Isopentenyl polyphenol compound as well as application and preparation method thereof

Abstract

The invention discloses an application of an isopentenyl polyphenol compound in preparation of a medicine for treating and / or preventing neurodegenerative diseases, the isopentenyl polyphenol compound can obviously promote the autophagy function of damaged mitochondria without influencing the autophagy function of normal mitochondria, promote the removal of the damaged mitochondria, and can be used for treating and / or preventing neurodegenerative diseases. The typical pathological characteristics of neurodegenerative diseases are improved, for example, phosphorylation of tau protein is inhibited, and learning and memory impairment caused by A beta is improved. Therefore, the isopentenyl polyphenol compound has wide application prospects in preparation of drugs for treating and / or preventing neurodegenerative diseases, preparation of preparations for promoting cell autophagy, preparation of preparations for inhibiting tau protein phosphorylation and preparation of preparations for removing A beta.

Description

[0001] This application is a divisional application of an application with an application date of October 8, 2023, application number 202311293553.6, and invention name “A kind of isopentenyl polyphenol compound, its application and preparation method”. Technical Field

[0002] The present invention relates to the technical field of plant natural products and biomedicine, and in particular to a method for extracting and separating isopentenyl polyphenol compounds in jackfruit plants and an application of the method in preparing medicines for treating and / or preventing neurodegenerative diseases and / or health foods for assisting in improving memory function. Background Art

[0003] Neurodegenerative diseases are a type of disease caused by the gradual loss of neuronal structure or function, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), different types of spinocerebellar ataxia (SCA), Pick's disease, cerebral ischemia (CI), brain injury (BI), epilepsy, primary age-related tauopathy (PART), etc. At present, the pathogenic mechanism of this type of disease is still unclear and there is no effective cure. The main pathogenesis hypotheses include the β-amyloid protein (Aβ) cascade hypothesis, the tau protein hypothesis, and the mitochondrial dysfunction and autophagy damage hypothesis that have been widely studied in recent years.

[0004] In recent years, more and more clinical studies have shown that there are a large number of damaged mitochondria (PMID: 33184293) and autophagosomes (PMID: 15751225) that have failed to be degraded by autophagy in brain cells of AD patients, proving that mitochondrial dysfunction and autophagic damage are early events in the pathogenesis of AD. Autophagy is a degradation mechanism of cytoplasmic components, including mitochondrial autophagy, which mainly removes damaged organelles, endoplasmic reticulum autophagy, aggregate autophagy, peroxisomal autophagy, and lysosomal autophagy, which mainly removes intracellular protein aggregates or DAMPs. Cellular autophagy disorders lead to the inability to clear abnormally folded proteins such as damaged mitochondria, inflammasomes, and tau proteins in a timely manner (PMID: 30116051), resulting in persistent mitochondrial dysfunction in neurons and glial cells, dysregulated cell metabolism, and insufficient energy (PMID: 30482941), triggering pathological neuroinflammation (PMID: 33691134, PMID: 33022416), and causing brain tissue damage. Therefore, enhancing cellular autophagy to clear damaged mitochondria and misfolded proteins provides new drug development targets for the treatment of neurodegenerative diseases (PMID: 33184293, PMID: 32333835, PMID: 35034905, PMID: 31577933 and PMID: 30742114).

[0005] Artocarpus is an important genus in the Moraceae family, with 52 species worldwide, distributed in Southeast Asia and some islands in the Pacific Ocean. There are about 15 species and 2 subspecies in my country. Plants of the genus Artocarpus have very important medicinal value, and many plants are used as traditional folk medicines in Indonesia, Thailand and Sri Lanka. Literature reports that more than 80 isopentenyl phenolic compounds were obtained from plants of the genus Artocarpus, and their skeleton types include isopentenyl polyphenols, dihydroisopentenyl polyphenols, chalcones, dihydrochalcones, bispyribacs, aurones, flavan-3-ols, 2-arylbenzofurans, and 1,2-stilbenes; these compounds have anti-inflammatory, antibacterial, antioxidant, anti-tumor, cytotoxic and anti-trypanosomal and malarial parasites, and inhibit pancreatic lipase, tyrosinase, xanthine oxidase, α-glucosidase, and melanin production. They have significant effects in antioxidant and cytotoxic aspects, but have not been involved in neurodegenerative diseases.

[0006] Jackfruit (Artocarpus heterophyllus Lam.) is not only a special tropical woody food crop, but also a special tropical fruit. It is included in the Compendium of Materia Medica and is mainly grown in Hainan, Guangxi, Guangdong, Yunnan and other regions in China. Jackfruit pulp is rich in functional components such as jackfruit polysaccharides, phenolic compounds, carotenoids, antioxidant components, etc., and has beneficial physiological effects such as antioxidant, anti-inflammatory, anti-cancer, auxiliary hypoglycemic, and immunity enhancement; the seeds are rich in functional active ingredients such as resistant starch, saponins, alkaloids, organic acids, and amino acids, and have physiological effects such as relieving spasms, stopping parasympathetic nervous system impulses, enhancing immunity, regulating emotions, and preventing aging, but none of them involve neurodegenerative diseases. Summary of the invention

[0007] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art. The present invention provides an isopentenyl polyphenol compound having the function of treating neurodegenerative diseases and improving memory function and its pharmaceutical use, which has good prospects for drug development.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] One object of the present invention is to provide an isopentenyl polyphenol compound, the chemical structure of which is shown in the following formula (I):

[0010]

[0011] (I)

[0012] Wherein, R2 and R5 each independently represent H or OH; R1, R3, R4, and R7 each independently represent H, C5-C 10 Alkenyl; R6 represents H or OH or C5 alkenyl.

[0013] The present invention also provides an isopentenyl polyphenol compound, the chemical structure of which is shown in the following formula (I):

[0014]

[0015] (I)

[0016] Wherein, the compound is Y1-10, and R1-R7 are shown in the following table:

[0017]

[0018] Another object of the present invention is to provide an application of an isopentenyl polyphenol compound in the preparation of a drug for treating and / or preventing neurodegenerative diseases.

[0019] The purpose of the present invention is to overcome the above-mentioned shortcomings of the prior art and provide an application of isopentenyl polyphenol compounds in the preparation of drugs for treating and / or preventing neurodegenerative diseases or health foods for assisting in improving memory function. The present invention finds that 8-geranylapigenin (Y1), sanggenon W (sanggenon W, Y2), 3'-geranyl-3-prenyl-2',4',5,7-tetrahydroxyflavone (Y3), 5'-geranyl-5,7,2',4'-tetrahydroxy-flavone (Y4), 5'-Z-geranyl-5,7,2',4'-tetrahydroxy-flavone (Y5), kuwanon S (kuwanon S, Y6), isokuwanon S (isokuwanon S, Y7), albanin D (Y8), 3',8-diprenyl-4',5,7-trihydroxy-flavone (Y9), kuwanon T (kuwanon Isopentenyl polyphenol compounds represented by β-actin (T, Y10) can enhance the autophagy function of damaged mitochondria, inhibit tau protein phosphorylation, and improve learning and memory disorders caused by Aβ.

[0020] Another object of the present invention is to provide an isopentenyl polyphenol compound in Jackfruit plants and an extraction and separation method thereof, which uses dried jackfruit branches and bark and root bark as starting materials, and obtains monomeric isopentenyl polyphenol compounds through solvent extraction and chromatographic column separation and purification methods.

[0021] The extraction solvents are 70%, water, petroleum ether, ethyl acetate and n-butanol extraction in sequence, and the chromatographic column separation methods are silica gel column, Sephadex LH-20 column and preparative liquid phase in sequence.

[0022] Another object of the present invention is to provide a pharmaceutical combination comprising isopentenyl polyphenol compounds for improving memory or cognitive function.

[0023] Another object of the present invention is to provide a pharmaceutical combination comprising isopentenyl polyphenol compounds for promoting autophagy function.

[0024] Another object of the present invention is to provide a pharmaceutical composition comprising isopentenyl polyphenol compounds for preparing a drug for reducing Aβ accumulation.

[0025] Another object of the present invention is to provide a pharmaceutical combination comprising isopentenyl polyphenol compounds for preparing the drug combination for inhibiting tau protein phosphorylation.

[0026] The neurodegenerative disease is caused by at least one of a decrease in autophagy, aggregation of amyloid β (Aβ) and aggregation of tau protein.

[0027] The neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinocerebellar ataxia, cerebral ischemia, brain injury, epilepsy, brain tumor and tauopathy.

[0028] The tauopathy is selected from frontotemporal dementia, progressive supranuclear palsy with dementia entanglement, corticobasal degeneration, Pick's disease or Creutzfeldt-Jakob disease, progressive subcortical gliosis, diffuse neurofibrillary tangles with calcification, argyrophilic grain dementia, amyotrophic lateral sclerosis-parkinsonism complex, dementia pugilistica, Down syndrome, Gerstmann-Straussler-Scheinker disease, Hallerworden-Spatz disease, inclusion body myositis, Creutzfeld-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-guanamian motor neuron disease with neurofibrillary tangles, chronic traumatic encephalopathy, postencephalitic parkinsonism and primary age-related tauopathy.

[0029] The medicine comprises one or more isopentenyl polyphenol compounds of the present invention, and / or pharmaceutically acceptable salts thereof, and / or stereoisomers thereof, and / or prodrug molecules thereof.

[0030] The term "prodrug" refers to a prodrug that is converted into a compound of the present application and a pharmaceutically acceptable salt thereof in vivo.

[0031] The medicine comprises one or more pharmaceutically acceptable carriers and / or diluents.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The isopentenyl polyphenol compounds of the present invention can significantly promote the autophagy function of damaged mitochondria without affecting the autophagy function of normal mitochondria, promote the removal of damaged mitochondria, and improve the typical pathological characteristics of neurodegenerative diseases, such as inhibiting the phosphorylation of tau protein, reducing Aβ deposition, and improving learning and memory functions. Therefore, isopentenyl polyphenol compounds have broad application prospects in the preparation of drugs for treating and / or preventing neurodegenerative diseases or health foods that assist in improving memory function, the preparation of preparations that promote cell autophagy or mitochondrial autophagy, the preparation of preparations that inhibit tau protein phosphorylation, and the preparation of preparations that remove Aβ. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the extraction and separation flow chart of isopentenyl polyphenol compounds Y1-10.

[0035] Figure 2 Isopentenyl polyphenol compounds have the effect of promoting the autophagy of damaged mitochondria in vitro. A: Flow cytometry detection of the promoting effect on the autophagy of damaged mitochondria. B: Statistical results.

[0036] Figure 3 Isopentenyl polyphenols have no effect on the autophagy function of normal mitochondria. A: Flow cytometry detection of the effect on autophagy of normal mitochondria. B: Statistical results.

[0037] Figure 4 The effect of isopentenyl polyphenols on Tau protein phosphorylation in vitro. A: Western blot method was used to detect the phosphorylated Tau protein at different sites in SHSY5Y-Tau cells. B: Statistical results.

[0038] Figure 5 Figure 3. The improving effect of isopentenyl polyphenol compounds on Morris water maze behavior of 5×FAD model mice. A: Schematic diagram of Morris water maze; B: Drug administration shortened the escape latency of 5×FAD mice during the first 7 training sessions; C: Movement trajectory of mice on the 8th day; D: The time mice stayed in the original platform quadrant on the 8th day; E: The number of times mice crossed the platform on the 8th day. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0040] Embodiment 1: Extraction and separation method of jackfruit

[0041] The dried branches and bark and root bark of jackfruit (24 kg) were crushed and extracted with 70% ethanol percolation, the extracts were combined, and the solvent was recovered under reduced pressure to obtain 2190 g of extract. After being suspended in water, they were extracted with petroleum ether, ethyl acetate, and n-butanol in turn to obtain petroleum ether fraction (7 g), ethyl acetate fraction (423 g), n-butanol fraction (603 g), and water fraction (1226 g), respectively. The ethyl acetate extract was separated and purified by silica gel, Sephadex LH-20 column chromatography, and preparative liquid phase separation to obtain compounds Y1 (427 mg), Y2 (828 mg), Y3 (43 mg), Y4 (218 mg), Y5 (89 mg), Y6 (47 mg), Y7 (56 mg), Y8 (77 mg), Y9 (59 mg), and Y10 (104 mg). The specific process is as follows Figure 1 shown.

[0042] Example 2: Structural Identification of Isopentenyl Polyphenols in Jackfruit

[0043] Through physicochemical data and spectral methods, the compounds separated in Example 1 were identified as 26 isopentenyl polyphenol compounds, namely: 8-geranylapigenin (Y1), sanggenon W (sanggenon W, Y2), 3′-geranyl-3-prenyl-2′,4′,5,7-tetrahydroxyflavone (Y3), 5′-geranyl-5,7,2′,4′-tetrahydroxy-flavone (Y4), 5′-Z-geranyl-5,7,2′,4′-tetrahydroxy-flavone (Y5), kuwanon S (kuwanon S, Y6), isokuwanon S (isokuwanon S, Y7), albanin D (Y8), 3′,8-diprenyl-4′,5,7-trihydroxy-flavone (Y9), and kuwanon T (kuwanon T, Y10).

[0044] Its chemical structure is shown in the following formula (I):

[0045]

[0046] (I)

[0047] Among them, R1 to R7 are as follows

[0048]

[0049]

[0050] Example 3 Effects of isopentenyl polyphenol compounds on the autophagy function of damaged mitochondria

[0051] 1. Experimental Methods

[0052] mt-keima is a pH-dependent fluorescent probe composed of the COX-VIII mitochondrial targeting sequence (Genebank GI: 1311703) and the coral-derived keima fluorescent protein (PDB: 2WHU), with a bimodal excitation spectrum (PMID: 21867919). The pH of mitochondria that have not undergone autophagy is nearly neutral, and mt-keima can excite green signals at 405nm. When the mitochondria undergo autophagy and are engulfed by lysosomes in an acidic pH environment, mt-keima can excite red signals at 561nm. It is a highly sensitive and specific quantitative analysis fluorescent probe. Hela-Parkin-mt keima cells, obtained by co-expressing Parkin (Gene ID: 5071) and mt-keima fluorescent reporter genes in Hela cells, are used as a tracer cell model for mitochondrial autophagy.

[0053] Hela-Parkin-mt keima cells in logarithmic growth state were seeded into 24-well plates, with 10 cells per well. 5 FCCP (uncoupler of mitochondrial oxidative phosphorylation) was used to induce mitochondrial damage, and then the drug to be tested was added to detect the effect of the drug on the autophagy function of damaged mitochondria.

[0054] Specifically, three groups were set up, namely: (1) mitochondrial damage model group: 2.5 μM FCCP; (2) drug administration group: 10 μM compounds Y1-Y10 + 2.5 μM FCCP; (3) blank solvent control group: final concentration of 0.1% (v / v) dimethylsulfoxide (DMSO).

[0055] Three replicate wells were set up in each group, with a final volume of 500 μL. After 24 h of drug intervention, the cells were digested and washed twice by centrifugation. The collected cells were detected by flow cytometry using two channels, Qdot 605 and PE-Texas Red. High Qdot605 Low The ratio of the cell population was used as the mitochondrial autophagy index.

[0056] 2. Experimental Results

[0057] The results are as follows Figure 2 As shown, Figure 2 The A in the figure shows the PE-Texas Red High Qdot 605Low The proportion of cell populations, Figure 2 B in the figure is a statistical graph. The results show that the FCCP mitochondrial damage model significantly increased the mitochondrial autophagy rate after administration. Therefore, isopentenyl polyphenol compounds have the function of increasing the autophagy of damaged mitochondria, can be used to prepare a drug combination that promotes autophagy function, and have the effect of treating and / or preventing neurodegenerative diseases caused by reduced autophagy.

[0058] Example 4 Effects of isopentenyl polyphenol compounds on normal mitochondrial autophagy function

[0059] 1. Experimental Methods

[0060] Hela-Parkin-mt keima cells in logarithmic growth state were seeded into 24-well plates, with 1×10 cells per well. 5 cells, cultured for 12 h, and intervened with compounds Y1 to Y10.

[0061] Specifically, three groups were set up, namely: (1) blank solvent control group: final concentration 0.1% (v / v) dimethyl sulfoxide (DMSO), (2) drug administration group: 10 μM compounds Y1-Y10, (3) positive control group: 10 μM FCCP.

[0062] Three replicate wells were set up in each group, with a final volume of 500 μL. After 24 h of drug intervention, the cells were digested and washed twice by centrifugation. The collected cells were detected by flow cytometry using two channels, Qdot 605 and PE-Texas Red. High Qdot605 Low The ratio of the cell population was used as the mitochondrial autophagy index.

[0063] 2. Experimental Results

[0064] The results are as follows Figure 3 As shown, Figure 2 The A in the figure shows the PE-Texas Red High Qdot 605 Low The proportion of cell populations, Figure 2 B is a statistical graph. The results show that isopentenyl polyphenol compounds have no effect on the autophagy function of normal mitochondria.

[0065] Example 5 Isopentenyl polyphenol compounds reduce hyperphosphorylation of tau protein in vitro

[0066] 1. Experimental Methods

[0067] SHSY5Y-Tau cells in logarithmic growth state were seeded into 6-well plates, with 20×104 cells, and cultured for 12 h. 10 μM isopentenyl polyphenols were administered for intervention, and the solvent control group was 0.1% DMSO.

[0068] After 24 hours of drug intervention, the cells were washed twice with PBS, and 200 μL of RIPA lysis buffer (containing 1% protease and phosphatase inhibitors) was added to each well to lyse the cells, extract proteins, and quantify the proteins using the BCA method.

[0069] Western blot method was used to detect the contents of total tau protein, Tau protein phosphorylated at Thr181, Ser202+Thr205, autophagy marker LC3B, and internal reference protein β-actin.

[0070] 2. Experimental Results

[0071] The results are as follows Figure 4 As shown in A and B, compounds Y1, Y2, Y4, Y6, Y7, Y8, Y9, and Y10 reduce the content of phosphorylated tau protein, and compounds Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, and Y10 reduce the proportion of tau protein phosphorylation. Therefore, isopentenyl polyphenol compounds have an in vitro pharmacodynamic effect of reducing tau protein phosphorylation, can be used to prepare a drug combination that inhibits tau protein phosphorylation, and have the effect of treating and / or preventing neurodegenerative diseases caused by tau protein aggregation.

[0072] Example 6 Isopentenyl polyphenol compounds improve learning and memory impairment in 5×FAD mice

[0073] 1. Experimental Methods

[0074] 5×FAD multiple transgenic mice (https: / / www.alzforum.org / research-models / 5xfad-b6sjl) carry five familial gene mutations (transgenic mice with five familial Alzheimer's disease, 5×FAD), among which the mutations related to the APP gene are K670N / M671L (Swedish), I716V (Florida) and V717I (London), and the mutations related to the PS1 gene are M146L and L286V. The transgenic promoter is Thy1. It is a relatively recognized AD model mouse.

[0075] Six-month-old 5×FAD multi-transgenic mice (5×FAD mice) and wild-type C57BL / 6 mice (wild-type mice) were selected. The 5×FAD mice were divided into two groups, each with six mice, namely: (1) a solvent control group and (2) a group receiving isopentenyl polyphenol compounds at 5 mg / kg·bw / day. Six wild-type mice were given normal saline.

[0076] Two months after intragastric administration, the Morris water maze test was performed. The Morris water maze test is a behavioral test that forces animals to swim and learn to find a platform hidden in the water. It is mainly used to test the learning and memory ability of experimental animals for spatial position and direction. The specific method is as follows: the mice were trained from three directions away from the platform, each time for 60 seconds or until the mice stood on the platform, for a total of 7 days. On the eighth day, the platform was removed and tested for 60 seconds. The platform search time during the training period and the number of platform crossings during the test period were counted.

[0077] 2. Experimental Results

[0078] like Figure 5 As shown in A, B, C and D in the figure, during the water maze training phase, the administration of compounds Y2, Y3, Y6 and Y8 significantly shortened the latency of 5×FAD mice to find the hidden platform. During the test period when the platform was removed on the 8th day, the administration of compounds Y2, Y3, Y6 and Y8 did not significantly change the residence time in the quadrant where the original platform was located, but increased the number of times the platform position was crossed, among which Y2 and Y6 had significant differences. Therefore, isopentenyl polyphenol compounds have the ability to enhance the learning and memory ability of AD model mice for the platform position, and the learning and memory disorders caused by Aβ can be used to prepare a drug combination for reducing Aβ accumulation, and have the effect of treating and / or preventing neurodegenerative diseases caused by the aggregation of β-amyloid protein (Aβ).

[0079] 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 the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. An isopentenyl polyphenol compound, wherein the isopentenyl polyphenol compound is represented by the following formula (I): Features: The compound is Y1-10, and R1-R7 are shown in the following table:

2. Use of an isopentenyl polyphenol compound according to claim 1 in the preparation of a medicament for treating and / or preventing neurodegenerative diseases.

3. Use of the isopentenyl polyphenol compound as claimed in claim 1 in the preparation of a drug for improving memory or cognitive function, promoting autophagy function, reducing Aβ accumulation or inhibiting tau protein phosphorylation.

4. The use according to claim 2, characterized in that: The neurodegenerative disease is selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinocerebellar ataxia, cerebral ischemia, brain injury, epilepsy, brain tumor or tauopathy.

5. Use as claimed in claim 4, wherein the tauopathy is selected from frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, Pick's disease or Creutzfeldt-Jakob disease, progressive subcortical gliosis, progressive supranuclear palsy with dementia entanglement, diffuse neurofibrillary tangles with calcification, argyrophilic grain dementia, amyotrophic lateral sclerosis Parkinson-dementia complex, pugilistic dementia, Down syndrome, Gerstmann-Straussler-Scheinker disease, Hallerworden-Spatz disease, inclusion body myositis, Creutzfeld-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-guanamian motor neuron disease with neurofibrillary tangles, chronic traumatic encephalopathy, postencephalitic parkinsonism or primary age-related tauopathy.

6. A pharmaceutical composition comprising one or more isopentenyl polyphenol compounds according to claim 1 and / or pharmaceutically acceptable salts thereof.

7. A pharmaceutical composition according to claim 6, characterized in that: The pharmaceutical composition comprises one or more pharmaceutically acceptable carriers and / or diluents.

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