Compound with anti-parkinson's disease activity in whole grass of saussurea involucrata and preparation method and application thereof

Abstract

This invention provides compounds with anti-Parkinson's disease (PD) activity, their sources, isolation and purification methods, and applications. The invention isolates and purifies the whole herb of *Saussurea involucrata* from the Tianshan Mountains of Xinjiang to obtain a new compound, Involucramide. Using the effect on the survival rate of H2O2-induced damaged SH-SY5Y cells—a PD model—as an indicator of anti-PD activity, it was found that Involucramide significantly improved the survival rate of H2O2-induced damaged SH-SY5Y cells and exhibited better activity than the first-line clinical anti-PD drug Rasagiline at 20 µM. This compound is expected to be developed into a drug for treating Parkinson's disease and other related neurodegenerative diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to compounds with anti-Parkinson's activity, their preparation methods and applications, specifically involving the separation and purification process, structural confirmation and anti-Parkinson's activity. Background Technology

[0002] Parkinson's disease (PD) was first described as "tremor paralysis" by the British physician James Parkinson in 1817. It is the second leading cause of central neurodegenerative disease after Alzheimer's disease. Clinically, it is mainly characterized by motor disorders such as rigidity, tremor, and bradykinesia, as well as non-motor symptoms such as decreased sense of smell, cognitive decline, sleep disturbances, and depression or anxiety. The number of PD patients worldwide is projected to increase from approximately 7 million in 2015 to 13 million in 2040, with my country accounting for more than half of the global total. With the increasing aging of the world's population, PD, as a representative neurodegenerative disease, has become a major health killer after malignant tumors and cardiovascular diseases, and the severity of its symptoms greatly impacts the quality of life of patients and their families.

[0003] Currently, clinical treatment for Parkinson's disease (PD) mainly includes surgical treatment and chemotherapy. Surgical treatment mainly includes deep brain stimulation (DBS, brain pacemaker surgery) and ablation of brain nuclei: the former can alleviate motor symptoms of PD, reduce drug dosage and side effects, but it is expensive, requires long-term maintenance, and carries risks such as cerebral hemorrhage and infection; the latter reduces symptoms by destroying specific brain tissue areas, but also carries risks such as cerebral hemorrhage, infection, electrode displacement, and nerve damage. In addition, neither of these surgical treatments can cure PD or stop its progression, and postoperative medication is still required. Drug treatment mainly falls into two categories: one is dopaminergic drugs, such as levodopa (L-DOPA), MAO-B inhibitors, and COMT inhibitors, which have some efficacy in early-stage PD patients, but long-term use can easily lead to dyskinesia, end-of-dose phenomenon, symptom fluctuations, and deterioration of motor function; the other is non-dopaminergic drugs, mainly adenosine A. 2A Long-term use of inhibitors, non-selective NMDA receptor antagonists, and anticholinergic drugs can easily cause adverse reactions such as dizziness, hallucinations, confusion, and cognitive impairment. Therefore, screening and developing novel chemical drugs to treat Parkinson's disease (PD) is of paramount importance.

[0004] Traditional Chinese medicine, with its long history, is characterized by its multi-component, multi-target, and low-toxicity properties. It has long been used by overseas Chinese communities to treat various diseases. Snow lotus whole plant ( Saussureainvolucrata*Saussurea involucrata* (Snow Lotus) is a plant belonging to the genus *Saussurea* in the family Compositae. It is a valuable medicinal herb native to Xinjiang, valued for both its medicinal and edible properties. It is believed to have effects such as tonifying the kidneys and promoting blood circulation, strengthening muscles and bones, and nourishing the nerves. Traditionally, it is used in folk medicine to treat conditions like wind-cold-dampness syndrome, lower abdominal pain due to cold, and irregular menstruation. Modern pharmacological research indicates that the active ingredients in the whole plant possess anti-inflammatory, analgesic, antiviral, antitumor, immune-enhancing, cardiovascular disease-preventing, and central nervous system-protective effects. It has now been developed into an injection and is clinically used primarily for inflammation and joint pain caused by acute and chronic rheumatoid arthritis and osteoarthritis. Summary of the Invention

[0005] The objective of this invention is to provide compounds with anti-PD activity, their sources, preparation methods, and applications.

[0006] In a first aspect, embodiments of the present invention disclose compounds having anti-PD activity, said compounds including compounds as shown in formula (I); or stereoisomers, tautomers or pharmaceutically acceptable salts of said compounds.

[0007]

[0008] Formula (I) Secondly, embodiments of this application disclose an anti-PD formulation, characterized in that the formulation comprises the compound described in the first aspect, and pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients include at least one of diluents, carriers, and excipients.

[0009] "Excipient" refers to a pharmaceutically acceptable material, composition, or medium that contributes to imparting form or consistency to a pharmaceutical composition. Each excipient must be compatible with the other components of the pharmaceutical composition when mixed to avoid interactions that would significantly reduce the efficacy of the compounds when administered to a patient, and to avoid interactions that would render the pharmaceutical composition pharmaceutically unacceptable. Furthermore, each excipient must have sufficiently high purity to be pharmaceutically acceptable.

[0010] Suitable pharmaceutically acceptable excipients will vary depending on the specific dosage form chosen. Additionally, suitable pharmaceutically acceptable excipients can be selected for their specific functions in the composition. For example, certain pharmaceutically acceptable excipients can be selected because they have the ability to promote the production of a homogeneous dosage form. Certain pharmaceutically acceptable excipients can be selected because they have the ability to produce a stable dosage form. Certain pharmaceutically acceptable excipients can be selected because they facilitate the carrying or transport of one or more compounds of the invention from one organ or part of the body to another organ or part of the body, once administered to a patient. Certain pharmaceutically acceptable excipients can be selected because they have the ability to enhance patient compliance.

[0011] Suitable pharmaceutically acceptable excipients include the following types: diluents, fillers, binders, disintegrants, lubricants, flow aids, granulators, coating agents, wetting agents, solvents, solubilizers, suspending agents, emulsifiers, sweeteners, flavoring agents, flavor masking agents, colorants, anti-caking agents, humectants, chelating agents, plasticizers, thickeners, antioxidants, preservatives, stabilizers, surfactants, and buffers. As those skilled in the art will understand, depending on the amount of excipient and other components present in the formulation, some pharmaceutically acceptable excipients can perform more than one function and can serve alternative functions.

[0012] The formulations provided in this application can be in forms suitable for use in the following ways: oral administration (e.g., as tablets, capsules, pouches, pills, lozenges, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and capsules), topical administration (e.g., as creams, ointments, emulsions, solutions, pastes, sprays, foams, and gels), transdermal administration (e.g., via transdermal patches), inhalation administration (e.g., as dry powders, aerosols, suspensions, and solutions), inhalation administration (e.g., as fine powders), or parenteral administration (e.g., as sterile aqueous or oily solutions for intravenous, subcutaneous, intramuscular, intraperitoneal, or intramuscular administration, or as suppositories for rectal administration).

[0013] Thirdly, embodiments of the present invention disclose a method for separating and preparing the compound described in the first aspect, comprising the following steps: Collect the whole plant of snow lotus from the Tianshan Mountains in Xinjiang; The whole herb was dried, pulverized, and extracted with 95% ethanol by percolation to obtain an extract; and The extract was subjected to further extraction and separation to obtain the compound; In this embodiment of the application, the extraction and separation step includes: A crude extract is obtained, which is obtained by concentrating the extract using a vacuum distillation apparatus; A refined extract is obtained by sequentially extracting the crude extract with petroleum ether-water, dichloromethane-water, and ethyl acetate-water; and The compound is obtained by first screening the refined extract for anti-PD activity, identifying the refined extract with anti-PD activity fraction, and then preparing the identified refined extract by normal-phase silica gel chromatography, reverse-phase silica gel chromatography, and high-performance liquid chromatography. The compound is the compound as described in claim 1.

[0014] The compound is the compound as described in claim 1.

[0015] In this application embodiment, the steps for obtaining the compound specifically include: The first component is obtained by loading the refined extract onto a normal-phase silica gel column and collecting the eluent. A second component is obtained by loading the first component onto a reversed-phase silica column and collecting the eluent. A third component is obtained by loading the second component onto a high-performance liquid chromatography column and collecting the eluent, the third component comprising a compound of formula (I).

[0016] Fourthly, embodiments of the present invention disclose the application of the compound described in the first aspect or the compound prepared by the preparation method described in the second aspect in the preparation of anti-PD drugs.

[0017] This invention relates to compounds with anti-PD activity, their preparation methods, and applications, which have at least the following beneficial effects: First, guided by anti-PD activity screening, the ethanol extract of the whole snow lotus plant from the Tianshan Mountains in Xinjiang was identified as having anti-PD activity. This process can be scaled up based on reproducible and sustainable development, without wasting resources.

[0018] Secondly, the compound provided in this application exhibits activity in improving the survival rate of Parkinson's disease model cells, and shows better activity than the positive control drug rasagiline at a concentration of 20 μM. This compound is expected to be developed into a drug for treating neurodegenerative diseases such as Parkinson's disease.

[0019] Furthermore, this invention only requires following the experimental protocol and optimizing the extraction, separation, and purification conditions to scale up production and selectively control the yield of the compound, ultimately obtaining the target product with anti-Parkinson's disease properties. Attached Figure Description

[0020] Figure 1 The image shows the results of screening anti-PD activity of refined extracts from different parts of the whole snow lotus herb provided in this embodiment of the invention.

[0021] Figure 2 High-resolution electrospray ionization mass spectrometry (HRESIMS) of the compound of formula (I) provided in the embodiments of the present invention.

[0022] Figure 3 The infrared spectrum of the compound represented by formula (I) provided in the embodiments of the present invention.

[0023] Figure 4 The ultraviolet spectrum of the compound represented by formula (I) provided in the embodiments of the present invention.

[0024] Figure 5 The following figures illustrate the experimental results of the compound of formula (I) provided in this embodiment of the invention on the treatment of H2O2-induced damaged SH-SY5Y cells: A shows the cytotoxicity of compound 1 and the positive control drug rasagiline on SH-SY5Y cells; B shows the effect of compound 1 and the positive control drug rasagiline on the survival rate of H2O2-induced damaged SH-SY5Y cells; C shows the microscopic effect of compound 1 and the positive control drug rasagiline on the survival of H2O2-induced damaged SH-SY5Y cells; D shows the effect of compound 1 on the intracellular GSH level of nerve cells; E and F show the effect of compound 1 on the intracellular HO-1 and NQO1 mRNA levels of nerve cells, respectively. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Reagents not specifically described in detail in this application are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be obtained from the prior art.

[0026] Example 1: Determination of the dichloromethane fraction of the whole herb extract of Saussurea involucrata with anti-PD activity 1. Snow lotus whole plant ( Saussureainvolucrata )collection The whole plant of snow lotus was collected from the Tianshan Mountains in Xinjiang: 5 kg of the whole plant was collected in October, dried in the sun, vacuum dried and then stored in the herbarium of the Key Laboratory of Special Medicinal and Edible Plant Resources Chemistry of Kashgar University.

[0027] 2. Extraction Obtaining crude and refined extracts: The whole herb of Saussurea involucrata was extracted with 95% ethanol, and the solvent was recovered under reduced pressure to obtain 95g of crude extract. Then, it was suspended in 5L of cold water and extracted sequentially with equal volumes of petroleum ether, dichloromethane and ethyl acetate. After being concentrated under reduced pressure, 20g of refined extract from the petroleum ether fraction, 15g of refined extract from the dichloromethane fraction and 30g of refined extract from the ethyl acetate fraction were obtained.

[0028] 3. Activity screening The effect of H2O2-induced damage on the survival rate of SH-SY5Y cells—a PD model—was used as an indicator of anti-PD activity. Using the CCK-8 assay, the dichloromethane fraction of the ethanol extract of *Saussurea involucrata* whole herb was screened for anti-PD activity. The steps included: SH-SY5Y cells or SH-SY5Y cells damaged by H2O2 (450 μM) were seeded in 96-well plates for 24 hours; Add 10 μL of 10% (v / v) CCK-8 solution and incubate the cells in a dark incubator for 2 hours; The absorbance (OD) value was recorded at 450 nm.

[0029] Cell viability was calculated using the following formula: Cell viability % = (Experimental group OD – Blank group OD) / (Normal group OD – Blank group OD) × 100%; the cell viability result was obtained from the average of the standard deviations (n=3), where, Blank group: Group with only culture medium added; Normal group (NormalControl): Unprocessed group; Model group (H2O2insult): Damage induced by 450 μM H2O2; Experimental group 1: After treatment with petroleum ether fraction extract (concentration: 1 mg / mL, solvent: DMSO) for 8 hours, H2O2 (450 μM) was added and the mixture was treated for 24 hours. Experimental group 2: After treatment with the refined extract of the dichloromethane fraction (concentration: 1 mg / mL, solvent: DMSO) for 8 hours, H2O2 (450 μM) was added and the treatment continued for 24 hours. Experimental Group 3: The refined extract of the ethyl acetate fraction (concentration: 1 mg / mL, solvent: DMSO) was treated for 8 hours, and then treated with H2O2 (450 μM) for 24 hours.

[0030] Result: As Figure 1 As shown, at a concentration of 1 mg / mL, the refined extract of the dichloromethane fraction can significantly improve the survival rate of SH-SY5Y cells-PD model cells damaged by H2O2, indicating that the dichloromethane fraction is the active anti-PD fraction of the ethanol extract of Saussurea involucrata.

[0031] Example 2: Preparation and structural identification of compounds with anti-PD activity (I) Preparation of the compound involucramide as shown in formula (I) Guided by anti-PD activity screening, we separated the refined extract from the dichloromethane fraction.

[0032] Obtaining the first fraction: 15g of the refined extract was loaded onto a normal-phase silica gel column (80-120 mesh) and eluted with a petroleum ether-acetone gradient (elution gradient 100:1-5:1). After TLC monitoring and combining identical fractions, 9 fractions were obtained, named Fr.1-Fr.9. Fraction Fr.5 was loaded again onto a normal-phase silica gel column and eluted with dichloromethane-methanol (elution gradient 100:1-5:1). After TLC monitoring and combining identical fractions, 8 fractions were obtained, named Fr.4.1-Fr.4.8. Among them, Fr.4.5 is the first fraction.

[0033] To obtain the second component: The first component Fr4.5 was loaded onto a reversed-phase silica column and eluted with a methanol-water gradient (elution gradient of 30% methanol to 100% methanol). After TLC monitoring and merging of identical fractions, five components were obtained, which were named Fr.4.5.1 to Fr.4.5.5 in sequence; among them, Fr.4.5.4 is the second component.

[0034] Obtain the third component: Load the second component Fr.4.5.4 onto a reversed-phase high-performance chromatography preparative column (Acclaim™ Trinity Q1LC, catalog number: 083244; mobile phase: acetonitrile:water = 60:40, 2 ml / min); to obtain the compound shown in formula (I).

[0035] (ii) Structural identification of compound 1 (involucramide) shown in formula (I) The structures of the compounds shown in formula (I) were obtained by nuclear magnetic resonance (NMR), high-resolution electrospray ionization mass spectrometry (HRESIMS), infrared spectroscopy, and ultraviolet spectroscopy, respectively.

[0036] Compound 1 (involucramide) shown in formula (I): white powder; high-resolution electrospray ionization mass spectrometry (HRESIMS) [M+Na] + m / z 281.1375 (Calculated value C) 12 H 22 N₂O₄Na, 281.1472); [ α ]20D+54.0( c 0.05, CH3OH); UV (CH3OH) λ max(logε) = 207(4.38) nm; IR(KBr) v max 3444,1643,1698,1055,1031,1014cm –1 ; 1 H and 13 CNMR data are shown in Table 1.

[0037]

[0038] The final compound obtained is the compound shown in formula (I).

[0039] Example 3: Application Study of Compounds with Anti-PD Activity 1. Cell viability was assessed using the CCK-8 assay, and the steps included: SH-SY5Y cells or SH-SY5Y cells damaged by H2O2 (450 μM) were seeded in 96-well plates for 24 hours; Add 10 μL of 10% (v / v) CCK-8 solution and incubate the cells in a dark incubator for 2 hours; The absorbance (OD) value was recorded at 450 nm.

[0040] Cell viability was calculated using the following formula: Cell viability % = (Experimental group OD – Blank group OD) / (Normal group OD – Blank group OD) × 100%; the cell survival rate was obtained by averaging the standard deviations (n=3), where, Blank group: Group with only culture medium added; Normal group (NormalControl): Unprocessed group; Experimental group 1: Treatment group with different concentration gradients of compound 1; Experimental group 2: Treatment groups with different concentrations of the positive control drug rasagiline; Compound 1 is the compound shown in formula (I).

[0041] Result: As Figure 5 As shown in Figure A, CCK-8 assay data analysis revealed that compound 1 and the positive control drug rasagiline had no significant cytotoxic effect on SH-SY5Y cells at a concentration range of 40 μM.

[0042] 2. Experiment on the anti-PD activity of the compound shown in formula (I) against H2O2-damaged SH-SY5Y cells (Parkinson's disease model): H2O2-damaged SH-SY5Y cells were used as a model cell for Parkinson's disease, and the anti-PD activity of the compounds was evaluated using the CCK-8 assay.

[0043] The steps for assessing cell viability using the CCK-8 assay include: SH-SY5Y cells or SH-SY5Y cells damaged by H2O2 (450 μM) were seeded in 96-well plates for 24 hours; Add 10 μL of 10% (v / v) CCK-8 solution and incubate the cells in a dark incubator for 2 hours; The absorbance (OD) value was recorded at 450 nm.

[0044] Cell viability was calculated using the following formula: Cell viability % = (OD of experimental group or model group – OD of blank group) / (OD of normal group – OD of blank group) × 100%; the cell survival rate was obtained by averaging the standard deviations (n=3), where, Blank group: Group with only culture medium added; Normal group (NormalControl): Unprocessed group; Model group (H2O2insult): Damage induced by 450 μM H2O2; Model group (H2O2insult): Damage induced by 450 μM H2O2; Experimental group 1: After treatment with compound 1 at different concentration gradients for 8 hours, H2O2 (450 μM) was added and the treatment continued for 24 hours. Experimental group 2: After treatment with different concentrations of the positive control drug rasagiline for 8 hours, H2O2 (450 μM) was added for 24 hours. Compound 1 is the compound shown in formula (I).

[0045] Result: As Figure 5 As shown in B, compound 1 protects SH-SY5Y cells from oxidative damage in a dose-dependent manner in the range of 1–40 μM; at a concentration of 20 μM, it has a higher level of recovery of cell viability than the positive control drug rasagiline.

[0046] 3. Compound 1 reversed the decrease in GSH in H2O2-damaged SH-SY5Y cells. SH-SY5Y cells were divided at a rate of 1×10 6 Cells were seeded in 100 μL of DMEM medium in a cell culture plate at a concentration per well, and divided into a normal control group, a model group, and an experimental group. The normal control group was the untreated group; the model group cells were added with 100 μL of 450 μM H2O2 solution and cultured for 24 h, serving as the PD model group; the experimental group cells were pretreated with 20 μM compound 1 for 8 hours, and then added with 100 μL of 450 μM H2O2 solution and cultured for a total of 24 h, serving as the experimental group.

[0047] GSH produced in the cells of the above groups was measured using a GSHELISA assay kit (catalog number QY-MB11514, Qiaoyu Biotechnology). Results are as follows: Figure 5 As shown in Figure D, compound 1 effectively reversed the decrease in GSH in PD model cells at 20 μM.

[0048] 4. Compound 1 upregulates HO-1 and NQO1 mRNA levels in H2O2-damaged SH-SY5Y cells. The cell culture and grouping methods are the same as those in section 3 above.

[0049] Total RNA was extracted from cells in each group using TRIPURE total RNA extraction reagent (Kelvin (Wuhan) Biotechnology Co., Ltd.). cDNA was then detected after reverse transcription using the EntiLink™ reverse transcriptase kit (Kelvin (Wuhan) Biotechnology Co., Ltd.). Quantitative real-time reverse transcription PCR was performed on the StepOne™ real-time PCR detection system according to the instructions of the EnTurbo™ SYBR Green PCR SuperMix kit (Kelvin (Wuhan) Biotechnology Co., Ltd.). Results are as follows: Figure 5 As shown in E and F, compound 1 upregulated the mRNA expression levels of HO-1 and NQO1 in PD model cells.

[0050] In summary, this invention targets the anti-PD active sites in the whole plant of Saussurea involucrata, explores its anti-PD monomeric compounds, and achieves efficient process scale-up based on process repeatability and sustainable development, without causing resource waste.

[0051] The compound exhibits activity in improving the survival rate of Parkinson's disease model cells, and shows better activity than the positive control drug rasagiline at a concentration of 20 μM. At a concentration of 20 μM, it can effectively reverse the decrease of GSH in PD model cells and upregulate the levels of HO-1 and NQO1 mRNA in PD model cells.

[0052] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. A compound with anti-Parkinson's disease activity, characterized in that, The structural formula of compound 1 is as shown in formula (I); Or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof of the compound.

2. A formulation with anti-Parkinson's disease activity, characterized in that, The formulation comprises an anti-Parkinson's disease agent made from the compound of claim 1 and pharmaceutically acceptable excipients.

3. The pharmaceutical preparation according to claim 2, characterized in that, The pharmaceutically acceptable excipients include at least one of diluents, carriers, and excipients.

4. A method for preparing the compound according to claim 1, characterized in that, The method for preparing compound 1 includes the following steps: Using solvent extraction, dried whole snow lotus herb is pulverized, and extracted four times with 95% ethanol via percolation. The extract is then concentrated under reduced pressure at approximately 40°C to obtain an extract. The total extract is then extracted sequentially with petroleum ether-water, dichloromethane-water, and ethyl acetate-water. Guided by anti-PD activity screening, the dichloromethane extract is concentrated, mixed with 80-120 mesh silica gel, and dry-packed onto a column. A petroleum ether-acetone gradient elution is performed at a gradient of 100:1-5:

1. TLC detection is performed, and fractions with similar characteristics are combined. The extract is then purified by repeated normal and reverse-phase silica gel column chromatography, gel chromatography, and high-performance liquid chromatography to obtain compound involucramide, i.e., compound 1.

5. The use of the compound of claim 1 or the compound prepared by the method of claim 4 in the preparation of anti-Parkinson's disease drugs.

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