Application of triterpenoid saponin compound and nervonic acid in preparation of medicine for preventing and treating Parkinson's disease and symptoms thereof

Abstract

The invention discloses application of a triterpenoid saponin compound and nervonic acid in preparation of a medicine for preventing and treating Parkinson's disease and symptoms thereof. The triterpenoid saponin compounds and the nervonic acid in different proportions are combined, and experiments prove that multiple indexes of Parkinson mice can be remarkably improved when the triterpenoid saponin compounds and the nervonic acid are combined in a specific proportion, and the effect of synergistically improving the Parkinson disease or the progress of the Parkinson disease is achieved. Therefore, the pharmaceutical composition disclosed by the invention can be used for more effectively treating or improving the Parkinson's disease, and has a wide application prospect in the field of treatment of the Parkinson's disease.

Description

[0001] Cross-references to related applications This application is a divisional application filed on February 20, 2025, with application number CN202510191557.6 and entitled "A pharmaceutical composition for treating or improving Parkinson's disease and its application thereof", which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention belongs to the field of biomedical technology, and in particular relates to the application of triterpenoid saponins and nervonic acid in the preparation of drugs for the prevention and treatment of Parkinson's disease and its symptoms. Background Technology

[0003] Parkinson's disease is a common neurodegenerative disease affecting middle-aged and elderly people. With the increasing aging of the global population, the number of people suffering from Parkinson's disease is rising annually. The most significant pathological change in Parkinson's disease is the degeneration and death of dopaminergic neurons in the substantia nigra of the midbrain, leading to a significant decrease in striatal dopamine levels and the accumulation of large numbers of Lewy bodies within neurons. The cause of Parkinson's disease remains unclear, and there is currently no effective cure, severely impacting the quality of life for patients.

[0004] Therefore, there is an urgent need to find an effective medicinal ingredient for the treatment of Parkinson's disease and related symptoms. Summary of the Invention

[0005] To address at least some of the technical problems in the prior art, the present invention provides the use of triterpenoid saponins and nervonic acid in the preparation of medicaments for the prevention and treatment of Parkinson's disease and its symptoms. Specifically, the present invention includes the following.

[0006] In a first aspect, the present invention provides the use of a combination of triterpenoid saponins and nervonic acid in the preparation of a medicament for the prevention and treatment of Parkinson's disease and its symptoms, wherein the mass ratio of the triterpenoid saponins to nervonic acid is 1:(2-10).

[0007] In some embodiments, the use of the combination of the triterpenoid saponin compound and nervonic acid according to the present invention in the preparation of a medicament for the prevention and treatment of Parkinson's disease and its symptoms, wherein the triterpenoid saponin compound comprises at least one of 3-O-(3′-O-α-L-arabinofuranosyl-2′-O-β-D-galactopyranosyl)-β-D-glucuronide-21,22-di-O-angeloyl-barrigenol and 3-O-β-D-glucopyranosyl-28-O-(2″-O-α-L-rhamnosyl)-β-D-glucopyranosyl-16-deoxybarringtogenol.

[0008] In some embodiments, the use of the combination of the triterpenoid saponin compound and nervonic acid according to the present invention in the preparation of a medicament for the prevention and treatment of Parkinson's disease and its symptoms is described, wherein the triterpenoid saponin compound and the nervonic acid are each of natural origin or artificial synthesis.

[0009] In some embodiments, the use of the combination of the triterpenoid saponin compound and nervonic acid according to the present invention in the preparation of a medicament for the prevention and treatment of Parkinson's disease and its symptoms is described, wherein both the triterpenoid saponin compound and the nervonic acid are derived from *Xanthoceras sorbifolium*, and the triterpenoid saponin compound is prepared by the following method: (I) Take the fruit shell, pedicel and / or seeds of *Sapindus mukorossi* after degreasing and crush them. Use a first solvent to extract at room temperature for 1-10 h to obtain an extract. Concentrate the extract to obtain a concentrate. Use a second solvent to extract the concentrate and remove the solvent to obtain a crude extract. (II) The crude extract was separated by silica gel column chromatography with gradient elution using dichloromethane, methanol and water, and further separated by high performance liquid chromatography with gradient elution using methanol and water to obtain the triterpenoid saponin compounds.

[0010] In some embodiments, the use of the combination of the triterpenoid saponin compound and nervonic acid according to the present invention in the preparation of a medicament for the prevention and treatment of Parkinson's disease and its symptoms, wherein the first solvent is the same as or different from the second solvent.

[0011] In some embodiments, the use of the combination of the triterpenoid saponin compound and nervonic acid according to the present invention in the preparation of a medicament for the prevention and treatment of Parkinson's disease and its symptoms, wherein the first solvent or the second solvent is selected from at least one of methanol, ethanol, acetone, n-butanol, ethyl acetate, chloroform, petroleum ether and dichloromethane.

[0012] In some embodiments, the use of the combination of the triterpenoid saponin compound and nervonic acid according to the present invention in the preparation of a medicament for the prevention and treatment of Parkinson's disease and its symptoms, wherein the nervonic acid is prepared by the following method: (1) Processing *Sapindus mukorossi* seeds to obtain *Sapindus mukorossi* oil; (2) Mix the *Sapindus mukorossi* oil with a third solvent, adjust the pH to 4-6, add a fourth solvent for extraction, wash with water, distill and dry to obtain crude product; (3) The crude product was purified to obtain the nervonic acid.

[0013] In some embodiments, according to the pharmaceutical composition of the present invention, wherein in step (3), the purification comprises dissolving the crude product in a fifth solvent, removing impurities using an adsorbent, filtering and concentrating to obtain a concentrate, adjusting the pH of the concentrate to 3-6, crystallizing, filtering, and drying to obtain the nervonic acid.

[0014] In some embodiments, the use of the combination of the triterpenoid saponin compound and nervonic acid according to the present invention in the preparation of a medicament for the prevention and treatment of Parkinson's disease and its symptoms, wherein the third, fourth and fifth solvents are the same or different.

[0015] In some embodiments, the use of the combination of the triterpenoid saponin compound and nervonic acid according to the present invention in the preparation of a medicament for the prevention and treatment of Parkinson's disease and its symptoms, wherein the third, fourth or fifth solvent is selected from at least one of ethanol, isopropanol, triethylamine, petroleum ether, chloroform, acetone and ethyl acetate.

[0016] In some embodiments, the use of the combination of triterpenoid saponins and nervonic acid according to the present invention in the preparation of a medicament for the prevention and treatment of Parkinson's disease and its symptoms, wherein the prevention and treatment includes at least one of the following: (1) Improves resting tremor in patients with Parkinson's disease; (2) Improve bradykinesia in patients with Parkinson's disease, including slow movements, difficulty initiating movements, and reduced facial muscle activity; (3) Improve muscle stiffness in patients with Parkinson's disease, or increase in both extensor and flexor muscle tone; (4) Improve postural balance disorders in patients with Parkinson's disease; (5) Improve sensory disturbances in patients with Parkinson's disease, including decreased sense of smell, numbness in the limbs, pain and other sensory abnormalities; (6) Improve autonomic dysfunction in patients with Parkinson's disease, including constipation, excessive sweating, drooling, urination disorders, orthostatic hypotension, and decreased sexual function; (7) Improve mental and cognitive impairments in patients with Parkinson's disease, including improving mental disorders such as anxiety and depression and / or cognitive impairments such as dementia and hallucinations.

[0017] In a second aspect, the present invention provides a pharmaceutical composition for treating or improving Parkinson's disease and its symptoms, comprising 1 part by weight of a triterpenoid saponin compound, 2-10 parts by weight of nervonic acid and 0-2000 parts by weight of a pharmaceutically acceptable carrier.

[0018] In some embodiments, the pharmaceutical composition according to the present invention includes, wherein the pharmaceutically acceptable carrier comprises at least one of a diluent, filler, absorbent, wetting agent, binder, disintegrant, lubricant, sweetener, preservative, and antioxidant.

[0019] In some embodiments, the pharmaceutical composition according to the present invention comprises an oral dosage form or an injectable dosage form.

[0020] This invention combines triterpenoid saponins and nervonic acid, and further research has revealed that these two compounds have a synergistic therapeutic effect on Parkinson's disease. This invention further validates that the composition can restore the propulsion index in gait analysis of Parkinson's mice, increase the suspension score in the suspension test, increase the number of spontaneous movements in Parkinson's mice, and inhibit the expression levels of inflammatory factors in Parkinson's disease mice. Therefore, the pharmaceutical composition of this invention has broad application prospects in the treatment of Parkinson's disease. Attached Figure Description

[0021] Figure 1 The effects of different drugs on the propulsion index in gait analysis of Parkinson's mice are shown.

[0022] Figure 2 The results of biotoxicity tests for different drugs are shown.

[0023] Figure 3 The effects of different drugs on the levels of dopamine and serotonin in the striatum of Parkinson's disease mice were shown.

[0024] Figure 4 The effects of different drugs on the expression levels of TH and DAT proteins in Parkinson's disease mice were shown.

[0025] Figure 5 The effects of different drugs on the expression of inflammatory factors in mice with Parkinson's disease were shown.

[0026] Figure 6 The effects of different drug combinations on the levels of dopamine and serotonin in the striatum of Parkinson's disease mice were shown.

[0027] Figure 7 The effects of different drug combinations on the expression of inflammatory factors in mice with Parkinson's disease were demonstrated. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Pharmaceutical Composition In one aspect, the present invention provides a pharmaceutical composition for treating or improving Parkinson's disease and its symptoms, comprising 1 part by weight of a triterpenoid saponin compound, 2-10 parts by weight of nervonic acid and 0-2000 parts by weight of a pharmaceutically acceptable carrier.

[0032] To better achieve synergistic treatment or improvement of Parkinson's disease and its symptoms, it is necessary to control the mass ratio of triterpenoid saponins to nervonic acid within a suitable range. In a preferred embodiment, the mass ratio of the triterpenoid saponins to nervonic acid is 1:(2-10), preferably 1:(2-9), and even more preferably 1:(2-8), for example 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8. Compared to using any single component alone, such as triterpenoid saponins or nervonic acid alone, compositions using the above-mentioned ratios can synergistically treat or improve Parkinson's disease and its related symptoms, conditions, or disease status.

[0033] In a preferred embodiment, the triterpenoid saponin compound comprises at least one of 3-O-(3′-O-α-L-furanarabinosyl-2′-O-β-D-galactopyranosyl)-β-D-glucuronide-21,22-di-O-angeloyl-barrigenol and 3-O-β-D-glucopyranosyl-28-O-(2″-O-α-L-rhamnosyl)-β-D-glucopyranosyl-16-deoxybarringtogenol.

[0034] In a preferred embodiment, both the triterpenoid saponin compound and the nervonic acid are derived from *Xanthoceras sorbifolium*, and the triterpenoid saponin compound is prepared by the following method: (I) Take the dried fruit shell, stalk and / or seeds of *Sapindus mukorossi* after degreasing and crush them. Use a first solvent to extract at room temperature for 1-10 h, preferably 2-9 h, and even more preferably 3-8 h, for example 3, 4, 5, 6, 7, 8 h to obtain an extract. Concentrate the extract to obtain a concentrate. Use a second solvent to extract the concentrate and remove the solvent to obtain a crude extract. (II) The crude extract was separated by silica gel column chromatography using a gradient elution of dichloromethane, methanol and water in a ratio of 30:1:1 to 1:1:1 (preferably 28:1:1 to 1:1:1, more preferably 25:1:1 to 1:1:1, even more preferably 20:1:1 to 1:1:1, such as 20:1:1, 15:1:1, 10:1:1, 5:1:1, 1:1:1). The extract was further separated by high performance liquid chromatography using a gradient elution of methanol and water in a ratio of 10:90 to 100:0 (preferably 10:80 to 100:1, more preferably 10:70 to 100:7, even more preferably 10:50 to 100:17, such as 1:5, 1:4, 1:3, 1:2, 1:1, 2:1) to obtain the triterpenoid saponins. It is understandable that, in order to better extract triterpenoid saponins, the steps of extraction, concentration, extraction, and separation can be performed several times, such as 1, 2, 3, 4, or 5 times.

[0035] In a preferred embodiment, the volume ratio of the residue after degreasing the pericarp, pedicel, or seeds of *Sapindus mukorossi* to the first solvent is 1:(1-10), preferably 1:(2-10), even more preferably 1:(3-10), further preferably 1:(4-10), and more preferably 1:(5-10), for example 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0036] In a preferred embodiment, the first solvent and the second solvent may be the same or different, and the first solvent or the second solvent includes at least one selected from methanol, ethanol, acetone, n-butanol, ethyl acetate, chloroform, petroleum ether, and dichloromethane. Those skilled in the art will understand that one or more combinations of the above solvents can be used, and when using a combination of multiple solvents, the volume ratio between the solvents can be adjusted as needed. In a specific embodiment, the first solvent is ethanol, and the second solvent is ethyl acetate and n-butanol.

[0037] In a preferred embodiment, the first solvent is 30-90% ethanol, preferably 35-85% ethanol, even more preferably 40-80% ethanol, and more preferably 45-75% ethanol, for example 45%, 50%, 55%, 60%, 65%, 70%, 75%. In a specific embodiment, the first solvent is 60% ethanol.

[0038] In a preferred embodiment, the nervonic acid is prepared by the following method: (1) Take the seeds or kernels of *Sapindus mukorossi*, dry and crush them, add 4-6 times the volume (e.g., 4, 4.5, 5, 5.5, 6 times the volume) of a mixed solvent of ethanol and ethyl acetate, adjust the pH to 10-12 (e.g., 10, 10.5, 11, 11.5, 12) for extraction, obtain the extract, concentrate it, and remove the solvent to obtain *Sapindus mukorossi* oil; (2) Mix the *Sapindus mukorossi* oil with a third solvent, adjust the pH to 4-6 (e.g., 4, 4.5, 5, 5.5, 6), add a fourth solvent for extraction, wash with water, distill, and dry to obtain the crude product; (3) The crude product is dissolved in a fifth solvent, impurities are removed using an adsorbent (including but not limited to activated carbon), filtered and concentrated to obtain a concentrated solution. The pH of the concentrated solution is adjusted to 3-6 (e.g., 3, 3.5, 4, 4.5, 5, 5.5, 6), crystallized, filtered, and dried to obtain the nervonic acid. It is understood that, in order to better extract nervonic acid, the extraction, concentration, and extraction steps can be performed several times, for example, 1, 2, 3, 4, 5 times.

[0039] In a preferred embodiment, the third, fourth, and fifth solvents may be the same or different, and the third, fourth, or fifth solvent includes at least one selected from ethanol, isopropanol, triethylamine, petroleum ether, chloroform, acetone, and ethyl acetate. In a specific embodiment, the third solvent is isopropanol, the fourth solvent is ethanol, and the fifth solvent is petroleum ether. Those skilled in the art will understand that one or more combinations of the above solvents can be used, and when using a combination of multiple solvents, the volume ratio between the solvents can be adjusted as needed. Unless otherwise stated, the triterpenoid saponins and nervonic acid of the present invention are extracted from the same batch of *Sapindus mukorossi* fruit.

[0040] In this invention, pharmaceutically acceptable carriers are used to transport or deliver a drug from one organ or part of the body to another organ or part of the body. Each carrier is "acceptable," meaning it is compatible with other components of the formulation (e.g., triterpenoid saponins and nervonic acid) and does not harm the patient. The pharmaceutically acceptable carriers include at least one of diluents, fillers, absorbents, wetting agents, binders, disintegrants, lubricants, sweeteners, preservatives, and antioxidants. Examples of diluents include, but are not limited to, polysorbate-80, phosphate buffer solution, physiological saline, aqueous buffer solution, solvent, and dispersion medium; fillers include, but are not limited to, starch, lactose, mannitol, and microcrystalline cellulose; absorbents include, but are not limited to, calcium sulfate, calcium hydrogen phosphate, and calcium carbonate; wetting agents include, but are not limited to, water and ethanol; binders include, but are not limited to, hydroxypropyl methylcellulose, povidone, and microcrystalline cellulose; disintegrants include, but are not limited to, croscarmellose sodium, croscarmellose, surfactants, and low-substituted hydroxypropyl cellulose; lubricants include, but are not limited to, magnesium stearate, talc, polyethylene glycol, sodium lauryl sulfate, micronized silica gel, and talc; sweeteners include, but are not limited to, sucralose, acetylsupan, saccharin, sucrose, xylitol, mannitol, sorbitol, glucose, fructose, and aspartame; preservatives include, but are not limited to, parabens, chlorobutanol, phenol, and sorbic acid; and antioxidants include, but are not limited to, ascorbic acid and methionine.

[0041] In a preferred embodiment, the treatment or improvement is achieved by administering a therapeutically effective amount of the pharmaceutical composition to the subject. The daily dose of the pharmaceutical composition is typically 1-5000 mg / kg, preferably 1-4000 mg / kg, even more preferably 1-3000 mg / kg, further preferably 1-2000 mg / kg, more preferably 1-1000 mg / kg, more preferably 1-900 mg / kg, more preferably 1-500 mg / kg, more preferably 1-300 mg / kg, more preferably 1-200 mg / kg, and even more preferably 1-100 mg / kg, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg / kg. It can be administered as a single dose once daily, or divided into multiple doses daily, or at intervals.

[0042] There are no particular limitations on the administration method of the pharmaceutical composition of the present invention. Representative administration methods include, but are not limited to, gavage, oral administration, enema, intravenous injection, intraperitoneal injection, intramuscular injection, intravenous drip, and spray. Accordingly, the pharmaceutical composition of the present invention can be formulated into various clinically acceptable dosage forms, including oral dosage forms, injectable dosage forms, topical dosage forms, or external dosage forms.

[0043] application In one aspect of the present invention, the use of a combination of triterpenoid saponins and nervonic acid in the preparation of a medicament for treating or improving Parkinson's disease and its symptoms is provided, wherein the mass ratio of the triterpenoid saponins to nervonic acid is 1:(2-10).

[0044] In this invention, the term "treatment or improvement" refers to therapeutic treatments and preventative or therapeutic measures aimed at preventing or slowing (reducing) undesirable physiological changes or disorders, such as the onset and progression of Parkinson's disease. Beneficial or desired clinical outcomes include, but are not limited to, the following, whether detectable or undetectable: symptom relief, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and reduction (whether partial or complete). Those requiring treatment include individuals who already have Parkinson's disease or related conditions, or those who require prevention or improvement of Parkinson's disease or related conditions.

[0045] In a preferred embodiment, the treatment or improvement includes at least one of the following: (1) Improves resting tremor in patients with Parkinson's disease; (2) Improve bradykinesia in patients with Parkinson's disease, including slow movements, difficulty initiating movements, and reduced facial muscle activity; (3) Improve muscle stiffness in patients with Parkinson's disease, or increase in both extensor and flexor muscle tone; (4) Improve postural balance disorders in patients with Parkinson's disease; (5) Improve sensory disturbances in patients with Parkinson's disease, including decreased sense of smell, numbness in the limbs, pain and other sensory abnormalities; (6) Improve autonomic dysfunction in patients with Parkinson's disease, including constipation, excessive sweating, drooling, urination disorders, orthostatic hypotension, and decreased sexual function; (7) Improve mental and cognitive impairments in patients with Parkinson's disease, including improving mental disorders such as anxiety and depression and / or cognitive impairments such as dementia and hallucinations.

[0046] Example 1 The following exemplarily illustrates the preparation of a medicine for treating or improving Parkinson's disease and its symptoms, as follows.

[0047] 1. Preparation of triterpenoid saponins 10 kg of dried *Xanthoceras sorbifolium* pericarp, pedicel, and seeds (after oil removal) residue was pulverized and extracted with 7 times its volume of 60% ethanol by reflux five times, 1.5 h each time. The extract was concentrated and then extracted four times sequentially with equal volumes of ethyl acetate and n-butanol to obtain the extract. The solvent was evaporated to obtain the crude extract. The crude extract was separated by silica gel column chromatography using a gradient elution of 30:1:1 dichloromethane, methanol, and water. Further separation was performed by high-performance liquid chromatography using a gradient elution of 4:1 methanol and water to obtain the triterpenoid saponins. GC-MS analysis showed that the purity of the triterpenoid saponins was 92.5%. Nuclear magnetic resonance analysis revealed the structures of the two most abundant compounds as follows: (1) 3-O-(3′-O-α-L-furanarabinose-2′-O-β-D-galactopyranose)-β-D-glucuronide-21,22-di-O-angeloyl-barrigenol, white needle crystals (methanol), turns purple in 10% concentrated sulfuric acid / ethanol solution. 1HNMR(400MHz,pridine-d5):δ3.27(1H,dd,J=11.6,4.6Hz,H-3),5.51(1H,brs,H-12);C-3-GlcUA:4.90(1H,d,J=7.6Hz,H-1′);C-2′-Gal:5.36(1H,d,J=7.7Hz,H-1″);C-3′-Ara:6.06(1H,d,J=2.0Hz,H-1″′).13CNMR(100MHz,pridine-d5):δ:39.4(C-1),26.4(C-2),89.6(C-3),38.6(C-4),55.3(C-5),18.5(C-6),36.1(C-7),40.7(C-8),46.8(C-9),36.7(C-10),23.7(C-11),125.1(C-12),143.4(C-13),47.4(C-14),67.2(C-15),73.1(C-16),48.1(C-17),41.2(C-18),46.6(C-19),36.5(C-20),78.3(C-21),73.3(C-22),27.6(C-23),16.5(C-24),15.5(C-25),17.3(C-26),21.0(C-27),62.8(C-28),29.2(C-29),19.9(C-30),104.9(C-3-GlcUA-1′),78.6(C-2′),86.1(C-3′),71.5(C-4′),77.0(C-5′),172.0(C-6′),104.6(C-2′-Gal-1″),73.2(C-2″),74.9(C-3″),69.5(C-4″),76.4(C-5″),61.5(C-6″),110.9(C-3′-Ara-1″′),83.3(C-2″′),77.3(C-3″′),85.2(C-4″′),62.0(C-5″′),167.4(C-21-Ang-1),128.6(C-21-Ang-2),137.1(C-21-Ang-3),15.6(C-21-Ang-4),20.7(C-21-Ang-5),167.8(C-22-Ang-1),128.8(C-22-Ang-2),136.2(C-22-Ang-3),15.4(C-22-Ang-4),20.4(C-22-Ang-5)。

[0048] (2) 3-O-β-D-glucopyranosyl-28-O-(2″-O-α-L-rhamnosyl-pyranosyl)-β-D-glucopyranosyl-16-deoxybarringtogenol, white powder (methanol), turns purple in 10% concentrated sulfuric acid / ethanol solution. 1 HNMR(400MHz,pridine-d5): δ3.38(1H,dd,J1=4.6Hz,J2=11.6Hz,H-3),5.54(1H,brs,H-12); C-3-Glc:4.93(1H, d, J=7.8Hz, H-1′); C-28-Glc: 4.73 (1H, d, J=7.5Hz, H-1″); C-2′-Rha: 6.61 (1H, brs, H-1″′). 13CNMR (100MHz, pri dine-d5): δ39.5(C-1),26.3(C-2),88.5(C-3),39.0(C-4),55.4(C-5),19.4(C-6),32.6(C-7),39.8(C-8),47.6 (C-9),36.6(C-10),23.7(C-11),123.5(C-12),142.9(C-13),41.6(C-14),26.0(C-15),18.2(C-16),42.9(C-17) ,41.9(C-18),46.1(C-19),36.1(C-20),76.6(C-21),72.6(C-22),28.0(C-23),16.5(C-24),15.5(C-25),16.8( C-26),25.5(C-27),74.5(C-28),30.1(C-29),18.4(C-30),106.6(C-3-glc-1′),74.5(C-2′),78.0(C-3′),71.5 (C-4′),78.5(C-5′),62.4(C-6′),103.3(C-28-glc-1″′),80.0(C-2″′),75.6(C-3″′),71.6(C-4″′),77.6(C-5″ ′), 62.7(C-6″″), 100.3(C-2″′-Rha1″″), 72.3(C-2″″), 72.0(C-3″″), 74.1(C-4″″), 68.8(C-5″″), 18.7(C-6″″).

[0049] 2. Preparation of nervonic acid (1) Take 5 kg of *Sapindus mukorossi* seeds, dry and crush them, add 4-6 times the volume of a mixed solvent of ethanol and ethyl acetate, adjust the pH to 10-12 and extract, obtain the extract, concentrate it and remove the solvent to obtain 1.5 kg of *Sapindus mukorossi* oil; (2) Mix the *Sapindus mukorossi* oil with 750 ml of isopropanol and stir. Let it stand at room temperature for 1 h, wash with water, adjust the pH to 4 with acetic acid, extract with 3 times the volume of ethanol, wash with water, distill and dry to obtain crude product. (3) The crude product was dissolved in an equal volume of petroleum ether, activated carbon was added, and the mixture was heated under reflux at 65°C for 2.5 h. The mixture was then filtered and concentrated to obtain a concentrated solution. The pH was adjusted to 5 with acetic acid, and the solution was crystallized at 0°C for 2.5 h. The crystallization was repeated 5 times, filtered, and dried to obtain 90 g of nervonic acid. GC-MS analysis showed that the purity of nervonic acid was 97.6%.

[0050] 3. Drug preparation A drug composition was obtained by dissolving triterpenoid saponins and nervonic acid in a mass ratio of 1:(2-10) with polysorbate-80 and PBS. The corresponding triterpenoid saponin drug was obtained by dissolving triterpenoid saponins in polysorbate-80 and PBS. The corresponding nervonic acid drug was obtained by dissolving nervonic acid in polysorbate-80 and PBS. The drugs were stored at low temperature for later use.

[0051] Example 2 The following illustrates the application of the drug prepared in Example 1 in the treatment of Parkinson's disease.

[0052] 1. Animal Model Construction Forty male C57BL / 6 mice, aged 8-10 weeks and weighing 20-22 g, were used. Eight mice were housed in separate cages under an environment with free access to food and water, a temperature of (25±2)℃, humidity of (60±10)%, and alternating light exposure for 12 hours (7:30 to 19:30). The mice were acclimatized for one week before the experiment. Subsequently, the mice were randomly divided into five groups (n=8) using a random number table: control group, model group, triterpenoid saponin group (triterpenoid saponin drug), nervonic acid group (nervonic acid drug), YWZ1 group, YWZ2 group, and YWZ3 group. YWZ1 group contained a drug combination of triterpenoid saponin and nervonic acid at a mass ratio of 1:2; YWZ2 group contained a drug combination of triterpenoid saponin and nervonic acid at a mass ratio of 1:5; and YWZ3 group contained a drug combination of triterpenoid saponin and nervonic acid at a mass ratio of 1:8. Except for the control group, all mice were given MPTP (20 mg / kg, dissolved in PBS) to establish a standard mouse model of Parkinson's disease. MPTP was injected intraperitoneally once a day for 7 consecutive days, while the control group was injected with an equal volume of physiological saline.

[0053] After the mouse model was successfully established, the control group and the model group were treated with equal amounts of physiological saline by gavage. The triterpenoid saponin group was treated with 40 mg / kg of triterpenoid saponin compound drug solution by gavage. The nervonic acid group was treated with 40 mg / kg of nervonic acid drug solution by gavage. The YWZ1 group, YWZ2 group and YWZ3 group were treated with 40 mg / kg of drug combination drug solution by gavage.

[0054] 2. Experimental Methods 2.1 Gait Analysis Experiment The mice were wetted and made to run continuously across a platform, with video automatically recorded by a camera. After data collection, the recorded video was analyzed using computer analysis software.

[0055] 2.2 Pole Climbing Test The pole-climbing test was used to assess the effects of different drug treatments on the motor coordination of mice. A wooden pole with a diameter of 9 mm and a length of 1 m was vertically fixed in the cage. Mice were allowed to acclimatize to the environment for at least 1 hour before the test. Then, the mice were placed on top of the pole, and the time it took for the animals to descend from the top of the pole to the ground was recorded.

[0056] 2.3 Suspension Test Gently place the mouse's forelimbs on the rope and observe the mouse. The scoring method is as follows: 3 points are given if the mouse can grasp the rope with all four paws, 2 points are given if it can grasp the rope with one hind paw, 1 point is given if it cannot grasp the rope with either hind paw, and 0 points are given if the mouse cannot grasp the rope and falls quickly.

[0057] 2.4 Open Field Experiment The open field test was used to assess the effects of drugs on spontaneous movement and exploratory behavior in mice. Mice were allowed at least 1 hour to acclimatize to the environment before the test. Each mouse was placed in a clean, bright, 60 cm × 60 cm × 40 cm square box (blue interior surface, white floor). The mouse's movements and behaviors were recorded over 5 minutes using a monitor, including the number of times the mouse stood upright (forelegs completely off the floor or climbing the wall) and grooming behavior (preening). Recordings were taken every 10 minutes. After each test, the experimental area was cleaned with 75% alcohol, and the open field video was analyzed using software.

[0058] 2.5 Drug toxicity testing Four mL of blood was drawn from the carotid artery of mice into a coagulation tube, allowed to stand at room temperature for 30 min, and then centrifuged (10,000 r / min, 4℃) to obtain serum. The levels of AST and ALT in the serum were detected using the microplate method.

[0059] 2.6 Determination of dopamine and serotonin levels in the striatum of Parkinson's disease mice To investigate whether the drug has the ability to prevent the degeneration of the dopaminergic system, the concentrations of dopamine (DA) and serotonin (5-HT) in the striatum were determined by HPLC.

[0060] 2.7 Detection of TH and DAT protein expression levels The protein expression levels of TH and DAT were detected by qRT-PCR.

[0061] 2.8 Detection of expression levels of inflammatory factors in Parkinson's disease mice To further investigate the effects of drugs on the inflammatory response in the striatum of Parkinson's mice, the expression levels of inflammatory factors such as IL-6, IL-8, and TNF-α in the striatum were analyzed by ELISA.

[0062] 3. Experimental Results 3.1 Effects of different treatment groups on propulsion index in gait analysis of MPTP model mice Patients with Parkinson's disease often exhibit significant changes in gait characteristics, such as gait instability, narrowing stride, and postural instability. Gait analysis experiments can quantify these changes by analyzing gait parameters (such as stride length, stride speed, and gait cycle). Quantitative analysis of these gait characteristics provides an important tool and method for the early diagnosis, treatment efficacy evaluation, and disease monitoring of Parkinson's disease. In gait analysis ( Figure 1 Compared with the control group, the model group showed significant differences in propulsion index gait indices at the left anterior, left posterior, right anterior, and right posterior positions. Compared with the model group, the application of triterpenoid saponins alone could restore the propulsion index to some extent, but its effect was weaker than that of the nervonic acid group, YWZ1 group, YWZ2 group, and YWZ3 group. Furthermore, the YWZ1, YWZ2, and YWZ3 groups significantly restored the propulsion index in mice. Therefore, the combined application of triterpenoid saponins and nervonic acid has a synergistic effect in restoring the propulsion index in mice.

[0063] 3.2 Effects of different drugs on pole-climbing time in MPTP model mice The main characteristics of Parkinson's disease are the loss of dopaminergic neurons and a decrease in dopamine levels, which affects motor control. The pole climbing test can help assess the effects of drugs on motor function in Parkinson's disease model animals. As shown in Table 1, compared with the control group, the pole climbing time of mice in the model group was significantly increased. Compared with the model group, the triterpenoid saponins alone could shorten the pole climbing time of mice to some extent, but their effect was weaker than that of the nervonic acid group, YWZ1 group, YWZ2 group, and YWZ3 group. Furthermore, the YWZ1, YWZ2, and YWZ3 groups significantly shortened the pole climbing time of mice. Therefore, the combined administration of triterpenoid saponins and nervonic acid has a synergistic effect in shortening the pole climbing time of mice.

[0064] Table 1. Effects of drugs on pole-climbing time in MPTP model mice 3.3 Effects of different drugs on the suspension score of MPTP model mice Parkinson's disease patients often experience muscle rigidity and difficulty moving, leading to significant impairment in their exercise endurance and muscle strength. The suspension test can be used to assess the effects of drugs on motor function in Parkinson's disease model animals. As shown in Table 2, the suspension scores of mice in the model group were significantly lower than those in the control group. Compared to the model group, the administration of triterpenoid saponins alone could increase the suspension scores of mice to some extent, but its effect was weaker than that of the nervonic acid group, YWZ1 group, YWZ2 group, and YWZ3 group. Furthermore, the YWZ1, YWZ2, and YWZ3 groups significantly increased the suspension scores of mice. Therefore, the combined administration of triterpenoid saponins and nervonic acid has a synergistic effect in increasing the suspension scores of mice.

[0065] Table 2 Effects of drugs on mouse suspension scores 3.4 Effects of different drugs on the mining test in MPTP model mice In Parkinson's disease research, animal models often exhibit motor characteristics such as slow movement and decreased motor ability. These characteristics can be observed and assessed through the animals' exploratory behavior and motor activity levels in an open-field experiment. As shown in Table 3, compared with the control group, the number of spontaneous movements in the model group was significantly reduced. Compared with the model group, the triterpenoid saponins alone could increase the number of spontaneous movements in mice to some extent, but their effect was weaker than that of the nervonic acid group, YWZ1 group, YWZ2 group, and YWZ3 group. Furthermore, the YWZ1 group, YWZ2 group, and YWZ3 group could significantly increase the number of spontaneous movements in mice. Therefore, the combined administration of triterpenoid saponins and nervonic acid has a synergistic effect in increasing the number of spontaneous movements in mice.

[0066] Table 3 Effects of the drug on the results of the mouse mining experiment 3.5 Effects of different drugs on mouse toxicity ALT and AST, as the most direct indicators of liver function, directly reflect whether liver function is normal. Therefore, serum AST and ALT levels in mice were measured to assess the toxic effects of triterpenoid saponins and nervonic acid on MPTP-induced Parkinson's disease in C57BL / 6 mice. Results are as follows: Figure 2 As shown, compared with the control group, triterpenoid saponins and nervonic acid had no significant effect on serum AST and ALT levels in mice.

[0067] 3.6 Effects of different drugs on dopamine and serotonin levels in the striatum of Parkinson's disease mice like Figure 3 As shown, compared with the control group, MPTP treatment significantly reduced the concentration levels of DA and 5-HT in the striatum. Compared with the model group, the triterpenoid saponins alone could restore the levels of dopamine and serotonin in the mouse striatum to some extent, but their effect was weaker than that of the nervonic acid group, YWZ1 group, YWZ2 group, and YWZ3 group. Furthermore, the YWZ1 group, YWZ2 group, and YWZ3 group could significantly restore the levels of dopamine and serotonin in the mouse striatum. Therefore, the combined application of triterpenoid saponins and nervonic acid has a synergistic effect in restoring the levels of dopamine and serotonin in the mouse striatum.

[0068] 3.7 Effects of different drugs on the expression levels of tyrosine hydroxylase (TH) and dopamine transporter (DAT) proteins like Figure 4 As shown, MPTP treatment significantly reduced the protein expression levels of TH and DAT. Compared with the model group, the triterpenoid saponins alone could restore the protein expression levels of TH and DAT in mice to some extent, but their effect was weaker than that of the nervonic acid group, YWZ1 group, YWZ2 group and YWZ3 group. Furthermore, the YWZ1 group, YWZ2 group and YWZ3 group could significantly restore the protein expression levels of TH and DAT in mice. Therefore, the combined application of triterpenoid saponins and nervonic acid has a synergistic effect on restoring the protein expression levels of TH and DAT in mice.

[0069] 3.8 The effect of different drugs on the inhibition of inflammatory factor expression in Parkinson's disease mice The results are as follows Figure 5 As shown, in the MPTP-induced model group mice, the levels of inflammatory factors IL-6, IL-8, and TNF-α were significantly higher than those in the control group mice. Compared with the model group, the triterpenoid saponins alone could inhibit the expression level of inflammatory factors in Parkinson's disease mice to a certain extent, but its effect was weaker than that of the nervonic acid group, YWZ1 group, YWZ2 group, and YWZ3 group. Furthermore, the YWZ1 group, YWZ2 group, and YWZ3 group could significantly inhibit the expression level of inflammatory factors in Parkinson's disease mice. Therefore, the combined application of triterpenoid saponins and nervonic acid has a synergistic effect in inhibiting the expression level of inflammatory factors in Parkinson's disease mice.

[0070] Comparative Example The following shows a comparison of drug compositions with different component ratios in the treatment of Parkinson's disease.

[0071] 1. Experimental Grouping Unlike Example 2, the groups included a control group, a model group, a YWZ4 group, a YWZ5 group, and a YWZ6 group. The YWZ4 group consisted of a drug composition of triterpenoid saponins and nervonic acid in a mass ratio of 1:3, the YWZ5 group consisted of a drug composition of triterpenoid saponins and nervonic acid in a mass ratio of 1:1, and the YWZ6 group consisted of a drug composition of triterpenoid saponins and nervonic acid in a mass ratio of 1:12.

[0072] 2. Experimental Methods 2.1 Pole Climbing Test The pole-climbing test was used to assess the effects of different drug treatments on the motor coordination of mice. A wooden pole with a diameter of 9 mm and a length of 1 m was vertically fixed in the cage. Mice were allowed to acclimatize to the environment for at least 1 hour before the test. Then, the mice were placed on top of the pole, and the time it took for the animals to descend from the top of the pole to the ground was recorded.

[0073] 2.2 Suspension Test Gently place the mouse's forelimbs on the rope and observe the mouse. The scoring method is as follows: 3 points are given if the mouse can grasp the rope with all four paws, 2 points are given if it can grasp the rope with one hind paw, 1 point is given if it cannot grasp the rope with either hind paw, and 0 points are given if the mouse cannot grasp the rope and falls quickly.

[0074] 2.3 Open Field Experiment The open field test was used to assess the effects of drugs on spontaneous movement and exploratory behavior in mice. Mice were allowed at least 1 hour to acclimatize to the environment before the test. Each mouse was placed in a clean, bright, 60 cm × 60 cm × 40 cm square box (blue interior surface, white floor). The mouse's movements and behaviors were recorded over 5 minutes using a monitor, including the number of times the mouse stood upright (forelegs completely off the floor or climbing the wall) and grooming behavior (preening). Recordings were taken every 10 minutes. After each test, the experimental area was cleaned with 75% alcohol, and the open field video was analyzed using software.

[0075] 2.4 Effects of different drug combinations on dopamine and serotonin levels in the striatum of MPTP model mice To investigate whether the drug has the ability to prevent the degeneration of the dopaminergic system, the concentrations of dopamine (DA) and its related metabolite 5-hydroxytryptamine (5-HT) in the striatum were determined by HPLC.

[0076] 2.5 Effects of different drug combinations on the expression levels of inflammatory factors in MPTP model mice To further investigate the effects of drugs on the inflammatory response in the striatum of Parkinson's mice, the expression levels of inflammatory factors such as IL-6, IL-8, and TNF-α in the striatum were analyzed by ELISA.

[0077] 3. Experimental Results 3.1 Effects of different drug compositions on pole-climbing time in MPTP model mice The results are shown in Table 4. Compared with the control group, the climbing time of mice in the model group was significantly increased. Although the drug intervention in the YWZ5 and YWZ6 groups could shorten the climbing time of mice to varying degrees, compared with the composition of Example 2 or the YWZ4 group, too much or too little nervonic acid component affected the shortening effect of the drug composition on the climbing time of mice.

[0078] Table 4. Effects of different drug compositions on pole-climbing time in MPTP model mice 3.2 Effects of different drug combinations on the hanging score of MPTP model mice As shown in Table 5, the suspension scores of mice in the model group were significantly lower than those in the control group. Compared with the model group, the suspension scores of mice in the YWZ5 and YWZ6 groups increased to varying degrees after drug intervention. However, compared with the composition of Example 2 or the YWZ4 group, too much or too little nervonic acid component affected the effect of the drug composition on increasing the suspension scores of mice.

[0079] Table 5. Effects of different drug compositions on mouse hanging scores 3.3 Effects of different drug compositions on the mining test in MPTP model mice As shown in Table 6, compared with the control group, the number of spontaneous movements in the model group decreased significantly. Compared with the model group, the YWZ5 and YWZ6 groups showed varying degrees of increase in the number of spontaneous movements in mice after drug intervention. However, compared with the composition of Example 2 or the YWZ4 group, too much or too little nervonic acid component affected the effect of the drug composition on increasing the number of spontaneous movements in mice.

[0080] Table 6. Effects of different drug compositions on the results of the mouse mining experiment. 3.4 Effects of different drug combinations on dopamine and serotonin levels in the striatum of MPTP model mice like Figure 6 As shown, MPTP treatment significantly reduced the concentration levels of striatal DA and 5-HT. Compared with the model group, drug intervention in the YWZ5 and YWZ6 groups increased the concentration levels of striatal DA and 5-HT to varying degrees. However, compared with the composition of Example 2 or the YWZ4 group, too much or too little nervonic acid component affected the effect of the drug composition on increasing the concentration levels of striatal DA and 5-HT.

[0081] 3.5 Effects of different drug combinations on the expression levels of inflammatory factors in MPTP model mice The results are as follows Figure 7 As shown, in the model group mice induced by MPTP, the levels of inflammatory factors IL-6, IL-8 and TNF-α were significantly higher than those in the control group mice. Drug intervention in the YWZ5 and YWZ6 groups could reduce the expression levels of inflammatory factors to varying degrees. However, compared with the composition of Example 2 or the YWZ4 group, too much or too little nervonic acid component affected the effect of the drug composition on reducing the expression level of inflammatory factors.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of the combination of triterpenoid saponins and nervonic acid in the preparation of drugs for the prevention and treatment of Parkinson's disease and its symptoms, characterized in that, The mass ratio of the triterpenoid saponins to nervonic acid is 1:(2-10).

2. The application of the combination of triterpenoid saponins and nervonic acid according to claim 1 in the preparation of a drug for preventing and treating Parkinson's disease and its symptoms, characterized in that, The triterpenoid saponin compounds include at least one of 3-O-(3′-O-α-L-arabinofuranosyl-2′-O-β-D-galactopyranosyl)-β-D-glucuronide-21,22-di-O-angeloyl-barrigenol and 3-O-β-D-glucopyranosyl-28-O-(2″-O-α-L-rhamnosyl)-β-D-glucopyranosyl-16-deoxybarringtogenol.

3. The application of the combination of triterpenoid saponins and nervonic acid according to claim 1 in the preparation of a drug for preventing and treating Parkinson's disease and its symptoms, characterized in that, The triterpenoid saponins and the nervonic acid are each of natural origin or artificial synthesis.

4. The application of the combination of triterpenoid saponins and nervonic acid according to claim 1 in the preparation of a drug for preventing and treating Parkinson's disease and its symptoms, characterized in that, Both the triterpenoid saponins and the nervonic acid are derived from *Xanthoceras sorbifolium*, and the triterpenoid saponins are prepared by the following method: (I) Take the fruit shell, fruit stalk and / or seeds of *Sapindus mukorossi* after degreasing and crush them. Extract them with a first solvent at room temperature for 1-10 h to obtain an extract. Concentrate the extract to obtain a concentrate. Extract the concentrate with a second solvent and remove the solvent to obtain a crude extract. (II) The crude extract was separated by silica gel column chromatography with gradient elution using dichloromethane, methanol and water, and further separated by high performance liquid chromatography with gradient elution using methanol and water to obtain the triterpenoid saponin compounds; Preferably, the first solvent and the second solvent are the same or different; Preferably, the first solvent or the second solvent is selected from at least one of methanol, ethanol, acetone, n-butanol, ethyl acetate, chloroform, petroleum ether, and dichloromethane.

5. The application of the combination of triterpenoid saponins and nervonic acid according to claim 4 in the preparation of a drug for preventing and treating Parkinson's disease and its symptoms, characterized in that, The nervonic acid is prepared by the following method: (1) Processing *Sapindus mukorossi* seeds to obtain *Sapindus mukorossi* oil; (2) Mix the *Sapindus mukorossi* oil with a third solvent, adjust the pH to 4-6, add a fourth solvent for extraction, wash with water, distill and dry to obtain crude product; (3) The crude product was purified to obtain the nervonic acid.

6. The application of the combination of triterpenoid saponins and nervonic acid according to claim 5 in the preparation of a drug for preventing and treating Parkinson's disease and its symptoms, characterized in that, In step (3), the purification includes dissolving the crude product in a fifth solvent, removing impurities using an adsorbent, filtering and concentrating to obtain a concentrated solution, adjusting the pH of the concentrated solution to 3-6, crystallizing, filtering and drying to obtain the nervonic acid; Preferably, the third solvent, the fourth solvent, and the fifth solvent are the same or different; Preferably, the third, fourth, or fifth solvent is selected from at least one of ethanol, isopropanol, triethylamine, petroleum ether, chloroform, acetone, and ethyl acetate.

7. The application of the combination of triterpenoid saponins and nervonic acid according to claim 1 in the preparation of a drug for preventing and treating Parkinson's disease and its symptoms, characterized in that, The prevention and control measures include at least one of the following: (1) Improves resting tremor in patients with Parkinson's disease; (2) Improve bradykinesia in patients with Parkinson's disease, including slow movements, difficulty initiating movements, and reduced facial muscle activity; (3) Improve muscle stiffness in patients with Parkinson's disease, or increase in both extensor and flexor muscle tone; (4) Improve postural balance disorders in patients with Parkinson's disease; (5) Improve sensory disturbances in patients with Parkinson's disease, including decreased sense of smell, numbness in the limbs, pain and other sensory abnormalities; (6) Improve autonomic dysfunction in patients with Parkinson's disease, including constipation, excessive sweating, drooling, urination disorders, orthostatic hypotension, and decreased sexual function; (7) Improve mental and cognitive impairments in patients with Parkinson's disease, including improving mental disorders such as anxiety and depression and / or cognitive impairments such as dementia and hallucinations.

8. A pharmaceutical composition for treating or improving Parkinson's disease and its symptoms, characterized in that, It comprises 1 part by weight of triterpenoid saponins, 2-10 parts by weight of nervonic acid, and 0-2000 parts by weight of a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8 for treating or improving Parkinson's disease and its symptoms, characterized in that, The pharmaceutically acceptable carriers include at least one of the following: diluents, fillers, absorbents, wetting agents, binders, disintegrants, lubricants, sweeteners, preservatives, and antioxidants.

10. The pharmaceutical composition according to claim 8 for treating or improving Parkinson's disease and its symptoms, characterized in that, The pharmaceutical composition includes oral or injectable dosage forms.

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