Compositions, compounds, wolfberry agmatine-enriched fractions and neuroprotective uses thereof

Abstract

The present application provides a composition, a compound, a wolfberry agmatine enriched part and its neuroprotective use. The wolfberry agmatine enriched part extracted from wolfberry fruit has excellent neurocyte protection effect and prevention and treatment effect on Parkinson's disease. The wolfberry agmatine enriched part has simple extraction process and excellent effect, and its neuroprotective effect is not only superior to wolfberry fruit water extract and wolfberry polysaccharide enriched part, but also superior to each wolfberry agmatine monomer.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine extraction. Specifically, this invention relates to a composition, a compound, and a spermidine-rich fraction of Lycium barbarum. This invention also relates to its use in nerve cell protection and in the prevention and treatment of diseases such as Parkinson's disease. Background Technology

[0002] Neurodegenerative diseases are a collective term for diseases caused by chronic, progressive degenerative changes in the central nervous system due to factors such as genetics, age, or oxidative stress. These include Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). Neurodegenerative diseases are chronic and prevalent, seriously threatening human health and quality of life.

[0003] Parkinson's disease (PD) is a common neurodegenerative disease, affecting 6.1 million people worldwide, with a prevalence rate of 1.7% in my country's population aged 65 and above. The main clinical symptoms of PD include muscle tremors, rigidity, difficulty moving, postural and motor incoordination, and further progression can lead to cognitive, perceptual, and memory impairments, as well as significant dementia. The primary pathological feature of PD is the degeneration of dopaminergic neurons in the substantia nigra compacta and a significant reduction in dopaminergic neurotransmitter levels in the striatum. The motor and non-motor symptoms of PD not only impact patients' quality of life but also impose a significant social and medical burden. The causes of dopaminergic neuronal degeneration remain unclear, but genetic factors, infections, abnormal immune function, aging, and in vitro and in vivo neurotoxins all play a role in the pathogenesis of PD.

[0004] Currently, the main treatments for Parkinson's disease (PD) include drug therapy, surgical treatment, and gene therapy. Drug therapy remains the primary treatment in clinical practice. PD medications are mainly divided into two categories: drugs that affect dopaminergic neurons and anticholinergic drugs. The representative drug affecting dopaminergic neurons is levodopa (L-dopa). This drug primarily works by supplementing insufficient dopamine in the brain. However, long-term use of L-dopa can also cause adverse reactions such as motor disorders and altered mental status. Therefore, clinically, selective monoamine oxidase B inhibitors such as selegiline are often used in combination with L-dopa to increase the local dopamine concentration in the striatum, thereby enhancing the efficacy of L-dopa. Anticholinergic drugs are mainly used for mild Parkinson's disease. These drugs block striatal cholinergic receptors and simultaneously block presynaptic dopaminergic cholinergic receptors, reducing cholinergic function and thus exerting their therapeutic effect. However, since the fundamental pathological change in PD is the degeneration of dopaminergic neurons, anticholinergic drugs can only be used as an adjunct therapy. The medications mentioned above for treating Parkinson's disease (PD) only improve symptoms and have limited effect on slowing disease progression, let alone curing PD. The development of drugs with new mechanisms of action is urgently needed.

[0005] Applying drugs with potential neuroprotective effects to salvage degenerated neurons—a strategy known as neuroprotective therapy—is currently the latest international approach to treating neurodegenerative diseases. This strategy involves using appropriate medications in the early stages of the disease to target the pathophysiological processes of neurodegenerative diseases, interfering with a series of pathological and biochemical reactions in brain tissue caused by various etiologies, delaying or blocking the process of neuronal degeneration, protecting neurons, and restoring their function. Early application of neuroprotective therapy can fundamentally reverse or halt the progression of the disease. Therefore, neuroprotection is a relatively ideal strategy for treating neurodegenerative diseases. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention provides a composition, a compound, and a spermidine-rich fraction of Lycium barbarum, which have excellent neuroprotective effects and good preventive and therapeutic effects on Parkinson's disease.

[0007] In one aspect, the present invention provides a composition comprising at least two of the following compounds: Lycium barbarum spermidine L(11)

[0008] 4′,4″-O-β-D-dipyranosel-N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylspermine (14)

[0009] Lycium barbarum spermidine M(21)

[0010] 4′,3″-O-β-D-dipyranosel-N1 -trans-caffeoyl-N 10 -Dihydrocaffeoylspermine (23)

[0011] 4′,4″-O-β-D-dipyranosel-N 1 -Dihydrocaffeoyl-N 10 -trans-caffeoyl spermidine (26)

[0012] Lycium barbarum spermidine H / I (28 / 29)

[0013] Spermine D (30) from wolfberry

[0014] Spermine E (31) from wolfberry

[0015] 4′,4″-O-β-D-dipyranosel-N 1 N 10 -ditrans-caffeoyl spermidine (32)

[0016] Lycium barbarum spermidine F(33)

[0017] 4′,3″-O-β-D-dipyranosel-N 1 -Dihydrocaffeoyl-N 10 -trans-caffeoyl spermidine (34)

[0018] Spermine A from wolfberry (38)

[0019] N 1 N 10 -Di(dihydrocaffeoyl)spermine (39)

[0020] N 1 -cis-caffeoyl-N 10 -Dihydrocaffeoylspermine (41)

[0021] N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylspermine (45)

[0022] N 1 -Dihydrocaffeoyl-N 10 -trans-caffeoyl spermidine (46)

[0023] N 1 N 10 -ditrans-caffeoylsemidine (48).

[0024] Preferably, the composition comprises:

[0025] Selected from at least one of group a and at least one of group b,

[0026] Group a:

[0027] Lycium barbarum spermidine L(11)

[0028] 4′,4″-O-β-D-dipyranosel-N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylspermine (14)

[0029] Lycium barbarum spermidine M(21)

[0030] Lycium barbarum spermidine H / I (28 / 29)

[0031] Spermine D (30) from wolfberry

[0032] Spermine E (31) from wolfberry

[0033] Lycium barbarum spermidine F(33)

[0034] Spermine A from wolfberry (38)

[0035] N 1 N 10 -Di(dihydrocaffeoyl)spermine (39)

[0036] N 1 -cis-caffeoyl-N 10 -Dihydrocaffeoylspermine (41)

[0037] N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylspermine (45)

[0038] N 1 -Dihydrocaffeoyl-N 10 -trans-caffeoyl spermidine (46)

[0039] N 1 N 10 -trans-caffeoyl spermidine (48);

[0040] Group b:

[0041] 4′,3″-O-β-D-dipyranosel-N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylspermine (23)

[0042] 4′,4″-O-β-D-dipyranosel-N 1 -Dihydrocaffeoyl-N 10 -trans-caffeoyl spermidine (26)

[0043] 4′,4″-O-β-D-dipyranosel-N1 N 10 -ditrans-caffeoyl spermidine (32)

[0044] 4′,3″-O-β-D-dipyranosel-N 1 -Dihydrocaffeoyl-N 10 -trans-caffeoyl spermidine (34).

[0045] Preferably, the composition comprises at least two of the following compounds:

[0046] Lycium barbarum spermidine L(11)

[0047] 4′,4v-O-β-D-dipyranosel-N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylspermine (14)

[0048] Lycium barbarum spermidine M(21)

[0049] 4′,3″-O-β-D-dipyranosel-N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylspermine (23)

[0050] Lycium barbarum spermidine H / I (28 / 29)

[0051] Spermine E (31) from wolfberry

[0052] Lycium barbarum spermidine F(33)

[0053] Spermine A from wolfberry (38)

[0054] N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylsemidine (45).

[0055] Preferably, the composition comprises at least two of the following compounds:

[0056] Lycium barbarum spermidine L(11)

[0057] 4′,4″-O-β-D-dipyranosel-N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylspermine (14)

[0058] 4′,3″-O-β-D-dipyranosel-N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylspermine (23)

[0059] 4′,4″-O-β-D-dipyranosel-N1 -Dihydrocaffeoyl-N 10 -trans-caffeoyl spermidine (26)

[0060] Spermine E (31) from wolfberry

[0061] 4′,4″-O-β-D-dipyranosel-N 1 N 10 -ditrans-caffeoyl spermidine (32)

[0062] 4′,3″-O-β-D-dipyranosel-N 1 -Dihydrocaffeoyl-N 10 -trans-caffeoyl spermidine (34)

[0063] N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylsemidine (45).

[0064] Preferably, the composition comprises at least two of the following compounds:

[0065] Lycium barbarum spermidine L(11)

[0066] 4′,4″-O-β-D-dipyranosel-N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylspermine (14)

[0067] Spermine E (31) from wolfberry

[0068] N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylsemidine (45).

[0069] More preferably, the composition comprises at least one of the following combinations:

[0070] Lycium barbarum spermidine L(11) and 4′,4″-O-β-D-dipyranose-N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoyl spermidine (14);

[0071] Lycium barbarum spermidine L(11) and Lycium barbarum spermidine E(31);

[0072] Lycium barbarum spermidine L(11) and N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoyl spermidine (45);

[0073] 4′,4″-O-β-D-dipyranosel-N 1-trans-caffeoyl-N 10 -Dihydrocaffeoyl spermidine (14) and lycium barbarum spermidine E (31);

[0074] 4′,4″-O-β-D-dipyranosel-N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylspermine (14) and N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoyl spermidine (45);

[0075] Lycium barbarum spermidine E(31) and N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylsemidine (45).

[0076] Preferably, in the composition, the content of each component is independently as follows:

[0077]

[0078] In another aspect, the present invention also provides a wolfberry spermidine compound, which is selected from:

[0079] 4′-O-β-D-glucopyranosyl-4″-O-(4-O-β-D-glucopyranosyl-β-D-glucopyranosyl)-N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylsperimidamine (20)

[0080] 4′-O-(4-O-β-D-glucopyranosyl-β-D-glucopyranosyl)-4″-O-β-D-glucopyranosyl-N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylsperimimine (22)

[0081] 4′,3″-O-β-D-dipyranosel-N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylspermine (23)

[0082] 4′,4″-O-β-D-dipyranosel-N 1 -Dihydrocaffeoyl-N 10 -trans-caffeoyl spermidine (26)

[0083] 4′-O-(3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)-4″-O-β-D-glucopyranosyl-N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylsperimimine (27)

[0084] 4′,4″-O-β-D-dipyranosel-N 1 N 10 -ditrans-caffeoyl spermidine (32)

[0085] 4′,3″-O-β-D-dipyranosel-N 1 -Dihydrocaffeoyl-N 10 -trans-caffeoyl spermidine (34)

[0086] 3′-O-(3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)-4″-O-β-D-glucopyranosyl-N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylspermine (35)

[0087] 4′,3″-O-β-D-dipyranosel-N 1 N 10 -ditrans-caffeoyl spermidine (40).

[0088] The present invention also provides a composition consisting of at least two of the above-mentioned wolfberry spermidine compounds.

[0089] In this invention, the "at least two" includes two, three, four, five, six, or more.

[0090] In another aspect, the present invention also provides a spermidine-rich fraction of wolfberry.

[0091] In one embodiment, the spermidine enrichment site of lycopene includes the composition of the present invention or any one of the compounds comprising spermidine of lycopene.

[0092] In one embodiment, the spermidine-rich fraction of wolfberry can be extracted from wolfberry.

[0093] Preferably, the HPLC characteristic chromatogram of the spermidine-enriched fraction of Lycium barbarum mainly contains 9 chromatographic peaks, including 4',4"-O-β-D-dipyranosel-N 1 -trans-caffeoyl-N 10 The retention time of dihydrocaffeoyl spermidine (14) was set to 1, and the relative retention times of the main chromatographic peaks were calculated. The relative retention time of lycopene spermidine L (11) was 0.96. 1 -trans-caffeoyl-N 10The relative retention time of dihydrocaffeoyl spermidine (14) was 1.00, and the relative retention time of lycium barbarum spermidine M (21) was 1.04. 1 -trans-caffeoyl-N 10 The relative retention times of dihydrocaffeoyl spermidine (23) were 1.08, those of lycopene spermidine H / I (28 / 29) were 1.12, those of lycopene spermidine E (31) were 1.17, those of lycopene spermidine F (33) were 1.29, and those of lycopene spermidine A (38) were 1.32. 1 -trans-caffeoyl-N 10 The relative retention time of -dihydrocaffeoylspermine (45) was 1.54.

[0094] The HPLC characteristic chromatogram of the spermidine-rich fraction of Lycium barbarum was established by reversed-phase high-performance liquid chromatography. The chromatographic conditions were as follows: octadecylsilane and silica gel were used as the stationary phase, and acetonitrile-water solution containing 0.2% formic acid was used as the mobile phase for gradient elution. The flow rate was 1 mL / min, the detection wavelength was 280 nm, and the column temperature was 30 °C.

[0095] Preferably, the spermidine-enriched fraction of wolfberry is prepared by the following method: extracting dried wolfberry fruit with ethanol, methanol or water, and concentrating the extract under reduced pressure to obtain a concentrated solution; centrifuging the concentrated solution, passing the supernatant through macroporous adsorption resin column chromatography, eluting an appropriate column bed volume with water and / or 10% to 95% ethanol-water, collecting the eluent, and concentrating the 20-40% ethanol-water eluent under reduced pressure to obtain the spermidine-enriched fraction of wolfberry.

[0096] Preferably, the extraction method includes thermal extraction or ultrasonic extraction.

[0097] Preferably, the extraction is performed 1-3 times.

[0098] Preferably, the amount of ethanol, methanol, or water is 5-15 times the weight of the dried wolfberry fruit.

[0099] Preferably, the 30% ethanol-water eluent is concentrated under reduced pressure to obtain the spermidine-rich fraction of wolfberry.

[0100] Preferably, the elution step includes elution sequentially with 2-5 column bed volumes of water, 2-5 column bed volumes of 5-15% ethanol-water, 3-6 column bed volumes of 20-40% ethanol-water, and 3-6 column bed volumes of 90-98% ethanol-water. More preferably, elution is performed with 3-4 column bed volumes of water, 3-4 column bed volumes of 10% ethanol-water, 4-5 column bed volumes of 30% ethanol-water, and 4-5 column bed volumes of 95% ethanol-water.

[0101] In another aspect, the present invention also provides a method for separating the aforementioned spermidine compounds from wolfberry, comprising:

[0102] The spermidine-rich fraction of Lycium barbarum was separated by medium-low pressure liquid chromatography with ODS column chromatography and preparative HPLC to obtain the aforementioned spermidine compound.

[0103] In one implementation, the separation method includes:

[0104] The spermidine-rich fraction of Lycium barbarum was subjected to medium-low pressure liquid chromatography (LC-ODS) column chromatography, eluted sequentially with methanol-water-formic acid at volume ratios of 5:95:0.2, 10:90:0.2, 15:85:0.2, 20:80:0.2, 30:70:0.2, 50:50:0.2, and 100:0:0.2, yielding six fractions: F3.1, F3.2, F3.3, F3.4, F3.5, and F3.6. Fraction F3.3 was further refined by reversed-phase preparative HPLC (Cosmosil C18). The sample was eluted with methanol-water-trifluoroacetic acid at a flow rate of 8 mL / min and a volume ratio of 18:82:0.1 to obtain five fractions: F3.3.1, F3.3.2, F3.3.3, F3.3.4, and F3.3.5. Fraction F3.3.5 was then prepared by reversed-phase semi-preparative HPLC (Phenomenex C18 column) using methanol-water-trifluoroacetic acid at a flow rate of 3 mL / min and a volume ratio of 20:80:0.1 to obtain seven fractions: F3.3.5.1, F3.3.5.2, F3.3.5.3, F3.3.5.4, F3.3.5.5, F3.3.5.6, and F3.3.5.7.

[0105] Fraction F3.3.5.1 was prepared by reversed-phase semi-preparative HPLC (Phenomenex C18 column) using acetonitrile-water-trifluoroacetic acid at a flow rate of 3 mL / min (v / v ratio 8:92:0.1) to obtain trifluoroacetate salts of compounds 20 and 22; fraction F3.3.5.2 was prepared by reversed-phase semi-preparative HPLC (YMC C18 column) using methanol-water-trifluoroacetic acid at a flow rate of 3 mL / min (v / v ratio 15:85:0.1) to obtain trifluoroacetate salts of compounds 23, 26, and 27; fraction F3.3.5.3 was prepared by reversed-phase semi-preparative HPLC (Phenomenex C18 column). The fraction F3.3.5.4 was eluted with methanol-water-trifluoroacetic acid at a flow rate of 3 mL / min and a volume ratio of 17:83:0.1 to obtain the trifluoroacetate of compound 32. The fraction F3.3.5.4 was prepared by reversed-phase semi-preparative HPLC (YMC C18 column) with acetonitrile-water-trifluoroacetic acid at a flow rate of 3 mL / min and a volume ratio of 11:89:0.1 to obtain the trifluoroacetate of compounds 34 and 35. The fractions F3.3.5.5 and F3.3.5.6 were prepared by reversed-phase semi-preparative HPLC (YMC C18 column) with acetonitrile-water-trifluoroacetic acid at a flow rate of 3 mL / min and a volume ratio of 12:88:0.1 to obtain the trifluoroacetate of compound 40.

[0106] Optionally, the separation method further includes a process for obtaining the target product from its trifluoroacetate. The raw materials, reagents, and operations required for this process are well known in the art.

[0107] Another aspect of the present invention is to provide a pharmaceutical composition comprising the composition described herein, a lycopene spermidine compound, or a lycopene spermidine enrichment fraction. The pharmaceutical composition may also comprise a pharmaceutically acceptable carrier.

[0108] Another aspect of the present invention is to provide a health product comprising the composition described in the present invention, a lycopene spermidine compound, or a lycopene spermidine-rich fraction.

[0109] Another aspect of the present invention is to provide a food or food additive comprising the composition described herein, a lycopene spermidine compound, or a lycopene spermidine enrichment fraction.

[0110] Another aspect of the present invention is to provide the use of the composition, the lycopene spermidine compound, or the lycopene spermidine enrichment fraction in the preparation of a medicament or health product, the medicament being used to protect nerve cells.

[0111] Another aspect of the present invention is to provide the use of the composition, the lycopene spermidine compound, or the lycopene spermidine enrichment fraction in the preparation of a food or food additive for the protection of nerve cells.

[0112] Another aspect of the present invention is to provide the use of the composition, the lycopene spermidine compound, or the lycopene spermidine enrichment fraction in the preparation of a medicament or health product for the prevention or treatment of neurodegenerative diseases.

[0113] Another aspect of the present invention is to provide the use of the composition, the lycopene spermidine compound, or the lycopene spermidine enrichment fraction in the preparation of food or food additives for the prevention or treatment of neurodegenerative diseases.

[0114] Preferably, the neurodegenerative disease includes diseases in the group consisting of Parkinson's disease, Alzheimer's disease, Lewy body dementia, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, prion diseases, HIV-related dementia, and any form of cognitive impairment associated with neurodegeneration, preferably Parkinson's disease.

[0115] Beneficial effects

[0116] This invention provides a composition, a compound, a spermidine-rich fraction of Lycium barbarum, and their uses. The composition of this invention comprises several compounds extracted from Lycium barbarum, particularly those derived from the spermidine-rich fraction of Lycium barbarum, some of which are novel spermidine compounds. The composition, compounds, and the spermidine-rich fraction of Lycium barbarum exhibit excellent neuroprotective effects and preventative and therapeutic effects against Parkinson's disease, among others. The extraction process of the spermidine-rich fraction of Lycium barbarum is simple and highly effective; its neuroprotective effect is superior not only to Lycium barbarum aqueous extract and Lycium barbarum polysaccharide-rich fractions, but also to individual spermidine monomers. Attached Figure Description

[0117] Figure 1 The HPLC chromatogram of the spermidine-rich fraction of wolfberry in Example 1 is shown, where 1-26 represent the peak numbers.

[0118] Figure 2 The HPLC chromatogram of the spermidine-rich fraction of wolfberry in Example 1 is shown, where 1-48 represents the compound number.

[0119] Figure 3 The effect of spermidine-rich region (XZ9-3) of Lycium barbarum on pole-climbing behavior in mice. (A) 21 days, (B) 27 days. Compared with the control group, ＊＊＊＊ P < 0.001; compared with MPTP mice, # P < 0.05 ###P < 0.005 #### P < 0.001.

[0120] Figure 4 The effect of spermidine-rich fraction (XZ9-3) from Lycium barbarum on grip strength in mice. (A) 21 days, (B) 27 days. Compared with the control group, ＊＊＊＊ P < 0.001; compared with MPTP mice, # P < 0.05 ## P < 0.01, #### P < 0.001.

[0121] Figure 5 The effect of spermidine-rich fraction (XZ9-3) from Lycium barbarum on twirling behavior in mice. (A) 21 days, (B) 27 days. Compared with the control group, ** P < 0.01, ＊＊＊* P < 0.001; compared with MPTP mice, # P < 0.05 ## P < 0.01, ### P < 0.005 #### P < 0.001.

[0122] Figure 6 The effects of spermidine-rich fraction (XZ9-3) from Lycium barbarum on DA, DOPACA, and HVA in the striatum of mice. (A) DA, (B) DOPAC, (C) HVA. Compared with the control group, ＊ P < 0.05 ＊＊ P < 0.01; compared with MPTP mice, # P<0.05, ## P < 0.01, ### P < 0.005.

[0123] Figure 7 The effect of spermidine-rich region (XZ9-3) of Lycium barbarum on the number of dopaminergic neurons and TH expression in mice. (A) Number of dopaminergic neurons, (B) TH expression. Compared with the control group, ＊＊ P < 0.01; compared with MPTP mice, # P < 0.05 ## P < 0.01. Detailed Implementation

[0124] The invention is described in more detail below to aid in understanding it.

[0125] It should be understood that the terms or words used in the specification and claims should not be construed as having the meaning defined in a dictionary, but rather as having a meaning consistent with their meaning in the context of the invention, based on the principle that the concept of a term may be appropriately defined by the inventor for the best explanation of the invention.

[0126] Preparation Example 1: Preparation Method of Spermine-Enriched Fraction from Lycium barbarum

[0127] The extraction method for spermidine-rich fractions from Lycium barbarum was as follows: 2.1 kg of Lycium barbarum was extracted twice by heating and reflux with 10 times the volume of water, 2 hours each time. The extract was filtered and concentrated to obtain approximately 10 L of concentrate. A small amount of the concentrate was evaporated to dryness, and the total weight of the Lycium barbarum aqueous extract was estimated to be 1.71 kg. Most of the concentrate (the corresponding weight of the aqueous extract was 1.63 kg) was centrifuged, and the supernatant was added to a prepared HP-20 macroporous adsorption resin column (13×120 cm). The sample solution outflow rate was controlled at 2-2.5 times the column bed volume / h by adjusting the valve. After repeated loading 3 times, the valve was closed, and static adsorption was allowed for 12 hours. After static adsorption, the solution was eluted sequentially with 3 column bed volumes of water, 3 column bed volumes of 10% ethanol-water, 4 column bed volumes of 30% ethanol-water, and 4 column bed volumes of 95% ethanol-water. The eluents were collected, and the solvents were recovered under reduced pressure. The resulting eluents were 1.41 kg of water, 35.2 g of 10% ethanol-water, 81.6 g of 30% ethanol-water, and 18.2 g of 95% ethanol-water. The water eluent was the enriched portion of Lycium barbarum polysaccharides, and the 30% ethanol-water eluent was the enriched portion of Lycium barbarum spermidine.

[0128] Preparation Example 2: Isolation, identification, and HPLC-MS analysis of new compounds from the spermidine-rich fraction of Lycium barbarum.

[0129] The HPLC characteristic chromatogram of the spermidine enriched fraction of Lycium barbarum prepared in Example 1 is as follows: Figure 1 and Figure 2 As shown in the characteristic chromatogram, compounds 1-19, 21, 24, 25, 28-31, 33, 36-39, and 41-48 were identified by comparison with standards and HPLC-MS. Notably, guided by the characteristic chromatogram, 4′-O-β-D-glucopyranosyl-4″-O-(4-O-β-D-glucopyranosyl-β-D-glucopyranosyl)-N were identified using chromatographic separation techniques such as ODS column chromatography and reversed-phase HPLC, and spectroscopic techniques such as UV, MS, and NMR. 1 -trans-caffeoyl-N 10-Dihydrocaffeoylsemidine (20), 4′-O-(4-O-β-D-glucopyranosyl-β-D-glucopyranosyl)-4"-O-β-D-glucopyranosyl-N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylsemidine (22), 4′,3″-O-β-D-dipyranose-N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylsemidine (23), 4′,4"-O-β-D-dipyranose-N 1 -Dihydrocaffeoyl-N 10 -trans-caffeoyl spermidine (26), 4′-O-(3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)-4"-O-β-D-glucopyranosyl-N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylsemine (27), 4′,4″-O-β-D-dipyranose-N 1 N 10 -ditrans-caffeoylspermine (32), 4′,3″-O-β-D-dipyranose-N 1 -Dihydrocaffeoyl-N 10 -trans-caffeoyl spermidine (34), 3"-O-(3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)-4"-O-β-D-glucopyranosyl-N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylspermine (35) and 4',3"-O-β-D-dipyranosel-N 1 N 10 -The structure of these 9 new compounds: trans-caffeoyl spermidine (40).

[0130] Under the same chromatographic conditions as the spermidine-rich fraction of Lycium barbarum, the newly isolated compounds 20, 22, 23, 26, 27, 32, 34, 35, and 40 were identified. Details of the identification are as follows: Figure 1 and Figure 2 .

[0131] 2.1 Separation Process

[0132] The spermidine-enriched fraction obtained from Lycium barbarum was subjected to medium-low pressure liquid chromatography (LC-ODS column chromatography) using methanol-water-formic acid at volume ratios of 5:95:0.2, 10:90:0.2, 15:85:0.2, 20:80:0.2, 30:70:0.2, 50:50:0.2, and 100:0:0.2, yielding six fractions: F3.1, F3.2, F3.3, F3.4, F3.5, and F3.6. Fraction F3.3 was then prepared by reversed-phase preparative HPLC (Cosmosil C18 column) using methanol-water-trifluoroacetic acid at a flow rate of 8 mL / min, yielding five fractions: F3.3.1, F3.3.2, F3.3.3, F3.3.4, and F3.3.5. Fraction F3.3.5 was prepared by reversed-phase semi-preparative HPLC (Phenomenex C18 column) using methanol-water-trifluoroacetic acid at a flow rate of 3 mL / min and a volume ratio of 20:80:0.1, yielding a total of seven fractions: F3.3.5.1, F3.3.5.2, F3.3.5.3, F3.3.5.4, F3.3.5.5, F3.3.5.6, and F3.3.5.7.

[0133] Fraction F3.3.5.1 was prepared by reversed-phase semi-preparative HPLC (Phenomenex C18 column) using acetonitrile-water-trifluoroacetic acid (8:92:0.1, v / v) at a flow rate of 3 mL / min to obtain trifluoroacetic acid salts of compounds 20 and 22. Fraction F3.3.5.2 was prepared by reversed-phase semi-preparative HPLC (YMC C18 column) using methanol-water-trifluoroacetic acid (15:85:0.1, v / v) at a flow rate of 3 mL / min to obtain trifluoroacetic acid salts of compounds 23, 26, and 27. Fraction F3.3.5.3 was prepared by reversed-phase semi-preparative HPLC (Phenomenex C18 column) using methanol-water-trifluoroacetic acid (17:83:0.1, v / v) at a flow rate of 3 mL / min to obtain trifluoroacetic acid salt of compound 32. Fraction F3.3.5.4 was prepared by reversed-phase semi-preparative HPLC (YMC C18 column) using acetonitrile-water-trifluoroacetic acid at a flow rate of 3 mL / min (v / v ratio 11:89:0.1) to obtain trifluoroacetate salts of compounds 34 and 35. Fractions F3.3.5.5 and F3.3.5.6 were prepared by reversed-phase semi-preparative HPLC (YMC C18 column) using acetonitrile-water-trifluoroacetic acid at a flow rate of 3 mL / min (v / v ratio 12:88:0.1) to obtain trifluoroacetate salt of compound 40.

[0134] 2.2 The physicochemical data of the new compound are as follows:

[0135] Trifluoroacetate of compound 20: a green oily liquid; -22.0(c 0.10, MeOH); UV(MeOH)λ max (log ε)203(4.15), 285(3.79), 319(3.65)nm; IR(KBr)v max 3324, 1685, 1440, 1207, 1141cm -1 ;ESI-MS(positive): m / z958.5[M+H] + ;ESI-MS(negative): m / z956.7[MH] - ;HR-ESI-MS(positive): m / z 958.4054[M+H] + (calcd.for C 43 H 64 N3O 21 (958.4032), the molecular formula of the compound was determined to be C 43 H 63 N3O 21 ;1H and 13 The C NMR data are shown in Table 1.

[0136] Trifluoroacetate of compound 22: a green oily liquid; -18.0(c 0.10, MeOH); UV(MeOH)λ max (log ε)204(4.03), 285(3.53), 318(3.39)nm; IR(KBr)v max 3296, 1686, 1438, 1210, 1141cm -1 ;ESI-MS(positive): m / z958.4[M+H] + ;ESI-MS(negative): m / z956.6[MH] - ; HR-ESI-MS(positive): m / z 958.4050[M+H] + (calcd.for C 43 H 64 N3O 21 (958.4032), the molecular formula of the compound was determined to be C 43 H 63 N3O 21 ; 1 H and 13 The C NMR data are shown in Table 1.

[0137] Trifluoroacetate of compound 23: a green oily liquid; -16.0(c 0.10, MeOH); UV(MeOH)λ max (log ε)203(4.06), 286(3.71), 315(3.58)nm; IR(KBr)v max 3271, 1685, 1438, 1210, 1135cm -1 ;ESI-MS(positive): m / z796.3[M+H] + ;ESI-MS(negative): m / z794.4[MH] - ; HR-ESI-MS(positive): m / z 796.3536[M+H] + (calcd.for C 37 H 54 N3O 16 (796.3504), the molecular formula of the compound was determined to be C 37 H 53 N3O 16 ; 1 H and 13 The C NMR data are shown in Table 1.

[0138] Trifluoroacetate of compound 26: a green oily liquid; -28.0(c 0.10, MeOH); UV(MeOH)λ max (log ε)204(4.22), 284(3.63), 317(3.46)nm; IR(KBr)v max 3309, 1689, 1438, 1207, 1144cm -1 ;ESI-MS(positive): m / z796.3[M+H] + ;ESI-MS(negative): m / z794.4[MH] - ; HR-ESI-MS(positive): m / z 796.3529[M+H] + (calcd.for C 37 H 54 N3O 16 (796.3504), the molecular formula of the compound was determined to be C 37 H 53 N3O 16 ; 1 H and 13 The C NMR data are shown in Table 1.

[0139] Trifluoroacetate of compound 27: a green oily liquid; -18.0(c 0.10, MeOH); UV(MeOH)λ max (log ε)204(4.08), 284(3.52), 319(3.35)nm; IR(KBr)v max 3321, 1689, 1442, 1207, 1138cm -1 ;ESI-MS(positive): m / z958.4[M+H] + ;ESI-MS(negative): m / z956.5[MH] - ; HR-ESI-MS(positive): m / z 958.4025[M + H] + (calcd.for C 43 H 64 N3O 21 (958.4032), the molecular formula of the compound was determined to be C 43 H 63 N3O 21 ; 1 H and 13 The C NMR data are shown in Table 1.

[0140] Trifluoroacetate of compound 32: a green oily liquid; -16.0(c 0.10, MeOH); UV(MeOH)λ max (log ε)209(3.98), 288(3.87), 316(3.76)nm; IR(KBr)v max 3436, 1685, 1438, 1205, 1142cm -1 ;ESI-MS(positive): m / z794.3[M+H] + ;ESI-MS(negative): m / z792.5[MH] - ; HR-ESI-MS(positive): m / z 794.3380[M + H]+(calcd.for C 37 H 52 N3O 16 (794.3348), the molecular formula of the compound was determined to be C 37 H 51 N3O 16 ; 1 H and 13The C NMR data are shown in Table 2.

[0141] Trifluoroacetate of compound 34: green oily liquid; -42.0(c 0.10, MeOH); UV(MeOH)λ max (log ε)203(4.46), 290(4.10), 313(4.12)nm; IR(KBr)v max 3287, 1685, 1438, 1207, 1138cm -1 ;ESI-MS(positive): m / z 796.3[M+H] + ;ESI-MS(negative): m / z 794.5[MH] - ;HR-ESI-MS(positive): m / z 796.3532[M+H] + (calcd.for C 37 H 54 N3O 16 (796.3504), the molecular formula of the compound was determined to be C 37 H 53 N3O 16 ; 1 H and 13 The C NMR data are shown in Table 2.

[0142] Trifluoroacetate of compound 35: a green oily liquid; -24.0(c 0.10, MeOH); UV(MeOH)λ max (log ε)203(4.37), 288(4.03), 314(4.09)nm; IR(KBr)v max 3296, 1682, 1438, 1207, 1135cm -1 ;ESI-MS(positive): m / z958.4[M+H] + ;ESI-MS(negative): m / z956.7[MH] - ; HR-ESI-MS(positive): m / z 958.4094[M+H]+(calcd.for C 43 H 64 N3O 21 (958.4032), the molecular formula of the compound was determined to be C 43 H 63 N3O 21 ; 1 H and 13 The C NMR data are shown in Table 2.

[0143] Trifluoroacetate of compound 40: a green oily liquid; -36.0(c 0.10, MeOH); UV(MeOH)λ max (log ε)213(4.28), 291(4.28), 313(4.26)nm; IR(KBr)v max 3288, 1683, 1435, 1205, 1139cm -1 ;ESI-MS(positive): m / z794.3[M+H] + ;ESI-MS(negative): m / z792.5[MH] - ;HR-ESI-MS(positive): m / z 794.3386[M+H] + (calcd.for C 37 H 52 N3O 16 (794.3348), the molecular formula of the compound was determined to be C 37 H 51 N3O 16 ; 1 H and 13 The C NMR data are shown in Table 2.

[0144] Table 1. New compounds 20, 22, 23, 26 and 27 1 H and 13 C NMR data and attribution.

[0145]

[0146]

[0147]

[0148] * Signal attribution may be interchangeable.

[0149] a Indistinguishable signals due to signal overlap or complex peak shapes are not indicated as peak multiplicity in reports.

[0150] b NMR data of the compound in DMSO-d6 (600 MHz for 1 H NMR, 150MHz for 13 (C NMR).

[0151] Table 2. New compounds 32, 34, 35 and 40 1 H and13 C NMR data and attribution.

[0152]

[0153]

[0154]

[0155] * Signal attribution may be interchangeable.

[0156] a Indistinguishable signals due to signal overlap or complex peak shapes are not indicated as peak multiplicity in reports.

[0157] b NMR data of the compound in DMSO-d6 (600 MHz for 1 H NMR, 150MHz for 13 (C NMR).

[0158] 2.3 HPLC-MS analysis of spermidine-rich fractions in Lycium barbarum

[0159] Chromatographic conditions

[0160] Capcell Pak C18 MG II (S-5), 250 × 4.6 mm; mobile phase: solvent A (water, 0.2% formic acid) and solvent B (acetonitrile), gradient elution (0 min - 5% B, 2 min - 5% B, 17 min - 7% B, 37 min - 10% B, 56 min - 15% B, 65 min - 25% B, 66 min - 100% B, 75 min - 100% B, 76 min - 5% B), flow rate: 1 mL / min, column temperature: 30 °C, detection wavelength: 280 nm.

[0161] Mass spectrometry conditions

[0162] Electrospray positive and negative ion modes; ion spray voltage: +3.5kV and -2.5kV; auxiliary temperature: 350℃; capillary temperature: 320℃; sheath gas flow rate: 30arb; auxiliary gas flow rate: 10arb; backflush gas flow rate: 2arb; scan range: m / z 100-1500; resolution: 70000, 17500; collision energy: 15, 37.5, 50eV.

[0163] The result is as follows Figure 1 and 2 As shown in Table 3-5.

[0164] Table 3. Correspondence between HPLC peaks and compound structures.

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174] Table 4. Content determination information of 17 spermidine components from Lycium barbarum in a spermidine-rich fraction of Lycium barbarum.

[0175]

[0176]

[0177]

[0178]

[0179]

[0180] Experimental Example 1: Protective effect of the test sample on rotenone-damaged SH-SY5Y neurons

[0181] 1 Experimental Methods

[0182] SH-SY5Y cells were cultured in DMEM medium. SH-SY5Y cells in the logarithmic growth phase were harvested, digested with trypsin, and then injected with 8 × 10⁻⁶ cells / mL. 4Cells were seeded at a density of 100 μL / well in 96-well plates and cultured at 37°C in a 5% CO2 incubator for approximately 24 hours. When cells reached approximately 60% confluence, the test samples were diluted with DMEM medium to the corresponding concentrations: 100 mg / L, 50 mg / L, 10 mg / L, 5 mg / L, 2.5 mg / L, and 1 mg / L. The cells were then incubated with the diluted DMEM medium for 2 hours. After 2 hours, the model group and the sample group were stimulated with 10 mM rotenone, while the control group was treated with an equal volume of DMEM medium. Culture was continued for approximately 22 hours. Cell viability was assessed using CCK-8 assay. CCK-8 solution was added to each well at a 1:10 ratio, and the plates were incubated. After 2-4 hours, the absorbance (OD value) was measured at 450 nm using a microplate reader. The results were calculated using the following formula: (OD value...) drug -OD Model ) / (OD Control -OD Model )×100%, by EC 50 The calculator calculates EC 50 .

[0183] 2 Experimental Results

[0184] As shown in Table 6, in rotenone-damaged SH-SY5Y cells, the spermidine-rich areas of Lycium barbarum significantly improved cell survival rate and EC50. 50 The concentration was 5.46 mg / L, which was significantly better than other tested samples, showing obvious neuroprotective activity. In addition, the following spermidines were also tested: Lycium barbarum spermidine A (38), Lycium barbarum spermidine B (42), Lycium barbarum spermidine D (30), Lycium barbarum spermidine E (31), Lycium barbarum spermidine F (33), Lycium barbarum spermidine H / I (28 / 29), Lycium barbarum spermidine L (11), Lycium barbarum spermidine M (21), Lycium barbarum spermidine N (5), and 4′,4"-O-β-D-dipyranose glucosyl-N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoylspermine (14), N 1 N 10 -Di(dihydrocaffeoyl)semine (39), N 1 -trans-caffeoyl-N 10 -Dihydrocaffeoyl spermidine (45) and the polysaccharide-rich fraction of Lycium barbarum have certain neuroprotective effects, while the water extract of Lycium barbarum has no obvious neuroprotective effect.

[0185] Table 6. Protective effect of the test samples on rotenone-damaged SH-SY5Y neurons (n=5).

[0186]

[0187] ＊ P < 0.05＊＊ P < 0.01 vs. the location of spermidine enrichment in wolfberry.

[0188] Table 7. Compound names, corresponding numbers, and structural information.

[0189]

[0190]

[0191]

[0192] Experimental Example 2: Pharmacodynamic Evaluation of the Treatment of a PD Mouse Model by Extraction of Spermine-Enriched Sites in Lycium barbarum

[0193] 1. Experimental Objective

[0194] Parkinson's disease (PD) is an age-related neurodegenerative disease characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta, ultimately leading to dopamine depletion in the midbrain striatum. Intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces dopaminergic neuronal degeneration and death in the substantia nigra of mice, along with a reduction in tyrosine hydroxylase (TH)-positive cells and nerve fibers, mimicking the pathological manifestations of PD and making it a common PD model induction drug. Therefore, this experiment used intraperitoneal injection of MPTP (20 mg / kg) for 14 consecutive days to induce a PD mouse model and evaluated the therapeutic effect of the spermidine-rich portion of Lycium barbarum on PD mice.

[0195] 2 Experimental Materials

[0196] 2.1 Laboratory Animals

[0197] SPF-grade male C57BL / 6J mice, weighing 22-24g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Five mice were housed in barrier-grade SPF animal enclosures at room temperature with free access to food and water. All procedures performed in accordance with the regulations of the Beijing Laboratory Animal Ethics Committee.

[0198] 2.2 Medicines and Reagents

[0199] MPTP was purchased from Sigma; the positive control drug selegiline tablets were provided by Orion (Finland) Pharmaceuticals Ltd.; the test substance, spermidine enrichment fraction of Lycium barbarum (XZ9-3), was prepared as in Preparation Example 1.

[0200] Drug preparation: MPTP was prepared into a 2 mg / mL solution using physiological saline; selegiline was prepared into a 1 mg / mL solution using sterile triple-distilled water; and XZ9-3 was prepared into 1 mg / mL, 5 mg / mL, and 10 mg / mL solutions using sterile triple-distilled water.

[0201] 3 Experimental Methods

[0202] 3.1 Establishment of PD mouse model and grouping and administration

[0203] C57 / BL6J mice were acclimatized for three days, then randomly divided into a blank control group (Control), an MPTP model group, an XZ9-3 10 mg / kg group, an XZ9-3 50 mg / kg group, an XZ9-3 100 mg / kg group, and a positive control selegiline (10 mg / kg) group. Mice in each group were administered the corresponding drug via gavage at a dose of 10 mL / kg. 30 minutes after administration, MPTP (20 mg / kg) was injected intraperitoneally once daily for 14 consecutive days. After MPTP injection was discontinued, mice were continuously administered the test drug and the positive control selegiline once daily for 14 consecutive days. On days 19, 20, 21, 25, 26, and 27, MPTP (15 mg / kg) was injected intraperitoneally 30 minutes after drug administration to maintain the mice's dyskinesia. Behavioral tests of stick spinning, pole climbing, and grip strength were performed on days 21 and 27. Animals were processed after the last behavioral test. The brains of 5 mice in each group were fixed in 4% paraformaldehyde, and the striatum and midbrain of the remaining mice were taken for testing relevant indicators.

[0204] 3.2 Detection Indicators

[0205] 3.2.1 Rotating Roller Experiment

[0206] The spinner test is a classic method for assessing motor coordination in mice. The spinner (Ugo Basie) consists of a horizontal metal rod (3 cm in diameter) approximately 50 cm long, divided into 5 segments by metal plates, with barriers separating the animals to prevent interference. The spinner rotates at a constant speed of 25 rpm. The mouse is placed on the rod, and the timer begins until the mouse falls off; this latency period (the time the mouse remains on the spinner) is recorded and represents its motor coordination ability. Each mouse is tested twice, 30 minutes apart, and the average of the two results is taken.

[0207] 3.2.2 Pole Climbing Experiment

[0208] The pole-climbing test is commonly used to evaluate limb coordination in mice. A pole-climbing apparatus (Institute of Materia Medica, Chinese Academy of Medical Sciences) with a diameter of 13 mm and a height of 150 mm is erected vertically. The mouse is placed head-down at the top of the metal pole and allowed to climb down naturally. The animal's behavior during the descent is observed. The mouse's behavior during the descent is scored according to a standard, as follows:

[0209] 5 points: Using all four limbs, crawling downwards in a coordinated manner step by step;

[0210] 4 points: Crawling downwards step by step, but also exhibiting gliding behavior with hind limbs;

[0211] 3 points: After climbing halfway, slide downhill, but you can hold on to the metal pole;

[0212] 2 points: Sliding behavior occurred before climbing more than half the distance;

[0213] 1 point: Do not fall off the pole after climbing halfway;

[0214] 0 points: Failed to grab the pole before climbing halfway and fell off.

[0215] 3.2.3 Grip strength test

[0216] The grip strength test is also an indicator of a mouse's motor ability. A nylon rope is suspended above the mouse cage, with sufficient bedding inside. Once the mouse grasps the rope with its front paws, it is immediately released. The mouse's behavior on the rope is recorded and scored. The scoring criteria are as follows:

[0217] 5 points: Attempting to move horizontally on the rope

[0218] 4 points: Limbs and tail wrapped around the rope

[0219] 3 points: Attempting to climb the rope or having its front paws and at least one hind paw hanging on the rope at the same time.

[0220] 2 points: Front paws hang on the rope (for more than 5 seconds)

[0221] 1 point: Front paws hooked onto the rope (less than 5 seconds)

[0222] 0 points: Fell directly off the rope (no gripping ability)

[0223] 3.2.4 Determination of monoamine neurotransmitter content in mouse striatum by HPLC-MS / MS method

[0224] High-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) is characterized by high separation capability, high selectivity, high sensitivity, and reliable qualitative analysis results, making it suitable for the analysis and determination of complex biological samples. After the final behavioral test, the striatum of each group of mice was collected, and the contents of the monoamine neurotransmitter dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) in the mouse striatum were determined by HPLC-MS / MS.

[0225] 3.2.5 Tissue Immunofluorescence

[0226] Following the final behavioral test, mice were anesthetized and perfused with paraformaldehyde (n=5 per group). The brains were then fixed in a 30% sucrose-PBS solution containing 4% paraformaldehyde. Serial sections of the substantia nigra were prepared coronally using a cryostat to a thickness of 20 μm. Sections were incubated with 3% H₂O₂ at room temperature for 30 min, washed with PBST, blocked with 5% goat serum for 30 min, and then incubated overnight with TH antibody at 4°C. After incubation with FITC fluorescent secondary antibody for 2 h, the colorimetric reaction was terminated with distilled water. The sections were dehydrated with a gradient of ethanol and xylene, and then mounted. Finally, the substantia nigra was observed under an upright microscope, and photographs were taken of selected compact regions to count the number of TH-immunopositive cells in the substantia nigra region.

[0227] 3.2.6 Western blot

[0228] Mouse midbrain was thoroughly homogenized with a mixture of RIPA lysis buffer, protease inhibitor, and protein phosphatase inhibitor. The homogenate was centrifuged at 12,000 rpm and 4°C for 30 min. The supernatant was collected and its protein concentration was determined using a BCA protein quantification kit. The protein concentration of all samples was adjusted to 5 mg / mL, and the mixture was boiled to denature the proteins. Proteins were separated by 8% SDS-PAGE gel electrophoresis, with a loading volume of 40 μg. After electrophoresis, the proteins were transferred to a PVDF membrane, blocked with 5% skim milk at room temperature with shaking for 2 h, and then incubated overnight at 4°C with TH primary antibody. The membrane was washed three times with TBST and then incubated for 2 h with the corresponding horseradish peroxidase-labeled secondary antibody. ECL chemiluminescence was performed using a LAS 4000, and the band grayscale was analyzed using Gelpro 32 software.

[0229] 3.3 Statistical Analysis

[0230] Experimental data are expressed as mean ± standard error (Mean ± SEM). Differences between different groups were compared using one-way ANOVA followed by the LSD-SNK test. P < 0.05 was considered statistically significant.

[0231] 4 Experimental Results

[0232] 4.1 Effect of the enrichment site of spermidine in Lycium barbarum (XZ9-3) on the pole climbing test in PD mice

[0233] PD mice underwent pole-climbing tests on days 21 and 27 after XZ9-3 administration, respectively. Figure 3 As shown, the difference in pole climbing test scores between the MPTP group and the Control group became increasingly significant over time. The pole climbing test results on day 27 showed that the pole climbing test scores of mice in all XZ9-3 dose groups and the selegiline group were significantly higher than those in the MPTP group.

[0234] 4.2 Effect of the spermidine-rich region of the test drug Lycium barbarum (XZ9-3) on grip strength in PD mice

[0235] Mice underwent grip strength tests on days 21 and 27 after drug administration. Figure 4 As shown, the difference in grip strength scores between the MPTP group and the Control group became increasingly significant over time. The final grip strength test results showed that the grip strength scores of mice in the XZ9-350mg / kg group, 100mg / kg group, and selegiline group were significantly higher than those in the MPTP group. The XZ9-310mg / kg group showed a trend towards improved grip strength, but no statistically significant difference was observed.

[0236] 4.3 Effects of the spermidine enrichment site (XZ9-3) of the test drug Lycium barbarum on the tumbling behavior of PD mice

[0237] PD mice underwent a spinner test on days 21 and 27 after drug administration, as follows: Figure 5 As shown, with increasing time, the differences in the spinner test results between the MPTP group and the Control group became increasingly significant, with a markedly shorter time spent on the spinner. On day 21, the spinner test results showed that, compared to MPTP mice, XZ9-3 50mg / kg, 100mg / kg, and selegiline all significantly increased the time mice spent on the spinner. On day 27, the spinner test results showed that the time spent on the spinner in all three XZ9-3 dose groups and the selegiline group was significantly longer than that in the MPTP group. These results indicate that XZ9-3 has a significant effect on improving motor coordination dysfunction in PD mice.

[0238] 4.4 Effects of the spermidine enrichment site (XZ9-3) of the test drug Lycium barbarum on monoamine neurotransmitters in the striatum of PD mice

[0239] After the final behavioral test, the striatum of mice was collected, and the contents of DA, DOPAC, and HVA in the striatum were determined by HPLC-MS / MS. Figure 6 As shown, the levels of DA, DOPAC, and HVA in the striatum of mice in the MPTP group were significantly lower than those in the Control group. The levels of DA in the striatum of mice in the XZ9-3 10 mg / kg and 100 mg / kg groups and the selegiline group were significantly higher than those in the MPTP group; the levels of DOPAC in the striatum of mice in all XZ9-3 dose groups and the selegiline group were slightly lower than those in the MPTP group; while the levels of HVA in the striatum of mice in all XZ9-3 dose groups and the selegiline group showed an increasing trend compared to the MPTP group, with the increase in the selegiline group being statistically significant.

[0240] 4.5 Effects of the spermidine-rich region (XZ9-3) of the test drug Lycium barbarum on the number of dopaminergic neurons and TH expression in PD mice

[0241] After the final behavioral test, mouse brains were harvested for immunofluorescence staining, such as... Figure 7 As shown, compared with the Control group, the number of TH-positive cells in the substantia nigra pars compacta of the MPTP group was significantly reduced; the number of TH-positive cells in the substantia nigra of the XZ9-3 50mg / kg, 100mg / kg, and selegiline groups was significantly increased compared with the MPTP group. Western blot analysis of TH expression in the mouse midbrain showed that compared with the Control group, the expression level of TH protein in the midbrain of the MPTP group was significantly reduced, while the expression level of TH protein in the midbrain of the XZ9-3 50mg / kg, 100mg / kg, and selegiline groups was significantly increased compared with the MPTP group. These results indicate that XZ9-3 can improve the function of dopaminergic neurons in the brain of PD mice.

[0242] 5. Experiment Summary

[0243] This experiment used an intraperitoneal injection of MPTP to induce a PD mouse model to evaluate the therapeutic effect of the spermidine-rich site of Lycium barbarum (XZ9-3) on PD. The behavioral results showed that the spermidine-rich site of Lycium barbarum (XZ9-3) significantly improved motor coordination dysfunction in the PD model mice. Simultaneously, XZ9-3 enhanced the function of dopaminergic neurons in PD mice, including increasing dopamine levels in the striatum, increasing the number of dopamine-positive cells in the substantia nigra, and increasing the expression of TH protein in the midbrain. In conclusion, XZ9-3 can improve dopaminergic neuronal function in PD mice, thereby improving motor coordination and showing promising potential as a novel drug for treating PD.

[0244] The preferred embodiments of the present invention have been described above, but are not intended to limit the invention. Those skilled in the art can make modifications and variations to the embodiments disclosed herein without departing from the scope and spirit of the invention.

Claims

1. A spermidine-enriched fraction from Lycium barbarum, wherein the spermidine-enriched fraction is prepared by the following method: Lycium barbarum dried fruit is extracted with water, and the extract is concentrated under reduced pressure to obtain a concentrated solution; the concentrated solution is centrifuged, and the supernatant is passed through a macroporous adsorption resin column for chromatography, eluting sequentially with 3-4 column bed volumes of water, 3-4 column bed volumes of 10% ethanol-water, 4-5 column bed volumes of 30% ethanol-water, and 4-5 column bed volumes of 95% ethanol-water; the eluent is collected, and the 30% ethanol-water eluent is concentrated under reduced pressure to obtain the spermidine-enriched fraction from Lycium barbarum.

2. The spermidine-rich fraction of wolfberry according to claim 1, characterized in that, The spermidine-rich region of wolfberry includes: 。 3. The spermidine-rich fraction of Lycium barbarum according to claim 1 or 2, characterized in that, The HPLC characteristic chromatogram of the spermidine-enriched fraction of Lycium barbarum mainly contains 9 chromatographic peaks, including 4', 4''- O - β -D-dipyranose- N 1 -trans-caffeoyl- N 10 The retention time of dihydrocaffeoyl spermidine (14) was set to 1, and the relative retention times of the main chromatographic peaks were calculated. The relative retention time of lycopene spermidine L (11) was 0.96, 4', 4''- O - β -D-dipyranose- N 1 -trans-caffeoyl- N 10 The relative retention time of dihydrocaffeoyl spermidine (14) was 1.00, and the relative retention time of lycopene spermidine M (21) was 1.04, 4', 3''- O - β -D-dipyranose- N 1 -trans-caffeoyl- N 10 The relative retention times of dihydrocaffeoyl spermidine (23) were 1.08, those of lycopene spermidine H / I (28 / 29) were 1.12, those of lycopene spermidine E (31) were 1.17, those of lycopene spermidine F (33) were 1.29, and those of lycopene spermidine A (38) were 1.

32. N 1 -trans-caffeoyl- N 10 The relative retention time of -dihydrocaffeoylspermine (45) was 1.

54.

4. A pharmaceutical composition for the prevention or treatment of Parkinson's disease, prepared from the spermidine-rich fraction of Lycium barbarum according to any one of claims 1-3.

5. The use of the spermidine-rich fraction of Lycium barbarum according to any one of claims 1-3 in the preparation of a medicament for the prevention or treatment of Parkinson's disease.

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