Pharmaceutical composition comprising padina arborescens extract as active ingredient for inhibiting LRRK2 kinase activity or for Anti-neuroinflammation

Abstract

The present invention relates to a pharmaceutical composition for inhibiting the activity of leucine-rich repeat kinase 2 (LRRK2) or for anti-neuroinflammation, and more specifically, to a pharmaceutical composition comprising a Padina arborescens extract as an active ingredient for inhibiting the activity of leucine-rich repeat kinase 2 (LRRK2) or for anti-neuroinflammation. According to the present invention, the risk of developing diseases and neuroinflammation due to the activity of the LRRK2 kinase is reduced, and the LRRK2 kinase can be utilized in medicines, foods, quasi-drugs, etc. for preventing, alleviating, and treating diseases and neuroinflammation due to the activity of the LRRK2 kinase.

Description

Pharmaceutical composition for inhibiting LRRK2 phosphorylation enzyme activity or for anti-neuroinflammatory purposes, containing extract of Buchatmal as an active ingredient

[0001] The present invention relates to a pharmaceutical composition for inhibiting LRRK2 (Leucine-rich repeat kinase 2) kinase activity or for anti-neuroinflammation, and more specifically, to a pharmaceutical composition for inhibiting LRRK2 kinase activity or for anti-neuroinflammation containing an extract of Buchatmal as an active ingredient.

[0002] As genome analysis technology advances, new genes related to degenerative brain diseases are discovered and the disease is understood more deeply, leading to the development of new drugs that target the cause of Parkinson's disease rather than just treating its symptoms.

[0003] LRRK2, the fourth gene identified as a Parkinson's disease-related gene following alpha-synuclein, PARK2 (Parkin), and PARK7 (DJ-1), is being actively studied as a new target for Parkinson's disease treatment. Mutations in LRRK2 lead to excessive activation of the kinase, increasing the incidence of Parkinson's disease. Furthermore, LRRK2 mutations are associated with various pathological mechanisms, including vesicle transport, lysosomal degradation, neurotransmission, and mitochondrial function.

[0004] Meanwhile, while microglia are necessary for maintaining neuronal activity, neuroinflammation induced by reactive microglia is associated with the progression of Parkinson's disease. Oxidative stress in dopaminergic neurons is also a pathological hallmark of Parkinson's disease. Since the release of proinflammatory cytokines from reactive microglia damages dopaminergic neurons, conversely, alleviating neuroinflammation may contribute to the treatment of Parkinson's disease. LRRK2 is also involved in neuroinflammation, and previous studies have demonstrated that inhibiting LRRK2 activity in reactive microglia can prevent dopaminergic neuronal degeneration.

[0005] Accordingly, interest in LRRK2 inhibitors as important pharmacological agents for the treatment of Parkinson's disease has increased in recent years. However, in the case of new LRRK2 inhibitors, development is often halted due to unexpected toxicity in the clinical trial stage, just like previously discovered alpha-synuclein inhibitors. That is, from the active substance development stage of new drugs to the clinical trial stage, researchers have selected and developed artificial compounds, antibody preparations, and aptamer preparations to find substances that simultaneously exhibit therapeutic effects on pathological phenomena such as alpha-synuclein polymerization, LRRK2 kinase activity, and neuroinflammation. However, it is presumed that these new drug candidates have already inducible properties that induce a certain degree of oxidative stress and neuroinflammation even in the cellular testing stage, which has caused problems.

[0006] However, despite these numerous studies, no research results have been reported to date showing that the extract of the natural raw material, Buchaetmal, is effective in inhibiting LRRK2 kinase activity and neuroinflammation.

[0007] Therefore, in order to solve these problems, the inventor of the present invention completed the present invention by measuring the inhibition of LRRK2 phosphorylation enzyme activity and the anti-neuroinflammatory effect using the extract of the Korean ginseng root, which suppresses the above-mentioned pathological phenomenon while being more biologically stable than artificial compounds, antibody preparations, and aptamer preparations, and additionally has antioxidant and anti-inflammatory effects, and by elucidating the mechanism of the effect.

[0008] The purpose of the present invention is to provide a pharmaceutical composition for inhibiting LRRK2 phosphorylation enzyme activity or for anti-neuroinflammation, which contains Padina extract as an active ingredient.

[0009] To achieve the above purpose, the present invention discloses a pharmaceutical composition for inhibiting LRRK2 phosphorylation enzyme activity or for anti-neuroinflammatory purposes, which comprises an extract of the plant as an active ingredient.

[0010] The above extract of the plant can suppress oxidative stress and simultaneously inhibit alpha-synuclein polymer formation and LRRK2 phosphorylation enzyme activity.

[0011] The above extract of the plant can reduce inflammatory cytokines and suppress neuroinflammation.

[0012] The above pharmaceutical composition is a pharmaceutical composition for preventing or treating a disease or condition mediated by or related to LRRK2, wherein the disease or condition mediated by or related to LRRK2 may be a degenerative brain disease.

[0013] The above degenerative brain disease may be at least one disease selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, and mild cognitive impairment.

[0014] In addition, in order to achieve the above purpose, the present invention further discloses a pharmaceutical preparation comprising the pharmaceutical composition as described above.

[0015] The above pharmaceutical preparation can be formulated and used in the form of any one type of oral formulation, external preparation, suppository, and sterile aqueous solution selected from the group consisting of powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols.

[0016] The above extract of the plant can be included in a solvent at a concentration of 0.1 to 10 μg / mL.

[0017] Meanwhile, in order to achieve the above purpose, the present invention additionally discloses a food composition for inhibiting LRRK2 phosphorylation enzyme activity or for anti-neuro-inflammation, which contains an extract of Buchatmal as an effective ingredient.

[0018] The above food composition may be any one selected from the group consisting of dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum and ice cream, soup, beverage, tea, drink, alcoholic beverage and vitamin complex.

[0019] The pharmaceutical composition for inhibiting LRRK2 kinase activity or for anti-neuroinflammatory activity, which comprises the extract of the present invention as an active ingredient, reduces the risk of developing diseases and neuroinflammation caused by the activity of LRRK2 kinase, and can be used as a medicine, food, or quasi-drug for preventing, improving, and treating diseases and neuroinflammation caused by the activity of LRRK2 kinase.

[0020] In addition, the pharmaceutical composition for inhibiting LRRK2 phosphorylation enzyme activity or for anti-neuroinflammatory purposes, which contains the extract of the present invention as an active ingredient, has the effect of simultaneously inhibiting alpha-synuclein polymer formation and LRRK2 phosphorylation enzyme activity.

[0021] In addition, the present invention has the advantage of having fewer side effects and being more biologically stable than artificial compounds, antibody preparations, and aptamer preparations, as it uses natural materials as raw materials.

[0022] Figure 1 illustrates the results of cell viability and cytotoxicity experiments according to the concentration of a composition according to one embodiment of the present invention, showing (a) the results of the cell viability experiment and (b) the results of the cytotoxicity experiment.

[0023] FIG. 2 illustrates the results of measuring the inhibitory effect of a composition according to one embodiment of the present invention on LRRK2 kinase activity, showing (a) the results of phosphorylation level analysis at pS1292, (b) the results of Western blot analysis, and (c) the results of phosphorylation level analysis at pS935.

[0024] FIG. 3 illustrates the results of measuring the inflammatory cytokine reduction effect of a composition according to one embodiment of the present invention in BV2 cells, including (a) the results of measuring the level of TNF-α expression using qPCR, (b) the results of analyzing the level of LRRK2 phosphorylation in BV2 cells using Western blot, and (c) the results of analyzing the level of phosphorylation of pS935.

[0025] FIG. 4 illustrates the results of measuring the inflammatory cytokine reduction effect of a composition according to one embodiment of the present invention in primary cultured mouse microglia, including (a) the results of measuring the level of TNF-α expression by qPCR, (b) the results of measuring the level of inducible iNOS expression by qPCR, (c) the results of TNF-α ELISA analysis of cell lysate supernatant, and (d) the results of Greiss analysis of cell lysate supernatant.

[0026] FIG. 5 illustrates the results of measuring the effect of a composition according to one embodiment of the present invention on reducing oxidative stress in primary cultured astrocytic cells of mice, showing (a) the results of measuring cellular ROS and (b) the results of measuring cellular NO.

[0027] FIG. 6 illustrates the results of measuring the inhibitory effect of a composition according to one embodiment of the present invention on alpha-synuclein polymer formation, including (a) thioflavin T analysis results, (b) Western blot analysis results, (c) alpha-synuclein density measurement results, and (d) total alpha-synuclein level measurement results in dopaminergic neurons.

[0028] FIG. 7 illustrates the results of measuring the effect of a composition according to one embodiment of the present invention on increasing lysosomal activity in dSH, including (a) lysosomal quantification results, (b) lysosomal GCase activity analysis results, and (c) lysosomal GCase activity analysis results based on lysosomal quantification values.

[0029] The terminology used in this specification is solely for the purpose of describing specific examples. Therefore, for example, singular expressions include plural expressions unless the context clearly dictates that they must be singular. In addition, it should be noted that terms such as "comprise" or "have" used in this specification are used to clearly indicate the presence of a feature, step, function, component, or combination thereof described in the specification, and are not used to preliminarily exclude the presence of other features, steps, functions, components, or combinations thereof.

[0030] Meanwhile, unless otherwise defined, all terms used herein should be considered to have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Therefore, unless explicitly defined herein, specific terms should not be interpreted in an overly idealistic or formal sense.

[0031] The term “treatment” as used in the present invention means any action by which symptoms related to neuroinflammation are improved or beneficially changed by administration of a pharmaceutical composition according to the present invention.

[0032] As used herein, the term "improvement" means any action that at least reduces a parameter associated with the condition being treated, for example, the severity of a symptom.

[0033] The term “prevention” used in the present invention means any act of inhibiting nerve damage or delaying its onset by administering a pharmaceutical composition or food composition according to the present invention.

[0034] Meanwhile, the food composition according to the present invention can be used simultaneously with or separately from a drug for treatment before or after the onset of the disease to prevent or improve neuroinflammation.

[0035] The term "neuroinflammation" used in the present invention refers to an inflammatory response that occurs in the nervous system, i.e., nerve cells, nerve tissue, etc. It may include a phenomenon in which microglia, immune cells present in the central nervous system, are activated by various exogenous and endogenous substances, producing and releasing substances such as inflammatory cytokines such as TNF-α and IL-1β, nitric oxide, and prostaglandins. While the production of these substances induces an immune response in the short term, excessive or continuous production is known to induce the death of nearby nerve cells, ultimately leading to neurodegeneration.

[0036]

[0037] The present specification discloses a pharmaceutical composition for inhibiting LRRK2 phosphorylation enzyme activity or for anti-neuroinflammatory purposes, comprising an extract of Buchatmal as an active ingredient.

[0038] The above extract of the plant can suppress oxidative stress and simultaneously inhibit alpha-synuclein polymer formation and LRRK2 phosphorylation enzyme activity.

[0039] The above extract of Buchaetmal is an effective substance for LRRK2 kinase inhibitor and can suppress oxidative stress-induced cytotoxicity caused by overexpression of LRRK2 mutant protein.

[0040] The above extract of the plant can reduce inflammatory cytokines and suppress neuroinflammation.

[0041] While early neuroinflammation appears to be a protective response of the brain, excessive inflammation is not only harmful, but can actually impair nerve regeneration, leading to neurodegenerative diseases and other neurological disorders.

[0042] Microglia, which regulate neuroinflammation, are activated continuously to produce cytokines and reactive oxygen species by single or chronic exposure to disease-related proteins, environmental toxins, cytokines, and neuronal damage, resulting in neuroinflammation and the progressive loss of neurons, i.e., neurodegenerative diseases.

[0043] The above pharmaceutical composition is a pharmaceutical composition for preventing or treating a disease or condition mediated by or related to LRRK2, wherein the disease or condition mediated by or related to LRRK2 may be a degenerative brain disease.

[0044] The above degenerative brain disease may be at least one disease selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, and mild cognitive impairment.

[0045] The above extract of the plant is an effective substance for a pharmaceutical composition for inhibiting LRRK2 phosphorylation enzyme activity or for anti-neuroinflammatory purposes. Since it is a natural raw material, it has fewer side effects and can be more biologically stable than artificial compounds, antibody preparations, and aptamer preparations.

[0046] The above-mentioned extract of the plant may be extracted with one or more organic solvents selected from the group consisting of ethanol, methanol, isopropyl alcohol, butanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), tetrahydrofuran (THF), acetonitrile, dichloromethane, ethyl acetate, hexane, diethyl ether, benzene, chloroform, and acetone, but is not limited thereto. The organic solvent is preferably, more specifically, ethanol, but is not limited thereto.

[0047]

[0048] Additionally, the present specification discloses a pharmaceutical preparation comprising a pharmaceutical composition as described above.

[0049] The above pharmaceutical preparation can be formulated and used in the form of any one type of oral formulation, external preparation, suppository, and sterile aqueous solution selected from the group consisting of powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols.

[0050] The pharmaceutical composition of the present invention may further comprise, in addition to the above-mentioned effective ingredient, appropriate carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. The pharmaceutical composition of the present invention may further comprise other pharmaceutically active ingredients or active mixtures.

[0051] Carriers, excipients, and thinners that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, the composition is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. with the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0052] The preferred dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, the severity of the disease, the drug form, the route of administration, and the duration of the administration, but can be appropriately selected by those skilled in the art. Administration may be administered once daily or in several divided doses. The above dosage does not limit the scope of the present invention in any way.

[0053] The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans via various routes. All modes of administration are contemplated, and for example, administration can be administered orally, transdermally, rectally, or intravenously, intramuscularly, subcutaneously, or by injection.

[0054] The definitions of terms for the excipients, binders, disintegrants, lubricants, flavoring agents, etc. of the present invention include those described in documents known in the art and having the same or similar functions.

[0055] The above-mentioned Buchaetmal extract may be included in the solvent at a concentration of 0.1 to 300 μg / mL. For example, when the Buchaetmal extract is included in the solvent at less than 0.1 μg / mL, the problem of not exhibiting the LRRK2 kinase activity inhibition or anti-neuroinflammatory effect of the Buchaetmal extract may occur. Conversely, when the Buchaetmal extract is included in the solvent at more than 300 μg / mL, the problem of exhibiting a cytotoxicity level that is not suitable as a pharmacological substance may occur. The Buchaetmal extract is more specifically preferably included in the solvent at a concentration of 1 to 10 μg / mL, but is not limited thereto. The Buchaetmal extract shows an increasing LRRK2 kinase activity inhibition effect within the concentration range of 1 to 10 μg / mL, and when the Buchaetmal extract is present at a concentration of 10 μg / mL or more, the LRRK2 kinase inhibition effect does not significantly increase compared to the concentration of 10 μg / mL and is maintained similarly. Therefore, the above-mentioned extract of the Korean ginseng root has the advantage of being cost-effective and economical in terms of process by exhibiting an inhibitory effect on LRRK2 kinase even when included in a low concentration of 1 to 10 μg / mL. In the cell viability and cytotoxicity experiments performed to process by setting the concentration of the Korean ginseng root to a smaller range in the experimental process of the present invention, it was confirmed that when 1 μg / mL was included in the solvent, the cell viability was higher and the cytotoxicity was lower than when 2 μg / mL and 4 μg / mL were included. Although the cytotoxicity at concentrations of 2 μg / mL and 4 μg / mL was not significant, the cell viability was the highest and the cytotoxicity was the lowest at a concentration of 1 μg / mL.

[0056]

[0057] Meanwhile, the present specification further discloses a food composition for inhibiting LRRK2 phosphorylation enzyme activity or for anti-neuroinflammatory purposes, which contains an extract of the plant as an active ingredient.

[0058] The above food composition may be any one selected from the group consisting of dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum and ice cream, soup, beverage, tea, drink, alcoholic beverage and vitamin complex.

[0059] The health functional food or food composition of the present invention may contain the extract of Atractylodes japonica alone or in combination with other foods or food ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing a food or beverage, the composition of the present invention may be added in an amount of 15 parts by weight or less, 10 parts by weight or less, 1 to 15 parts by weight, 1 to 10 parts by weight, 1 to 5 parts by weight, or 0.1 to 15 parts by weight based on the total weight. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may also be used in an amount above the above range.

[0060] There is no particular limitation on the type of the above food. Examples of foods to which the above substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, dairy products including gum and ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and all foods in the conventional sense are included. The above food composition can be used as a food material such as milk or yogurt, food and beverage, or functional food. The food composition can contain various flavoring agents or natural carbohydrates as additional ingredients. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Sweeteners can include natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame.

[0061] The food composition of the present invention may also contain nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages, or combinations thereof. The food composition of the present invention may also contain fruit pulp for the production of natural fruit juices, fruit juice drinks, vegetable drinks, or combinations thereof. Such additives may be selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition.

[0062] The effective ingredient of the present invention, the extract of the burdock root, can also be used as a quasi-drug, but is not limited thereto.

[0063] The quasi-drug composition of the present invention refers to a product that exhibits the effect of treating, alleviating, managing, or preventing a disease, but has a milder effect on the human body than a pharmaceutical product. This excludes products used for pharmaceutical purposes under the Pharmaceutical Affairs Act, and includes products classified according to the separate classification standards established by the Ministry of Health and Welfare. Specifically, this may include, but is not limited to, topical skin preparations or personal hygiene products.

[0064] When adding the composition of the present invention to a quasi-drug composition, the composition may be added as is or used together with other quasi-drug ingredients, and may be used appropriately according to conventional methods. The amount of active ingredient mixed may be appropriately determined depending on the intended use.

[0065] Duplicate contents are omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention belongs.

[0066] Hereinafter, the claims of the present specification will be described in more detail with reference to the attached drawings and embodiments. However, the drawings and embodiments presented in this specification may be modified in various ways by those skilled in the art and may have various forms, and therefore, the description in this specification should not be considered to limit the present invention to a specific disclosed form, but to include all equivalents and substitutes included in the spirit and technical scope of the present invention. In addition, the attached drawings are presented to help those skilled in the art understand the present invention more accurately, and may be depicted in an exaggerated or reduced form compared to the actual form.

[0067] {Examples and Evaluation}

[0068] In order to investigate the simultaneous inhibitory effect of Padina arborescens, an extract of Padina arborescens, an effective ingredient of the present invention, on LRRK2 phosphorylation enzyme activity and alpha-synuclein polymer formation, as well as on the anti-neuroinflammatory effect, the inventors of the present invention confirmed the effect of Padina arborescens, a seaweed, on neuroinflammation in the BV2 microglial cell line and primary cultured mouse microglia and astroglial cells.

[0069] 1. Materials and Methods

[0070] (1) Materials

[0071] 1) P. arborescens extract

[0072] The P. arborescens extract was applied for and transferred from the Marine Life Resources Integrated System under the Ministry of Oceans and Fisheries. The P. arborescens extract was extracted using ethanol as the extraction solvent. The P. arborescens extract was dissolved in DMSO (Dimethyl Sulfoxide) to a concentration of 2 mg / mL and then filtered through a 0.2 μg nylon membrane to remove large structures and bacteria.

[0073]

[0074] (2) Method

[0075] 1) Cell culture and reagent treatment

[0076] Mouse microglial cell line BV2 was cultured and maintained in growth medium consisting of Dulbecco's modified essential medium (DMEM, LM001-07; Welgene, Gyeongsan-si, Republic of Korea), 1X antibiotic-antimycotic solution (15240062, Gibco, Carlsbad, CA, USA), and 5% EqualFETAL (EF-0500-A; Atlas Biologicals, Inc., Fort Collins, CO, USA) in an incubator at 37°C and 5% CO2. For induction of proinflammatory cytokines, lipopolysaccharide (LPS, 200 ng / mL; L4391, Sigma-Aldrich, St. Louis, MO, USA) mixed in EqualFETAL depleted growth medium was used for 4 or 18 h, and 1 μg / mL of P. arborescens extract (PA_EXT) was co-treated for 4 or 18 h. The human dopaminergic neuronal cell line SH-SY5Y was maintained in DMEM (LM001-07; Welgene) with 10% EqualFETAL (EF-0500-A; Atlas Biologicals, Inc.) and 1X antibiotic-antimycotic solution (15240062; Gibco) in an incubator at 37°C and 5% CO2. Differentiation of SH-SY5Y was achieved by treatment with 10 μM all-trans retinoic acid (R2625; Sigma-Aldrich) for 7 days, followed by treatment with DMSO and PA_EXT for 7 days.

[0077]

[0078] 2) Preparation and culture of primary cultured microglia and astroglia from rats

[0079] For culture, primary mouse microglia and astroglia were collected from the fetal brain (embryonic day 17 [E17]), dissected, and the cortex was isolated. The isolated tissue was incubated with trypsin-ethylenediaminetetraacetic acid (EDTA, 0.25%), phenol red (25200056, Gibco), and deoxyribonuclease I from bovine pancreas (DN-25-100MG; Sigma-Aldrich) for 30 min and dissociated by pipetting. Cell debris and myelin were removed by centrifugation at 200 x g for 10 min at room temperature (RT). Cells were seeded into T75 flasks (11090; SPL, Pocheon-si, Republic of Korea) through a nylon mesh filter and stored in 10% fetal bovine serum (BFS-1000; T&I, Chuncheon-si, South Korea) and 1X antibiotic-antimycotic solution (LM002-04; Welgene). After 1 week, TrypLE TM Cells were dissociated using Express enzyme (1X), phenol red-free (12604013; Gibco), re-dissociated into T75 flasks, and amplified. After 2 weeks, microglia were dissociated by vigorous tapping and purified using TrypLE without phenol red. TM Astroglial cells were isolated using the Express enzyme (1X). Afterwards, primary cultured mouse microglia or astrocytes 5X10 were used for experiments. 5 Dogs were seeded in 12-well plates (30012; SPL). 100 ng / mL LPS or 0.8 μg / mL PA_EXT was mixed in FBS-depleted growth medium and administered for 24 h, after which cells and culture medium were harvested for subsequent analysis.

[0080]

[0081] 3) In vitro kinase assay method

[0082] For in vitro kinase assay, a mixture of 65 ng of full-length LRRK2 G2019S recombinant protein (A15200; Thermo Fisher Scientific) and 50 μM adenosine triphosphate (ATP, A1852-1VL; Sigma-Aldrich) in 1X kinase buffer (A15200; Thermo Fisher Scientific, Waltham, MA, USA) was incubated at 35 °C for 1 h. The reaction was then stopped by adding 4X Laemmli sample buffer (L1100-001; GenDEPOT Katy, TX, USA), and the phosphorylation level of full-length LRRK2 G2019S was assessed using Western blot analysis.

[0083]

[0084] 4) Western blot analysis

[0085] To quantify proteins, cells were washed twice with cold Dulbecco's phosphate-buffered saline (DPBS LB001-02; Welgene) and harvested into 1x sample buffer diluted with 4x Laemmli sample buffer, reducing agent (GenDEPOT), and sterile water. Samples were sonicated for 20 s at 10% amplification using an ultrasonic processor (VCX 130; Sonics & Materials, Inc., Newtown, CT, USA) and boiled at 95 °C for 5 min. Each sample was loaded onto a 4–20% MINI152 PROTEAN® TGX precast protein gel, 15-well (15 μL; 4561096; Bio-Rad, Hercules, CA, USA) and electrophoresed at 100 V for 100 min. After transferring the proteins to a nitrocellulose membrane (10600004; Cytiva, Marlborough, MA, USA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed at 300 mA for 80 min. The nitrocellulose membrane containing the protein of interest was soaked in 5% skim milk in osmotic buffered saline containing 0.1% Tween-20 (TBST) for 30 min at room temperature. The primary antibodies listed in Table 1 were mixed with 1% 158 bovine serum albumin (BSA) in TBST overnight. The membrane was washed three times with TBST and incubated with secondary antibodies (Table 1) to detect protein bands. Luminata Crescendo Western horseradish-peroxidase (HRP, WBLUR0500; Merck & Co., Inc., Kenilworth, NJ, USA) was used to detect immunoreactive signals on nitrocellulose membranes, and a MicroChemi 4.2 camera (Shimadzu, Kyoto, Japan) was used to obtain images of protein bands.

[0086]

[0087]

[0088] 5) mRNA isolation and cDNA synthesis

[0089] Cells were washed twice with cold DPBS, and mRNA was isolated from the harvested cells using the RNeasy Plus mini kit (74134; Qiagen, Germantown, MD, USA).

[0090] A TORscript cDNA synthesis kit (EZ005S; Enzynomics, Daejeon, Republic of Korea) was used for cDNA synthesis. For cDNA synthesis and amplification, a mixture containing 2 μL 10X TORscript RT buffer, 1 μL TORscript reverse transcriptase (200 U / μL), 2 μL dNTP mixture (2 mM), 2 μL total RNA, 1 μL oligodT, 0.5 μL RNase inhibitor (40 U / μL), 11.5 μL RNase-free sterile water, and 1 μg total RNA was incubated at 55°C for 60 min. The reaction was then stopped by incubating the sample at 95°C for 5 min.

[0091]

[0092] 6) qPCR analysis

[0093] qPCR analysis was performed with 0.5 μL of synthesized cDNA and 5 μL of TOPreal TMPCR was performed using SYBR Green qPCR PreMIX (RT500S; Enzynomics, Daejeon, Republic of Korea), 0.25 μL of primers, and 4.25 μL of RNase-free sterile water with the primers listed in Table 2. The assay was also performed using a magnetic induction cycler Cycler (MIC; BioMolecular Systems, Upper Coomera QLD, Australia). mRNA levels were measured using the following formula: 2^(-delta CT).

[0094]

[0095]

[0096] 7) ELISA analysis for enzyme-linked immunosorbent assay of cytokines and neurostimulating factors

[0097] To detect the levels of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and inducible nitric oxide (iNOS), culture fluids from BV2 cells and primary murine microglia were collected by centrifugation at 4,000 rpm for 10 min at 4°C. For each experiment, 100 μL of culture fluid was applied to a commercial murine TNF-α ELISA kit (DY510-05; R&D System, Minneapolis, MN, USA). All assays were performed according to the manufacturer's instructions. Additionally, a Synergy 2 microplate reader (Biotek Instruments, Inc., Winooski, VT, USA) was used to measure absorbance at 450 nm.

[0098]

[0099] 8) Griess assay for measuring intracellular nitric oxide (NO) levels

[0100] NO levels were measured using the Griess assay (G7921, Thermo Fisher Scientific), which evaluates two major stable and nonvolatile breakdown products of NO. Fifty μL of the collected cell lysate supernatant was centrifuged at 4,000 rpm for 10 min at 4°C and used for the Griess assay. Equal amounts of f N-(1-naphthyl) ethylenediamine (component A) and sulfanilic acid (component B) were mixed to obtain the Griess reagent, and 20 μL of the Griess reagent was mixed with a mixed sample of deionized water (280 μL) in a dark 96-well plate. After incubation at room temperature for 30 min, the Synergy TM The absorbance of the sample was recorded at 548 nm using a 2 system (Biotek).

[0101]

[0102] 9) Measurement of cellular reactive oxygen species (ROS)

[0103] Primary rat astrocytes seeded in dark 96-well plates were treated with 3 μM rotenone (RTN; 557368; Sigma-Aldrich) or DMSO (D2650; Sigma-Aldrich) and co-treated with 1 μg / mL PA_EXT. After 24 h, 5 μM CellRox (C10444; Thermo Fisher Scientific) and 2 μM Hoechst 33342 (62249; Thermo Fisher Scientific) were added, and the cells were incubated for 30 min. The cells were then washed twice with cold DPBS and fixed with cold 4% paraformaldehyde (161-20141; Fujifilm Wako Pure 201 Chemical Corporation, Tokyo, Japan) for 15 min. Afterwards, cells were washed three times with cold DPBS, and the absorbance of CellROX and Hoechst33342 was recorded using a FlexStation 3 multimode microplate reader (Molecular Devices, San Jose, CA, USA) at excitation wavelengths of 485 nm and 361 nm and emission wavelengths of 520 nm and 497 nm, respectively.

[0104]

[0105] 10) Analysis of seed-accelerated fibrogenesis of alpha-synuclein

[0106] Recombinant alpha-synuclein monomers were purified and isolated from BL21 Escherichia coli transformed with a human alpha-synuclein plasmid provided by Dr. Seungjae Lee. Alpha-synuclein monomers (1 mg / mL) were then incubated with or without 5 μg / mL fibrillar alpha-synuclein (fibrillar seeds) at 37°C with shaking at 150 rpm. During incubation for the indicated times, vector DMSO or PA_EXT was added to the alpha-synuclein monomer pool along with fibrillar seeds.

[0107]

[0108] 11) Thioflavin T analysis

[0109] Each sample (100 μL) was collected at the designated time and immediately frozen for thioflavin T analysis. Thioflavin T analysis was performed according to previously described procedures (Bae et al., 2013).

[0110]

[0111] 12) Sandwich-enzyme-linked immunosorbent assay (ELISA) for alpha-synuclein

[0112] Differentiated SH-SY5Y (dSH) cells treated with DMSO or PA_EXT were lysed in phosphate-buffered saline (PBS; Gibco, 18912014) containing 1% Triton-X 100 (85111, Thermo Fisher Scientific) and 1Х Xpert protease inhibitor cocktail (P3100-005, GenDEPOT). The lysates were centrifuged at 12,000 × g for 10 min at 4 °C, and the supernatants of the separated lysates were subjected to sandwich ELISA as described in our previous study (Nam et al., 2020).

[0113]

[0114] 13) Lysosomal activity analysis

[0115] Differentiated SH-SY5Y (dSH) cells treated with DMSO or PA_EXT were stained with 1 μM LysoTracker Blue DND-22 (L7525; Thermo Fisher Scientific), 2 μM 5-(pentafluorobenzoamino) Fluorescein Di-β-D-Glucopyranoside (PFB-FDGlu, P11947; Thermo Fisher Scientific), and 0.5 μM SYTO 59 red fluorescent nucleic acid stain (S11341; Thermo Fisher Scientific) for 1 h, washed twice with PBS, and images were acquired using a FLoid Cell Imaging Station (4471136; Thermo Fisher Scientific). Intensity was analyzed using Multigauge software (Fujifilm, Tokyo, Japan).

[0116]

[0117] 14) Data estimation and statistical analysis

[0118] Western blot images were analyzed by densitometry using a MultiGage (Fujifilm), and the significance and composition of data charts were estimated using Prism 8 software (GraphPad). The statistical analyses and significance levels used in this study are indicated in the figure legends.

[0119]

[0120] <Example>

[0121] The present invention will be described in more detail through the following examples; however, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0122]

[0123] Example 1

[0124] The P. arborescens extract, obtained from the Marine Life Resources Integrated Information System under the Ministry of Oceans and Fisheries, was dissolved in DMSO at a concentration of 2 mg / mL. The extract was then filtered through a 0.2 μm nylon membrane to remove large structures and bacteria before use.

[0125] In Example 1, a filtered P. arborescens extract solution was diluted to a concentration of 0.1 μg / mL to prepare a composition of extract of P. arborescens (PA_EXT) according to one embodiment of the present invention (hereinafter referred to as “Example 1”).

[0126]

[0127] Example 2

[0128] A composition of the extract of P. arborescens according to one embodiment of the present invention was prepared in the same manner as in Example 1, except that the filtered P. arborescens extract solution was diluted to a concentration of 0.2 μg / mL (hereinafter referred to as “Example 2”).

[0129]

[0130] Example 3

[0131] A composition of the extract of P. arborescens according to one embodiment of the present invention was prepared in the same manner as in Example 1, except that the filtered P. arborescens extract solution was diluted to a concentration of 0.4 μg / mL (hereinafter referred to as “Example 3”).

[0132]

[0133] Example 4

[0134] A composition of the extract of P. arborescens according to one embodiment of the present invention was prepared in the same manner as in Example 1, except that the filtered P. arborescens extract solution was diluted to a concentration of 10 μg / mL (hereinafter referred to as “Example 4”).

[0135]

[0136] Example 5

[0137] A composition of the extract of P. arborescens according to one embodiment of the present invention was prepared in the same manner as in Example 1, except that the filtered P. arborescens extract solution was diluted to a concentration of 100 μg / mL (hereinafter referred to as “Example 5”).

[0138]

[0139] Example 6

[0140] A composition of the extract of P. arborescens according to one embodiment of the present invention was prepared in the same manner as in Example 1, except that the filtered P. arborescens extract solution was diluted to a concentration of 200 μg / mL (hereinafter referred to as “Example 6”).

[0141]

[0142] Example 7

[0143] A composition of the extract of P. arborescens according to one embodiment of the present invention was prepared in the same manner as in Example 1, except that the filtered P. arborescens extract solution was diluted to a concentration of 300 μg / mL (hereinafter referred to as “Example 7”).

[0144]

[0145] The materials and compositions of Examples 1 to 7 are summarized in Table 3 below.

[0146]

[0147]

[0148] Comparative Example 1

[0149] This is a DMSO solution to which no P. arborescens extract solution is added (hereinafter referred to as “Comparative Example 1”).

[0150]

[0151] Comparative Example 2

[0152] MLi-2 is a LRRK2 inhibitor (hereinafter referred to as “Comparative Example 2”).

[0153]

[0154] 2. Experiment and Evaluation

[0155] (1) Evaluation of cell viability and cytotoxicity according to PA-EXT concentration

[0156] Figure 1 illustrates the results of cell viability and cytotoxicity experiments according to the concentration of a composition according to one embodiment of the present invention, showing (a) the results of the cell viability experiment and (b) the results of the cytotoxicity experiment.

[0157] According to Fig. 1, the concentration of PA_EXT was adjusted to 1 ㎍ / mL, 2 ㎍ / mL, 4 ㎍ / mL, 10 ㎍ / mL, 100 ㎍ / mL, 200 ㎍ / mL, and 400 ㎍ / mL as in Examples 1 to 7, and the cell viability and cytotoxicity were tested, and the results shown in Table 4 below were obtained.

[0158]

[0159] Referring to Table 4, the cell viability and cytotoxicity results of Examples 1 and 4 to 7 can be confirmed. The cell viability and cytotoxicity of Examples 1 and 4 to 7 did not show significant values ​​that were difficult to utilize as pharmacological substances, and all showed biostable results. However, in Example 7, the decrease in cell viability and the increase in cytotoxicity were found to be greater than the differences between the other examples.

[0160] Referring to FIGS. 1A and 1B, in order to avoid high concentrations that may cause cytotoxicity, a concentration-dependent cell viability and cytotoxicity test was performed according to an embodiment of the present invention, and as a result, it can be confirmed that Example 1 exhibits higher cell viability and lower cytotoxicity than Comparative Examples 1, 2, and 3. Although Examples 1 to 3 and Comparative Example 1 did not exhibit cytotoxicity that could significantly affect cells, Example 1 exhibited the lowest cytotoxicity result. In the case of Examples 2 and 3, there was no significant difference compared to Comparative Example 1, but since they exhibited lower cell viability and higher cytotoxicity than Example 1, it was decided to treat PA_EXT in the remaining experiments at a concentration of 1 ㎍ / mL.

[0161]

[0162] (2) Inhibitory effect of PA_EXT on LRRK2 kinase activity in BV2 cells

[0163] FIG. 2 illustrates the results of measuring the inhibitory effect of a composition according to one embodiment of the present invention on LRRK2 kinase activity, showing (a) the results of phosphorylation level analysis at pS1292, (b) the results of Western blot analysis, and (c) the results of phosphorylation level analysis at pS935.

[0164] To confirm the inhibitory effect of the composition according to one embodiment of the present invention on LRRK2 phosphorylation enzyme activity, in vitro LRRK2 analysis and LPS-based neuroinflammation induction were performed in BV2 cells.

[0165] According to FIG. 2, the concentration of PA_EXT was adjusted to 1 ㎍ / mL, 2 ㎍ / mL, 4 ㎍ / mL, 10 ㎍ / mL, 100 ㎍ / mL, 200 ㎍ / mL, and 400 ㎍ / mL as in Examples 1 to 7, and the inhibitory effect on LRRK2 phosphorylation enzyme activity was tested, and the results shown in Table 5 below were obtained.

[0166]

[0167] Referring to Table 5, the inhibitory effects of LRRK2 kinase activity of Examples 1 and 4 to 7 can be confirmed. Compared to Example 1, Example 4 showed a lower LRRK2 level, and for Examples 5 to 7, it can be confirmed that the values ​​did not change significantly from those of Example 4.

[0168] Referring to FIG. 2a, it can be confirmed that Example 1 significantly inhibited LRRK2 kinase activity and reduced the phosphorylation activity at the S1292 site of the LRRK2 G2019S recombinant protein. In addition, it can be confirmed that the degree of inhibition of LRRK2 kinase activity of Example 1 is similar to the degree of inhibition by MLi-2, a LRRK2 inhibitor of Comparative Example 2.

[0169] Referring to Figures 2b and 2c, it can be confirmed that Example 1 is consistent with the phosphorylation of the pS935 site of mouse LRRK2 by LPS in BV2 cells. However, Example 1 shows a result of significantly inhibiting LKK2 kinase activity without increasing it, which suggests that the pharmacological activity of P. arborescens extract on LRRK2 kinase activity needs to be further studied.

[0170]

[0171] (3) Inflammatory cytokine inhibitory effect of PA_EXT in BV2 cells

[0172] FIG. 3 illustrates the results of measuring the inflammatory cytokine reduction effect of a composition according to one embodiment of the present invention in BV2 cells, including (a) the results of measuring the level of TNF-α expression using qPCR, (b) the results of analyzing the level of LRRK2 phosphorylation in BV2 cells using Western blot, and (c) the results of analyzing the level of phosphorylation of pS935.

[0173] To confirm the TNF-α expression reduction effect of the composition according to one embodiment of the present invention, TNF-α mRNA moisture was evaluated and compared between LPS treatment and combined treatment with LPS and PA_EXT.

[0174] Referring to Figures 3a to 3c, it can be confirmed that Example 1 reduced inflammatory cytokines through inhibition of LRRK2 kinase activity. Here, LPS treatment significantly increased LRRK2 kinase activity and increased TNF-α expression. Example 1 showed a result of reducing both LRRK2 kinase activity and TNF-α expression. These results indicate that the alleviation of inflammatory cytokine expression was mediated by inhibition of LRRK2 kinase activity through the therapeutic component of the P. arborescens extract.

[0175]

[0176] (4) Effect of reducing TNF-α and iNOS gene expression in primary cultured mouse microglia

[0177] FIG. 4 illustrates the results of measuring the inflammatory cytokine reduction effect of a composition according to one embodiment of the present invention in primary cultured mouse microglia, including (a) the results of measuring the level of TNF-α expression by qPCR, (b) the results of measuring the level of inducible iNOS expression by qPCR, (c) the results of TNF-α ELISA analysis of cell lysate supernatant, and (d) the results of Greiss analysis of cell lysate supernatant.

[0178] To confirm that a composition according to one embodiment of the present invention exhibits an inhibitory effect on neuroinflammation in microglia, neuroinflammation was induced by LPS treatment in primary cultured microglia from mice, and then the attenuation of neuroinflammation by PA_EXT was tested.

[0179] Referring to FIGS. 4a and 4b, it can be confirmed that Example 1 significantly reduced the expression of TNF-α and iNOS genes induced by LPS treatment.

[0180] Referring to Figures 4c and 4d, it can be confirmed that the high levels of pro-inflammatory cytokine TNF-α and NO release from cells after LPS treatment were reduced when LPS and Example 1 were co-treated. These results indicate that the P. arborescens extract can reduce neuroinflammation in microglia.

[0181]

[0182] (5) Effect of alleviating oxidative stress in primary cultured mouse astrocytic cells

[0183] FIG. 5 illustrates the results of measuring the effect of a composition according to one embodiment of the present invention on reducing oxidative stress in primary cultured astrocytic cells of mice, showing (a) the results of measuring cellular ROS and (b) the results of measuring cellular NO.

[0184] Astroglial cells are associated with neuroinflammation, and increased levels of ROS and NO in reactive astroglial cells have been observed under neuroinflammatory conditions. Accordingly, to determine the oxidative stress-reducing effect of a composition according to one embodiment of the present invention, the effect of PA_EXT on ROS and NO levels in primary cultured rat astroglial cells after rotenone (RTN) treatment was evaluated.

[0185] Referring to Figures 5a and 5b, it can be confirmed that cellular ROS and NO levels increased after RTN treatment, but cellular ROS and NO levels decreased when Example 1 and RTN were combined. In other words, the P. arborescens extract is effective in both inhibiting LRRK2 and regulating neuroinflammation.

[0186]

[0187] (6) Inhibitory effect on alpha-synuclein polymer formation in differentiated SH-SYS5Y cells

[0188] FIG. 6 illustrates the results of measuring the inhibitory effect of a composition according to one embodiment of the present invention on alpha-synuclein polymer formation, including (a) thioflavin T analysis results, (b) Western blot analysis results, (c) alpha-synuclein density measurement results, and (d) total alpha-synuclein level measurement results in dopaminergic neurons.

[0189] Polymer formation, i.e., aggregation, of alpha-synuclein is related to oxidative stress, and PA_EXT alleviated oxidative stress in mouse primary cultured cells. Accordingly, an experiment was conducted on the hypothesis that a composition according to an embodiment of the present invention affects the aggregation of alpha-synuclein in dopaminergic neurons. To confirm the aggregation-inhibiting effect of the composition according to an embodiment of the present invention on alpha-synuclein, seed-induced fibrillation of monomeric synuclein was analyzed using fibril seeds of alpha-synuclein with or without PA_EXT for 14 days, and samples were collected every two days and thioflavin T analysis was performed at the end of the culture.

[0190] Referring to Figure 6a, it can be confirmed that when there is a seed (DMSO), the fibrillation of alpha synuclein is accelerated compared to when there is no seed, but when Example 1 is added, the fibrillation of alpha synuclein is significantly suppressed.

[0191] For Fig. 6b, the remaining samples after culture were analyzed by Western blot.

[0192] Referring to FIGS. 6b and 6c, Comparative Example 1 showed a result in which high molecular weight α-synuclein (HMW) of the polymer increased along with a decrease in alpha-synuclein monomers compared to the case without seeds, and it can be confirmed that Example 1 significantly suppresses the production and increase of HMW compared to the case without seeds and Comparative Example 1.

[0193] For Fig. 6d, the inhibitory effect of PA_EXT on alpha-synuclein aggregation was confirmed by testing endogenous alpha-synuclein aggregation through RA differentiation in SH-SYSY, a human dopaminergic neuronal cell line.

[0194] Referring to FIG. 6d, it can be confirmed that Example 1 inhibited the production of alpha-synuclein fibrils in dopaminergic neurons, and the total alpha-synuclein level in dopaminergic neurons was significantly reduced after treatment with Example 1.

[0195] FIG. 7 illustrates the results of measuring the effect of a composition according to one embodiment of the present invention on increasing lysosomal activity in dSH, including (a) lysosomal quantification results, (b) lysosomal GCase activity analysis results, and (c) lysosomal GCase activity analysis results based on lysosomal quantification values.

[0196] Because previous studies have shown that lysosomal GCase is involved in the degradation of alpha-synuclein, we sought to analyze GCase activity using pfb-fdglu.

[0197] Referring to FIG. 7, it can be confirmed that PA-EXT is useful for inhibiting alpha-synuclein polymer formation in dopaminergic neurons and increasing the degradation of alpha-synuclein in lysosomes.

[0198] That is, the P. arborescens extract exhibits the effect of inhibiting alpha-synuclein polymerization and dSH in vitro. In addition, the P. arborescens extract exhibits the effect of preventing alpha-synuclein accumulation by increasing lysosomal activity.

[0199]

[0200] The pharmaceutical composition for inhibiting LRRK2 kinase activity or for anti-neuroinflammatory activity, which comprises the extract of the present invention as an active ingredient, reduces the risk of developing diseases and neuroinflammation caused by the activity of LRRK2 kinase, and can be used as a medicine, food, or quasi-drug for preventing, improving, and treating diseases and neuroinflammation caused by the activity of LRRK2 kinase.

[0201] In addition, the pharmaceutical composition for inhibiting LRRK2 phosphorylation enzyme activity or for anti-neuroinflammatory purposes, which contains the extract of the present invention as an active ingredient, has the effect of simultaneously inhibiting alpha-synuclein polymer formation and LRRK2 phosphorylation enzyme activity.

[0202] In addition, the present invention has the advantage of having fewer side effects and being more biologically stable than artificial compounds, antibody preparations, and aptamer preparations, as it uses natural materials as raw materials.

[0203] The above description is merely an example of the technical idea of ​​the present invention, and those skilled in the art will appreciate that various modifications and variations can be made without departing from the essential characteristics of the present invention.

[0204] Accordingly, the embodiments disclosed in the present invention are intended to illustrate, rather than limit, the technical concept of the present invention, and the scope of the technical concept of the present invention is not limited by these embodiments. The scope of protection of the present invention should be interpreted by the following claims, and all technical concepts within the scope equivalent thereto should be construed as being included within the scope of the present invention.

Claims

1. A pharmaceutical composition for inhibiting LRRK2 phosphorylation enzyme activity or for anti-neuroinflammatory purposes, comprising Padina extract as an active ingredient.

2. In paragraph 1, The above extract of the plant is a pharmaceutical composition for inhibiting LRRK2 kinase activity or for anti-neuroinflammation, which inhibits oxidative stress and simultaneously inhibits alpha-synuclein polymer formation and LRRK2 (Leucine-rich repeat kinase 2) kinase activity.

3. In paragraph 1, The above extract of the plant is a pharmaceutical composition for inhibiting LRRK2 phosphorylation enzyme activity or for anti-neuroinflammation, which reduces inflammatory cytokines and suppresses neuroinflammation.

4. In paragraph 1, The pharmaceutical composition is a pharmaceutical composition for preventing or treating a disease or condition mediated by or related to LRRK2, wherein the disease or condition mediated by or related to LRRK2 is a degenerative brain disease, a pharmaceutical composition for inhibiting LRRK2 phosphorylation enzyme activity or for anti-neuroinflammation.

5. In paragraph 4, A pharmaceutical composition for inhibiting LRRK2 phosphorylation enzyme activity or for anti-neuroinflammatory purposes, wherein the above-mentioned degenerative brain disease is at least one selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, and mild cognitive impairment.

6. A pharmaceutical preparation comprising a pharmaceutical composition according to any one of claims 1 to 5.

7. In paragraph 6, The above pharmaceutical preparation is a pharmaceutical preparation used by being formulated in the form of any one type of oral dosage form, external preparation, suppository, and sterile aqueous solution selected from the group consisting of powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols.

8. In paragraph 6, A pharmaceutical preparation wherein the above extract of the plant is contained in a solvent at a concentration of 0.1 to 10 μg / mL.

9. A food composition for inhibiting LRRK2 phosphorylation enzyme activity or for anti-neuroinflammation, comprising Padina extract as an active ingredient.

10. In paragraph 9, The food composition is any one selected from the group consisting of dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum and ice cream, soup, beverage, tea, drink, alcoholic beverage and vitamin complex, a food composition for inhibiting LRRK2 phosphorylation enzyme activity or for anti-neuroinflammation.

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