fluorescence intensity (83.36%) increased significantly (P < 0.05)( Figure 3 A, B).

[0056] 2.4 Barlerin significantly improves the motor ability of PD model mice

[0057] The open field experiment results showed that compared with the Con group, the movement trajectories of the mice in the PD group were significantly sparser and concentrated around the four sides, while the trajectories of the mice intervened with different concentrations of Barlerin gradually became denser, especially in the group of mice intervened with 8 mg / kg Barlerin( Figure 4A). Compared with the Con group (64.71 mm / s), the open-field movement speed of PD group mice decreased significantly to 31.76 mm / s (P < 0.0001); while the average open-field movement speed of mice in the 1 mg / kg Barlerin intervention group increased to 38.41 mm / s, that of mice in the 4 mg / kg Barlerin intervention group increased to 42.35 mm / s, and that of mice in the 8 mg / kg Barlerin intervention group increased to 56.79 mm / s. Among them, the average open-field movement speed of mice in the 8 mg / kg Barlerin intervention group increased most significantly (P < 0.001). In addition, the rotarod test results showed that compared with the Con group (265.8 s), the falling latency of the rotarod test of PD group mice was significantly shortened to 133.8 s (P < 0.001); while the falling latency of the rotarod test of mice in the 1 mg / kg Barlerin intervention group increased to 159.4 s, that of mice in the 4 mg / kg Barlerin intervention group increased to 162.4 s, and that of mice in the 8 mg / kg Barlerin intervention group increased to 227.6 s. Among them, the falling latency of the rotarod test of mice in the 8 mg / kg Barlerin intervention group increased most significantly (P < 0.05)( Figure 4 B, C).

[0058] In summary, treatment with 5 μM methyl 8-O-acetylshanzhiside can effectively increase the cell viability of PD cells, reduce cytotoxicity, and effectively increase the mitochondrial membrane potential of PD cells, rescuing mitochondrial damage. Treatment with 8 mg / kg methyl 8-O-acetylshanzhiside can effectively increase the average open-field movement speed of PD mice and prolong the falling latency of the rotarod test. The above results prove that methyl 8-O-acetylshanzhiside can increase cell viability, reduce cytotoxicity, and improve the motor ability of PD mice by reducing mitochondrial damage in PD cells, playing a neuroprotective role in PD and having good clinical application prospects.

[0059] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. Application of terpenoid compounds in neuroprotective drugs for Parkinson's disease.

2. The use of the drug for protecting Parkinson's disease neurons according to claim 1, characterized in that: The iridoid compound is 8-O-acetyl jasminoidin methyl ester.

3. The use of the drug for protecting Parkinson's disease neurons according to claim 2, characterized in that: The 8-O-acetyl shanzhioside methyl ester plays a protective role in neuronal damage.

4. The use of the drug for protecting Parkinson's disease neurons according to claim 3, characterized in that: The 8-O-acetyl shanzhioside methyl ester reduces the damage and cytotoxicity of dopaminergic neuron MES23.5 cells induced by overexpression of α-Syn protein, improves the mitochondrial dysfunction caused by overexpression of α-Syn protein in dopaminergic neuron MES23.5, alleviates the movement disorder of Parkinson's disease mice, and thus plays a protective role in Parkinson's disease nerve damage.

5. A drug for protecting Parkinson's disease nerves, characterized in that: The main component of the medicine is 8-O-acetyl shanzhioside methyl ester.

6. The drug for protecting Parkinson's disease nerves according to claim 5, characterized in that: The effective concentration of 8-O-acetyl shanzhioside methyl ester in reducing the damage and cytotoxicity of dopaminergic neuron MES23.5 cells induced by overexpression of α-Syn protein in vitro is 1.25 μM-5 μM.

7. The drug for protecting Parkinson's disease nerves according to claim 5, characterized in that: The drug is prepared into a clinically acceptable drug preparation with 8-O-acetyl shanzhioside methyl ester as the main component and pharmaceutically acceptable excipients or auxiliary components.

8. The drug for protecting Parkinson's disease nerves according to claim 5, characterized in that: The pharmaceutical preparation includes any one of tablets, pills, capsules, granules, syrups, powders, granules, suppositories, drops, emulsions, solutions and suspensions.

9. A pharmaceutical composition for treating and / or preventing Parkinson's disease, characterized in that: The pharmaceutical composition is prepared into a clinically acceptable pharmaceutical preparation by using 8-O-acetyl shanzhioside methyl ester as a main component and adding pharmaceutically acceptable excipients or auxiliary components.

10. The pharmaceutical composition for treating and / or preventing Parkinson's disease according to claim 9, characterized in that: The content of the 8-O-acetyl shanzhioside methyl ester in the pharmaceutical composition is 1 mg / kg-8 mg / kg.

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