WO2024039197A1

WO2024039197A1 - Composition for preventing or treating neurodegenerative disease comprising compounds originating from aspergillus sp. sf-7402

Info

Publication number: WO2024039197A1
Application number: PCT/KR2023/012186
Authority: WO
Inventors: 임정한; 김일찬; 한세종; 오현철; 손재학; 조동규
Original assignee: 한국해양과학기술원
Priority date: 2022-08-18
Filing date: 2023-08-17
Publication date: 2024-02-22
Also published as: KR20240025326A

Abstract

The present invention relates to a composition for preventing or treating neurodegenerative disease, the composition comprising a compound isolated from the Antarctic fungus Aspergillus sp. SF-7402 and, particularly, a composition for preventing or treating neurodegenerative disease, the composition comprising sterigmatocystin which is a xanthone compound, or a derivative thereof.

Description

ASPERGILLUS SP. Composition for preventing or treating neurodegenerative diseases containing a compound derived from SF-7402

The present invention relates to the Antarctic fungus Aspergillus sp. A composition for preventing or treating neurodegenerative diseases containing a compound isolated from SF-7402, specifically, neurodegenerative diseases containing sterigmatocystin, a xanthone compound, or a derivative thereof. It relates to a composition for the prevention or treatment of.

Neuroinflammation, an inflammatory response within the brain or spinal cord, is mediated by the production of cytokines, chemokines, reactive oxygen species, and second messengers. The role of these inflammatory mediators in the central nervous system (CNS) has been investigated in various neurodegenerative diseases, including Parkinson’s disease (PD), Huntington’s disease (HD), and Alzheimer’s disease (AD). Microglia play an active role in immune surveillance of the CNS by producing factors that affect surrounding astrocytes and neurons, especially in response to pathogen invasion and tissue damage. Rapid microglial activation reflects the tissue's response to injury to initiate wound healing and protect neurons from further damage. Therefore, activated microglia are known to have a neuroprotective effect. Furthermore, once activated, inflammatory microglia produce pro-inflammatory molecules, including nitric oxide (NO), inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX)-2. Secretes increased levels of mediators and pro-inflammatory cytokines such as TNF-α and IL-6, which are associated with activation of the nuclear factor-κ light chain enhancer of the activated B cell (NF-κB) pathway. Secrete. Various studies have shown that activation of NF-κB protects neurons from various damages such as oxidative stress, excitotoxicity, and amyloid-β peptide toxicity. NF-κB has also been demonstrated to be a key signaling agent affecting cell permeability, endocytosis, and intracellular transport at the blood-brain barrier level. Therefore, NF-κB plays an important role in regulating the causes of diseases related to neuroinflammation (Shih, R.H.; Wang, C.Y.; Yang, C.M. NF-kappaB signaling pathways in neurological inflammation: A mini review. Front. Mol. Neurosci. 2015 , 8, 77.)

To explore new bioactive natural products, researchers have made great efforts in recent years to discover new sources in various environments. The polar regions, where various animal groups and microorganisms such as bacteria, actinomycetes, and fungi live, are attracting attention as a rich source of various secondary metabolites with antibacterial, antitumor, antiviral, and anti-inflammatory effects. Antarctica, with its cold, dry climate accompanied by strong solar radiation, has fostered many unique microbial resources that are attracting attention.

Antarctic microorganisms, especially fungi, have proven to be a rich source of new secondary metabolites. Metabolites derived from Antarctic bacteria are classified into polyketides, peptides, alkaloids, and terpenoids, which exhibit strong anti-inflammatory effects, and exhibit anticancer, antibacterial, and protein tyrosine phosphatase 1B (PTP1B) inhibitory activities.

Under this technical background, the inventors of the present application have developed the Antarctic fungus Aspergillus sp. Among the metabolites of SF-7402, compounds such as sterigmatocystin or derivatives thereof were isolated, and it was confirmed that these compounds could be used for the prevention or treatment of neurodegenerative diseases, and the present invention was completed.

발명의 요약Summary of the Invention

The purpose of the present invention is to provide a composition for preventing or treating neurodegenerative disease containing a xanthone compound.

The purpose of the present invention is to provide a method for preventing or treating neurodegenerative disease including a xanthone compound.

The purpose of the present invention is to provide a xanthone compound for use in the prevention or treatment of neurodegenerative disease.

The object of the present invention is to provide a method for producing the composition.

In order to achieve the above object, the present invention provides a composition for preventing or treating neurodegenerative disease, comprising a compound represented by the following structural formula 1 as an active ingredient:

In structural formula 1, R is alkoxy or hydrogen.

The present invention provides a method for preventing or treating neurodegenerative diseases, comprising administering a compound represented by structural formula 1 to a patient.

The present invention provides the use of the compound represented by Structural Formula 1 in the production of a composition for preventing or treating neurodegenerative diseases.

The present invention relates to the fungus Aspergillus sp. Culturing SF-7402 to extract metabolites; and separating xanthone compounds from metabolites.

Figure 1 shows the structure of the isolated metabolite.

Figure 2 shows the results of confirming the effect of compounds on cell viability (A) and nitrite content (B). BV2 cells were incubated with various concentrations of compounds 1-4 for 24 hours. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. BV2 cells were pretreated with the indicated concentrations of compounds for 3 h and stimulated with lipopolysaccharide (LPS) (0.5 μg/mL) for 24 h. Bars represent the mean standard deviation of three independent experiments. ***p<0.001 compared to LPS treatment group.

Figure 3 shows inducible nitric oxide synthase (iNOS) (A, C) and cyclooxygenase-2 (COX-2) induced in BV2 cells stimulated with lipopolysaccharide (LPS). )(B,D) shows the results of confirming the protein expression level. Cells were pretreated with the indicated concentrations of

compounds

1 and 2 for 3 h and stimulated with LPS (0.5 g/mL) for 24 h. Representative blots from three independent experiments are shown. Immunoblots were quantified using ImageJ software. Band intensity was normalized to β-actin. *p<0.05, **p<0.01 compared to LPS treatment group.

Figure 4 shows the effects of the isolated compounds on TNF-α (A), IL-6 (B), and PGE2 (C) in BV2 cells stimulated with LPS. Cells were pretreated with the indicated concentrations of compounds 1-4 for 3 h and stimulated with LPS (0.5 μg/mL) for 24 h. Bars represent the mean standard deviation of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 compared to LPS treatment group.

Figure 5 shows the effects of isolated compounds on the NF-κB (p65) pathway in BV2 cells (A-D). Cells were pretreated with the indicated concentrations of

compounds

1 and 2 for 3 hours and stimulated with lipopolysaccharide (LPS, 1 g/mL) for 1 hour. Representative blots from three independent experiments are shown. Immunoblots were quantified using ImageJ software. IκB-α intensity was normalized to β-actin. P65 intensity was normalized to PCNA. *p<0.05, **p<0.01, ***p<0.001 compared to LPS treatment group.

발명을 실시하기 위한 구체적인 내용Specific details for carrying out the invention

Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

Microglia play an important role in immune defense and tissue repair in the central nervous system (CNS). Activation of microglia and resulting neuroinflammation play an important role in the pathogenesis of neurodegenerative diseases. Recently, inflammation-alleviating strategies have received increasing attention in neurodegenerative diseases. In BV2 cells stimulated with lipopolysaccharide (LPS), the Antarctic fungus Aspergillus sp. The anti-neuroinflammatory potential of SF-7402 derived compounds was discovered and evaluated.

Through chemical investigation, four metabolites were identified from the fungus, namely 5-methoxysterigmatocystin, sterigmatocystin, aversin, and 6,8-O-dimethylver. Cicolorin A (6,8-O-dimethylversicolorin A) was isolated. Their chemical structures were revealed by extensive spectroscopic analysis, HR-ESI-MS, and comparison with those reported in the literature. The anti-neuroinflammatory effects of the isolated metabolites were assessed by measuring the production of nitric oxide (NO), TNF-α, and IL-6 at non-cytotoxic concentrations in LPS-activated microglia. 5-methoxysterigmatocystin and sterigmatocystin showed a significant effect on NO production and a moderate effect on suppressing TNF-α and IL-6 expression. The underlying molecular mechanisms were investigated using Western blot analysis. Treatment with 5-methoxysterigmatocystin and sterigmatocystin inhibited NO production through downregulation of nitric oxide synthase (iNOS) expression induced in LPS-stimulated BV2 cells. Additionally, 5-methoxysterigmatocystin and sterigmatocystin reduced the nuclear translocation of NF-κB. These results suggest that sterigmatocystin present in the fungal strain Aspergillus sp. is a promising candidate for the treatment of neuroinflammatory diseases.

Based on this, the present invention relates to a composition for preventing or treating neurodegenerative disease, which contains a xanthone compound represented by the following structural formula 1 as an active ingredient:

In structural formula 1, R is alkoxy or hydrogen.

The present invention relates to a method for preventing or treating neurodegenerative disease, comprising administering a xanthone compound represented by structural formula 1 to a patient.

The present invention relates to the use of a xanthone compound represented by structural formula 1 in the production of a composition for preventing or treating neurodegenerative diseases.

Neuroinflammation is an early protective mechanism, and inflammatory mediators play a role in restoring damaged neurons and glial cells during acute neuroinflammation. However, chronic neuroinflammation tends to cause more nerve damage and ultimate degradation. Finding treatments for neuroinflammation could potentially reduce the progression of neurodegenerative diseases.

Antarctic microorganisms, especially fungi, have proven to be a rich source of new secondary metabolites. Metabolites derived from Antarctic bacteria are classified into polyketides, peptides, alkaloids, and terpenoids, which exhibit strong anti-inflammatory effects, and exhibit anticancer, antibacterial, and protein tyrosine phosphatase 1B (PTP1B) inhibitory activities. Using BV2 cells, Aspergillus sp. The anti-neuroinflammatory effects of four metabolites isolated from SF-7402 (KCTC 14992BP) were investigated, and may provide a new strategy for the treatment of neuroinflammation.

In ongoing attempts to study bioactive metabolites isolated from Antarctic fungi, the fungal strain Aspergillus sp. Four secondary metabolites (1-4) were isolated from SF-7402. The isolated metabolites were 5-methoxysterigmatocystin (1), sterigmatocystin (2), aversin (3), and 6,8-O-dimethylversicolo. It was identified as Lin A (6,8-O-dimethylversicolorin A: 4). In a previous study, 5-methoxysterigmatocystin did not affect the expression of heme oxygenase (HO)-1 induced by cobalt protoporphyrin (CoPP) expression. However, there has been no clear study on the therapeutic mechanism of 5-methoxysterigmatocystin in BV2 cells stimulated with LPS.

The anti-neuroinflammatory effects of sterigmatocystin (2), aversin (3), and 6,8-O-dimethylversicolorin A (6,8-O-dimethylversicolorin A: 4) have been reported. It didn't work. Here, we describe the structural properties of the isolated metabolites 1–4 and their anti-neuroinflammatory activity in LPS-stimulated BV2 microglia.

Nitric oxide is formed enzymatically from arginine by three nitric oxide synthase (NOS) isoforms. Inducible NOS (iNOS), an isoform of the NOS family, can be induced by LPS and various cytokines. iNOS is expressed in many cell types, including microglia. In a healthy brain, microglia do not express iNOS. Nevertheless, under ischemic, traumatic, neurotoxic, or inflammatory injury, they are activated to produce iNOS and release large amounts of NO. Afterwards, cell membrane structures can be damaged, affecting DNA transcription and protein synthesis, and directly damaging neurons.

NO causes neurodegeneration by changing intracellular Fe2+ concentration. Therefore, knockdown of iNOS in microglia reduces CNS degeneration by suppressing NO production. Two xanthones, 5-methoxysterigmatocystin and sterigmatocystin, showed significant inhibition of NO production (Figure 2B). Subsequently, the effect of sterigmatocystin (1 and 2) on LPS-induced expression of iNOS and COX-2 proteins in BV2 cells was examined by Western blot analysis. Both sterigmatocystins (1 and 2) inhibited LPS-induced iNOS expression but had no effect on COX-2 expression ( Fig. 3 ). Although the signaling pathways of COX-2 and iNOS expression are complex, these different effects may be due to the dependence of the iNOS and COX-2 promoters on various transcription factors. The iNOS promoter contains several cis-acting elements, including NF-κB, AP-1, C/EBPβ, and Stat, while the COX-2 promoter contains NF-κB, C/EBPβ, and CRE cis-acting elements. Promoter activity varies depending on cell type and applied stimulus.

In support of this notion, the expression of other proinflammatory cytokines, such as TNF-α and IL-6, which are known to be critically dependent on the activation of NF-κB, were significantly inhibited by compounds 1 and 2 (Figure 4A, B). Among the immune mediators released from activated microglia, NO and proinflammatory cytokines, including TNF-α and IL-6, are important mediators in the pathogenesis of neuroinflammation. A large body of evidence shows that cytokines such as TNF-α and IL-6 can be indicators of microglial activation. TNF-α released from brain cells is at the center of neuroinflammatory responses under pathological conditions. IL-6 is one of the factors responsible for the acute neuroinflammatory response to inflammatory injury. PGE _2, a product of COX-2 enzyme, is also regulated by NF-κB and plays an important role in neuroinflammation. However, compounds 1 and 2 were unable to inhibit PGE ₂ production, reflecting the same effect of

compounds

1 and 2 on COX-2 expression (Figure 4C). These results suggest that the regulatory effects of

compounds

1 and 2 in LPS-stimulated BV2 cells are less dependent on the COX-2 promoter compared to the iNOS promoter.

Based on these results, the present invention includes, for example, 5-methoxysterigmatocystin in structural formula 1, where R is methoxy, or sterigmatocystin, where R is hydrogen. It can be included.

Chronic neuroinflammation is mainly regulated by brain microglia and immune cells. Chronic neuroinflammation is known to be the main reason for neuronal loss during PD and forms the basis of neurodegeneration. NF-κB is a member of the Rel family of transcription factors. RelA (p65) is one of the five members of the Rel family. Post-transcriptional modification of p65 plays an important role in pathogenesis. Post-transcriptional modification of p65 plays an important role in the initiation of neurodegenerative processes activated by ischemic injury and glutamate or β-amyloid toxicity. The most important IκB protein is considered to be IκB-α because it interacts with the nuclear localization signal of p65.

Compound

1 and 2 pretreatment reduced p65 expression in a concentration-dependent manner (Figure 5). IκB-α phosphorylation in the cytoplasmic fraction was increased by LPS stimulation, but compounds 1 and 2 inhibited the response in a concentration-dependent manner (Figure 5). These results confirm that sterigmatocystin exhibits an anti-neuroinflammatory effect by inhibiting NF-κB.

Sterigmatocystin is a xanthone containing a xanthone and a bisfuran group. Sterigmatocystin derivatives are reported to have numerous biological activities, including antitumor, anti-inflammatory, antiviral and antibacterial activities. Among the isolated sterigmatocystins, compound 2, which differs from compound 1 by the absence of a methoxy group at the C-5 position, has a greater effect on the expression of p65 protein and IκB-α phosphorylation at the indicated concentrations in LPS-stimulated BV2 cells. shown (Figure 5B). This shows the important role of methoxy among the aromatic ring groups of sterigmatocystin.

Here, the compounds are derived from the Antarctic fungus Aspergillus sp. It may have been separated from SF-7402. The Antarctic strain Aspergillus sp. Two xanthones, sterigmatocystin (1, 2) and two anthraquinones (3, 4), present in SF-7402 were identified. It was confirmed that sterigmatocystin exhibits excellent anti-neuroinflammatory effects by inhibiting the production of NO, TNF-α, and IL-6 and the expression of iNOS protein. These effects were mediated through activation of the NF-κB pathway in LPS-stimulated BV2 microglia.

The neurodegenerative diseases include, for example, Parkinson's disease, Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, and Angelman syndrome. , Liddle syndrome, Pick disease, Paget disease, cancer, or traumatic brain injury, but are not limited thereto.

The composition of the present invention may be prepared to include one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration. The pharmaceutically acceptable carrier must be compatible with the active ingredient of the present invention, and may include saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or two or more of these ingredients. It can be used by mixing, and other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed.

In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc. In particular, it is preferable to formulate and provide it in a lyophilized form. To prepare a freeze-dried formulation, methods commonly known in the art to which the present invention pertains may be used, and a stabilizer for freeze-drying may be added. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the art or a method disclosed by Remington's Pharmaceutical Science (Mack Publishing company, Easton PA).

The pharmaceutical composition of the present invention is preferably administered parenterally, and the dosage varies depending on the patient's weight, age, gender, health status, diet, administration time, method, excretion rate, or severity of the disease. An expert in the technical field can easily determine this.

A single dosage of the composition according to the present invention may be 1 μg/kg to 100 mg/kg, preferably 5 μg/kg to 50 mg/kg, and is administered once a day or 1-3 times a week. However, the dosage and administration interval are not limited to this.

It relates to a method for preventing or treating neurodegenerative diseases, including administration to animals according to the present invention.

The animal may be a human, a primate such as a monkey, a dog, a pig, a cow, a sheep, a goat, a mouse, or a rat, but is not limited thereto. The animal may be, for example, a human.

The method of administering the composition can be determined by an expert in the art based on the patient's symptoms and the severity of the disease.

The pharmaceutical composition of the present invention can be administered parenterally. The route of administration of the composition according to the present invention is not limited to these, but includes, for example, oral cavity, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intestinal, sublingual. Alternatively, topical administration is possible. The pharmaceutical composition of the present invention can be administered to the central nervous system (CNS). The pharmaceutical composition of the present invention can be administered by intrathecal injection.

The dosage of the composition according to the present invention varies depending on the patient's weight, age, gender, health condition, diet, administration time, method, excretion rate, or severity of the disease, and is easily understood by an expert in the art. You can decide. Additionally, the composition of the present invention can be formulated into a suitable dosage form for clinical administration using known techniques.

“Dose” means a specified amount of pharmaceutical agent given in a single administration or over a specified period of time. Doses may be administered as one, two or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous administration is desired, the desired dose requires a volume that is not readily accommodated by a single injection, and two or more injections may be used to achieve the desired dose. Medications are administered by infusion over a long period of time or continuously. Dosage may be stated as the amount of pharmaceutical agent per hour, day, week, or month.

Example

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Materials and Methods

1. General experimental method

Optical rotation was recorded using a Jasco P-2000 digital polarimeter (JASCO Corp., Tokyo, Japan). HR-ESI-MS data were acquired using an electrospray ionization (ESI) quadrupole time-of-flight (Q-TOF) tandem mass spectrometry (MS/MS) system (ABSCRIEX Triple). Nuclear magnetic resonance (NMR) spectra (1D and 2D) were recorded in CDCl3 (δH/δC =7.26/77.16) using a JEOL JNM ECP-400 spectrometer (JEOL Ltd., Tokyo, Japan), and chemical shifts were recorded as those of residual solvent. Reference was made to the peak. Heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond correlation (HMBC) experiments were optimized for ¹ JCH=140Hz and ⁿ JCH=8Hz. Thin-layer chromatography (TLC) was performed on Kieselgel 60 F ₂₅₄ (1.05715; Merck, Darmstadt, Germany) or RP-18 F _254s (Merck, Darmstadt, Germany) plates. The spots were visualized by spraying a 10% aqueous solution of sulfuric acid (H ₂ SO ₄ ) on the plate and then heating it. Column chromatography was performed with silica gel (Kieselgel 60, 70-230 mesh and 230-400 mesh Merck, Darmstadt, Germany) and YMC octadecyl functionalized silica gel (C ₁₈ ). High-performance liquid chromatography (HPLC) was performed on a preparative C ₁₈ column (10 .

2. Fungal material and culture

Fungal strain SF-7402 was isolated from lichen collected at King George Island, Antarctica (62°1406.15’’S, 58°46’22.68’’W) in January 2017. 1 g of sample was ground with a mortar and pestle and mixed with sterile seawater (10 mL). A portion of the sample (0.1 mL) was processed using the spread plate method in potato dextrose agar (PDA) medium containing seawater and incubated at 25°C for 14 days. After subculturing the isolate several times, the final pure culture was selected and stored at -70°C. Fungal strain SF-7402 was identified using ribosomal RNA (rRNA) sequencing. Through GenBank search using the ITS gene of SF-7402 (GenBank accession number MZ267531), Aspergillus jensenii (NR_135444), A. creber (NR_135442), A. versicolor (NR_131277), and A. protuberus (NR_135353) were identified at 99.62%, respectively. The closest matches showing sequence homology of 99.62%, 99.43%, and 99.43% were selected. Therefore, the fungal strain SF-7402 is Aspergillus sp. It was deposited on 2022.06.08 as A voucher specimen (SF-7402) (KCTC 14992BP).

3. Extraction and separation of metabolites

The fungal strain Aspergillus sp. SF-7402 was cultured in 20 Fernbach flasks each containing 650 mL of PDA medium containing 3% NaCl (v/v). Flasks were individually inoculated with 2 mL seed cultures of fungal strains and incubated at 25°C for 14 days. Fermentation culture media were combined and extracted with EtOAc (20 L). The combined EtOAc extracts were then filtered through filter paper and evaporated to dryness to obtain the crude extract (4.8 g). The crude extract was fractionated by reversed-phase (RP) C ₁₈ flash column chromatography ₍ 30 Five fractions of SF-7402-1 to SF-7402-5 were obtained by eluting with a stepwise gradient of MeOH (500 mL each). Fraction SF-7402-5 (568.8 mg) was applied to a _SephadexLH -20 column (33 7402-5.4) was obtained. Fraction SF-7402-5.2 (521.6 mg) was chromatographed on a silica gel column (33 1, SF7402-5.2.10) was obtained. Finally, subfraction SF-7402-5.2.7 (153.9 mg) was separated using C ₁₈ prep HPLC (45-65% CH ₃ CN in H ₂ O, over 45 min) and compound 1 (25.3 mg, tR = 30 min), 2 (15.8 mg, tR = 34 min), 3 (9.5 mg, tR = 39 min) and 4 (1.1 mg, tR = 43 min).

4. Cell culture and preparation

Tissue culture reagents, such as Roswell Park Memorial Institute 1640 (RPMI1640) and fetal bovine serum, were purchased from Gibco BRL Co. (Grand Island, NY, USA). All chemicals were purchased from Sigma-Aldrich Chemical Co. Major antibodies, including anti-iNOS, anti-COX-2, anti-pIκBα, anti-actin, anti-p65 and anti-PCNA antibodies, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All primary antibodies were of rabbit origin. Anti-rabbit secondary antibody was purchased from Millipore (Billerica, MA, USA). Enzyme-linked immunosorbent assay (ELISA) kits for IL-6 and TNF-α were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

5. Cell viability assay

BV2 cells were cultured in RPMI1640 containing 1% antibiotic (penicillin-streptomycin) and 10% heat-inactivated _FBS at 5 Mitochondrial reductase reduced the tetrazolium salt to 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) into formazan crystals. This was used to measure the effect of

compounds

1, 2, 3 and 4 on cell viability. To measure cell viability, 5 mg/mL MTT was treated with each cell suspension (1105 cells/mL) to form formazan for 4 hours. The formed formazan was dissolved in DMSO and the absorbance was measured at 540 nm.

6. Measurement of NO production

The concentration of nitrite in the conditioned medium was determined using a method based on the Griess reaction. After mixing equal amounts of cell culture medium and Griess reagent and reacting, the nitrite level was measured at 570 nm.

7. IL-6 and TNF-α production assay

Culture media were collected and the levels of IL-6 and TNF-α present in each sample were determined using commercial kits (BioLegend, San Diego, CA, USA). The analysis was performed according to the manufacturer's instructions. BV2 cells were seeded in a 24-well culture plate at a density of 2×10 ⁵ cells/well. After incubation, the supernatant was recovered and used in a cytokine ELISA kit to measure the concentrations of IL-6 and TNF-α.

8. Determination of PGE ₂ (Prostaglandin E2) levels

Media was collected to determine the level of PGE2 present in each sample. The analysis was performed according to the manufacturer's instructions. BV2 cells were seeded in a 24-well culture plate at a density of 2×10 ⁵ cells/well. After incubation, the supernatant was collected. PGE ₂ levels were then measured using a specific ELISA kit from R&D Systems, Inc. (Minneapolis, MN, USA).

9. iNOS and COX-2 protein expression analysis

Pelleted BV2 cells were washed with phosphate-buffered saline and dissolved in RIPA buffer. Protein assay dye reagent concentrate obtained from Bio-Rad Laboratories (#5000006; Hercules, CA, USA) was mixed in sample loading buffer to quantify equal amounts of protein and separated by SDS-PAGE. The separated proteins were transferred to a nitrocellulose membrane. Non-specific binding to the membrane was blocked by incubation in a solution of skim milk. The membrane was incubated with primary antibody overnight at 4°C and then reacted with horseradish peroxidase-labeled secondary antibody (Millipore).

10. NF-κB (p65) expression analysis

To study the localization of NF-κB, BV2 cells were seeded in 24-well culture plates at a density of ⁴⁰ and 2. Cytoplasmic and nuclear fractions were separated using a nuclear extraction kit (Cayman, Ann Arbor, MI, USA). Each extracted fraction was dissolved according to the protocol provided by the manufacturer. The expression of the NF-κB (p65) pathway was then determined by Western blot analysis. Equivalent amounts of protein were quantified with protein assay dye reagent concentrate obtained from Bio-Rad Laboratories (#5000006; Hercules, CA, USA), mixed with sample loading buffer, and separated by SDS-PAGE. The separated proteins were then transferred to a nitrocellulose membrane. Non-specific binding to the membrane was blocked by incubation in a solution of skim milk. Membranes from cytoplasmic fraction samples were incubated with pIκBα antibody overnight at 4°C, whereas membranes from nuclear fraction samples were incubated with p65 antibody and then reacted with horseradish peroxidase-labeled secondary antibody (Millipore).

11. Statistical analysis

Data are expressed as the mean ± standard deviation of three independent experiments. The three groups were compared using centralized analysis of variance followed by Tukey's multiple comparison test. Statistical analyzes were performed using GraphPad Prism software version 5.01 (GraphPad Software Inc., San Diego, CA, USA).

Example 1. Confirmation of structure and impact of isolated metabolites on BV2 cell survival

To identify bioactive metabolites that cause anti-neuroinflammatory effects, the fungal strain Aspergillus sp. An efficient chromatographic separation technique was adopted to separate the four compounds (1-4) in SF-7402 (Figure 1). By comparing the spectral data with values reported in the literature, 5-methoxysterigmatocystin (1), sterigmatocystin (2), aversin (3), and 6,8-O-dimethylversicolorin A( It was characterized as 4).

To avoid toxic effects of the isolated metabolites, the survival rate of BV2 cells was measured after treatment for 24 hours at concentrations ranging from 10 to 40 μM using the MTT assay. As shown in Figure 2A, all four compounds (1-4) showed cytotoxicity to BV2 cells at concentrations of 20 and 40 μM. Therefore, additional experiments were performed using each compound in the non-toxic concentration range (2-10 μM). NO, a neurotransmitter second messenger molecule, mediates a variety of physiological and pathological processes in many organ systems, including the brain. To investigate the inhibitory effect of the isolated metabolites (1-4) on NO production in LPS-induced BV2 cells, cell culture medium was collected and nitrite concentration was measured using the Griess reaction. The results showed that two xanthones, 5-methoxysterigmatocystin (1) and sterigmatocystin (2), significantly inhibited NO production in LPS-induced BV2 cells, whereas two anthraquinones, aversin (3), significantly inhibited NO production in LPS-induced BV2 cells. ) and 6,8-O-dimethylversicolorin A (4) did not inhibit NO production (Figure 2B).

Example 2. Confirmation of inhibition of LPS-induced expression of iNOS and COX-2 proteins in BV2 cells by sterigmatocystin

The effect of sterigmatocystin (1 and 2) on LPS-induced expression of iNOS and COX-2 proteins in BV2 cells was investigated by Western blot analysis.

Two sterigmatocystins (1 and 2) were found to inhibit LPS-induced iNOS expression, but had no effect on COX-2 expression (Figure 3). This suggests that sterigmatocystin with a xanthone skeleton is a promising compound for selective inflammation inhibition in LPS-stimulated BV2 cells.

Example 3. Sterigmatocystin inhibits the production of LPS-induced inflammatory cytokines in BV2 cells.

To evaluate the inhibitory effect on the expression of pro-inflammatory cytokines TNF-α, IL-6 and PGE ₂ , isolated metabolites were treated with LPS stimulated BV2 cells. (1-4) was used. The experiment was performed using an ELISA kit. As shown in Figure 4, only sterigmatocystin (1 and 2) showed moderately effective inhibition of TNF-α and IL-6 expression, while other compounds did not inhibit expression.

Compound 1 reduced the expression of intracellular TNF-α and IL-6 by approximately 25% and 20%, respectively, at a concentration of 10 μM. Compound 2 showed a strong effect, inhibiting TNF-α expression by more than 30% in activated BV2 cells at a concentration of 10 μM and inhibiting IL-6 expression by more than 30% at all concentrations tested (2, 5, and 10 M). However, pretreatment with sterigmatocystin (1 and 2) could not eliminate PGE2 production in LPS-stimulated BV2 cells, which is consistent with the same effect of sterigmatocystin (1 and 2) on the expression of iNOS and COX-2. reflects. These data demonstrate a role for sterigmatocystins (1 and 2) in suppressing proinflammatory cytokine expression.

Example 4. Sterigmatocystin inhibits LPS-induced activation of the NF-kB pathway in BV2 cells

Because abnormal regulation of NO, TNF-α, and IL-6 in activated microglia is associated with activation of the NF-kB (p65) pathway, sterigmatocystins (1 and 2) promote NF-kB activation. We further evaluated whether to up-regulate. BV2 cells were pretreated with the indicated concentrations of

compounds

1 and 2 for 3 hours and then stimulated with LPS (1 μg/mL) for 1 hour. Cytoplasmic and nuclear fractions were extracted from cell lysates, and the protein level of p65 was increased in the nuclear fraction of the LPS treatment group. Pretreatment with

compounds

1 and 2 reduced p65 expression in a concentration-dependent manner (Figure 5). Phosphorylation of IκB-α in the cytosolic fraction was also increased by LPS stimulation. However, compounds 1 and 2 inhibited this reaction in a concentration-dependent manner (Figure 5). These results suggest that sterigmatocystin exerts an anti-neuroinflammatory effect by inhibiting the activation of the NF-κB pathway.

According to the present invention, sterigmatocystin or a derivative thereof, a xanthone compound, exhibits excellent anti-neuroinflammatory effects by inhibiting the production of NO, TNF-α, and IL-6 and the expression of iNOS protein. It was confirmed that this was mediated through activation of the NF-κB pathway in LPS-stimulated BV2 microglial cells. Through this, it can be effectively used in the prevention or treatment of neurodegenerative diseases, and provides a promising basis for the development of therapeutic agents for the treatment of neurodegenerative diseases.

As above, specific parts of the content of the present invention have been described in detail, and for those skilled in the art, it is clear that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. It will be obvious. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims

A composition for preventing or treating neurodegenerative disease comprising a xanthone compound represented by the following structural formula 1 as an active ingredient:

In structural formula 1, R is alkoxy or hydrogen.
The composition of claim 1, wherein R is methoxy, comprising 5-methoxysterigmatocystin.
The composition of claim 1, wherein R is hydrogen and contains sterigmatocystin.
The method of claim 1, wherein the compound is an Antarctic fungus Aspergillus sp. A composition characterized in that it is derived from SF-7402.
The method of claim 1, wherein the neurodegenerative disease includes Parkinson's disease, Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, and Angelman's disease. ) syndrome, Liddle syndrome, Pick's disease, Paget's disease or traumatic brain injury.
The fungus Aspergillus sp. Culturing SF-7402 to extract metabolites; and

A method for producing the composition of claim 1, comprising the step of separating xanthone compounds from metabolites.