Pharmaceutical composition for treating or improving Parkinson's disease and application thereof

Abstract

The invention discloses a pharmaceutical composition for treating or improving Parkinson's disease and application of the pharmaceutical composition. 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

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a pharmaceutical composition for treating or improving Parkinson's disease and its applications. Background Art

[0002] Parkinson's disease is a neurodegenerative disease commonly found in the middle-aged and elderly. With the increasing degree of global aging, the number of Parkinson's disease patients is increasing year by year. The most important pathological change in Parkinson's disease is the degenerative death of dopaminergic neurons in the substantia nigra of the midbrain, resulting in a significant decrease in striatal dopamine content and the aggregation of a large number of Lewy bodies in neurons, which causes the disease. The cause of Parkinson's disease is still unclear at present, and there is no effective cure method, which seriously affects the normal quality of life of patients.

[0003] Therefore, there is an urgent need to seek an effective medicinal ingredient for treating Parkinson's disease and its related diseases. Summary of the Invention

[0004] To solve at least some of the above-mentioned technical problems in the prior art, the present invention provides a pharmaceutical composition for treating or improving Parkinson's disease and its applications. Specifically, the present invention includes the following content.

[0005] In a first aspect of the present invention, there is provided a pharmaceutical composition for treating or improving Parkinson's disease and its symptoms, which 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.

[0006] In certain embodiments, for the pharmaceutical composition according to the present invention, wherein the triterpenoid saponins comprise at least one of 3 - O-(3′ - O-α - L - arabinofuranosyl - 2′ - O-β - D - galactopyranosyl)-β - D - glucuronopyranosyl - 21,22 - di - O - angeloyl - barrigenol and 3 - O-β - D - glucopyranosyl - 28 - O-(2″ - O-α - L - rhamnopyranosyl)-β - D - glucopyranosyl - 16 - deoxybarringtogenol.

[0007] In certain embodiments, for the pharmaceutical composition according to the present invention, wherein the triterpenoid saponins and the nervonic acid are each respectively of natural origin or synthetic.

[0008] In certain embodiments, for the pharmaceutical composition according to the present invention, wherein the triterpenoid saponins and the nervonic acid are both from Xanthoceras sorbifolium Bunge, and the triterpenoid saponins are prepared by the following method:

[0009] (I) taking the de-oiled husk, pedicel and / or seed residue of Xanthoceras sorbifolia and crushing them, extracting them with a first solvent at room temperature for 1-10 hours to obtain an extract, concentrating the extract to obtain a concentrate, extracting the concentrate with a second solvent, and removing the solvent to obtain a crude extract;

[0010] (II) separating the crude extract by silica gel column chromatography, using dichloromethane, methanol and water for gradient elution, and further separating by high performance liquid chromatography, using methanol and water for gradient elution, to obtain the triterpenoid saponin compounds.

[0011] In certain embodiments, according to the pharmaceutical composition of the present invention, the first solvent is the same as or different from the second solvent.

[0012] In certain embodiments, according to the pharmaceutical composition of the present invention, 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.

[0013] In certain embodiments, according to the pharmaceutical composition of the present invention, the nervonic acid is prepared by the following method:

[0014] (1) processing Xanthoceras sorbifolia seeds to obtain Xanthoceras sorbifolia oil;

[0015] (2) mixing the Xanthoceras sorbifolia oil with a third solvent, adjusting the pH to 4-6, adding a fourth solvent for extraction, washing, distilling, and drying to obtain a crude product;

[0016] (3) Purify the crude product to obtain the nervonic acid.

[0017] In certain embodiments, according to the pharmaceutical composition of the present invention, in step (3), the purification includes dissolving the crude product using 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 neuraminic acid.

[0018] In certain embodiments, according to the pharmaceutical composition of the present invention, the third, fourth and fifth solvents are the same or different.

[0019] In certain embodiments, according to the pharmaceutical composition of the present invention, the third, fourth or fifth solvent is selected from at least one of ethanol, isopropanol, triethylamine, petroleum ether, chloroform, acetone and ethyl acetate.

[0020] In certain embodiments, for the pharmaceutical composition according to the present invention, wherein the pharmaceutically acceptable carrier comprises at least one of a diluent, a filler, an absorbent, a wetting agent, a binder, a disintegrant, a lubricant, a sweetening agent, a preservative, and an antioxidant.

[0021] In certain embodiments, for the pharmaceutical composition according to the present invention, wherein the treatment or improvement is achieved by administering a therapeutically effective amount of the pharmaceutical composition to a subject.

[0022] In certain embodiments, for the pharmaceutical composition according to the present invention, wherein the effective amount is 0.1 - 5000 mg / Kg.

[0023] A second aspect of the present invention provides the use of a combination of triterpenoid saponins and nervonic acid in the preparation of a drug for treating or improving Parkinson's disease and its symptoms, wherein the mass ratio of the triterpenoid saponins to the nervonic acid is 1:(2 - 10).

[0024] By combining triterpenoid saponins and nervonic acid and through further research, it has been found that triterpenoid saponins and nervonic acid have a synergistic effect in treating Parkinson's disease. The present invention further verifies that this composition can restore the propulsion index in gait analysis of Parkinson's mice, increase the hanging score of Parkinson's mice in the hanging test, increase the spontaneous movement times of Parkinson's mice, inhibit the expression level of inflammatory factors in Parkinson's disease mice, etc. Therefore, the pharmaceutical composition of the present invention has broad application prospects in the treatment of Parkinson's disease. Description of the Drawings

[0025] Figure 1 Shows the effects of different drugs on the propulsion index in gait analysis of Parkinson's mice.

[0026] Figure 2 Shows the results of the biological toxicity detection of different drugs.

[0027] Figure 3 Shows the effects of different drugs on the contents of dopamine and 5 - hydroxytryptamine in the striatum of Parkinson's disease mice.

[0028] Figure 4 Shows the effects of different drugs on the protein expression levels of TH and DAT in Parkinson's disease mice.

[0029] Figure 5 Shows the effects of different drugs on the expression of inflammatory factors in Parkinson's disease mice.

[0030] Figure 6 Shows the effects of different pharmaceutical compositions on the contents of dopamine and 5 - hydroxytryptamine in the striatum of Parkinson's disease mice.

[0031] Figure 7Shows the effects of different pharmaceutical compositions on the expression of inflammatory factors in Parkinson's disease mice. Detailed Description of the Invention

[0032] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be construed as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0035] Drug composition

[0036] One aspect of the present invention provides a pharmaceutical composition for treating or improving Parkinson's disease and its symptoms, which 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.

[0037] In order to better achieve the synergistic treatment or improvement of Parkinson's disease and its symptoms, it is necessary to control the mass ratio of triterpenoid saponins and nervonic acid within a suitable range. In a preferred embodiment, the mass ratio of the triterpenoid saponins to the nervonic acid is 1:(2 - 10), preferably 1:(2 - 9), more preferably 1:(2 - 8), such as 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 with the use of any single component alone, such as the use of triterpenoid saponins or nervonic acid alone, the use of the composition with the above ratio can synergistically treat or improve Parkinson's disease and its related symptoms, conditions, or disorders.

[0038] In a preferred embodiment, the triterpenoid saponin compound comprises at least one of 3-O-(3′-O-α-L-arabinofuranosyl-2′-O-β-D-galactopyranosyl)-β-D-glucuronopyranosyl-21,22-di-O-angeloyl-barrigenol and 3-O-β-D-glucopyranosyl-28-O-(2″-O-α-L-rhamnopyranosyl)-β-D-glucopyranosyl-16-deoxybarringtogenol.

[0039] In a preferred embodiment, both the triterpenoid saponin compound and the nervonic acid are derived from Xanthoceras sorbifolium Bunge, and the triterpenoid saponin compound is prepared by the following method:

[0040] (I) Take the dried shell, stalk and / or defatted residue of the seed of Xanthoceras sorbifolium Bunge, crush it, and extract it with a first solvent at room temperature for 1 - 10 h, preferably 2 - 9 h, more preferably 3 - 8 h, such as 3, 4, 5, 6, 7, 8 h to obtain an extract. Concentrate the extract to obtain a concentrate, and extract the concentrate with a second solvent, and remove the solvent to obtain a crude extract;

[0041] (II) Separate the crude extract by silica gel column chromatography, and perform gradient elution with dichloromethane, methanol and water in a ratio of 30:1:1 - 1:1:1 (preferably 28:1:1 - 1:1:1, more preferably 25:1:1 - 1:1:1, even more preferably 20:1:1 - 1:1:1, such as 20:1:1, 15:1:1, 10:1:1, 5:1:1, 1:1:1). Further separate it by high performance liquid chromatography, and perform gradient elution with methanol and water in a ratio of 10:90 - 100:0 (preferably 10:80 - 100:1, more preferably 10:70 - 100:7, even more preferably 10:50 - 100:17, such as 1:5, 1:4, 1:3, 1:2, 1:1, 2:1) to obtain the triterpenoid saponin compound. It can be understood that in order to better extract the triterpenoid saponin compound, steps such as extraction, concentration, extraction, and separation can be carried out several times, such as 1, 2, 3, 4, 5 times.

[0042] In a preferred embodiment, the volume ratio of the defatted residue of the shell, stalk or seed of Xanthoceras sorbifolium Bunge to the first solvent is 1:(1 - 10), preferably 1:(2 - 10), more preferably 1:(3 - 10), further preferably 1:(4 - 10), even more preferably 1:(5 - 10), such as 1:5, 1:6, 1:7, 1:8, 1:9, 1:10.

[0043] In a preferred embodiment, the first solvent and the second solvent are the same or different, and the first solvent or the second solvent includes at least one of methanol, ethanol, acetone, n-butanol, ethyl acetate, chloroform, petroleum ether, and dichloromethane. Those skilled in the art will understand that one or a combination 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 solvents are ethyl acetate and n-butanol.

[0044] In a preferred embodiment, the first solvent is 30-90% ethanol, preferably 35-85% ethanol, more preferably 40-80% ethanol, even more preferably 45-75% ethanol, such as 45%, 50%, 55%, 60%, 65%, 70%, 75%. In a specific embodiment, the first solvent is 60% ethanol.

[0045] In a preferred embodiment, the nervonic acid is prepared by the following method:

[0046] (1) Take the seeds or kernels of Xanthoceras sorbifolium, dry and crush them, add a mixed solvent of ethanol and ethyl acetate with a volume 4-6 times (such as 4, 4.5, 5, 5.5, 6 times) the volume, adjust the pH to 10-12 (such as 10, 10.5, 11, 11.5, 12) for extraction to obtain an extract, concentrate it, and remove the solvent to obtain Xanthoceras sorbifolium oil;

[0047] (2) Mix the Xanthoceras sorbifolium oil with a third solvent, adjust the pH to 4-6 (such as 4, 4.5, 5, 5.5, 6), add a fourth solvent for extraction, wash with water, distill, and dry to obtain a crude product;

[0048] (3) Dissolve the crude product with a fifth solvent, use an adsorbent (including but not limited to activated carbon) to remove impurities, filter and concentrate to obtain a concentrated solution, adjust the pH of the concentrated solution to 3-6 (such as 3, 3.5, 4, 4.5, 5, 5.5, 6), crystallize, filter, and dry to obtain the nervonic acid. It can be understood that in order to better extract nervonic acid, steps such as extraction, concentration, and extraction can be carried out several times, such as 1, 2, 3, 4, 5 times.

[0049] In a preferred embodiment, the third solvent, the fourth solvent and the fifth solvent are the same or different, and the third, fourth or fifth solvent includes at least one of 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 a combination 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 specified, the triterpenoid saponins and nervonic acid of the present invention are extracted from the same batch of Xanthoceras sorbifolium Bunge.

[0050] In the present invention, a pharmaceutically acceptable carrier is involved in transporting or delivering a drug from one organ or a part of the body to another organ or another part of the body. Each carrier is "acceptable", that is, it is compatible with other components of the formulation (such as triterpenoid saponins and nervonic acid) and does not harm the patient. The pharmaceutically acceptable carrier includes at least one of diluents, fillers, absorbents, wetting agents, binders, disintegrants, lubricants, sweeteners, preservatives and antioxidants. Examples of the diluent include but are not limited to polysorbate-80, phosphate buffer solution, physiological saline, aqueous buffer solution, solvent, dispersion medium, etc.; the filler includes but is not limited to starch, lactose, mannitol, microcrystalline cellulose, etc.; the absorbent includes but is not limited to calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent includes but is not limited to water, ethanol, etc.; the binder includes but is not limited to hypromellose, povidone, microcrystalline cellulose, etc.; the disintegrant includes but is not limited to croscarmellose sodium, crospovidone, surfactant, low-substituted hydroxypropyl cellulose, etc.; the lubricant includes but is not limited to magnesium stearate, talc, polyethylene glycol, sodium lauryl sulfate, colloidal silica, talc, etc.; the sweetener includes but is not limited to sucralose, acesulfame, saccharin, sucrose, xylitol, mannitol, sorbitol, glucose, fructose, aspartame, etc.; the preservative includes but is not limited to parabens, chlorobutanol, phenol, sorbic acid, etc.; the antioxidant includes but is not limited to ascorbic acid, methionine, etc.

[0051] In a preferred embodiment, the treatment or improvement is achieved by administering a therapeutically effective amount of the pharmaceutical composition to a subject. The daily dosage of the pharmaceutical composition is generally 1 - 5000 mg / kg, preferably 1 - 4000 mg / kg, more preferably 1 - 3000 mg / kg, further preferably 1 - 2000 mg / kg, still more preferably 1 - 1000 mg / kg, even more preferably 1 - 900 mg / kg, even more preferably 1 - 500 mg / kg, even more preferably 1 - 300 mg / kg, even more preferably 1 - 200 mg / kg, even more preferably 1 - 100 mg / kg, such as 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 a day, administered in multiple doses per day, or administered at intervals.

[0052] There is no particular limitation 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, spraying, etc. Correspondingly, the drug of the present invention can be made into various clinically acceptable dosage forms, including oral dosage forms, injection dosage forms, topical dosage forms or external use dosage forms, etc.

[0053] Application

[0054] One aspect of the present invention provides the use of a combination of triterpenoid saponins and nervonic acid in the preparation of a drug for treating or improving Parkinson's disease and its symptoms, wherein the mass ratio of the triterpenoid saponins to the nervonic acid is 1:(2 - 10).

[0055] In the present invention, the term "treatment or improvement" refers to therapeutic treatment and preventive or prophylactic measures, the purpose of which is to prevent or slow down (reduce) undesired physiological changes or disorders, such as the occurrence and progression of Parkinson's disease. Beneficial or desired clinical outcomes include but are not limited to the following whether detectable or undetectable outcomes, including relief of symptoms, reduction in the degree of disease, stabilization of the disease state (i.e., not worsening), delay or slowdown in disease progression, improvement or alleviation of the disease state, and alleviation (whether partial or complete). Those in need of treatment include those who already have Parkinson's disease or related diseases or those who need to prevent or improve Parkinson's disease or related diseases.

[0056] In a preferred embodiment, the treatment or improvement includes at least one of the following situations:

[0057] (1) Improving the resting tremor of Parkinson's disease patients;

[0058] (2) Improving the bradykinesia of Parkinson's disease patients, including slow movement, difficulty in initiating movement, and reduced activity of facial expression muscles;

[0059] (3) Improve muscle stiffness in patients with Parkinson's disease, or simultaneous increase in extensor and flexor muscle tone;

[0060] (4) Improve postural balance disorders in patients with Parkinson's disease;

[0061] (5) Improve sensory disturbances in patients with Parkinson's disease, including sensory abnormalities such as hyposmia, limb numbness, pain, etc.;

[0062] (6) Improve autonomic dysfunction in patients with Parkinson's disease, including constipation, hyperhidrosis, sialorrhea, urinary disorders, orthostatic hypotension, sexual dysfunction, etc.;

[0063] (7) Improve mental and cognitive disorders in patients with Parkinson's disease, including improving mental disorders such as anxiety, depression, and / or cognitive disorders such as dementia, hallucinations, etc.

[0064] Example 1

[0065] The preparation of a drug for treating or improving Parkinson's disease and its symptoms is exemplarily shown below, as follows.

[0066] 1. Preparation of triterpenoid saponin compounds

[0067] Take 10 kg of the residue after defatting the dried shell, stalk, and seeds of Xanthoceras sorbifolium Bunge, crush it, add 7 times the volume of 60% ethanol, reflux and extract 5 times, each time for 1.5 h to obtain an extract, concentrate it to obtain a concentrated solution, extract it 4 times successively with equal volumes of ethyl acetate and n-butanol to obtain an extract, evaporate to remove the solvent to obtain a crude extract; separate the crude extract by silica gel column chromatography, use a gradient elution of dichloromethane, methanol, and water in a ratio of 30:1:1, and further separate it by high-performance liquid chromatography, use a gradient elution of methanol and water in a ratio of 4:1 to obtain the triterpenoid saponin compounds. Analyzed by GC-MS, the purity of the triterpenoid saponin compounds is 92.5%. Analyzed by nuclear magnetic resonance, the structures of the two compounds with the highest content are as follows:

[0068] (1) 3-O-(3′-O-α-L-arabinofuranosyl-2′-O-β-D-galactopyranosyl)-β-D-glucuronopyranosyl-21,22-di-O-angeloyl-barrigenol, white needle crystals (methanol), showing purple in 10% sulfuric acid / ethanol solution. 1HNMR (400 MHz, pyridine-d5): δ 3.27 (1H, dd, J = 11.6, 4.6 Hz, H-3), 5.51 (1H, brs, H-12); C-3-GlcUA: 4.90 (1H, d, J = 7.6 Hz, H-1′); C-2′-Gal: 5.36 (1H, d, J = 7.7 Hz, H-1″); C-3′-Ara: 6.06 (1H, d, J = 2.0 Hz, H-1″′). 13C NMR (100 MHz, pyridine-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).

[0069] (2) 3-O-β-D-glucopyranosyl-28-O-(2″-O-α-L-rhamnopyranosyl)-β-D-glucopyranosyl-16-deoxybarringtogenol, white powder (methanol), showing purple color with 10% sulfuric acid / ethanol solution. 1 HNMR (400 MHz, pyridine-d5): δ 3.38 (1H, dd, J1 = 4.6 Hz, J2 = 11.6 Hz, H-3), 5.54 (1H, brs, H-12); C-3-Glc: 4.93 (1H, d, J = 7.8 Hz, H-1′); C-28-Glc: 4.73 (1H, d, J = 7.5 Hz, H-1″); C-2′-Rha: 6.61 (1H, brs, H-1″′). 13CNMR (100 MHz, pyridine-d5): δ 39.5 (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″″).

[0070] 2. Preparation of nervonic acid

[0071] (1) Take 5 kg of Xanthoceras sorbifolia Bunge kernels, dry and crush them, add a mixed solvent of ethanol and ethyl acetate with a volume 4 - 6 times that of the kernels, adjust the pH to 10 - 12 for extraction to obtain an extract, concentrate it, and remove the solvent to obtain 1.5 kg of Xanthoceras sorbifolia Bunge oil;

[0072] (2) Mix the Xanthoceras sorbifolia Bunge 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 using acetic acid, extract with ethanol with a volume 3 times that of the mixture, wash with water, distill, and dry to obtain a crude product;

[0073] (3) Dissolve the crude product with petroleum ether of equal volume, add activated carbon, heat and reflux at 65 °C for 2.5 h, filter and concentrate to obtain a concentrated solution, adjust the pH to 5 using acetic acid, crystallize at 0 °C for 2.5 h, repeat crystallization 5 times, filter, and dry to obtain 90 g of nervonic acid. Analyzed by GC - MS, the purity of nervonic acid is 97.6%.

[0074] 3. Preparation of drugs

[0075] Take triterpenoid saponin compounds and nervonic acid with a mass ratio of 1:(2 - 10), add polysorbate - 80 and PBS to dissolve them to obtain a drug composition. Take triterpenoid saponin compounds, add polysorbate - 80 and PBS to dissolve them to obtain the corresponding triterpenoid saponin drug. Take nervonic acid, add polysorbate - 80 and PBS to dissolve it to obtain the corresponding nervonic acid drug, and store them at low temperature for standby.

[0076] Example 2

[0077] The following shows the application of the drug prepared in Example 1 in the treatment of Parkinson's disease.

[0078] 1. Construction of animal model

[0079] Forty 8- to 10-week-old male C57BL / 6 mice, weighing 20-22 g, were housed in cages of eight and fed ad libitum with food and water at a temperature of (25±2)°C, a humidity of (60±10)%, and a 12-hour light-dark cycle (light on from 7:30 to 19:30). The mice were adaptively fed for one week before the experiment. Subsequently, the mice were divided into 5 groups (n = 8) according to the random number table method, including a control group, a model group, a triterpenoid saponin group (triterpenoid saponin drug), a nervonic acid group (nervonic acid drug), YWZ1 group, YWZ2 group, and YWZ3 group. Among them, the YWZ1 group was a drug combination of triterpenoid saponin and nervonic acid with a mass ratio of 1:2, the YWZ2 group was a drug combination of triterpenoid saponin compound and nervonic acid with a mass ratio of 1:5, and the YWZ3 group was a drug combination of triterpenoid saponin compound and nervonic acid with a mass ratio of 1:8. Except for the control group, all mice were used to establish a conventional Parkinson's disease mouse model with MPTP (20 mg / kg, dissolved in PBS). MPTP was intraperitoneally injected continuously for 7 days, once a day, and the control group was injected with an equal volume of normal saline.

[0080] After the mouse model was successfully constructed, the control group and the model group were given intragastric intervention with an equal volume of normal saline. The triterpenoid saponin group was given intragastric intervention with a 40 mg / kg triterpenoid saponin compound drug solution. The nervonic acid group was given intragastric intervention with a 40 mg / kg nervonic acid drug solution. The YWZ1 group, YWZ2 group, and YWZ3 group were respectively given intragastric intervention with a 40 mg / kg drug combination drug solution.

[0081] 2. Experimental methods

[0082] 2.1 Gait analysis experiment

[0083] The feet of the mice were wetted, and the mice were made to pass continuously through a treadmill, and the video was automatically recorded by a camera. After the collection was completed, the recorded video was analyzed using computer analysis software.

[0084] 2.2 Pole climbing test

[0085] The pole climbing test was used to evaluate the effect of different drug treatments on the motor coordination ability of mice. A wooden pole with a diameter of 9 mm and a length of 1 m was vertically fixed in the cage. The mice were allowed to adapt to the environment for at least 1 h before the test. Subsequently, the mice were placed on the top of the pole, and the time for the animals to descend from the top of the pole to the ground was recorded.

[0086] 2.3 Hanging experiment

[0087] The forelimbs of the mice were gently placed on the rope, and the mice were observed. The scoring method was as follows: If all four paws of the mice could grasp the hanging rope, it was recorded as 3 points; if one hind paw was used to grasp the hanging rope, it was recorded as 2 points; if both hind paws could not grasp the hanging rope, it was 1 point; if the mice could not grasp the hanging rope and fell quickly, it was recorded as 0 points.

[0088] 2.4 Open field test

[0089] The open field test is used to evaluate the effects of drugs on the spontaneous locomotor ability and exploratory behavior of mice. Mice were allowed to acclimate to the environment for at least 1 h before the test. A single mouse was placed in a clean and bright square box (60 cm × 60 cm × 40 cm) with a blue inner surface and a white floor. The movement behavior of the mouse within 5 min was recorded using a monitor, and the number of times the mouse stood upright (both front limbs completely off the floor or climbing the wall) and the grooming behavior (self-grooming) were also recorded. Recordings were made every 10 min. After each experiment, the experimental site was cleaned with 75% alcohol, and the open field video was analyzed using software.

[0090] 2.5 Drug toxicity detection

[0091] 4 mL of carotid artery blood was drawn from the mouse into a clot-promoting tube, allowed to stand at room temperature for 30 min, and then centrifuged (10000 r / min, 4 °C) to obtain serum. The levels of AST and ALT in the serum were detected using the microplate method.

[0092] 2.6 Determination of dopamine and 5-hydroxytryptamine contents in the striatum of Parkinson's disease mice

[0093] To explore whether the drug has the ability to prevent the degeneration of the dopaminergic system, the concentrations of dopamine (DA) and 5-hydroxytryptamine (5-HT) in the striatum were determined using HPLC method.

[0094] 2.7 Detection of TH and DAT protein expression levels

[0095] The protein expression levels of TH and DAT were detected by qRT-PCR method.

[0096] 2.8 Detection of the expression levels of inflammatory factors in Parkinson's disease mice

[0097] To further study the effects of the drug 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 method.

[0098] 3. Experimental results

[0099] 3.1 Effects of different treatment groups on the propulsion index in gait analysis of MPTP model mice

[0100] Parkinson's disease patients often show obvious changes in gait characteristics, such as unsteady gait rhythm, narrowed stride, and postural instability. Gait analysis experiments can quantify these changes by analyzing gait parameters (such as step length, walking speed, gait cycle, etc.). By quantitatively analyzing the changes in gait characteristics, it provides important tools and methods for the early diagnosis, treatment effect evaluation, and disease monitoring of Parkinson's disease. In gait analysis (Figure 1 ) Compared with the control group, there were significant differences in the gait indexes of the propulsion index in the left front, left rear, right front, and right rear of the model group. Compared with the model group, the administration of triterpenoid saponins alone could restore the propulsion index to a certain extent, but its effect was weaker than that of the nervonic acid group, YWZ1 group, YWZ2 group, and YWZ3 group, and the YWZ1 group, YWZ2 group, and YWZ3 group could significantly restore the propulsion index of mice. Therefore, the combined administration of triterpenoid saponins and nervonic acid had a synergistic effect on restoring the propulsion index of mice.

[0101] 3.2 Effects of different drugs on the pole climbing time of MPTP model mice

[0102] The main characteristics of Parkinson's disease are the loss of dopaminergic neurons and the decrease in dopamine levels, which affect motor control. The pole climbing experiment can help evaluate the effects of drugs on the motor function of Parkinson's disease model animals. As shown in Table 1, compared with the control group, the pole climbing time of the model group mice increased significantly. Compared with the model group, the administration of triterpenoid saponins alone could shorten the pole climbing time of mice to a certain extent, but its effect was weaker than that of the nervonic acid group, YWZ1 group, YWZ2 group, and YWZ3 group, and the YWZ1 group, YWZ2 group, and YWZ3 group could significantly shorten the pole climbing time of mice. Therefore, the combined administration of triterpenoid saponins and nervonic acid had a synergistic effect on shortening the pole climbing time of mice.

[0103] Table 1 Effects of drugs on the pole climbing time of MPTP model mice

[0104]

[0105]

[0106] 3.3 Effects of different drugs on the hanging score of MPTP model mice

[0107] Parkinson's disease patients often suffer from muscle stiffness and movement difficulties, which lead to obvious impairment of their exercise endurance and muscle strength. The hanging experiment can be used to evaluate the effects of drugs on the motor function of Parkinson's disease model animals. As shown in Table 2, compared with the control group, the hanging score of the model group mice decreased significantly. Compared with the model group, the administration of triterpenoid saponins alone could increase the hanging score of mice to a certain extent, but its effect was weaker than that of the nervonic acid group, YWZ1 group, YWZ2 group, and YWZ3 group, and the YWZ1 group, YWZ2 group, and YWZ3 group could significantly increase the hanging score of mice. Therefore, the combined administration of triterpenoid saponins and nervonic acid had a synergistic effect on increasing the hanging score of mice.

[0108] Table 2 Effects of drugs on the hanging score of mice

[0109]

[0110]

[0111] 3.4 Effects of Different Drugs on the Open Field Test in MPTP Model Mice

[0112] In Parkinson's disease research, animal models often exhibit motor characteristics such as slow movement and decreased motor ability, which can be observed and evaluated through the exploratory behavior and motor activity level of animals in the open field experiment. As shown in Table 3, compared with the control group, the number of spontaneous movements in the model group decreased significantly; compared with the model group, the triterpenoid saponin compounds administered alone could increase the number of spontaneous movements in mice to a certain extent, but their effects were weaker than those of the nervonic acid group, YWZ1 group, YWZ2 group, and YWZ3 group, and the YWZ1 group, YWZ2 group, and YWZ3 group could significantly increase the number of spontaneous movements in mice. Therefore, the combined administration of triterpenoid saponin compounds and nervonic acid has a synergistic effect on increasing the number of spontaneous movements in mice.

[0113] Table 3 Effects of Drugs on the Results of the Open Field Test in Mice

[0114]

[0115] 3.5 Effects of Different Drugs on the Toxicity of Mice

[0116] As the most direct liver function indicators, ALT and AST directly reflect whether the liver level is normal. Therefore, the serum levels of AST and ALT in mice were detected to evaluate whether triterpenoid saponin compounds and nervonic acid have toxic effects on MPTP-induced Parkinson's disease C57BL / 6 mice. The results are as Figure 2 shown. Compared with the control group, triterpenoid saponin compounds and nervonic acid had no significant effect on the serum levels of AST and ALT in mice.

[0117] 3.6 Effects of Different Drugs on Dopamine and 5-Hydroxytryptamine in the Striatum of Parkinson's Disease Mice

[0118] As Figure 3 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 saponin compounds administered alone could restore the levels of dopamine and 5-hydroxytryptamine in the striatum of mice to a certain extent, but their effects were weaker than those of the nervonic acid group, YWZ1 group, YWZ2 group, and YWZ3 group, and the YWZ1 group, YWZ2 group, and YWZ3 group could significantly restore the levels of dopamine and 5-hydroxytryptamine in the striatum of mice. Therefore, the combined administration of triterpenoid saponin compounds and nervonic acid has a synergistic effect on restoring the levels of dopamine and 5-hydroxytryptamine in the striatum of mice.

[0119] 3.7 Effects of Different Drugs on Protein Expression Levels of Tyrosine Hydroxylase (TH) and Dopamine Transporter (DAT)

[0120] As Figure 4 shown, MPTP treatment significantly reduced the protein expression levels of TH and DAT. Compared with the model group, the triterpenoid saponin compounds administered alone could restore the protein expression levels of TH and DAT in mice to a certain extent, but their effects were weaker than those of the nervonic acid group, YWZ1 group, YWZ2 group, and YWZ3 group. Moreover, the YWZ1 group, YWZ2 group, and YWZ3 group could significantly restore the protein expression levels of TH and DAT in mice. Therefore, the combined administration of triterpenoid saponin compounds and nervonic acid had a synergistic effect on restoring the protein expression levels of TH and DAT in mice.

[0121] 3.8 Inhibitory Effects of Different Drugs on Expression of Inflammatory Factors in Parkinson's Disease Mice

[0122] The results were as Figure 5 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. Compared with the model group, the triterpenoid saponin compounds administered alone could inhibit the expression levels of inflammatory factors in Parkinson's disease mice to a certain extent, but their effects were weaker than those of the nervonic acid group, YWZ1 group, YWZ2 group, and YWZ3 group. Moreover, the YWZ1 group, YWZ2 group, and YWZ3 group could significantly inhibit the expression levels of inflammatory factors in Parkinson's disease mice. Therefore, the combined administration of triterpenoid saponin compounds and nervonic acid had a synergistic effect on inhibiting the expression levels of inflammatory factors in Parkinson's disease mice.

[0123] Comparative Example

[0124] The following shows the comparison of pharmaceutical compositions with different group ratios in the treatment of Parkinson's disease.

[0125] 1. Experimental Grouping

[0126] Different from Example 2, the grouping included a control group, a model group, a YWZ4 group, a YWZ5 group, and a YWZ6 group. Among them, the YWZ4 group was a pharmaceutical composition with a mass ratio of triterpenoid saponin compounds to nervonic acid of 1:3, the YWZ5 group was a pharmaceutical composition with a mass ratio of triterpenoid saponin compounds to nervonic acid of 1:1, and the YWZ6 group was a pharmaceutical composition with a mass ratio of triterpenoid saponin compounds to nervonic acid of 1:12.

[0127] 2. Experimental Methods

[0128] 2.1 Pole-Climbing Test

[0129] The pole climbing test was used to evaluate the effect of different drug treatments on the motor coordination ability of mice. A wooden pole with a diameter of 9 mm and a length of 1 m was vertically fixed in the cage. The mice were allowed to adapt to the environment for at least 1 h before the test, and then the mice were placed on the top of the pole, and the time for the animals to descend from the top of the pole to the ground was recorded.

[0130] 2.2 Hanging experiment

[0131] The forelimbs of the mice were gently placed on the rope, and the mice were observed. The scoring method was as follows: If all four paws of the mice could grasp the hanging rope, it was recorded as 3 points; if one hind paw was used to grasp the hanging rope, it was recorded as 2 points; if both hind paws could not grasp the hanging rope, it was 1 point; if the mice could not grasp the hanging rope and fell quickly, it was recorded as 0 points.

[0132] 2.3 Open field experiment

[0133] The open field experiment was used to evaluate the effect of drugs on the spontaneous motor ability and exploratory behavior of mice. The mice were allowed to adapt to the environment for at least 1 h before the test. A single mouse was placed in a clean and bright square box (60 cm × 60 cm × 40 cm, the inner surface color of the box was blue and the floor was white), and the movement behavior of the mouse within 5 min was recorded using a monitor, and the number of times the mouse stood upright (both forelimbs completely left the bottom plate or climbed the wall) and the grooming behavior (grooming) were recorded. Record once every 10 min. After each experiment, the experimental site was cleaned with 75% alcohol, and the open field video was analyzed by software.

[0134] 2.4 Effects of different drug compositions on the contents of dopamine and 5-hydroxytryptamine in the striatum of MPTP model mice

[0135] To explore 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 method.

[0136] 2.5 Effects of different drug compositions on the expression levels of inflammatory factors in MPTP model mice

[0137] To further study the effect of the drug 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 method.

[0138] 3. Experimental results

[0139] 3.1 Effects of different drug compositions on the pole climbing time of MPTP model mice

[0140] The results are shown in Table 4. Compared with the control group, the pole climbing time of the mice in the model group increased significantly. Although the drug intervention in the YWZ5 group and the YWZ6 group could shorten the pole climbing time of the mice to varying degrees, however, compared with the composition with the ratio 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 pole climbing time of the mice.

[0141] Table 4 Effects of different drug compositions on the pole climbing time of MPTP model mice

[0142]

[0143] 3.2 Effects of different drug compositions on the hanging score of MPTP model mice

[0144] As shown in Table 5, compared with the control group, the hanging score of the mice in the model group decreased significantly. Compared with the model group, the drug intervention in the YWZ5 group and the YWZ6 group could increase the hanging score of the mice to varying degrees. However, compared with the composition with the ratio of Example 2 or the YWZ4 group, too much or too little nervonic acid component affected the increasing effect of the drug composition on the hanging score of the mice.

[0145] Table 5 Effects of different drug compositions on the hanging score of mice

[0146] Group Hanging score Control group 2.7 Model group 1.2 YWZ4 group 2.4 YWZ5 group 2.2 YWZ6 group 2.2

[0147] 3.3 Effects of different drug compositions on the open field test of MPTP model mice

[0148] As shown in Table 6, compared with the control group, the number of spontaneous movements of the model group decreased significantly; compared with the model group, the drug intervention in the YWZ5 group and the YWZ6 group could increase the number of spontaneous movements of the mice to varying degrees. However, compared with the composition with the ratio of Example 2 or the YWZ4 group, too much or too little nervonic acid component affected the increasing effect of the drug composition on the number of spontaneous movements of the mice.

[0149] Table 6 Effects of different drug compositions on the results of the open field test of mice

[0150]

[0151] 3.4 Effects of different drug compositions on dopamine and 5-hydroxytryptamine in the striatum of MPTP model mice

[0152] As Figure 6As shown, after MPTP treatment, the concentration levels of striatal DA and 5-HT were significantly decreased. Compared with the model group, after drug intervention in the YWZ5 group and the YWZ6 group, the concentration levels of striatal DA and 5-HT could be increased to varying degrees. However, compared with the composition of the ratio in Example 2 or the YWZ4 group, too much or too little nervonic acid component affected the increasing effect of the drug composition on the concentration levels of striatal DA and 5-HT.

[0153] 3.5 Effects of Different Drug Compositions on the Expression Levels of Inflammatory Factors in MPTP Model Mice

[0154] The results are as Figure 7 shown. In the model group of 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 of mice. After drug intervention in the YWZ5 group and the YWZ6 group, the expression levels of inflammatory factors could be decreased to varying degrees. However, compared with the composition of the ratio in Example 2 or the YWZ4 group, too much or too little nervonic acid component affected the decreasing effect of the drug composition on the expression levels of inflammatory factors.

[0155] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features. And these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pharmaceutical composition for treating or improving Parkinson's disease and its symptoms, characterized in that: The invention comprises 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.

2. The pharmaceutical composition according to claim 1, characterized in that The triterpenoid saponin compound comprises at least one of 3-O-(3′-O-α-L-arabinofuranosyl-2′-O-β-D-galactopyranosyl)-β-D-pyranoglucuronic acid-21,22-di-O-angeloyl-barrigenol and 3-O-β-D-pyranoglucosyl-28-O-(2″-O-α-L-rhamnopyranosyl)-β-D-pyranoglucosyl-16-deoxybarringtogenol.

3. The pharmaceutical composition according to claim 1, characterized in that The triterpenoid saponin compounds and the nervonic acid are respectively of natural origin or artificially synthesized.

4. The pharmaceutical composition according to claim 1, characterized in that The triterpenoid saponin compound and the nervonic acid are both from Xanthoceras sorbifolia, and the triterpenoid saponin compound is prepared by the following method: (I) taking the de-oiled husk, pedicel and / or seed residue of Xanthoceras sorbifolia and crushing them, extracting them with a first solvent at room temperature for 1-10 hours to obtain an extract, concentrating the extract to obtain a concentrate, extracting the concentrate with a second solvent, and removing the solvent to obtain a crude extract; (II) separating the crude extract by silica gel column chromatography, using dichloromethane, methanol and water for gradient elution, and further separating by high performance liquid chromatography, using methanol and water for gradient elution, to obtain the triterpenoid saponin compound; Preferably, the first solvent is the same as or different from the second solvent; 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 pharmaceutical composition according to claim 4, characterized in that The nervonic acid is prepared by the following method: (1) processing Xanthoceras sorbifolia seeds to obtain Xanthoceras sorbifolia oil; (2) mixing the Xanthoceras sorbifolia oil with a third solvent, adjusting the pH to 4-6, adding a fourth solvent for extraction, washing, distilling, and drying to obtain a crude product; (3) Purify the crude product to obtain the nervonic acid.

6. The pharmaceutical composition according to claim 5, characterized in that In step (3), the purification includes dissolving the crude product with a fifth solvent, removing impurities with 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, fourth and fifth solvents 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 pharmaceutical composition according to claim 1, characterized in that The pharmaceutically acceptable carrier includes at least one of a diluent, a filler, an absorbent, a wetting agent, a binder, a disintegrant, a lubricant, a sweetener, a preservative and an antioxidant.

8. The pharmaceutical composition according to claim 1, characterized in that The treatment or improvement is achieved by administering a therapeutically effective amount of the pharmaceutical composition to the subject.

9. The pharmaceutical composition according to claim 8, characterized in that The effective amount is 0.1-5000 mg / Kg.

10. Use of a combination of triterpenoid saponins and neuraminic acid in the preparation of a drug for treating or improving Parkinson's disease and its symptoms, characterized in that: The mass ratio of the triterpenoid saponin compound to nervonic acid is 1:(2-10).

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