A kind of isopentenyl polyphenol compound and its application and preparation method

Abstract

The present invention discloses the use of an isopentenyl polyphenol compound in the preparation of a drug for treating and / or preventing neurodegenerative diseases. The isopentenyl polyphenol compound of the present invention can significantly promote the autophagy function of damaged mitochondria without affecting the autophagy function of normal mitochondria, promote the clearance of damaged mitochondria, and improve the typical pathological characteristics of neurodegenerative diseases, such as inhibiting the phosphorylation of tau protein and improving learning and memory impairment caused by Aβ. Therefore, the isopentenyl polyphenol compound has broad application prospects in the preparation of drugs for treating and / or preventing neurodegenerative diseases, the preparation of preparations for promoting cell autophagy, the preparation of preparations for inhibiting tau protein phosphorylation, and the preparation of preparations for clearing Aβ.

Description

[0001] This application is a divisional application of the application with 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 from Jackfruit plants and their application in preparing drugs for treating and / or preventing neurodegenerative diseases and / or health foods for assisting in improving memory function. Background Art

[0003] Neurodegenerative diseases are a class of diseases caused by the gradual loss of neuronal structure or function. They primarily include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), various types of spinocerebellar ataxia (SCA), Pick's disease, cerebral ischemia (CI), brain injury (BI), epilepsy, and primary age-related tauopathy (PART). The pathogenic mechanisms of these diseases remain unclear, and there are no effective cures. Major hypotheses for their pathogenesis include the β-amyloid protein (Aβ) cascade hypothesis, the tau protein hypothesis, and, more recently, the mitochondrial dysfunction and autophagy impairment hypotheses, which have been widely studied.

[0004] In recent years, an increasing number of clinical studies have demonstrated the presence of numerous damaged mitochondria (PMID: 33184293) and autophagosomes (PMID: 15751225) in brain cells from patients with Alzheimer's disease (AD), demonstrating that mitochondrial dysfunction and autophagic impairment are early events in the pathogenesis of AD. Autophagy is a cytoplasmic degradation mechanism that includes mitophagy (primarily for the clearance of damaged organelles), endoplasmic reticulum (ER) autophagy, aggregophagy (primarily for the clearance of intracellular protein aggregates or depleted AMPs), peroxisomal autophagy, and lysosomal autophagy. Impaired autophagy prevents the timely clearance of damaged mitochondria, inflammasomes, and abnormally folded proteins such as tau (PMID: 30116051), leading to persistent mitochondrial dysfunction in neurons and glial cells, dysregulated cellular metabolism, and energy deficiency (PMID: 30482941), triggering pathological neuroinflammation (PMID: 33691134, PMID: 33022416), and ultimately causing brain 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 across Southeast Asia and several Pacific islands. Approximately 15 species and two subspecies are found in my country. Artocarpus plants have significant medicinal value, and many are used in traditional folk medicines in Indonesia, Thailand, and Sri Lanka. Literature reports indicate that over 80 prenylphenolic compounds have been isolated from Artocarpus plants, with skeletons including prenyl polyphenols, dihydroprenyl polyphenols, chalcones, dihydrochalcones, bisphenylpyrazols, aurones, flavan-3-ols, 2-arylbenzofurans, and 1,2-stilbenes. These compounds exhibit anti-inflammatory, antibacterial, antioxidant, antitumor, cytotoxic, antitrypanosomal and anti-malarial activities, as well as inhibitory effects on pancreatic lipase, tyrosinase, xanthine oxidase, α-glucosidase, and melanin production. Their antioxidant and cytotoxic properties are particularly significant, but these compounds have not been implicated in neurodegenerative diseases.

[0006] Jackfruit (Artocarpus heterophyllus Lam.) is both a unique tropical woody crop and a unique tropical fruit, listed in the Compendium of Materia Medica. It is primarily cultivated in Hainan, Guangxi, Guangdong, and Yunnan provinces of China. Jackfruit flesh is rich in functional components such as jackfruit polysaccharides, phenolic compounds, carotenoids, and antioxidants, exhibiting beneficial physiological effects such as antioxidant, anti-inflammatory, anti-cancer, hypoglycemic, and immune-enhancing properties. The seeds are rich in functional active ingredients such as resistant starch, saponins, alkaloids, organic acids, and amino acids, exhibiting physiological benefits such as alleviating spasms, suppressing parasympathetic nervous system impulses, enhancing immunity, regulating mood, and preventing aging. However, none of these benefits have been linked to 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 drug development prospects.

[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, 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 a use of an isopentenyl polyphenol compound in the preparation of a medicament for treating and / or preventing neurodegenerative diseases.

[0019] The present invention aims to overcome the above-mentioned deficiencies of the prior art and to provide an application of an isopentenyl polyphenol compound in the preparation of a drug for treating and / or preventing neurodegenerative diseases or a health food for assisting in improving memory function. The present invention has found 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-catenin (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 a method for extracting and separating the same. The method 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 composition for reducing Aβ accumulation.

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

[0026] The neurodegenerative disease is caused by at least one of a decrease in autophagy, aggregation of amyloid beta (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 tangles, corticobasal degeneration, Pick's disease or Creutzfeldt-Jakob disease, progressive subcortical gliosis, diffuse neurofibrillary tangles with calcification, argyrophilic grain dementia, amyotrophic lateral sclerosis-Parkinson-dementia 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, postencephalitis 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 described herein can significantly promote the autophagy of damaged mitochondria without affecting the autophagy of normal mitochondria, promote the clearance of damaged mitochondria, and improve typical pathological features of neurodegenerative diseases, such as inhibiting tau protein phosphorylation, 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 cellular autophagy or mitochondrial autophagy, the preparation of preparations that inhibit tau protein phosphorylation, and the preparation of preparations that clear Aβ. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the extraction and separation flow chart of isopentenyl polyphenol compound 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 normal mitochondrial autophagy. A: Flow cytometry analysis of their effect on normal mitochondrial autophagy. B: Statistical results.

[0037] Figure 4 Figure 3. Effects of isopentenyl polyphenols on Tau protein phosphorylation in vitro. A: Western blot analysis of Tau protein phosphorylation at different sites in SHSY5Y-Tau cells. B: Statistical results.

[0038] Figure 5 Figure 3. The improvement effect of isopentenyl polyphenol compounds on the Morris water maze behavior of 5×FAD model mice. A: Schematic diagram of the 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 day 8; D: The time mice stayed in the original platform quadrant on day 8; E: The number of times mice crossed the platform on day 8. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

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

[0041] The dried branches and bark roots of jackfruit (24 kg) were crushed and extracted with 70% ethanol by percolation. The extracts were combined and the solvent was recovered under reduced pressure to obtain 2190 g of extract. After being suspended in water, the extracts were extracted with petroleum ether, ethyl acetate, and n-butanol in sequence 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 isolated in Example 1 were identified as 26 isopentenyl polyphenol compounds, namely: 8-geranylapigenin (Y1), 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 (Y6), isokuwanon S (Y7), albanin D (Y8), 3′,8-diprenyl-4′,5,7-trihydroxy-flavone (Y9), and kuwanon T (Y10).

[0044] Its chemical structural formula 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 autophagy in 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). It exhibits a bimodal excitation spectrum (PMID: 21867919). When the pH of mitochondria not undergoing autophagy is near neutral, mt-keima emits a green signal at 405 nm. However, when mitochondria undergo autophagy and are engulfed by lysosomes, mt-keima emits a red signal at 561 nm, making it a highly sensitive and specific fluorescent probe for quantitative analysis. Hela cells co-expressing Parkin (Gene ID: 5071) and the mt-keima fluorescent reporter gene, resulting in Hela-Parkin-mt-keima cells, serve as a cell model for tracing 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 (an 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 the 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] Each group was divided into 3 replicate wells with a final volume of 500 μL. After 24 hours of drug intervention, the cells were digested and washed twice by centrifugation. The collected cells were detected by flow cytometry using Qdot 605 and PE-Texas Red channels. The PE-Texas Red High Qdot605 Low The ratio of the cell population was used as the mitochondrial autophagy rate 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 of each group. High Qdot 605Low The proportion of cell populations, Figure 2 Figure B is a statistical graph. The results show that FCCP administration significantly increased the rate of mitochondrial autophagy in the mitochondrial injury model. Therefore, isopentenyl polyphenols have the ability to enhance autophagy in damaged mitochondria and can be used to prepare drug combinations that promote autophagy, potentially treating and / or preventing neurodegenerative diseases caused by decreased autophagy.

[0058] Example 4 Effects of Isopentenyl Polyphenols on Normal Mitochondrial Autophagy

[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 of 0.1% (v / v) dimethyl sulfoxide (DMSO), (2) drug administration group: 10 μM compounds Y1-Y10, (3) positive control group: 10 μM FCCP.

[0062] Each group was divided into 3 replicate wells with a final volume of 500 μL. After 24 hours of drug intervention, the cells were digested and washed twice by centrifugation. The collected cells were detected by flow cytometry using Qdot 605 and PE-Texas Red channels. The PE-Texas Red High Qdot605 Low The ratio of the cell population was used as the mitochondrial autophagy rate 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 of each group. High Qdot 605 Low The proportion of cell populations, Figure 2 B is a statistical graph. The results show that isopentenyl polyphenols 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 20×10 cells per well.4 The cells were cultured for 12 h. 10 μM isopentenyl polyphenols were administered as intervention, while 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. Protein was extracted and quantified using the BCA method.

[0069] Western blot was used to detect the levels 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 in Figure 3, compounds Y1, Y2, Y4, Y6, Y7, Y8, Y9, and Y10 reduced the content of phosphorylated tau protein, and compounds Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, and Y10 reduced the proportion of tau protein phosphorylation. Therefore, isopentenyl polyphenol compounds have an in vitro pharmacodynamic effect of reducing tau protein phosphorylation and can be used to prepare a drug combination that inhibits tau protein phosphorylation, thereby having 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, with 6 mice in each group, namely: (1) solvent control group, (2) isopentenyl polyphenol compound administration group (5 mg / kg·bw / day), and 6 wild-type mice were given normal saline.

[0076] Two months after oral administration, the Morris water maze test was performed. The Morris water maze test is a behavioral test in which animals are forced to swim and learn to locate a platform hidden in water. It is primarily used to test the animals' learning and memory of spatial position and direction. The specific method is as follows: mice are trained starting from three directions away from the platform, each for 60 seconds or until the mouse stands on the platform. Training is carried out for a total of 7 days. On the eighth day, the platform is removed and the test is conducted for 60 seconds. The platform search time during the training period and the number of platform crossings during the test period are recorded.

[0077] 2. Experimental Results

[0078] like Figure 5 As shown in Figures A, B, C, and D, during the water maze training phase, administration of compounds Y2, Y3, Y6, and Y8 significantly shortened the latency for 5×FAD mice to find the hidden platform. During the test phase, when the platform was removed on day 8, administration of compounds Y2, Y3, Y6, and Y8 did not significantly change the time spent in the quadrant where the original platform was located, but increased the number of times the platform location was crossed, with Y2 and Y6 showing significant differences. Therefore, isopentenyl polyphenol compounds have the ability to enhance the learning and memory ability of AD model mice regarding the location of the platform, and can be used to prepare drug combinations that reduce Aβ accumulation and are effective in treating and / or preventing neurodegenerative diseases caused by amyloid beta (Aβ) aggregation.

[0079] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection 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): （I） Its characteristics are: The compound is Y1, wherein R1-R7 are shown in the following table: 。 2. A pharmaceutical composition comprising the isopentenyl polyphenol compound according to claim 1 and / or its pharmaceutically acceptable salt.

3. A pharmaceutical composition according to claim 2, characterized in that: The pharmaceutical composition comprises one or more pharmaceutically acceptable carriers and / or diluents.

4. Use of an isopentenyl polyphenol compound in the preparation of a medicament for treating and / or preventing neurodegenerative diseases, wherein the isopentenyl polyphenol compound is represented by the following formula (I): （I） Its characteristics are: The compounds are Y1 and Y6, and R1-R7 are shown in the following table: 。 5. The use according to claim 4, 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 or epilepsy.

6. Use of an isopentenyl polyphenol compound in the preparation of a drug for improving memory or cognitive function and promoting autophagy of damaged mitochondria, wherein the isopentenyl polyphenol compound is represented by the following formula (I): （I） Its characteristics are: The compounds are Y1 and Y6, and R1-R7 are shown in the following table: 。

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