KR20200136085A - Composition for preventing or treating neuronal disorders comprising phlorotannins - Google Patents

Abstract

The present invention relates to a composition for preventing or treating neurological disorders containing phlorofucofuroeckol A or dieckol as an active component, wherein the phlorofucofuroeckol A or dieckol noncompetitively inhibits monoamine oxidase A (MAO-A) or monoamine oxidase B (MAO-B) activity, promotes signal transduction by a dopamine D_3 receptor or a dopamine D_4 receptor, and inhibits signal transduction by a dopamine D_1 receptor, thereby being able to be usefully used in the prevention or treatment of neurological disorders.

Description

Composition for preventing or treating neuronal disorders comprising phlorotannins {Composition for preventing or treating neuronal disorders comprising phlorotannins}

The present invention relates to a composition for the prevention or treatment of neurological diseases comprising a florofucofuroexcol-A or diexol as an active ingredient.

Studies on antidepressant drugs or treatments for Parkinson's disease have been extensively conducted in animal models, and one of the common characteristics of these drugs is serotonin (5-HT) or dopamine (a neurotransmitter in the central nervous system). dopamine) (Evrard et al., 2002; Finberg and Rabey, 2016).

MAO (monoamine oxidases, EC 1.4.3.4) is a flavin-containing enzyme that is involved in the catabolism of neurotransmitters (Finberg and Rabey, 2016). Two isoforms, MAO-A and MAO-B exist. The isoform has about 70% of the same sequence, but has different binding power to neurotransmitters. MAO-A selectively degrades dopamine, serotonin and norepinephrine, and MAO-B degrades dopamine, phenethylamine and benzylamine (Ramsay and Albreht, 2018).

Inhibition of MAO-A can increase the concentration of serotonin in the synaptic cleft, while acute systemic injection of MAO inhibitors can induce inhibition of serotonin (Evrard et al., 2002). In previous studies, it was reported that WAY 100635, a potent serotonin receptor antagonist (5-HT _1A receptor antagonist), inhibited MAO inhibitor-induced serotonin cell firing when treated with MAO inhibitors and increased the efficiency of MAO inhibitors ( Sharp et al., 1997).

Dopamine, a catecholamine-based neurotransmitter, is known to act by promoting dopamine receptors, a member of the G-protein-coupled receptors (GPCRs) superfamily involved in a variety of neurological processes including motivation, emotion, and reward. Yes (Newman et al., 2012). Dopamine receptors are classified into D ₁ -like (D ₁ R, D ₅ R) and D ₂ -like (D ₂ R, D ₃ R, D ₄ R) subtypes based on their similarity in signaling (Butini et al., 2016).

D ₂ -like D ₂ R, D ₃ R and D ₄ R stimulate G a _i/o to inhibit adenylate cyclase activity and cAMP (cyclic adenosine monophosphate) production. Whole or partial D ₂ R and D ₃ R agonists are widely used in the treatment of Parkinson's disease, and D ₂ R or D ₃ R antagonists are effective in the treatment of schizophrenia (Bello et al., 2019). D ₄ R, mainly expressed in the hippocampus and prefrontal cortex, influences exploratory behavior, attention, and suppression avoidance tasks. The activity of D ₄ R can be used to treat cognitive impairments associated with schizophrenia and attention deficit/hyperactivity disorder (Keck et al., 2019).

On the other hand, D ₁ -like D ₁ R is the most dopamine receptor in the forebrain, and is known to stimulate G a _s/olf to enhance adenylate cyclase activity and cAMP production (Butini et al., 2016). In addition, compared to L-DOPA (L-3,4-dihydroxyphenylalanine) alone treatment in Parkinson's disease patients, blocking of D ₁ R by SCH23390, a D ₁ R antagonist, increased brain AADH (aromatic L-amino acid decarboxylase). It has been reported to significantly enhance the conversion of L-DOPA to dopamine by (Bourne et al., 2001).

Phlorotannins are abundantly present in algae such as Ecklonia stolonifera , Ecklonia cava , Ecklonia bicyclis and Eisenia bicyclis . Phlorotannin is a polymer of phloroglucinol units and is biosynthesized by the polyketide pathway (Li et al., 2011). Florotannin is an antioxidant, anti-inflammatory (Kim et al., 2009), anti-HIV (Artan et al., 2008), anti-breast cancer (Kim et al., 2015), anti-allergy (Sugiura et al., 2006) and anti-hyperpigmentation (Heo et al., 2009).

Korean Patent Registration No. 10-0542751

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of neurological diseases comprising phlorofucofuroeckol-A or dieckol as an active ingredient.

Another object of the present invention is to provide a health functional food for preventing or improving neurological diseases comprising phlorofucofuroeckol-A or dieckol as an active ingredient.

Another object of the present invention is MAO-A (monoamine oxidase A) or h MAO-B (human monoamine oxidase B) comprising the step of treating phlorofucofuroeckol-A on individuals other than humans It is to provide a method of inhibiting the activity of.

Another object of the present invention is signal transduction by dopamine D ₃ receptor or dopamine D ₄ receptor comprising the step of treating phlorofucofuroeckol-A or dieckol to individuals other than humans. It is to provide a way to promote it.

Another object of the present invention is a method of inhibiting signaling by dopamine D ₁ receptor comprising the step of treating phlorofucofuroeckol-A or dieckol to individuals other than humans. To provide.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of neurological diseases comprising phlorofucofuroeckol-A or dieckol as an active ingredient.

In one embodiment of the present invention, the fluorofucofuroexcol-A or diexol may be derived from Ecklonia stolonifera , but is not limited thereto, and is chemically synthesized or commercially available by a method known in the art. You can use any material.

In an embodiment of the present invention, the florofucofuroexcol-A may have a non-competitive inhibitory activity against monoamine oxidase A (MAO-A) or monoamine oxidase B (MAO-B).

In one embodiment of the present invention, the fluorofucofuroexcol-A or diexol may be an agonist of dopamine D ₃ receptor or dopamine D ₄ receptor, and is an antagonist of dopamine D ₁ receptor. Can be.

The term "agonist" of the present invention is a substance that activates a receptor in order to produce a biological response by binding to a receptor, and "antagonist" binds to a receptor like the above agonist, but a biological response opposite to the agonist It is a substance that inhibits the receptor to inhibit.

In an embodiment of the present invention, the fluorofucofuroexcol-A or diexol may be administered at a concentration of 0.1 to 200 μM, preferably at a concentration of 1 to 100 μM, More preferably, it may be administered at a concentration of 10 to 100 μM, but is not limited thereto.

In one embodiment of the present invention, the neurological disease is any one selected from the group consisting of neurodegenerative diseases, schizophrenia, schizophrenia, attention deficit hyperactivity disorder, personality disorder, autism, depression, and delusional disorder. The neurodegenerative disease may be any one selected from the group consisting of Parkinson's disease, Alzheimer's disease, senile dementia, diabetic dementia, alcoholic dementia, and vascular dementia, but is not limited thereto.

In addition, the present invention provides a health functional food for preventing or improving neurological diseases comprising phlorofucofuroeckol-A or dieckol as an active ingredient.

In addition, the present invention relates to the activity of MAO-A (monoamine oxidase A) or h MAO-B (human monoamine oxidase B) comprising the step of treating phlorofucofuroeckol-A on an individual other than humans. It provides a way to inhibit.

In addition, the present invention promotes signaling by dopamine D ₃ receptor or dopamine D ₄ receptor comprising the step of treating phlorofucofuroeckol-A or dieckol to individuals other than humans. Provide a way to make

In addition, the present invention provides a method for inhibiting signal transduction by dopamine D ₁ receptor comprising the step of treating phlorofucofuroeckol-A or dieckol to individuals other than humans. .

The compounds according to the present invention, florofucofuroexcol-A or diexol, noncompetitively inhibit the activity of MAO-A (monoamine oxidase A) or MAO-B (monoamine oxidase B), and dopamine D ₃ receptor or dopamine D ₄ It has the effect of increasing dopamine by promoting signal transduction by receptors and inhibiting signal transduction by dopamine D ₁ receptors, so it can be usefully used in the prevention or treatment of neurological diseases.

FIG. 1 is a diagram including Eckol, dioxinodehydroeckol, phloroglucinol, dieckol, and phlorofucofuroeckol-A (PFF-A). It shows the chemical structure of phlorotannin.
2A and B show a Lineweaver-Burk plot and a secondary plot for h MAO-A of fluorofucofuroexcol-A, and FIGS.2C and D are a Lineweaver for h MAO-B of fluorofucofuroexcol-A. -Burk plot and secondary plot are shown.
Figure 3A shows the binding site of the florofucofuroexcol-A docked in h MAO-A and the positive control, and Figure 3B shows the interaction of the h MAO-A and the florofucofuroexcol-A complex in 2D. Figure 3C shows the binding site of the florofucofuroexcol-A docked in h MAO-B and the positive control, and Figure 3D shows the interaction of the h MAO-B and the florofucofuroexcol-A complex 2D (Florofucofuroexcol-A, orange; harmine, magenta; deprenyl, black; and FAD, yellow bar).
Figure 4 shows the agonist effect of fluoroglucinol, diecol and fluorofucofuroexcol-A by concentration on dopamine D ₃ and D ₄ receptors.
Figure 5A shows the binding site of the positive control and the florotannin docked to the dopamine D ₁ receptor, Figures 5B and E show the interaction of the dopamine D ₁ receptor-bound fluoroglucinol, Figures 5C and F Fig. 5D and G show the interaction of the dopamine D ₁ receptor-bound diexol, and FIGS. 5D and G show the interaction of the dopamine D ₁ receptor-bound fluorofucofuroexcol-A (fluoroglucinol, yellow; Dieccol, green; fluorofucofuroexcol-A, orange; dopamine, blue; and SCH 23390, black bars).
Figure 6A shows the binding site of the positive control and the florotannin docked to the dopamine D ₃ receptor, Figures 6B and E show the interaction of the dopamine D ₃ receptor-bound fluoroglucinol, Figures 6C and F Fig. 6D and G show the interaction of dopamine D ₃ receptor-bound diexol, and FIGS. 6D and G show the interaction of fluorofucofuroexcol-A bound to dopamine D ₃ receptor (fluoroglucinol, yellow; Dieccol, green; fluorofucofuroexcol-A, orange; dopamine, blue; and eticlopride, black bar).
Figure 7A shows the binding site of the dopamine D ₄ receptor docked with fluorotannin and the positive control, Figures 7B and E show the interaction of the dopamine D ₄ receptor-bound fluoroglucinol, Figures 7C and F Fig. 7D and G show the interaction of the dopamine D ₄ receptor-bound diexol, and Figs. 7D and G show the interaction of the dopamine D ₄ receptor-bound fluorofucofuroexcol-A (fluoroglucinol, yellow; Dieccol, green; Florofucofuroekcol-A, orange; dopamine, blue; and clozapine, black bar).

The compounds according to the present invention, phlorofucofuroeckol-A or dieckol, may be derived from extracts or fractions of Ecklonia stolonifera .

The extract may be extracted and separated from nature using a method known in the art for extraction and separation, and the "extract" as defined in the present invention is extracted from Ecklonia stolonifera using an appropriate solvent. It will, for example, include all of the crude extract, polar solvent soluble extract, or non-polar solvent soluble extract of Gompi ( Ecklonia stolonifera ).

As an appropriate solvent for extracting the extract from the gompi ( Ecklonia stolonifera ), any organic solvent that is pharmaceutically acceptable may be used, and water or an organic solvent may be used, but is not limited thereto, for example, Alcohols having 1 to 4 carbon atoms, including purified water, methanol, ethanol, propanol, isopropanol, butanol, etc., acetone, ether, benzene ), chloroform, ethyl acetate, methylene chloride, hexane, and cyclohexane may be used alone or in combination. Preferably, ethanol may be used.

As the extraction method, any one of methods such as hot water extraction, cold precipitation extraction, reflux cooling extraction, solvent extraction, steam distillation method, ultrasonic extraction method, elution method, and compression method may be used. In addition, the desired extract may be further subjected to a conventional fractionation process, or may be purified using a conventional purification method. There is no limitation on the method for preparing the extract of Gompi ( Ecklonia stolonifera ) of the present invention, and any known method may be used.

The compounds of the present invention, fluorofucofuroexcol-A or diekcol, are derived from Ecklonia stolonifera , and refer to compounds represented by the following formulas 1 or 2, respectively. The fluorofucofuro-excol-A or diexol may be isolated from a natural source such as Ecklonia stolonifera , but is not limited thereto, and chemically synthesized or commercially available materials may be used by methods known in the art. .

[Formula 1]

[Formula 2]

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. In the present invention, the term "pharmaceutically acceptable" means exhibiting properties that are not toxic to cells or humans exposed to the composition. The carrier may be used without limitation as long as it is known in the art such as a buffering agent, a preservative, a painless agent, a solubilizing agent, an isotonic agent, a stabilizer, a base agent, an excipient, and a lubricant.

In addition, the pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories and sterile injectable solutions according to a conventional method I can. Further, it may be used in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel or gel for external skin. Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, such as starch, calcium carbonate, in the Cycotria rubra extract, It is prepared by mixing sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. have. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like can be used.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administration" of the present invention means introducing a predetermined substance to an individual by an appropriate method, and the route of administration of the composition may be administered through any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration may be administered, but are not limited thereto.

The term "individual" refers to all animals including humans, such as mice, mice, and domestic animals. Preferably, it may be a mammal including a human.

The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not cause side effects, and the effective dose level is the sex, age, and Weight, health status, type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration, and rate of excretion, duration of treatment, factors including drugs used in combination or concurrently, and other fields of medicine. It can be readily determined by a person skilled in the art according to well known factors. Administration may be administered once a day at the recommended dosage, or may be divided several times.

The food composition of the present invention may include all foods in a conventional sense, and may be mixed with terms known in the art, such as functional foods and health functional foods.

The term "functional food" of the present invention refers to a food manufactured and processed using raw materials or ingredients having useful functions for the human body according to the Health Functional Food Act No.6727, and the term "functional" It means ingestion for the purpose of obtaining useful effects for health purposes such as controlling nutrients for structure and function or for physiological effects.

The term "health functional food" of the present invention refers to a food manufactured and processed by extracting, concentrating, refining, mixing, or extracting, concentrating, refining, and mixing specific ingredients contained in food ingredients or using specific ingredients for the purpose of health supplementation. It refers to foods designed and processed to sufficiently exert biological control functions such as biological defense, biological rhythm control, disease prevention and recovery, etc. by ingredients. The health food composition is used to prevent diseases and recover diseases. Can perform functions related to etc.

There is no limitation on the kind of food in which the composition of the present invention can be used. In addition, the composition of the present invention may be prepared by mixing suitable other auxiliary ingredients and known additives that may be included in food according to the choice of a person skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and There are vitamin complexes and the like, and can be prepared by adding the extract according to the present invention and a fraction thereof as a main component to juice, tea, jelly, and juice.

In addition, foods that can be applied to the present invention include, for example, special nutritional foods (e.g., formulas, infant foods, etc.), processed meat products, fish meat products, tofu, rice cakes, noodles (e.g., ramen, noodles, etc.), health supplement foods. , Seasoned foods (e.g. soy sauce, miso, red pepper paste, mixed sauce, etc.), sauces, confectionery (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc.) ), beverages (e.g. fruit, vegetable beverages, soy milk, fermented beverages, etc.), natural seasonings (e.g., ramen soup, etc.).

When the health functional food composition of the present invention is used in the form of a beverage, it may contain various sweetening agents, flavoring agents, natural carbohydrates, and the like as an additional component, like a normal beverage. In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin , Alcohol, carbonated beverages, etc. may contain. In addition, it may contain flesh for the manufacture of natural fruit juice, fruit juice beverage and vegetable beverage.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention more specifically, and the scope of the present invention is not limited to these examples.

Example 1. Experimental materials and methods

1.1. Chemicals and reagents

Phlorotannin, including phloroglucinol, dioxinodehydroeckol, dieckol, and phlorofucofuroeckol-A (PFF-A) ( Ecklonia stolonifera ) was isolated from the ethanol extract (Yoon N. et al ., Fish Sci. 2008, 74, 200-207). The chemical structural formula of fluorotannin is shown in FIG. 1.

1.2. h MAO-A (human monoamine oxidase A) and h MAO-B (human monoamine oxidase B) enzyme activity assay

In order to measure h MAO-A and h MAO-B inhibitory activity by fluorotannin, the present inventors performed an experiment using a MAO-Glo ^TM assay kit (Promega, Madison, WI, USA). Briefly, 12.5 μL of luciferin derivative was pre-incubated with an equal volume of fluorotannin or positive control (R-(-)-diprenyl HCl) by concentration, and then 25 μL of enzyme solution ( h MAO-A and h MAO-B enzyme was diluted with 2X reaction buffer (pH 7.4)) and incubated for 1 hour. To terminate the enzymatic reaction, 50 μL of RLDR (reconstituted luciferin detection reagent) was added and incubated for 20 to 30 minutes, and then ALU (arbitrary) using a Luminometer (Spectra Max L, Molecular Devices, Sunnyvale, CA, USA). light unit) was measured.

1.3. h MAO-A (huma monoamine oxidase A) and h Kinetic analysis of MAO-B (human monoamine oxidase B)

For the kinetic analysis of h MAO-A and h MAOB inhibition by fluorofucofuroexcol-A, the present inventors described various concentrations of substrate ( h MAO-A: 40, 80, 160 μM; and h MAO-B: 4 , 8, 16 μM) and fluorofucofuroexcol-A ( h MAO-A: 0, 2, 10, 20 μM; and h MAO-B: 0, 2.5, 5, 10 μM). Is shown as a Lineweaver-Burk plot.

1.4. Functional GPCRs (G-protein-coupled receptor) screening

Functional GPCRs cell-based assays include cAMP for G _i /G _s -coupled receptors; And the reading of multiple secondary messengers comprising IP ₁ /IP ₃ and calcium flux to G _{q -linked} receptors. Functional assays were performed in Eurofins Cerep (Le Bois I'Eveque, France) using transfected cells expressing human cloned receptors. The protocol of the in-house assay is shown in www.eurofinsdiscoveryservices.com/cms/cms-content/services/in-vitro-assays/gpcrs/functional, and specific experimental conditions are shown in Table 1 below. In the present invention, a stabilized cell line expressing recombinant GPCRs was used.

1.5. Measurement of cAMP levels

GPCR gene of interest (dopamine D ₁ , D ₃ or D ₄ receptor) was introduced into CHO (chinese hamster ovary) cells and transfected. The transfected CHO-GPCR cell line was suspended in HBSS/20 mM HEPES buffer (pH 7.4) to which 500 μM IBMX was added, dispensed on a microplate, and then treated without fluorotannin (control) or treated (25, 50 and 100 μM) and incubated at room temperature for 30 minutes. After incubation, the cells were lysed, and a fluorescent acceptor (D2-labeled cAMP) and a fluorescent donor (anti-cAMP antibody with europium cryptate) were added. After 1 hour, fluorescence transfer was measured at λex=337 nm and λem=620 and 665 nm using an Envision™ microplate reader (Perkin Elmer, Waltham, MA, USA). The cAMP concentration was determined by dividing the signal measured at 665 nm by the signal measured at 620 nm. The results of the agonist effect were expressed as the percent of the control response to dopamine, and the results of the antagonist effect were expressed as the percent inhibition of the control response to dopamine.

1.6. Intracellular [Ca ²⁺ ] Level measurement

Cells expressing other receptors were transfected with an expression vector encoding the receptor polypeptide and grown for a time sufficient for the receptor to be expressed. Then, a fluorescent probe (Fluo8 Direct, Invitogen, Carlsbad, CA, USA) mixed with probencid in HBSS/2 M HEPES buffer (pH 7.4) was added to each well and equilibrated at 37° C. for 1 hour. Made it. Thereafter, the assay plate was placed in a CellLux™ microplate reader (PerkinElmer, Waltham, MA, USA), and after adding fluorotannin (25 and 50 μM), a reference agonist or a buffer (blank), The fluorescence intensity which changes in proportion to the Ca ²⁺ ion concentration of the cytosol was measured. The experimental conditions carried out in each experiment are shown in Table 1 above. Accordingly, a concentration-response curve was generated to calculate the EC ₅₀ value.

The agonist effect was calculated as the percentage of the control response (measured response / control response × 100) to the known reference agonist of each target, and the antagonist effect was calculated as the control reference agonist in each target. ) Was calculated as the percent inhibition (100-(measured reaction / control reaction) × 100)

1.7. Homology modeling

The primary sequence (ID: P21728, DRD1_HUMAN) of the human dopamine D ₁ receptor (D ₁ R) was obtained from UniProt. As a template for modeling human D ₁ R, β ₂ adrenergic receptor (β ₂ R) was used, and the crystal structure of β ₂ R (PDB ID: 2RH1) was obtained from RCSB Protein Data Bank (PDB). Homology modeling was done through the SWISS-MODEL program and refined through the ModRefiner server.

1.8. In silico Docking simulation

The automated docking simulation was performed using the AutoDock 4.2 program. h MAO-A-harmine complex (PDB ID: 2Z5X), h MAO-B-deprenyl complex (PDB ID: 2BYB), h D ₃ R-eticlopride complex ( PDB ID: 3PBL) and the X-ray crystal structure of the h D ₄ R-nemonapride complex (PDB ID: 5WIU) were obtained from RCSB Protein Data Bank (PDB). The 3D structures of fluoroglucinol, diecol and fluorofucofuroexcol-A were respectively generated and transformed by Marvin Sketch program (v17,1,30, ChemAxon, Budapest, Hungary). Automated docking simulations were run using AutoDockTools (ADT) to evaluate the proper mating direction. For the docking calculation, Gasteiger charges were added by default, the rotatable bond was set to ADT, and all torsions were rotatable. The grid maps were created with AutoGrid. The docking protocol for docking non-bent or bendable ligands consisted of 15 independent genetic algorithms. Other parameters used are ADT defaults. Results were analyzed and visualized using Discovery Studio (v17.2, Accelrys, San Diego, CA, USA) and UCSF Chimera (http://www.cgl.ucsf.edu/chimera/).

1.8. Statistical analysis

The IC ₅₀ values (50% inhibitory concentration, μM) and EC ₅₀ values (50% effective concentration, μM) obtained from the logarithmic concentration inhibition curve were expressed as the mean ± standard deviation of three independent experiments.

Example 2. In vitro ( in vitro ) From fluorotannin h MAO-A and h Inhibitory effect of MAO-B

The present inventors used R-(-)-diprenyl HCl as a positive control group and used phloroglucinol, dioxinodehydroeckol, dieckol, and fluorofucofuro belonging to fluorotannin. An experiment was conducted to measure the inhibitory effect of h MAO-A and h MAO-B by phlorofucofuroeckol-A (PFF-A). As a result, it was confirmed that treatment with fluorofucofuroexcol-A showed an inhibitory effect on the activity of MAO, and in particular, for h MAO-B, the IC ₅₀ value was 4.89 ± 0.32, indicating a remarkable inhibitory effect on activity (Table 2). Selective index (SI) was calculated as the ratio of h MAO-A IC ₅₀ / h MAO-B IC ₅₀ , and the SI of fluorofucofuroexcol-A was 1.89 (Table 2). In contrast, fluoroglucinol or dioxynodehydroexcol did not show any effect at the tested concentration.

Example 3. h MAO-A (huma monoamine oxidase A) and h Kinetic analysis results of MAO-B (human monoamine oxidase B)

The present inventors performed an enzyme kinetic analysis through a Lineweaver-Burk plot and a secondary plot thereof in order to confirm the h MAO inhibition mechanism. As a result, the Lineweaver-Burk plot for the inhibition of the activity of h MAO-A and h MAO-B by fluorofucofuroexcol-A was drawn in a straight line and crossed in the x-axis (FIGS. 2A and 2C ). Thus, it was confirmed that Florofucofuroexcol-A is a noncompetitive inhibitor of h MAO-A and h MAO-B. In addition, the inhibitory constants ( K _i ) of h MAO-A and h MAO-B by the florofucofuroexcol-A through a secondary plot of the concentration of fluorofucofuroexcol-A versus 1/ V max were 5.18 and It was identified as 2.69 μM (Figures 2B and 2D).

Example 4. h Docking simulation results for MAO

The present inventors performed a docking simulation to demonstrate the mechanism of inhibition of h MAO-A and h MAO-B by fluorofucofuroexcol-A. As a result, as shown in Fig. 3 and Table 3 (binding energy and binding position), fluorofucofuroexcol-A is -7.71 and -7.22 at the non-catalytic positions of h MAO-A and h MAO-B, respectively. It was combined with a binding energy of kcal/mol, which was consistent with the results of kinetic analysis.

Specifically, 6 hydrogen bonds were observed between the hydroxyl moiety of fluorofucofuroexcol-A and the polar residue of h MAO-A and the Gly110 residue at the position where h MAO-A was bound to fluorofucofuroexcol-A. (Figures 3A and 3B). In addition, fluorofucofuroexcol-A interacted with the Lys316, Lys90 and Val83 residues of h MAO-A through pi-bonding (Fig. 3B). In the complex in which fluorofucofuroexcol-A was bound to h MAO-B, 6 hydrogen bonds were observed between the hydroxyl moiety of fluorofucofuroexcol-A and the hydrophilic residue of h MAO-B (Figs. 3C and 3D). . In addition, several hydrophobic interactions were observed between the phenol ring of fluorofucofuroexcol-A and the residues Glu483, Phe101, Trp119, Pro102 and Arg120 of h MAO-B (FIG. 3D ).

h MAO-A and MAO-B h in a flow of Foucault furo ekkol -A and the binding position is a non-product was confirmed to be similar to the bonding position of competitive h MAO-A and MAO-B h of crocin (crocin) inhibitors of (Monte CD et al ., Eur. J. Med. Chem. 2014, 82, 164-171).

Example 5. Functional GPCRs assay results

The present inventors analyzed dopamine D ₁ , D ₃ and D ₄ receptors (D ₁ , D ₃ and D ₄ ) through in vitro cell-based functional assay assays; Muscarinic acetylcholine receptor (M ₅ ); Neurokinin-1 receptor (NK ₁ ); Serotonin 1A receptor (serotonin 1A receptor, 5HT _1A ); And vasopressin V _1A receptor (V _1A ) was carried out an experiment to confirm the effect of fluoroglucinol, diecol and fluorofucofuroexcol-A.

As a result, it was confirmed that DEXCOL and FLOROFUCOFUROEXCOL-A were concentration-dependently as potent agonists for dopamine D ₃ and D ₄ receptors (FIG. 4). In particular, when treated with 100 μM of Diecol and Florofucofuroexcol-A for dopamine D ₃ receptor, agonist activity of 81.10 ± 0.66 and 98.57 ± 2.14%, respectively, and EC ₅₀ values were 44.21 ± 3.25 and 19.21, respectively. It was found to be ± 0.48 μM (Fig. 4A). In addition, when treated with 100 μM of Diecol and Florofucofuroexcol-A for dopamine D ₄ receptor, agonist activity of 74.43 ± 6.37 and 98.50 ± 12.50%, respectively, and EC ₅₀ values were 34.0 ± 8.62 and 23.47, respectively. It was confirmed as ± 1.55 μM (Fig. 4B).

On the other hand, as shown in Table 4 (promoting or inhibiting effect of fluorotannin on each receptor), for dopamine D ₁ receptor, Dieekchol and Florofucofuroexcol-A completely acted as antagonists, and 100 In the case of treatment with μM of Diecol and Florofucofuroexcol-A, the inhibitory effects were shown at 60.60 ± 2.97 and 81.40 ± 1.41%. In addition, in the case of treatment with 100 μM of fluorofucofuroexcol-A, the antagonist effect was also shown in the M ₅ , NK ₁ , 5HT _1A and V _1A receptors, and the agonist effect was partially shown on the M ₅ , NK ₁ and V _1A receptors. Confirmed. In addition, in the case of treatment with 100 μM of DEXchol, antagonist effects of 77.7 and 76.8% were shown for NK ₁ and 5HT _1A receptors, respectively, and partial agonist effects of 54.7% were shown for NK ₁ receptors. Unlike fluorofucofuroexcol-A, 100 μM of Diexol showed an agonist effect on the V _1A receptor. However, fluoroglucinol did not show any stimulating or inhibitory effect on any of the target receptors.

From the above results, fluorofucofuroexcol-A is an antagonist of dopamine D ₁ receptor (D ₁ R); Agonists of dopamine D ₃ and D ₄ receptors (D ₃ R and D ₄ R); Antagonists and partial agonists of muscarinic receptors (M ₅ ); Antagonists and partial agonists of neurokinin-1 receptor (NK ₁ ); Antagonists of serotonin 1A receptor (5HT _1A ); And vasopressin V _1A receptor (V _1A ) antagonist and partial agonist.

In addition, DEXchol is an antagonist of the dopamine D ₁ receptor (D ₁ R); Agonists of dopamine D ₃ and D ₄ receptors (D ₃ R and D ₄ R); Antagonists and partial agonists of neurokinin-1 receptor (NK ₁ ); And serotonin 1A receptor (5HT _1A ) antagonist.

Therefore, fluorofucofuroexcol-A or diexol can be used in the treatment of schizophrenia as an antagonist of dopamine D ₁ receptor, and as an agonist of dopamine D ₃ and D ₄ receptors, treatment of Parkinson's disease or schizophrenia Can be used for

Example 6. Simulation results of docking of fluorotannin to dopamine receptor

The present inventors have conducted a simulation to determine the molecular docking of the tannin combined with the flow in the dopamine D ₁ receptor by homology modeling of the dopamine D ₁ receptors (D ₁ R) based on the structure of the beta 2-adrenergic receptor. As a result, as shown in Table 5 and FIG. 5, diexol and florofucofuroexcol-A were docked at the active site of the dopamine D ₁ receptor, and hydrogen bonding with the preserved Asp103 residue at the third transmembrane site Formed. The two dibenzo-1,4-dioxin functional groups of Diecchol were surrounded by the hydrophobic residues of the dopamine D ₁ receptor and formed a pi-interaction with the Phe288, Leu190, Ile104, Ile154 and Pro158 residues. In addition, the fluoroglucinol moiety located on the inner side of the diekchol was bonded to the Ser198 residue of the fifth transmembrane site through a pi-isolation electron pair interaction.

Similarly, the dibenzo-1,4-dioxin and dibenzofuran functional groups of fluorofurofucoexcol-A formed a pi-pi stacking bond with the Phe288 residue of the dopamine D ₁ receptor, and with the Val317 and Ile104 residues. Pi-interactions were formed. In addition to the hydrophobic interaction, the hydroxyl groups of fluorofucofuroexcol-A were strongly bound to the dopamine D ₁ receptor through five hydrogen bonds. However, fluoroglucinol showed low binding affinity with conserved aspartic acid and serine residues of the dopamine D ₁ receptor.

In addition, the present inventors performed a docking simulation for the complex of D. Exol and fluorofucofuroexcol-A bound to dopamine D ₃ receptor (D ₃ R). As a result, as shown in Table 6 and FIG. 6, the predicted dopamine D ₃ receptor-dieccol complex and dopamine D ₃ receptor-florofucofuroexcol-A complex had important interactions that could stabilize the complex, It was confirmed that this was mainly due to the strong interaction with the Asp110 residue at the conserved active site and the pi-pi interaction with the surrounding hydrophobic residues.

In particular, the hydroxyl group of fluorofucofuroexcol-A formed five hydrogen bonds with the OBP (orthosteric binding pocket) residue of the dopamine D ₃ receptor, and the phenolic rings of fluorofucofuroexcol-A were Phe346, Cys114 and Asp110. The residues interacted with each other through pi-pi stacking bonds, pi-sulfur bonds, and pi-anionic bonds (FIGS. 6D and 6G).

In addition, in the dopamine D ₃ receptor-dieccol complex, four hydrogen bonds were observed between the hydroxyl of Diecol and the OBP and Val86 residues of the dopamine D ₃ receptor (FIGS. 6C and 6F ). The inner fluoroglucinol site of Dieccol formed an electrostatic bond and a pi-pi stack bond with Asp110 and Phe345 residues, respectively. In addition, the dibenzo-1,4-dioxin moiety of Dieccol was bonded to Tyr365, Cys114, Val111, Leu89, Val189 and His349 residues through pi-interaction.

In addition, the present inventors performed a docking simulation for the complex of D. Exol and fluorofucofuroexcol-A bound to dopamine D ₄ receptor (D ₄ R). As a result, as shown in Table 7 and FIG. 7, Dexekol and Florofucofuroexcol-A were docked with strong binding force to the active site of the dopamine D ₄ receptor. Particularly, the hydroxyl group of Dieccol and Florofucofuroexcol-A formed hydrogen bonds with Ser197 and Asp115 residues, which are important for activation in the dopamine D ₄ receptor. In addition, Diecol and Florofucofuroexcol-A formed a pi-interaction with surrounding residues of His414, Val193, Arg186, Cys185, Leu187 and Met122.

On the other hand, fluoroglucinol showed low avidity to the dopamine D ₃ receptor and the dopamine D ₄ receptor, unlike the diecol and fluorofucofuroexcol-A. The dopamine D ₃ receptor and the dopamine D ₄ receptor have a relatively large binding site for binding to fluoroglucinol, and thus the interaction with the OBP residue of the dopamine receptor was limited.

Claims (12)

A pharmaceutical composition for the prevention or treatment of neurological diseases comprising phlorofucofuroeckol-A or dieckol as an active ingredient. The method of claim 1,
The composition characterized in that the florofucofuro-ekol-A or D-ekol is derived from Gompi ( Ecklonia stolonifera ). The method of claim 1,
The composition characterized in that the florofucofuroexcol-A has a non-competitive inhibitory activity against MAO-A (monoamine oxidase A) or MAO-B (monoamine oxidase B). The method of claim 1,
The composition characterized in that the florofucofuroexcol-A or dieccol is an agonist of dopamine D ₃ receptor or dopamine D ₄ receptor. The method of claim 1,
The composition characterized in that the fluorofucofuroexcol-A or diexol is an antagonist of a dopamine D ₁ receptor. The method of claim 1,
The composition characterized in that the florofucofuro-ekol-A or diec-col is administered at a concentration of 10 to 200 μM. The method of claim 1,
The neurological disease is any one selected from the group consisting of neurodegenerative diseases, schizophrenia, schizophrenia, attention deficit hyperactivity disorder, personality disorder, autism, depression, and delusional disorder. The method of claim 7,
The neurodegenerative disease is a composition, characterized in that any one selected from the group consisting of Parkinson's disease, Alzheimer's disease, senile dementia, diabetic dementia, alcoholic dementia and vascular dementia. A health functional food for the prevention or improvement of neurological diseases containing phlorofucofuroeckol-A or dieckol as an active ingredient. A method of inhibiting the activity of MAO-A (monoamine oxidase A) or h MAO-B (human monoamine oxidase B), comprising the step of treating phlorofucofuroeckol-A to non-human individuals. A method for promoting signaling by dopamine D ₃ receptor or dopamine D ₄ receptor, comprising the step of treating a subject other than humans with phlorofucofuroeckol-A or dieckol. A method of inhibiting signaling by dopamine D ₁ receptor, comprising the step of treating an individual other than humans with phlorofucofuroeckol-A or dieckol.

Images