WO2023229332A1 - Health food composition and pharmaceutical composition comprising trehalose for preventing, alleviating or treating retinal diseases or degenerative brain diseases - Google Patents

Health food composition and pharmaceutical composition comprising trehalose for preventing, alleviating or treating retinal diseases or degenerative brain diseases Download PDF

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WO2023229332A1
WO2023229332A1 PCT/KR2023/006972 KR2023006972W WO2023229332A1 WO 2023229332 A1 WO2023229332 A1 WO 2023229332A1 KR 2023006972 W KR2023006972 W KR 2023006972W WO 2023229332 A1 WO2023229332 A1 WO 2023229332A1
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disease
trehalose
degenerative brain
health food
preventing
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French (fr)
Korean (ko)
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장완진
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줄리아 연구소 주식회사
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/385—Concentrates of non-alcoholic beverages
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/15—Vitamins

Definitions

  • the present invention relates to health food compositions and pharmaceutical compositions for preventing, improving or treating retinal diseases or degenerative brain diseases, and specifically, to health food compositions containing trehalose for preventing, improving or treating retinal diseases or degenerative brain diseases. and pharmaceutical compositions.
  • Aging is known to be an important risk factor for many neurodegenerative diseases, including age-related macular degeneration (AMD) and Alzheimer's disease (AD). These diseases significantly reduce the quality of life of millions of older people every year.
  • AMD age-related macular degeneration
  • AD Alzheimer's disease
  • a ⁇ amyloid beta
  • amyloid structures thought to be toxic oligomers, are known to have a typical round shape with a hollow center (Quist et al. 2005). In most cases, research oligomers were found in laboratory-generated samples or mutant proteins. Recent studies have extracted amyloid fibers and oligomers from Alzheimer's brain (Shankar et al. 2008; Paravastu et al. 2009). Using cell culture techniques to control amyloid beta expression, oligomers can be extracted from different stages of the disease and the structures formed can be characterized. Identification of the oligomeric structure responsible for cytotoxicity in neurodegenerative diseases is important in determining toxicity.
  • Amyloid precursor protein is mostly localized in retinal pigment epithelial (RPE) cells of the eye and pituitary neurons of the brain (Yoshida et al. 2005). Neurotoxic amyloid beta peptide oligomers can be seen as key mediators of these aging diseases.
  • RPE retinal pigment epithelial
  • the present inventor confirmed that oxidative stress and nitric oxide (NO) caused by light-induced and hypoxia in retinal cells and nerve cells are involved in amyloid beta aggregation and upregulates its concentration, and discovered a composition that decomposes such amyloid beta aggregation.
  • NO oxidative stress and nitric oxide
  • the problem to be solved by the present invention is to provide a health food composition that can effectively prevent or improve retinal diseases such as age-related macular degeneration or degenerative brain diseases such as Alzheimer's disease.
  • Another problem to be solved by the present invention is to provide a pharmaceutical composition that can effectively prevent or treat the retina disease or degenerative brain disease.
  • a health food composition for preventing or improving retinal disease or degenerative brain disease according to an embodiment of the present invention for solving the above problems includes trehalose.
  • Trehalose may be included as an active ingredient.
  • Trehalose is a disaccharide and may have a structure as shown in Chemical Formula 1 or 2 below.
  • health food may include all health foods, health functional foods, health supplements, and health promoting foods.
  • the health food may mean a health functional food or a health supplement, but is not limited thereto.
  • the retinal disease may include one or more selected from the group consisting of age-related macular degeneration (AMD), retinitis pigmentosa (RP), diabetic retinopathy (DR), retinopathy of prematurity (ROP), and glaucoma.
  • AMD age-related macular degeneration
  • RP retinitis pigmentosa
  • DR diabetic retinopathy
  • ROP retinopathy of prematurity
  • Degenerative brain diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, mild cognitive impairment, cerebral amyloid angiopathy, and amyloid stroke. ), systemic amyloid disease, Dutch amyloidosis, Niemann-Pick disease, amyotrophic lateral sclerosis, Spinocerebellar Atrophy, Tourette's Syndrome, Friedrich's Ataxia, Machado-Joseph's disease, Lewy Body Dementia, Dystonia, and Progressive Supranuclear Palsy. It may contain one or more of your choice.
  • the retina disease or degenerative brain disease may specifically be a light-induced disease or a disease caused by amyloid beta (A ⁇ ).
  • a light-induced disease may refer to a disease that is caused, worsened, or developed by exposure to light, and a disease caused by amyloid beta may be caused, worsened, or developed by the increase (or upregulation), aggregation, or accumulation of amyloid beta in the body. It may mean a disease.
  • the retina disease or degenerative brain disease may be a disease that is caused, worsened, or developed by increased (or upregulated), aggregation, or accumulation of amyloid beta due to oxidative stress (or nitric oxide) caused by light exposure.
  • the health food composition of the present invention may prevent or improve the disease by decomposing amyloid beta (or amyloid beta oligomer) or improving the aggregation state.
  • the retinal disease may be wet or dry age-related macular degeneration, and in particular may be dry age-related macular degeneration.
  • Dry age-related macular degeneration can be a disease caused or worsened by light, such as increased oxidative stress, retinal degeneration, and retinal cell death.
  • embodiments of the present invention are not limited thereto.
  • the degenerative brain disease may be Alzheimer's disease, Parkinson's disease, cerebral amyloid angiopathy, amyloid stroke, systemic amyloid disease, or Dutch amyloidosis, and more specifically, may be Alzheimer's disease.
  • embodiments of the present invention are not limited thereto.
  • the retinal disease may be a disease related to retinal pigment epithelial (RPE).
  • RPE retinal pigment epithelial
  • Retinal pigment epithelium-related diseases may be diseases that occur or worsen when the retinal pigment epithelium is damaged or worsened by oxidative stress, etc.
  • dry age-related macular degeneration, wet age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy It may include one or more selected from the group consisting of retinopathy of prematurity and glaucoma.
  • Oxidative stress is known to be one of the major factors causing structural defects and functional damage in retinal pigment epithelial cells. Continuous oxidative stress causes damage to mitochondrial DNA in the retina, destroying mitochondrial function and leading to apoptosis, which can lead to vision impairment due to retinal damage. Vision impairment due to retinal damage in humans is one of the main causes of blindness, and increased oxidative stress causes diseases such as dry age-related macular degeneration, wet age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, retinopathy of prematurity, and glaucoma. It is believed to be a cause of retinal disease.
  • the health food composition of the present invention can be used to prevent or improve retinal pigment epithelium-related diseases by protecting the retinal pigment epithelium from oxidative stress or delaying cell damage.
  • the health food composition of the present invention can be manufactured and administered in various dosage forms. When formulated, it can be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations include tablets, pills, powders, granules, capsules, etc. These solid preparations are prepared by mixing one or more compounds with at least one excipient. Additionally, lubricants other than simple excipients may also be used. Liquid preparations include suspensions, oral solutions, emulsions, and syrups, and may include commonly used diluents and excipients.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
  • Solid preparations include tablets, pills, powders, granules, capsules, etc. These solid preparations are prepared by mixing one or more compounds with at least one excipient. Additionally, lubricants other than
  • trehalose can be added directly to food or used together with other foods or food ingredients, and can be used appropriately according to conventional methods.
  • the mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention or improvement).
  • composition when the composition is a health functional beverage composition, it may contain various flavoring agents or natural carbohydrates as additional ingredients like ordinary beverages. Furthermore, it may further contain various nutrients, vitamins, flavors, colorants, thickeners, thickeners, pH adjusters, stabilizers, preservatives, carbonating agents, etc. In exemplary embodiments, it may further contain pulp for the production of fruit or vegetable beverages.
  • the health food composition of the present invention may further include one or more selected from the group consisting of fruit concentrate, malic acid, aloe vera powder, sugarcane powder, vitamins, calcium lactate, thickener, sucralose, and purified water.
  • the fruit concentrate may include one or more selected from the group consisting of red grape concentrate, white grape concentrate, carrot concentrate, and blueberry concentrate
  • the thickener may include one or more of gellan gum and xanthan gum.
  • the vitamins may include one or more of vitamin C and vitamin A.
  • it may further include a natural fruit flavor including one or more of natural blueberry flavor and natural green grape flavor, and may further include bilberry powder.
  • the health food composition of the present invention may have the following composition.
  • trehalose 0.5-10 wt% (or parts by weight) of trehalose
  • Aloe vera powder 0.05-1 wt% (or parts by weight);
  • Thickener 0.01-0.2 wt% (or parts by weight);
  • Remaining amount of purified water (or 75-90 wt%, or parts by weight)
  • it may further include 0.1-1 wt% (or part by weight) of natural fruit flavor and 0.05-1 wt% (or part by weight) of bilberry powder.
  • the health food composition of the present invention may contain some of the above ingredients, and in an exemplary embodiment, one or more of fruit concentrate, malic acid, and natural fruit flavor; One or more of aloe vera powder, sugar cane powder, and sucralose; One or more of vitamins and calcium lactate; thickener; and purified water.
  • the health food composition of the present invention may further include one or more selected from the group consisting of enzyme-treated stevia, carnitine, and taurine.
  • a pharmaceutical composition for preventing or treating retinal disease or degenerative brain disease according to an embodiment of the present invention to solve the above other problems includes trehalose, and trehalose may be included as an active ingredient.
  • composition of the present invention can be prepared and administered in various oral and parenteral dosage forms.
  • Oral administration is as described above, and parenteral administration may include subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.
  • Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, and emulsions.
  • the pharmaceutical composition may include trehalose, for example, 0.1-10 wt%, but is not limited thereto.
  • the present invention provides a method for preventing or treating retinal disease or degenerative brain disease, comprising administering a pharmaceutical composition containing trehalose to a patient in need of prevention or treatment of retinal disease or degenerative brain disease. You can.
  • retinal diseases such as age-related macular degeneration or degenerative brain diseases such as Alzheimer's disease can be effectively prevented, improved, and treated through a health food composition or pharmaceutical composition containing trehalose.
  • Figure 1 is an image visually showing that amyloid beta is up-regulated under oxidative stress according to an experimental example of the present invention.
  • Figure 2 is a graph showing light-induced oxygen-dependent NO formation according to an experimental example of the present invention.
  • Figure 3 shows the intracellular NO status using fluorescent substances during light exposure and in a cancer environment according to an experimental example of the present invention.
  • Figure 4 is an image confirming the state of amyloid beta oligomer aggregation before and after treatment with the composition of the present invention according to an experimental example of the present invention.
  • Figure 5 shows cells through (a) cytotoxicity evaluation, (b) cell viability evaluation, and (c) LDH (lactate dehydrogenase) measurement of trehalose in ARPE-19 cell damage caused by oxidative stress according to an experimental example of the present invention. This is a graph of the toxicity evaluation results.
  • Figure 6 is a graph confirming the cytoprotective effect of low concentration trehalose (50 ⁇ M) on ARPE-19 cell damage caused by oxidative stress according to an experimental example of the present invention.
  • Figure 7 is a graph confirming the protective effect of trehalose on mitochondrial function damaged by oxidative stress according to an experimental example of the present invention.
  • Figure 8 is an evaluation of the efficacy of trehalose on ROS (reactive oxygen species) production and mitochondrial damage in ARPE-19 cells damaged by oxidative stress according to an experimental example of the present invention, mitochondrial staining, and confocal microscopy images.
  • ROS reactive oxygen species
  • first, second, A, B, (a), and (b) may be used. These terms are only used to distinguish the component from other components, and the nature, sequence, or order of the component is not limited by the term.
  • 'prevention' means suppressing or delaying the occurrence of symptoms or diseases in an individual who does not yet have symptoms or diseases but may develop such symptoms or diseases.
  • 'treatment' or 'improvement' means any action that improves or beneficially changes symptoms in an individual, such as (a) suppressing the development (worsening) of a symptom or disease, (b) suppressing the development (worsening) of a symptom or disease means relief, or (c) elimination of symptoms or disease.
  • 'individual' or 'subject' means a cell, tissue, animal, or human for which a specific disease or condition is to be diagnosed, prevented, or treated using the present invention.
  • 'biological sample' refers to the subject's blood, plasma, serum, cells, tissues, body fluids, saliva, urine, aqueous humor, anterior chamber fluid, etc.
  • 'derived from' may mean not only separation from a specific object or part, but also the object or part itself.
  • 'control group' refers to normal cells or patients that do not have or are not caused by a specific disease or disease.
  • 'level' may also mean content, concentration, etc. that can be confirmed by measuring or analyzing the presence of a specific factor.
  • a liquid formulation composition containing trehalose was prepared with the following composition.
  • a liquid composition containing trehalose was prepared with the following composition.
  • a liquid composition containing trehalose was prepared with the following composition.
  • a liquid composition containing trehalose was prepared with the following composition.
  • a liquid composition containing trehalose was prepared with the following composition.
  • Retinal Pigment Epithelial (RPE) cells were cultured and exposed to various light stresses, including light/dark cycles, continuous illumination, and various light intensities (200-5,000 lux), and nitric oxide (NO) production was measured.
  • the measurements were performed using a Sievers 280i nitric oxide analyzer (NOA) equipped with a custom sampling cell housing a T-25 tissue culture flask.
  • NOA Sievers 280i nitric oxide analyzer
  • a ⁇ oligomers were measured using a known method under cell culture conditions known to induce A ⁇ formation, including hypoxia and light-induced oxidative stress on retinal pigment epithelial cells (Yoshida et al. 2005).
  • Nerve growth factor (NGF) and serum were extracted from rat neural cell line PC-12 using a known method, and biochemical methods were used to determine the size, structure, and hydrophobicity of amyloid beta oligomers (Matrone et al. 2008).
  • different amyloid beta oligomers were formed using synthetic peptides under different conditions of oxidative stress, pH, and ionic strength and compared with toxic amyloid beta oligomers. The oligomeric structure was determined and toxicity tested in cell culture using PC-12 and retinal pigment epithelial cells.
  • Figure 2 is a graph showing light-induced oxygen-dependent NO formation.
  • Figure 2 when retinal pigment epithelial cells (ARPE-19 cells) are exposed to light and arginine and oxygen are added, NO is formed due to oxidative stress.
  • This shows the potential interaction between NO and amyloid beta through synthase or arginine metabolism, where NO is a messenger for phagocytosis in the retinal pigment epithelium.
  • Figure 3 shows the intracellular NO status during light exposure and in a cancer environment using a fluorescent substance (4,5-diaminofluorescein diacetate).
  • proteomic techniques including 2D electrophoresis and MALDI-TOF-TOF mass spectrometry, were used to identify early protein markers of oxidative stress in the retinal pigment epithelium.
  • Proteome changes in retinal pigment epithelial cells reveal proteins involved in key cellular processes, including retinoid trafficking (IRBP), calcium signaling (calreticulin), G protein signaling (transducin), and protein aggregation (amyloid beta). It was.
  • IRBP retinoid trafficking
  • calreticulin calcium signaling
  • G protein signaling transducin
  • protein aggregation protein aggregation
  • amyloid beta induces cell death signals and protein nitration in the retinal pigment epithelium through the formation of amyloid beta aggregates under certain pH, ionic strength, and oxidative stress.
  • Photoreceptor apoptosis is the final cell death pathway in age-related macular degeneration (AMD), retinitis pigmentosa (RP), glaucoma, and Alzheimer's disease.
  • Figure 1 is a visual image showing upregulation of amyloid beta under oxidative stress.
  • amyloid beta increased in primary retinal pigment epithelial cells under oxidative stress (red) conditions (200 ⁇ M H 2 O 2 ) compared to the control group (green).
  • Proteins were separated by 2D electrophoresis and visualized by Coomassie staining. Artificial green and red colors were added to compare gels. Merged images showed differential expression of target proteins identified by mass spectrometry. Light-induced nitric oxide appears to trigger nitration of amyloid beta and accelerate aggregate formation.
  • retinal diseases such as macular degeneration and brain diseases such as Alzheimer's disease are worsened by the increase and aggregation of amyloid beta.
  • NO nitric oxide
  • amyloid beta oligomers were first extracted. Retinal pigment epithelium and PC-12 cells grow under different conditions that induce amyloid beta formation. Referring to known methods, cells were harvested at different points during apoptosis and oligomers were extracted using TBS and guanidine Hal to obtain soluble and insoluble oligomers, respectively (Shankar et al. 2008; Paravastu et al. 2009). .
  • Thioflavin-T is a reagent used to detect amyloid structures (Wu et al. 2008; Biancalana et al. 2009). Congo red was used as an amyloid dye (Klunk et al. 1999) to be used with ANS, a hydrophobic detection.
  • FIG. 4 is an image confirming the state of amyloid beta oligomer aggregation before and after processing the composition of Example 1.
  • retinal pigment epithelium and PC-12 cells with elevated amyloed beta expression were directly treated with the compositions of Examples 1 to 5 containing trehalose. It was confirmed that amyloid beta oligomers were similarly decomposed in cells treated with the compositions of Examples 1 to 5.
  • composition containing trehalose decomposed the treated amyloid beta oligomer By confirming that the composition containing trehalose decomposed the treated amyloid beta oligomer, it is expected that the composition of the present invention will be effective in preventing, improving and treating retinal diseases such as macular degeneration and Alzheimer's disease and brain diseases. You can.
  • a solution containing 0.021 g of trehalose was prepared.
  • the solubility of trehalose in water is 0.1 g/mL, and the solution is clear and colorless.
  • Other single compounds were also prepared for comparison.
  • Human retinal pigment epithelial cell line (ARPE-19) cells were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM (Dulbecco's modified Eagle's medium) and Ham's F12 1:1 mixed medium DMEM/F12 (Gibco, Carlsdad, CA, USA), 10% fetal bovine serum (Gibco, Burlington, ON, Canada) and 1% penicillin-streptomycin (Gibco, Life Technologies Limited, Paisley, UK) were added and cultured in wet conditions at 37°C and 5% CO2 . When the cells grew to a density of 70% or more between passages 8 and 11, they were subcultured and used for experiments.
  • DMEM Dynabecco's modified Eagle's medium
  • Ham's F12 1:1 mixed medium DMEM/F12 Gib's F12 1:1 mixed medium
  • fetal bovine serum Gibl bovine serum
  • penicillin-streptomycin Gibco, Life Technologies Limited, Paisley,
  • a cell counting kit-8 (CCK-8; Dojindo, Japan) assay was performed.
  • CCK-8 Dojindo, Japan
  • ARPE- 19 cells were distributed in a 96 well plate (1 did. 20 ⁇ l of CCK-8 solution was added to each well and incubated for 2 hours in a 5% CO 2 incubator at 37°C.
  • the absorbance value was measured at 450 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).
  • tBHP tert-Butyl Hydroperoxide
  • OCR oxygen consumption rate
  • Figure 5 shows (a) cytotoxicity evaluation of trehalose, (b) cell viability evaluation of trehalose in oxidative stress-induced cells, and (c) LDH (lactate dehydrogenase) measurement in ARPE-19 cell damage caused by oxidative stress. This is a graph of the results of cytotoxicity evaluation.
  • experimental substance number 18 refers to trehalose.
  • Figure 6 is a graph confirming the cytoprotective effect of low concentration trehalose (50 ⁇ M) on ARPE-19 cell damage caused by oxidative stress.
  • Figure 6 evaluates the cell viability of trehalose (50 ⁇ M, No. 18) in oxidative stress-induced cells (#, P ⁇ 0.05 vs. CTR; * P ⁇ 0.05 vs. tBHT), and
  • Figure 7 shows oxidative stress Cell survival rate of trehalose (JK-18) was evaluated in induced cells (#, P ⁇ 0.05 vs. CTR; * P ⁇ 0.05 vs. tBHT).
  • numbers 18 and JK-18 refer to trehalose.
  • Figure 7 is a graph confirming the protective effect of trehalose on mitochondrial function damaged by oxidative stress. Specifically, (a) assessment of oxygen consumption rate (OCR) changes in trehalose, (b) non-mitochondrial oxygen consumption (#, P ⁇ 0.05 vs. CTR; * P ⁇ 0.05 vs. tBHT), (c) basal Oxygen consumption (#, P ⁇ 0.05 vs. CTR; * P ⁇ 0.05 vs. tBHT), (d) maximal respiratory rate (#, P ⁇ 0.05 vs. CTR; * P ⁇ 0.05 vs. tBHT), (e) ATP production (#, P ⁇ 0.05 vs. CTR; * P ⁇ 0.05 vs. tBHT) and (f) amount of electrons leaked (#, P ⁇ 0.05 vs. CTR; * P ⁇ 0.05 vs. tBHT).
  • OCR oxygen consumption rate
  • JK-18 refers to trehalose.
  • the function of mitochondria was confirmed by analyzing the amount of respiration that occurred.
  • the basal respiratory interval in the control group stimulated with oxidative stress was found to be 46.92 ⁇ 1.6 pmole/min (a, c), and in the trehalose-treated group, it was significantly recovered to 67.63 ⁇ 0.96 pmole/min.
  • the maximum respiratory interval was lowered to 24.34 ⁇ 8.3 pmole/min, and after FCCP administration, the maximum respiratory interval was lowered to 70.46 ⁇ 6.26 pmole/min (d).
  • the maximum respiration of the trehalose and normal groups was 131.24 ⁇ 2.49 pmole/min for the normal control group, and the maximum respiration for trehalose (JK-18) was significantly increased to 93.19 ⁇ 5.7 pmole/min.
  • the basal respiration section and ATP production section (e) were decreased by oxidative stress (26.59 ⁇ 5.11 pmole/min), but recovered by trehalose (JK-18, 47.12 ⁇ 1.2 pmole/min).
  • oxidative stress 26.59 ⁇ 5.11 pmole/min
  • trehalose JK-18, 47.12 ⁇ 1.2 pmole/min
  • Proton leak increased by oxidative stress was significantly reduced by trehalose (f).
  • Figure 8 shows evaluation of the efficacy of trehalose on ROS (reactive oxygen species) production and mitochondrial damage in ARPE-19 cells damaged by oxidative stress, mitochondrial staining, and confocal microscopy images. Specifically, (a) DCFDA fluorescence measurement (#, P ⁇ 0.05 vs. CTR; * P ⁇ 0.05 vs. tBHT) and (b) Mitotracker average expression measurement (#, P ⁇ 0.05 vs. CTR; * P ⁇ 0.05 vs. tBHT) results are shown.
  • JK-18 refers to trehalose.
  • the intensity of mitotracker decreased by tBHP (1472.33 ⁇ 224.46 IU) compared to the normal group (2884.67 ⁇ 903.55 IU), qualitatively showing that the mitochondrial intensity was significantly restored by trehalose. , confirmed quantitatively (JK-18, 2591.33 ⁇ 343.43 IU).
  • ARPE-19 cell damage caused by tBHP was accompanied by impaired mitochondrial function, which was related to ROS generation due to oxidative stress.
  • trehalose since trehalose has been confirmed to have an inhibitory and protective effect against oxidative stress, mitochondrial damage, and DNA damage, it has been shown to be effective in treating retinal diseases associated with them (wet age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, retinopathy of prematurity, It can be seen that it is suitable for use in the prevention, improvement or treatment of (glaucoma, etc.).

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Abstract

The present invention relates to a health food composition and a pharmaceutical composition for preventing, alleviating or treating retinal diseases or degenerative brain diseases and, specifically, to a health food composition and a pharmaceutical composition comprising trehalose for preventing, alleviating or treating retinal diseases or degenerative brain diseases.

Description

트레할로스를 포함하는 망막 질환 또는 퇴행성 뇌 질환의 예방, 개선 또는 치료용 건강식품 조성물 및 약학적 조성물Health food compositions and pharmaceutical compositions for preventing, improving or treating retinal diseases or degenerative brain diseases containing trehalose
본 발명은 망막 질환 또는 퇴행성 뇌 질환의 예방, 개선 또는 치료용 건강식품 조성물 및 약학적 조성물에 관한 것으로서, 구체적으로는 트레할로스를 포함하는 망막 질환 또는 퇴행성 뇌 질환의 예방, 개선 또는 치료용 건강식품 조성물 및 약학적 조성물에 관한 것이다.The present invention relates to health food compositions and pharmaceutical compositions for preventing, improving or treating retinal diseases or degenerative brain diseases, and specifically, to health food compositions containing trehalose for preventing, improving or treating retinal diseases or degenerative brain diseases. and pharmaceutical compositions.
노화는 노인황반변성(age-related macular degeneration, AMD) 및 알츠하이머병(Alzheimer's disease, AD)을 비롯한 많은 신경 퇴행성 질환의 중요한 위험 요소로 알려져 있다. 이러한 질병은 매년 수백만 명의 노인 삶의 질을 크게 떨어뜨린다. 이러한 질병에서 중첩되는 현상 중 하나는 뇌와 눈에서 아밀로이드 베타(Amyloid beta, Aβ)의 형성이다. Aging is known to be an important risk factor for many neurodegenerative diseases, including age-related macular degeneration (AMD) and Alzheimer's disease (AD). These diseases significantly reduce the quality of life of millions of older people every year. One of the overlapping phenomena in these diseases is the formation of amyloid beta (Aβ) in the brain and eyes.
독성을 가진 올리고머로 생각되는 다중 아밀로이드 구조는 속이 빈 중앙이 있는 일반적인 둥근 모양을 갖는 것으로 알려져 있다(Quist et al. 2005). 대부분의 경우 연구용 올리고머는 실험실에서 만든 샘플이나 돌연변이 단백질에서 발견되었다. 최근 연구에서는 알츠하이머의 뇌에서 아밀로이드 섬유와 올리고머를 추출하였다(Shankar et al. 2008; Paravastu et al. 2009). 아밀로이드 베타 발현을 조절하는 세포 배양 기술을 사용하여 질병의 여러 단계에서 올리고머를 추출하여 형성된 구조를 특성화할 수 있다. 신경퇴행성 질환에서 세포 독성을 담당하는 올리고머 구조의 규명은 독성을 결정하는 데 중요하다.Multiple amyloid structures, thought to be toxic oligomers, are known to have a typical round shape with a hollow center (Quist et al. 2005). In most cases, research oligomers were found in laboratory-generated samples or mutant proteins. Recent studies have extracted amyloid fibers and oligomers from Alzheimer's brain (Shankar et al. 2008; Paravastu et al. 2009). Using cell culture techniques to control amyloid beta expression, oligomers can be extracted from different stages of the disease and the structures formed can be characterized. Identification of the oligomeric structure responsible for cytotoxicity in neurodegenerative diseases is important in determining toxicity.
아밀로이드 전구체 단백질(APP)은 대부분 눈의 망막색소상피(Retinal Pigment Epithelial, RPE) 세포와 뇌의 하수체 신경 세포에 국한되어 있다(Yoshida et al.2005). 신경독성 아밀로이드 베타 펩타이드 올리고머는 이러한 노화 질환의 핵심 매개체로 볼 수 있다.Amyloid precursor protein (APP) is mostly localized in retinal pigment epithelial (RPE) cells of the eye and pituitary neurons of the brain (Yoshida et al. 2005). Neurotoxic amyloid beta peptide oligomers can be seen as key mediators of these aging diseases.
본 발명자는 망막 세포와 신경 세포에서 광 유도, 저산소 등에 의한 산화 스트레스 및 산화질소(NO)가 아밀로이드 베타 응집에 관여하고 농도를 상향 조절함을 확인하고, 이러한 아밀로이드 베타 응집을 분해하는 조성물을 발견하여 상기 노화 질환의 예방, 개선 또는 치료에 사용할 수 있음을 확인함으로써 본 발명을 완성하였다.The present inventor confirmed that oxidative stress and nitric oxide (NO) caused by light-induced and hypoxia in retinal cells and nerve cells are involved in amyloid beta aggregation and upregulates its concentration, and discovered a composition that decomposes such amyloid beta aggregation. The present invention was completed by confirming that it can be used to prevent, improve, or treat the above-mentioned aging disease.
[특허문헌][Patent Document]
국내등록특허공보 제10-1298651호Domestic Patent Publication No. 10-1298651
이에, 본 발명이 해결하고자 하는 과제는 노인황반변성과 같은 망막 질환 또는 알츠하이머병과 같은 퇴행성 뇌 질환을 효과적으로 예방 또는 개선할 수 있는 건강식품 조성물을 제공하는 것이다.Accordingly, the problem to be solved by the present invention is to provide a health food composition that can effectively prevent or improve retinal diseases such as age-related macular degeneration or degenerative brain diseases such as Alzheimer's disease.
본 발명이 해결하고자 하는 다른 과제는 상기 망막 질환 또는 퇴행성 뇌 질환을 효과적으로 예방 또는 치료할 수 있는 약학적 조성물을 제공하는 것이다.Another problem to be solved by the present invention is to provide a pharmaceutical composition that can effectively prevent or treat the retina disease or degenerative brain disease.
본 발명의 과제들은 이상에서 언급한 기술적 과제로 제한되지 않으며, 언급되지 않은 또 다른 기술적 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.The problems of the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned will be clearly understood by those skilled in the art from the description below.
상기 과제를 해결하기 위한 본 발명의 일 실시예에 따른 망막 질환 또는 퇴행성 뇌 질환의 예방 또는 개선용 건강식품 조성물은 트레할로스(trehalose)를 포함한다. 트레할로스는 유효성분으로서 포함될 수 있다. 트레할로스는 이당체(disaccharide)로서 하기 화학식 1 또는 2와 같은 구조를 갖는 것일 수 있다.A health food composition for preventing or improving retinal disease or degenerative brain disease according to an embodiment of the present invention for solving the above problems includes trehalose. Trehalose may be included as an active ingredient. Trehalose is a disaccharide and may have a structure as shown in Chemical Formula 1 or 2 below.
[화학식 1][Formula 1]
Figure PCTKR2023006972-appb-img-000001
Figure PCTKR2023006972-appb-img-000001
[화학식 2][Formula 2]
Figure PCTKR2023006972-appb-img-000002
Figure PCTKR2023006972-appb-img-000002
본 명세서에서, 건강식품이란 건강식품, 건강기능식품, 건강보조식품 및 건강증진식품을 모두 포함하는 의미일 수 있다. 일 실시예에서, 상기 건강식품은 건강기능식품 또는 건강보조식품을 의미할 수 있으나, 이에 제한되는 것은 아니다.In this specification, health food may include all health foods, health functional foods, health supplements, and health promoting foods. In one embodiment, the health food may mean a health functional food or a health supplement, but is not limited thereto.
망막 질환은 노인황반변성(AMD), 색소성 망막염(RP), 당뇨병성 망막병증(DR), 미숙아 망막병증(ROP) 및 녹내장(glaucoma)으로 이루어진 군에서 선택되는 하나 이상을 포함할 수 있다.The retinal disease may include one or more selected from the group consisting of age-related macular degeneration (AMD), retinitis pigmentosa (RP), diabetic retinopathy (DR), retinopathy of prematurity (ROP), and glaucoma.
퇴행성 뇌 질환은 알츠하이머병(Alzheimer's disease), 파킨슨병(Parkinson's disease), 헌팅턴병(huntington's disease), 다발성경화증(multiple sclerosis),　경도인지장애(mild cognitive impairment), 대뇌　아밀로이드　맥관병증, 아밀로이드성 뇌졸증(stroke), 전신성　아밀로이드병, 더취(Dutch)형　아밀로이드증, 니만-픽병(니만-피크 병(Niemann­Pick disease), 근위축성 측삭 경화증 (amyotrophic lateral sclerosis), 척수소뇌성 운동실조증(Spinocerebellar Atrophy), 뚜렛 증후군(Tourette's Syndrome), 프리드리히 보행실조(Friedrich's Ataxia), 마차도-조셉 병(Machado-Joseph's disease), 루이 소체 치매(Lewy Body Dementia), 근육긴장이상(Dystonia) 및 진행성 핵상 마비(Progressive Supranuclear Palsy)로 이루어진 군에서 선택되는 하나 이상을 포함할 수 있다.Degenerative brain diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, mild cognitive impairment, cerebral amyloid angiopathy, and amyloid stroke. ), systemic amyloid disease, Dutch amyloidosis, Niemann-Pick disease, amyotrophic lateral sclerosis, Spinocerebellar Atrophy, Tourette's Syndrome, Friedrich's Ataxia, Machado-Joseph's disease, Lewy Body Dementia, Dystonia, and Progressive Supranuclear Palsy. It may contain one or more of your choice.
상기 망막 질환 또는 퇴행성 뇌 질환은 구체적으로, 광에 의한 질환이거나 아밀로이드 베타(Amyloid beta, Aβ)에 의한 질환일 수 있다. 광에 의한 질환이란 광 노출에 의해 유발, 악화 또는 발전하는 질환을 의미할 수 있고, 아밀로이드 베타에 의한 질환은 체내 아밀로이드 베타의 증가(또는 상향 조절), 응집 또는 축적에 의해 유발, 악화 또는 발전하는 질환을 의미할 수 있다. The retina disease or degenerative brain disease may specifically be a light-induced disease or a disease caused by amyloid beta (Aβ). A light-induced disease may refer to a disease that is caused, worsened, or developed by exposure to light, and a disease caused by amyloid beta may be caused, worsened, or developed by the increase (or upregulation), aggregation, or accumulation of amyloid beta in the body. It may mean a disease.
보다 구체적으로는, 상기 망막 질환 또는 퇴행성 뇌 질환은 광 노출에 의한 산화 스트레스(또는 산화질소)로 인해 아밀로이드 베타가 증가(또는 상향 조절), 응집 또는 축적되어 유발, 악화 또는 발전하는 질환일 수 있다. 즉, 본 발명의 건강식품 조성물은 아밀로이드 베타(또는 아밀로이드 베타 올리고머)를 분해하거나 응집 상태를 개선하여 상기 질환을 예방 또는 개선하는 것일 수 있다.More specifically, the retina disease or degenerative brain disease may be a disease that is caused, worsened, or developed by increased (or upregulated), aggregation, or accumulation of amyloid beta due to oxidative stress (or nitric oxide) caused by light exposure. . That is, the health food composition of the present invention may prevent or improve the disease by decomposing amyloid beta (or amyloid beta oligomer) or improving the aggregation state.
이러한 질환으로서, 망막 질환은 습식(wet) 또는 건식(dry) 노인황반변성일 수 있고, 특히 건식 노인황반변성일 수 있다. 건식 노인황반변성은 광에 의해 산화 스트레스 증가, 망막 변성, 망막세포 사멸 등이 유발 또는 악화되는 질환일 수 있다. 다만 본 발명의 실시예가 이에 제한되는 것은 아니다.As such a disease, the retinal disease may be wet or dry age-related macular degeneration, and in particular may be dry age-related macular degeneration. Dry age-related macular degeneration can be a disease caused or worsened by light, such as increased oxidative stress, retinal degeneration, and retinal cell death. However, embodiments of the present invention are not limited thereto.
또한, 퇴행성 뇌 질환은 알츠하이머병, 파킨슨병, 대뇌　아밀로이드　맥관병증, 아밀로이드성 뇌졸증, 전신성　아밀로이드병 또는 더취형　아밀로이드증일 수 있고, 보다 구체적으로는 알츠하이머병일 수 있다. 다만 본 발명의 실시예가 이에 제한되는 것은 아니다.Additionally, the degenerative brain disease may be Alzheimer's disease, Parkinson's disease, cerebral amyloid angiopathy, amyloid stroke, systemic amyloid disease, or Dutch amyloidosis, and more specifically, may be Alzheimer's disease. However, embodiments of the present invention are not limited thereto.
본 발명의 다른 실시예에서, 망막 질환은 망막색소상피(retinal pigment epithelial, RPE) 관련 질환일 수 있다. 망막색소상피 관련 질환이란 망막색소상피가 산화적 스트레스 등에 의해 손상 또는 악화됨으로써 발병하거나 악화되는 질환일 수 있고, 구체적으로는 건식 노인황반변성, 습식 노인황반변성, 색소성 망막염, 당뇨병성 망막병증, 미숙아 망막병증 및 녹내장으로 이루어진 군에서 선택되는 하나 이상을 포함할 수 있다.In another embodiment of the present invention, the retinal disease may be a disease related to retinal pigment epithelial (RPE). Retinal pigment epithelium-related diseases may be diseases that occur or worsen when the retinal pigment epithelium is damaged or worsened by oxidative stress, etc. Specifically, dry age-related macular degeneration, wet age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, It may include one or more selected from the group consisting of retinopathy of prematurity and glaucoma.
망막색소상피 세포의 구조적 결함 및 기능손상을 초래하는 주요 요인 중 하나는 산화적 스트레스로 알려져 있다. 지속적인 산화적 스트레스는 망막의 미토콘드리아 DNA 손상을 유발함으로써 미토콘드리아의 기능을 파괴하고 세포사멸(apotosis)에까지 이르러 망막손상으로 인한 시력 장애를 초래할 수 있다. 인간의 망막손상으로 인한 시력 장애는 실명의 주요한 원인 중 하나이며, 증가된 산화적 스트레스는 상기 건식 노인황반변성, 습식 노인황반변성, 색소성 망막염, 당뇨병성 망막병증, 미숙아 망막병증 및 녹내장과 같은 망막질환의 원인으로 여겨지고 있다. Oxidative stress is known to be one of the major factors causing structural defects and functional damage in retinal pigment epithelial cells. Continuous oxidative stress causes damage to mitochondrial DNA in the retina, destroying mitochondrial function and leading to apoptosis, which can lead to vision impairment due to retinal damage. Vision impairment due to retinal damage in humans is one of the main causes of blindness, and increased oxidative stress causes diseases such as dry age-related macular degeneration, wet age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, retinopathy of prematurity, and glaucoma. It is believed to be a cause of retinal disease.
이에 따라, 본 발명의 건강식품 조성물은 산화적 스트레스로부터 망막색소상피를 보호하거나 세포손상을 지연시킴으로써 상기 망막색소상피 관련 질환의 예방 또는 개선 용도로 사용할 수 있다.Accordingly, the health food composition of the present invention can be used to prevent or improve retinal pigment epithelium-related diseases by protecting the retinal pigment epithelium from oxidative stress or delaying cell damage.
본 발명의 건강식품 조성물은 여러 가지 제형으로 제조 및 투여될 수 있다. 제제화할 경우에는 통상적으로 사용되는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제를 섞어 조제된다. 또한 단순한 부형제 이외에 윤활제들도 사용될 수 있다. 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 희석제, 부형제 등이 포함될 수 있다. The health food composition of the present invention can be manufactured and administered in various dosage forms. When formulated, it can be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations include tablets, pills, powders, granules, capsules, etc. These solid preparations are prepared by mixing one or more compounds with at least one excipient. Additionally, lubricants other than simple excipients may also be used. Liquid preparations include suspensions, oral solutions, emulsions, and syrups, and may include commonly used diluents and excipients.
또한, 트레할로스는 식품에 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합량은 사용 목적(예방 또는 개선용)에 따라 적합하게 결정될 수 있다. Additionally, trehalose can be added directly to food or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention or improvement).
또한, 상기 조성물이 건강기능성 음료 조성물일 경우 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 나아가, 여러 가지 영양제, 비타민, 풍미제, 착색제, 중진제, 증점제, pH 조절제, 안정화제, 방부제, 탄산화제 등을 더 함유할 수 있다. 예시적인 실시예에서는, 과일 또는 야채 음료의 제조를 위한 과육을 더 함유할 수 있다.In addition, when the composition is a health functional beverage composition, it may contain various flavoring agents or natural carbohydrates as additional ingredients like ordinary beverages. Furthermore, it may further contain various nutrients, vitamins, flavors, colorants, thickeners, thickeners, pH adjusters, stabilizers, preservatives, carbonating agents, etc. In exemplary embodiments, it may further contain pulp for the production of fruit or vegetable beverages.
몇몇 실시예에서, 본 발명의 건강식품 조성물은 과일농축액, 사과산, 알로에베라분말, 사탕수수분말, 비타민, 젖산칼슘, 증점제, 수크랄로스 및 정제수로 이루어진 군에서 선택되는 하나 이상을 더 포함할 수 있다.In some embodiments, the health food composition of the present invention may further include one or more selected from the group consisting of fruit concentrate, malic acid, aloe vera powder, sugarcane powder, vitamins, calcium lactate, thickener, sucralose, and purified water.
구체적인 실시예에서, 상기 과일농축액은 적포도농축액, 백포도농축액, 당근농축액 및 블루베리농축액으로 이루어진 군에서 선택되는 하나 이상을 포함할 수 있고, 상기 증점제는 젤란검 및 잔탄검 중 하나 이상을 포함할 수 있으며, 상기 비타민은 비타민 C 및 비타민 A 중 하나 이상을 포함할 수 있다.In a specific embodiment, the fruit concentrate may include one or more selected from the group consisting of red grape concentrate, white grape concentrate, carrot concentrate, and blueberry concentrate, and the thickener may include one or more of gellan gum and xanthan gum. The vitamins may include one or more of vitamin C and vitamin A.
또한, 천연블루베리향 및 천연청포도향 중 하나 이상을 포함하는 천연과일향을 더 포함할 수 있고, 빌베리분말을 더 포함할 수 있다.In addition, it may further include a natural fruit flavor including one or more of natural blueberry flavor and natural green grape flavor, and may further include bilberry powder.
구체적인 실시예에서, 본 발명의 건강식품 조성물은 다음과 같은 조성을 갖는 것일 수 있다.In a specific example, the health food composition of the present invention may have the following composition.
트레할로스 0.5-10 wt% (또는 중량부);0.5-10 wt% (or parts by weight) of trehalose;
과일농축액 5-15 wt% (또는 중량부);5-15 wt% (or parts by weight) of fruit concentrate;
사과산 0.1-1 wt% (또는 중량부);0.1-1 wt% (or parts by weight) of malic acid;
알로에베라분말 0.05-1 wt% (또는 중량부);Aloe vera powder 0.05-1 wt% (or parts by weight);
사탕수수분말 0.01-0.2 wt% (또는 중량부);Sugarcane powder 0.01-0.2 wt% (or parts by weight);
비타민 0.01-0.2 wt% (또는 중량부);Vitamin 0.01-0.2 wt% (or parts by weight);
젖산칼슘 0.01-0.2 wt% (또는 중량부);Calcium lactate 0.01-0.2 wt% (or parts by weight);
증점제 0.01-0.2 wt% (또는 중량부);Thickener 0.01-0.2 wt% (or parts by weight);
수크랄로스 0.005-0.2 wt% (또는 중량부); 및Sucralose 0.005-0.2 wt% (or parts by weight); and
정제수 잔량 (또는 75-90 wt%, 또는 중량부)Remaining amount of purified water (or 75-90 wt%, or parts by weight)
또한, 천연과일향 0.1-1 wt%(또는 중량부)를 더 포함할 수 있고, 빌베리분말 0.05-1 wt%(또는 중량부)를 더 포함할 수 있다.In addition, it may further include 0.1-1 wt% (or part by weight) of natural fruit flavor and 0.05-1 wt% (or part by weight) of bilberry powder.
본 발명의 건강식품 조성물은 상기 성분 중 일부를 포함하는 것일 수 있는데, 예시적인 실시예에서 과일농축액, 사과산 및 천연과일향 중 하나 이상; 알로에베라분말, 사탕수수분말 및 수크랄로스 중 하나 이상; 비타민 및 젖산칼슘 중 하나 이상; 증점제; 및 정제수를 포함하는 것일 수 있다.The health food composition of the present invention may contain some of the above ingredients, and in an exemplary embodiment, one or more of fruit concentrate, malic acid, and natural fruit flavor; One or more of aloe vera powder, sugar cane powder, and sucralose; One or more of vitamins and calcium lactate; thickener; and purified water.
예시적인 실시예에서, 본 발명의 건강식품 조성물은 효소처리스테비아, 카르니틴 및 타우린으로 이루어진 군에서 선택되는 하나 이상을 더 포함할 수 있다.In an exemplary embodiment, the health food composition of the present invention may further include one or more selected from the group consisting of enzyme-treated stevia, carnitine, and taurine.
상기 다른 과제를 해결하기 위한 본 발명의 일 실시예에 따른 망막 질환 또는 퇴행성 뇌 질환 예방 또는 치료용 약학적 조성물은 트레할로스를 포함하고, 트레할로스는 유효성분으로서 포함될 수 있다.A pharmaceutical composition for preventing or treating retinal disease or degenerative brain disease according to an embodiment of the present invention to solve the above other problems includes trehalose, and trehalose may be included as an active ingredient.
본 발명의 약학적 조성물은 경구 및 비경구의 여러 가지 제형으로 제조 및 투여될 수 있다. 경구 투여는 전술한 바와 같으며, 비경구 투여는 피하주사, 정맥주사, 근육 내 주사 또는 흉부 내 주사를 포함할 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제가 포함된다.The pharmaceutical composition of the present invention can be prepared and administered in various oral and parenteral dosage forms. Oral administration is as described above, and parenteral administration may include subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, and emulsions.
비제한적인 실시예에서, 상기 약학적 조성물은 트레할로스를 예를 들어 0.1-10 wt%로 포함할 수 있으나, 이에 제한되는 것은 아니다.In a non-limiting example, the pharmaceutical composition may include trehalose, for example, 0.1-10 wt%, but is not limited thereto.
본 발명은 다른 실시예에 따라 망막 질환 또는 퇴행성 뇌 질환의 예방 또는 치료가 필요한 환자에게 트레할로스를 포함하는 약학적 조성물을 투여하는 단계를 포함하는 망막 질환 또는 퇴행성 뇌 질환의 예방 또는 치료 방법을 제공할 수 있다. According to another embodiment, the present invention provides a method for preventing or treating retinal disease or degenerative brain disease, comprising administering a pharmaceutical composition containing trehalose to a patient in need of prevention or treatment of retinal disease or degenerative brain disease. You can.
기타 실시예의 구체적인 사항들은 상세한 설명에 포함되어 있다.Specific details of other embodiments are included in the detailed description.
본 발명의 실시예들에 따르면, 트레할로스를 포함하는 건강식품 조성물 또는 약학적 조성물을 통해 노인황반변성과 같은 망막 질환 또는 알츠하이머병과 같은 퇴행성 뇌 질환을 효과적으로 예방, 개선 및 치료할 수 있다.According to embodiments of the present invention, retinal diseases such as age-related macular degeneration or degenerative brain diseases such as Alzheimer's disease can be effectively prevented, improved, and treated through a health food composition or pharmaceutical composition containing trehalose.
본 발명의 실시예들에 따른 효과는 이상에서 예시된 내용에 의해 제한되지 않으며, 더욱 다양한 효과들이 본 명세서 내에 포함되어 있다.Effects according to embodiments of the present invention are not limited to the contents exemplified above, and further various effects are included in the present specification.
도 1은 본 발명의 실험예에 따라 산화 스트레스 하에서 아밀로이드 베타가 상향 조절된 것을 시각적으로 나타낸 이미지이다. Figure 1 is an image visually showing that amyloid beta is up-regulated under oxidative stress according to an experimental example of the present invention.
도 2는 본 발명의 실험예에 따라 광 유도된 산소 의존적 NO 형성을 나타낸 그래프이다.Figure 2 is a graph showing light-induced oxygen-dependent NO formation according to an experimental example of the present invention.
도 3은 본 발명의 실험예에 따라 광 노출 시와 암 환경에서의 세포내 NO 상태를 형광물질을 사용하여 나타낸 것이다.Figure 3 shows the intracellular NO status using fluorescent substances during light exposure and in a cancer environment according to an experimental example of the present invention.
도 4는 본 발명의 실험예에 따라 본 발명의 조성물을 처리하기 전과 후의 아밀로이드 베타 올리고머 응집 상태를 확인한 이미지이다.Figure 4 is an image confirming the state of amyloid beta oligomer aggregation before and after treatment with the composition of the present invention according to an experimental example of the present invention.
도 5는 본 발명의 실험예에 따라 산화적 스트레스에 의한 ARPE-19 세포 손상에서 트레할로스의 (a) 세포독성 평가, (b) 세포생존율 평가 및 (c) LDH(젖산탈수소 효소) 측정을 통한 세포 독성 평가 결과 그래프이다.Figure 5 shows cells through (a) cytotoxicity evaluation, (b) cell viability evaluation, and (c) LDH (lactate dehydrogenase) measurement of trehalose in ARPE-19 cell damage caused by oxidative stress according to an experimental example of the present invention. This is a graph of the toxicity evaluation results.
도 6은 본 발명의 실험예에 따라 산화적 스트레스에 의한 ARPE-19 세포 손상에서 저농도 트레할로스(50 μM)의 세포보효 효과를 확인한 그래프이다. Figure 6 is a graph confirming the cytoprotective effect of low concentration trehalose (50 μM) on ARPE-19 cell damage caused by oxidative stress according to an experimental example of the present invention.
도 7은 본 발명의 실험예에 따라 산화적 스트레스에 의해 손상된 미토콘드리아 기능에 대한 트레할로스의 보호 효과를 확인한 그래프이다. Figure 7 is a graph confirming the protective effect of trehalose on mitochondrial function damaged by oxidative stress according to an experimental example of the present invention.
도 8은 본 발명의 실험예에 따라 산화적 스트레스에 의해 손상된 ARPE-19 세포의 ROS (reactive oxygzen species) 생산과 미토콘드리아 손상에 대한 트레할로스의 효능 평가, 미토콘드리아 염색 및 공초점현미경 이미지이다. Figure 8 is an evaluation of the efficacy of trehalose on ROS (reactive oxygen species) production and mitochondrial damage in ARPE-19 cells damaged by oxidative stress according to an experimental example of the present invention, mitochondrial staining, and confocal microscopy images.
본 발명의 이점 및 특징, 그리고 그것들을 달성하는 방법은 상세하게 후술되어 있는 실시예들을 참조하면 명확해질 것이다. 그러나 본 발명은 이하에서 개시되는 실시예들에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 것이며, 단지 실시예들은 본 발명의 개시가 완전하도록 하며, 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이며, 본 발명은 청구항의 범주에 의해 정의될 뿐이다.The advantages and features of the present invention and methods for achieving them will become clear with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below and will be implemented in various different forms, and only the embodiments serve to ensure that the disclosure of the present invention is complete, and those skilled in the art It is provided to fully inform the person of the scope of the invention, and the present invention is only defined by the scope of the claims.
본 명세서에서 사용된 용어는 실시예들을 설명하기 위한 것이며 본 발명을 제한하고자 하는 것은 아니다. 본 명세서에서, '및/또는'은 언급된 아이템들의 각각 및 하나 이상의 모든 조합을 포함한다. 또, 단수형은 문구에서 특별히 언급하지 않는 한 복수형도 포함한다. 명세서에서 사용되는 '포함한다(comprises)' 및/또는 '포함하는(comprising)'은 언급된 구성요소 외에 하나 이상의 다른 구성요소의 존재 또는 추가를 배제하지 않는다. '-' 또는 '내지'를 사용하여 나타낸 수치 범위는 다른 언급이 없는 한 그 앞과 뒤에 기재된 값을 각각 하한과 상한으로서 포함하는 수치 범위를 나타낸다. '약' 또는 '대략'은 그 뒤에 기재된 값 또는 수치 범위의 20% 이내의 값 또는 수치 범위를 의미한다.The terminology used herein is for describing embodiments and is not intended to limit the invention. As used herein, 'and/or' includes each and every combination of one or more of the mentioned items. Additionally, the singular form also includes the plural form unless specifically stated in the phrase. As used in the specification, 'comprises' and/or 'comprising' does not exclude the presence or addition of one or more other components in addition to the mentioned components. The numerical range indicated using '-' or 'to' indicates a numerical range that includes the values written before and after it as the lower and upper limits, respectively, unless otherwise specified. ‘About’ or ‘approximately’ means a value or numerical range within 20% of the value or numerical range stated thereafter.
또한, 본 발명의 실시예의 구성 요소를 설명하는 데 있어서, 제1, 제2, A, B, (a), (b) 등의 용어를 사용할 수 있다. 이러한 용어는 그 구성 요소를 다른 구성 요소와 구별하기 위한 것일 뿐, 그 용어에 의해 해당 구성 요소의 본질이나 차례 또는 순서 등이 한정되지 않는다.Additionally, when describing the components of an embodiment of the present invention, terms such as first, second, A, B, (a), and (b) may be used. These terms are only used to distinguish the component from other components, and the nature, sequence, or order of the component is not limited by the term.
다른 정의가 없다면, 본 명세서에서 사용되는 모든 용어(기술 및 과학적 용어를 포함)는 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 공통적으로 이해될 수 있는 의미로 사용될 수 있을 것이다. 또 일반적으로 사용되는 사전에 정의되어 있는 용어들은 명백하게 특별히 정의되어 있지 않는 한 이상적으로 또는 과도하게 해석되지 않는다.Unless otherwise defined, all terms (including technical and scientific terms) used in this specification may be used with meanings that can be commonly understood by those skilled in the art to which the present invention pertains. Additionally, terms defined in commonly used dictionaries are not interpreted ideally or excessively unless clearly specifically defined.
그리고 본 발명의 실시예를 설명함에 있어, 관련된 공지 구성 또는 기능에 대한 구체적인 설명이 본 발명의 실시예에 대한 이해를 방해한다고 판단되는 경우에는 그 상세한 설명은 생략한다.Also, in describing embodiments of the present invention, if detailed descriptions of related known configurations or functions are judged to impede understanding of the embodiments of the present invention, the detailed descriptions will be omitted.
본 명세서에서, '예방'이란 증상 또는 질환은 아직 없으나, 이러한 증상 또는 질환에 걸릴 수 있는 개체에서 증상 또는 질환의 발생을 억제하거나 지연시키는 것을 의미한다. In this specification, 'prevention' means suppressing or delaying the occurrence of symptoms or diseases in an individual who does not yet have symptoms or diseases but may develop such symptoms or diseases.
본 명세서에서, '치료' 또는 '개선'이란 개체에서 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미하며, 예를 들어 (a) 증상 또는 질환의 발전(악화)의 억제, (b) 증상 또는 질환의 경감, 또는 (c) 증상 또는 질환의 제거를 의미한다. In this specification, 'treatment' or 'improvement' means any action that improves or beneficially changes symptoms in an individual, such as (a) suppressing the development (worsening) of a symptom or disease, (b) suppressing the development (worsening) of a symptom or disease means relief, or (c) elimination of symptoms or disease.
본 명세서에서, '개체' 또는 '대상'이란 본 발명을 이용하여 특정 질병 또는 질환을 진단, 예방 또는 치료하려는 세포, 조직, 동물 또는 인간을 의미한다.In this specification, 'individual' or 'subject' means a cell, tissue, animal, or human for which a specific disease or condition is to be diagnosed, prevented, or treated using the present invention.
본 명세서에서, '생물학적 시료'란 대상의 혈액, 혈장, 혈청, 세포, 조직, 체액, 타액, 뇨, 안방수, 전방수 등을 의미한다.In this specification, 'biological sample' refers to the subject's blood, plasma, serum, cells, tissues, body fluids, saliva, urine, aqueous humor, anterior chamber fluid, etc.
본 명세서에서, '유래'란 특정 대상 또는 특정 부위에서 분리되는 것뿐만 아니라 대상 또는 부위 그 자체를 의미할 수도 있다.In this specification, 'derived from' may mean not only separation from a specific object or part, but also the object or part itself.
본 명세서에서, '대조군'이란 특정 질병 또는 질환이 없거나 유발되지 않은 정상 세포 또는 환자 등을 의미한다.In this specification, 'control group' refers to normal cells or patients that do not have or are not caused by a specific disease or disease.
본 명세서에서, '수준'이란 특정 인자의 존재량을 측정 또는 분석하여 확인할 수 있는 함량, 농도 등도 의미할 수 있다.In this specification, 'level' may also mean content, concentration, etc. that can be confirmed by measuring or analyzing the presence of a specific factor.
이하, 본 발명의 실시예들을 제조예와 실험예를 통해 상세하게 설명하나, 본 발명의 효과가 하기 실험예에 의해 제한되지 아니함은 자명하다.Hereinafter, embodiments of the present invention will be described in detail through manufacturing examples and experimental examples, but it is obvious that the effect of the present invention is not limited by the following experimental examples.
제조예: 본 발명의 조성물 제형 제조Preparation Example: Preparation of composition formulation of the present invention
실시예 1Example 1
다음과 같은 조성으로 트레할로스를 포함하는 액제(liquid formulation) 조성물을 제조하였다.A liquid formulation composition containing trehalose was prepared with the following composition.
트레할로스 0.5 wt%; 적포도농축액 10 wt%; 사과산 0.3 wt%; 동결건조알로에베라(100:1) 0.1 wt%; 사탕수수분말 0.05 wt%; 비타민C 0.05 wt%; 젖산칼슘 0.05 wt%; 젤란검 0.03 wt%; 수크랄로스 0.015 wt%; 정제수 잔량Trehalose 0.5 wt%; Red grape concentrate 10 wt%; Malic acid 0.3 wt%; Freeze-dried aloe vera (100:1) 0.1 wt%; Sugarcane powder 0.05 wt%; Vitamin C 0.05 wt%; Calcium lactate 0.05 wt%; Gellan gum 0.03 wt%; Sucralose 0.015 wt%; Purified water remaining
실시예 2Example 2
다음과 같은 조성으로 트레할로스를 포함하는 액제 조성물을 제조하였다.A liquid composition containing trehalose was prepared with the following composition.
트레할로스 3 wt%; 적포도농축액 10 wt%; 사과산 0.3 wt%; 동결건조알로에베라(100:1) 0.1 wt%; 사탕수수분말 0.05 wt%; 비타민C 0.05 wt%; 젖산칼슘 0.05 wt%; 젤란검 0.03 wt%; 수크랄로스 0.015 wt%; 정제수 잔량; Trehalose 3 wt%; Red grape concentrate 10 wt%; Malic acid 0.3 wt%; Freeze-dried aloe vera (100:1) 0.1 wt%; Sugarcane powder 0.05 wt%; Vitamin C 0.05 wt%; Calcium lactate 0.05 wt%; Gellan gum 0.03 wt%; Sucralose 0.015 wt%; Remaining amount of purified water;
실시예 3Example 3
다음과 같은 조성으로 트레할로스를 포함하는 액제 조성물을 제조하였다.A liquid composition containing trehalose was prepared with the following composition.
트레할로스 5 wt%; 적포도농축액 10 wt%; 사과산 0.3 wt%; 동결건조알로에베라(100:1) 0.1 wt%; 사탕수수분말 0.05 wt%; 비타민C 0.05 wt%; 젖산칼슘 0.05 wt%; 젤란검 0.03 wt%; 수크랄로스 0.015 wt%; 정제수 잔량 Trehalose 5 wt%; Red grape concentrate 10 wt%; Malic acid 0.3 wt%; Freeze-dried aloe vera (100:1) 0.1 wt%; Sugarcane powder 0.05 wt%; Vitamin C 0.05 wt%; Calcium lactate 0.05 wt%; Gellan gum 0.03 wt%; Sucralose 0.015 wt%; Purified water remaining
실시예 4Example 4
다음과 같은 조성으로 트레할로스를 포함하는 액제 조성물을 제조하였다.A liquid composition containing trehalose was prepared with the following composition.
트레할로스 10 wt%; 적포도농축액 10 wt%; 사과산 0.3 wt%; 동결건조알로에베라(100:1) 0.1 wt%; 사탕수수분말 0.05 wt%; 비타민C 0.05 wt%; 젖산칼슘 0.05 wt%; 젤란검 0.03 wt%; 수크랄로스 0.015 wt%; 정제수 잔량Trehalose 10 wt%; Red grape concentrate 10 wt%; Malic acid 0.3 wt%; Freeze-dried aloe vera (100:1) 0.1 wt%; Sugarcane powder 0.05 wt%; Vitamin C 0.05 wt%; Calcium lactate 0.05 wt%; Gellan gum 0.03 wt%; Sucralose 0.015 wt%; Purified water remaining
실시예 5Example 5
다음과 같은 조성으로 트레할로스를 포함하는 액제 조성물을 제조하였다.A liquid composition containing trehalose was prepared with the following composition.
트레할로스 0.5 wt%; 정제수 잔량Trehalose 0.5 wt%; Purified water remaining
실험예 1: 산화 스트레스에 의한 아밀로이드 베타 상향 조절 확인Experimental Example 1: Confirmation of amyloid beta upregulation by oxidative stress
망막색소상피(Retinal Pigment Epithelial, RPE) 세포를 배양하고 명암 주기, 연속 조명 및 다양한 광도(200-5,000 lux)를 포함한 다양한 광 스트레스에 노출하여 산화질소(NO) 생성을 측정하였다. 상기 측정은 T-25 조직 배양 플라스크를 수용하는 맞춤형 샘플링 셀이 장착된 Sievers의 280i 산화질소 분석기(NOA)를 사용하여 수행되었다. 광에 의해 스트레스를 받는 망막색소상피 세포에 의해 생성된 NO의 수준을 정량적으로 측정하고 새로운 NO를 사용하여 배양된 망막색소상피 세포에 정밀하게 제어된 NO의 표면 플럭스를 전달함으로써 망막색소상피 세포에서 아밀로이드 베타(Amyloid beta, Aβ)의 상향 조절을 유도하는 NO의 농도를 결정하였다. Retinal Pigment Epithelial (RPE) cells were cultured and exposed to various light stresses, including light/dark cycles, continuous illumination, and various light intensities (200-5,000 lux), and nitric oxide (NO) production was measured. The measurements were performed using a Sievers 280i nitric oxide analyzer (NOA) equipped with a custom sampling cell housing a T-25 tissue culture flask. By quantitatively measuring the level of NO produced by retinal pigment epithelial cells stressed by light and using fresh NO to deliver a precisely controlled surface flux of NO to cultured retinal pigment epithelial cells. The concentration of NO that induces upregulation of amyloid beta (Aβ) was determined.
다음으로, 망막색소상피 세포에 대한 저산소 및 광 유도 산화 스트레스를 포함하여 Aβ 형성을 유도하는 것으로 알려진 세포 배양 조건에서, 공지된 방법을 이용하여 Aβ 올리고머를 측정하였다(Yoshida et al. 2005). 공지된 방법으로 쥐 신경 세포주 PC-12에서 신경 성장 인자(NGF)와 혈청을 추출하고 아밀로이드 베타 올리고머의 크기, 구조 및 소수성을 파악하기 위한 생화학적 방법을 사용하였다(Matrone et al. 2008). 이어서, 산화 스트레스, pH 및 이온 강도가 다른 조건에서 합성 펩타이드를 사용하여 다른 아밀로이드 베타 올리고머를 형성하여 독성 아밀로이드 베타 올리고머와 비교하였다. 올리고머 구조를 결정하고 PC-12 및 망막색소상피 세포를 사용한 세포 배양에서 독성을 테스트하였다.Next, Aβ oligomers were measured using a known method under cell culture conditions known to induce Aβ formation, including hypoxia and light-induced oxidative stress on retinal pigment epithelial cells (Yoshida et al. 2005). Nerve growth factor (NGF) and serum were extracted from rat neural cell line PC-12 using a known method, and biochemical methods were used to determine the size, structure, and hydrophobicity of amyloid beta oligomers (Matrone et al. 2008). Then, different amyloid beta oligomers were formed using synthetic peptides under different conditions of oxidative stress, pH, and ionic strength and compared with toxic amyloid beta oligomers. The oligomeric structure was determined and toxicity tested in cell culture using PC-12 and retinal pigment epithelial cells.
도 2는 광 유도된 산소 의존적 NO 형성을 나타낸 그래프이다. 도 2와 같이, 망막색소상피 세포(ARPE-19 cell)가 광에 노출되고 아르기닌과 산소가 첨가되면 산화 스트레스에 의한 NO가 형성된다. 이는 망막색소상피에서 NO가 식균 작용의 메신저로서 합성효소 또는 아르기닌 대사를 통한 NO-아밀로이드 베타간의 잠재적인 상호작용을 보여준다. 도 3은 광 노출 시와 암 환경에서의 세포내 NO 상태를 형광물질(4,5-diaminofluorescein diacetate)을 사용하여 나타낸 것이다.Figure 2 is a graph showing light-induced oxygen-dependent NO formation. As shown in Figure 2, when retinal pigment epithelial cells (ARPE-19 cells) are exposed to light and arginine and oxygen are added, NO is formed due to oxidative stress. This shows the potential interaction between NO and amyloid beta through synthase or arginine metabolism, where NO is a messenger for phagocytosis in the retinal pigment epithelium. Figure 3 shows the intracellular NO status during light exposure and in a cancer environment using a fluorescent substance (4,5-diaminofluorescein diacetate).
또한, 2D 전기영동 및 MALDI-TOF-TOF 질량 분석을 포함한 단백질체 기술을 사용하여 망막색소상피에서 산화 스트레스의 초기 단백질 마커를 식별하였다. 망막색소상피 세포의 프로테옴 변화는 레티노이드 트래피킹(IRBP), 칼슘 신호 전달(칼레티쿨린), G 단백질 신호 전달(트랜스듀신) 및 단백질 응집 (아밀로이드 베타)을 포함한 주요 세포 과정에 관여하는 단백질을 나타내었다. 상기 결과는 망막색소상피 초기 신호 분자와 산화 스트레스로 인한 신경변성의 표적 단백질을 보여주며, 특히 산화 스트레스로 인한 세포 배양에서 아밀로이드 베타가 상향 조절됨을 의미할 수 있다. 즉, 아밀로이드 베타는 특정 pH, 이온 강도 및 산화 스트레스 하에서 아밀로이드 베타 응집체 형성을 통해 망막색소상피에서 세포 사멸 신호 및 단백질 질화를 유도한다. 광 수용체의 세포사멸(apoptosis)은 노화 관련 황반변성(AMD), 색소성 망막염(RP), 녹내장, 알츠하이머병 등의 최종 세포 사멸 경로이다. Additionally, proteomic techniques, including 2D electrophoresis and MALDI-TOF-TOF mass spectrometry, were used to identify early protein markers of oxidative stress in the retinal pigment epithelium. Proteome changes in retinal pigment epithelial cells reveal proteins involved in key cellular processes, including retinoid trafficking (IRBP), calcium signaling (calreticulin), G protein signaling (transducin), and protein aggregation (amyloid beta). It was. The above results show retinal pigment epithelium early signaling molecules and target proteins of neurodegeneration due to oxidative stress, and may indicate that amyloid beta is specifically upregulated in cell culture due to oxidative stress. That is, amyloid beta induces cell death signals and protein nitration in the retinal pigment epithelium through the formation of amyloid beta aggregates under certain pH, ionic strength, and oxidative stress. Photoreceptor apoptosis is the final cell death pathway in age-related macular degeneration (AMD), retinitis pigmentosa (RP), glaucoma, and Alzheimer's disease.
도 1은 산화 스트레스 하에서 아밀로이드 베타가 상향 조절된 것을 시각적으로 나타낸 이미지이다. 도 1과 같이, 아밀로이드 베타는 1차 망막색소상피 세포에서 대조군(녹색)에 비해 산화 스트레스(적색) 조건(200 μM H2O2)에서 증가하였다. 단백질은 2D 전기영동으로 분리하고 Coomassie 염색으로 시각화하였다. 겔을 비교하기 위해 인공적인 녹색과 적색을 첨가하였다. 병합된 이미지는 질량 분석에 의해 식별된 표적 단백질의 차별적인 발현을 보여주었다. 광 유도 산화질소는 아밀로이드 베타의 니트로화를 유발하고 응집체 형성을 가속화하는 것으로 나타난다.Figure 1 is a visual image showing upregulation of amyloid beta under oxidative stress. As shown in Figure 1, amyloid beta increased in primary retinal pigment epithelial cells under oxidative stress (red) conditions (200 μM H 2 O 2 ) compared to the control group (green). Proteins were separated by 2D electrophoresis and visualized by Coomassie staining. Artificial green and red colors were added to compare gels. Merged images showed differential expression of target proteins identified by mass spectrometry. Light-induced nitric oxide appears to trigger nitration of amyloid beta and accelerate aggregate formation.
즉, 황반변성과 같은 망막 질환이나 알츠하이머병 등의 뇌 질환은 아밀로이드 베타의 증가 및 응집에 의해 악화되는데, 광 노출 등으로 발생하는 산화 스트레스와 이에 의해 생성되는 산화 질소(NO)는 아밀로이드 베타를 상향 조절하고 세포 독성을 유발하는 원인이 됨을 알 수 있다.In other words, retinal diseases such as macular degeneration and brain diseases such as Alzheimer's disease are worsened by the increase and aggregation of amyloid beta. Oxidative stress caused by light exposure and nitric oxide (NO) generated thereby upregulate amyloid beta. It can be seen that it causes cytotoxicity.
실험예 2: 본 발명의 조성물의 아밀로이드 베타 분해 효능 확인Experimental Example 2: Confirmation of amyloid beta decomposition efficacy of the composition of the present invention
상기 제조예에서 제조된 본 발명의 조성물들의 아밀로이드 베타 분해 효능을 확인하기 위해, 먼저 아밀로이드 베타 올리고머를 추출하였다. 망막색소상피 및 PC-12 세포는 아밀로이드 베타 형성을 유도하는 서로 다른 조건에서 성장한다. 공지된 방법을 참조하여, 세포는 사멸(apoptosis) 동안 다른 지점에서 채취되고 올리고머는 TBS 및 구아니딘 Hal을 사용하여 추출되어 각각 가용성 및 불용성 올리고머를 얻었다(Shankar et al. 2008; Paravastu et al. 2009). In order to confirm the amyloid beta decomposition efficacy of the compositions of the present invention prepared in the above Preparation Example, amyloid beta oligomers were first extracted. Retinal pigment epithelium and PC-12 cells grow under different conditions that induce amyloid beta formation. Referring to known methods, cells were harvested at different points during apoptosis and oligomers were extracted using TBS and guanidine Hal to obtain soluble and insoluble oligomers, respectively (Shankar et al. 2008; Paravastu et al. 2009). .
추출된 올리고머의 화학적 속성은 염료와 FTIR로 분석된다. Thioflavin-T(ThT)는 아밀로이드 구조를 감지하는 데 사용되는 시약이다(Wu et al. 2008; Biancalana et al. 2009). 콩고 레드는 소수성 검출인 ANS와 함께 사용될 아밀로이드 염료(Klunk et al. 1999)로 사용되었다. The chemical properties of the extracted oligomers are analyzed by dye and FTIR. Thioflavin-T (ThT) is a reagent used to detect amyloid structures (Wu et al. 2008; Biancalana et al. 2009). Congo red was used as an amyloid dye (Klunk et al. 1999) to be used with ANS, a hydrophobic detection.
다음으로, 트레할로스를 포함하는 상기 실시예 1 내지 5의 조성물을 아밀로이드 베타 올리고머 추출물에 처리하고, 처리된 아밀로이드 베타 올리고머 또는 그 응집 상태가 분해되는 것을 확인하였다. 도 4는 실시예 1의 조성물을 처리하기 전과 후의 아밀로이드 베타 올리고머 응집 상태를 확인한 이미지이다. Next, the compositions of Examples 1 to 5 containing trehalose were treated with amyloid beta oligomer extract, and it was confirmed that the treated amyloid beta oligomer or its aggregated state was decomposed. Figure 4 is an image confirming the state of amyloid beta oligomer aggregation before and after processing the composition of Example 1.
또한, 아밀로에드 베타 발현이 상향된 망막색소상피 및 PC-12 세포에 트레할로스를 포함하는 상기 실시예 1 내지 5의 조성물을 직접 처리하였다. 상기 실시예 1 내지 5의 조성물이 처리된 세포에서는 마찬가지로 아밀로이드 베타 올리고머가 분해된 것이 확인되었다.In addition, retinal pigment epithelium and PC-12 cells with elevated amyloed beta expression were directly treated with the compositions of Examples 1 to 5 containing trehalose. It was confirmed that amyloid beta oligomers were similarly decomposed in cells treated with the compositions of Examples 1 to 5.
이와 같이 트레할로스를 포함하는 상기 조성물이 처리된 아밀로이드 베타 올리고머를 분해한 것을 확인함으로써, 본 발명의 조성물이 황반변성, 알츠하이머병 등의 망막 질환 및 뇌 질환의 예방, 개선 및 치료에 효능을 나타낼 것으로 기대할 수 있다.By confirming that the composition containing trehalose decomposed the treated amyloid beta oligomer, it is expected that the composition of the present invention will be effective in preventing, improving and treating retinal diseases such as macular degeneration and Alzheimer's disease and brain diseases. You can.
실험예 3: 본 발명의 조성물의 망막색소상피세포 보호 효능 확인Experimental Example 3: Confirmation of the retinal pigment epithelial cell protective effect of the composition of the present invention
<1. 실험물질의 준비><1. Preparation of experimental materials>
트레할로스 0.021 g이 용해된 용액을 준비하였다. 트레할로스의 물에 대한 용해도는 0.1 g/mL이며, 용액은 투명한 무색이다. 그 외 비교를 위한 다른 단일 화합물들을 함께 준비하였다.A solution containing 0.021 g of trehalose was prepared. The solubility of trehalose in water is 0.1 g/mL, and the solution is clear and colorless. Other single compounds were also prepared for comparison.
<2. 세포 배양><2. Cell Culture>
인간 망막색소상피세포주 (ARPE-19) 세포는 American Type Culture Collection (Manassas, VA, USA)에서 구입하였고 DMEM (Dulbecco's modified Eagle's medium)과 Ham's F12 1:1 혼합 배지인 DMEM/F12 (Gibco, Carlsdad, CA, USA)에 10% fetal bovine serum(Gibco, Burlington, ON, Canada), 1% penicillin-streptomycin (Gibco, Life Technologies Limited, Paisley, UK)를 첨가해 37 ℃, 5% CO2 조건에서 습윤 배양했고, passage 8-11 사이의 세포가 70% 이상의 밀도로 성장했을 때 계대 배양하여 실험에 사용하였다.Human retinal pigment epithelial cell line (ARPE-19) cells were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM (Dulbecco's modified Eagle's medium) and Ham's F12 1:1 mixed medium DMEM/F12 (Gibco, Carlsdad, CA, USA), 10% fetal bovine serum (Gibco, Burlington, ON, Canada) and 1% penicillin-streptomycin (Gibco, Life Technologies Limited, Paisley, UK) were added and cultured in wet conditions at 37°C and 5% CO2 . When the cells grew to a density of 70% or more between passages 8 and 11, they were subcultured and used for experiments.
<3. 세포 생존율 평가><3. Cell viability evaluation>
실험물질들에 대한 세포 독성을 평가하기 위하여 cell counting kit-8 (CCK-8; Dojindo, Japan) assay를 실시하였다. 우선, 실험물질 자체의 세포 독성을 확인하기 위해 96 well plate (1 x 104 cells/well)에 ARPE-19 세포를 분주하여 70 %의 confluence에 도달하면, 농도별 실험물질을 처리하여 24시간 배양하였다. 각 well에 20 ㎕씩 CCK-8 용액을 넣은 후 37 ℃, 5% CO2 incubator에서 2시간 배양하였다. 다음 microplate reader (Molecular Devices, Sunnyvale, CA, USA)를 이용하여 450 nm에서 흡광도 값을 측정하였다. 한편, tert-Butyl Hydroperoxide (tBHP, Sigma-Aldrich Chemical Co., USA) 처리에 따른 산화적 스트레스에 대한 실험물질의 세포 보호 효과를 확인하기 위하여 tBHP (300 μM)과 실험물질을 24시간 동안 동시 처리 후, 각 well에 20 ㎕씩 CCK-8 처리하여 microplate reader를 이용하여 450 nm에서 흡광도 값을 측정하였다. NAC (N-Acetyl-L-cysteine)은 양성 대조군으로서 1.0 mM이 사용되었다.To evaluate the cytotoxicity of the test substances, a cell counting kit-8 (CCK-8; Dojindo, Japan) assay was performed. First, to check the cytotoxicity of the test substance itself, ARPE- 19 cells were distributed in a 96 well plate (1 did. 20 ㎕ of CCK-8 solution was added to each well and incubated for 2 hours in a 5% CO 2 incubator at 37°C. Next, the absorbance value was measured at 450 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). Meanwhile, to confirm the cytoprotective effect of the test substance against oxidative stress following treatment with tert-Butyl Hydroperoxide (tBHP, Sigma-Aldrich Chemical Co., USA), tBHP (300 μM) and the test substance were simultaneously treated for 24 hours. Afterwards, 20 ㎕ of CCK-8 was treated in each well, and the absorbance value was measured at 450 nm using a microplate reader. NAC (N-Acetyl-L-cysteine) was used at 1.0 mM as a positive control.
<4. 산소 소비율(Oxygen consumption rate, OCR) 측정><4. Oxygen consumption rate (OCR) measurement>
세포내 산소 소비율을 측정하기 위해, 1 x 104 cells/well 개의 ARPE-19 세포를 XF analyzer (Seahorse Bioscience, USA)용 plate에 10% fetal bovine serum (GIBCO, USA) 이 포함된 DMEM/F-12 medium을 사용하여 배양하였다. 세포의 산소 소비율(Oxygen consumption rate, OCR)을 측정하기 위해, bicarbonate-free medium으로 교체한 뒤, CO2 없는 세포 배양기에서 1시간 배양하였다. 20분 동안 세포의 basal level의 OCR 값을 측정한 뒤 세포를 300 μM tBHP 또는 각 실험물질을 4시간 동안 동시 처리 후 60분간 OCR 변화를 측정하였다. 후에 0.5 μM oligomycin, 1 μM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), 1 μM rotenone, 1 μg/ml antimycin A 등의 4가지 호흡 사슬 억제제를 순서대로 처리하면서, 다시 OCR 변화량을 측정하였다.To measure the intracellular oxygen consumption rate, 1 x 104 cells/well ARPE-19 cells were cultured on a plate for an Cultured using 12 medium. To measure the oxygen consumption rate (OCR) of cells, the medium was replaced with bicarbonate-free medium and cultured in a cell incubator without CO 2 for 1 hour. After measuring the OCR value at the basal level of the cells for 20 minutes, the cells were simultaneously treated with 300 μM tBHP or each test substance for 4 hours, and the OCR changes were measured for 60 minutes. Later, four types of respiratory chain inhibitors, including 0.5 μM oligomycin, 1 μM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), 1 μM rotenone, and 1 μg/ml antimycin A were sequentially treated, and the change in OCR was measured again.
<5. 세포내 미토콘드리아 모양 및 활성산소 측정><5. Measurement of intracellular mitochondrial shape and active oxygen>
세포내 산화적 스트레스에 의한 미토콘드리아 모양 및 세포 내 ROS (reactive oxygzen species, 활성산소)를 측정하기 위해, 1.5 x 104 cells/well 개의 ARPE-19 세포를 μ-slide well plate에 10% fetal bovine serum (GIBCO, USA) 이 포함된 DMEM/F-12 medium을 사용하여 배양하였다. 그리고 세포에 300 μM tBHP 또는 각 실험물질을 4시간 동안 동시 처리 후 ROS 생성 변화를 측정하기 위해 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA; Invitrogen) 염색법을 이용하였고, 미토콘드리아 모양을 확인하기 위해 MitoTracker™ Red CMXRos (Themo fisher scientific, USA) 염색을 40분간시켰다. 이 후 PBS로 2회 세척하고, DAPI를 함유하는 배지로 교환하였다. 30분 뒤 PBS로 2회 세척 후 배지로 교체 후 공초점 레이저 스캐닝 현미경 (LSM 710; Carl Zeiss, Inc.)을 사용하여 이미지를 얻었다. ROS intensity는 ROS detection cell-based assay kit (Cayman chemical, USA)로 측정하였고, mitochondria intensity는 cell lysis 시킨 후 EX. 556 nm/EM. 575 nm 범위에서 fluorenscence microplate reader로 측정하였다.To measure mitochondrial shape and intracellular ROS ( reactive oxygen species) caused by intracellular oxidative stress, 1.5 (GIBCO, USA) was cultured using DMEM/F-12 medium containing . And after simultaneous treatment of cells with 300 μM tBHP or each test substance for 4 hours, 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA; Invitrogen) staining was used to measure changes in ROS production, and to confirm the shape of mitochondria. MitoTracker™ Red CMXRos (Themo fisher scientific, USA) staining was performed for 40 minutes. Afterwards, it was washed twice with PBS and replaced with a medium containing DAPI. After 30 minutes, the cells were washed twice with PBS, replaced with medium, and images were obtained using a confocal laser scanning microscope (LSM 710; Carl Zeiss, Inc.). ROS intensity was measured using a ROS detection cell-based assay kit (Cayman chemical, USA), and mitochondria intensity was measured using EX. 556nm/EM. It was measured with a fluorescence microplate reader in the 575 nm range.
<6. 통계처리><6. Statistical processing>
GraphPad Prism® version 7.04(Graphpad Inc., San Diego, CA, USA) 의 one-way ANOVA를 사용하여 통계분석을 실시하였으며, T-test로 사후 검정하여 p<0.05 값을 유의한 값으로 보고 통계 처리하였다.Statistical analysis was performed using one-way ANOVA with GraphPad Prism ® version 7.04 (Graphpad Inc., San Diego, CA, USA), and post-hoc testing was performed with T-test, with p<0.05 considered significant and statistical processing performed. did.
<7. 실험 결과><7. Experiment results>
도 5는 산화적 스트레스에 의한 ARPE-19 세포 손상에서 (a) 트레할로스의 세포독성 평가, (b) 산화적 스트레스 유도된 세포에서 트레할로스의 세포생존율 평가 및 (c) LDH(젖산탈수소 효소) 측정을 통한 세포 독성 평가 결과 그래프이다. 도 5에서 18번 실험물질이 트레할로스를 의미한다.Figure 5 shows (a) cytotoxicity evaluation of trehalose, (b) cell viability evaluation of trehalose in oxidative stress-induced cells, and (c) LDH (lactate dehydrogenase) measurement in ARPE-19 cell damage caused by oxidative stress. This is a graph of the results of cytotoxicity evaluation. In Figure 5, experimental substance number 18 refers to trehalose.
도 5에 나타난 바와 같이, 트레할로스의 세포내 독성을 확인하기 위해 인간의 망막상피세포주(ARPE-19) 세포에 100 μM 24시간 동안 배양한 후 세포 생존율을 확인하였는데, 100 μM에서 세포 독성이 나타나지 않았다(a). 상기 결과를 바탕으로 세포에 산화적 스트레스를 주기 위해 tBHP(tert-Butyl hydroperoxide)를 사용하였고, 세포 보호 효과가 있는지 확인하기 위해 tBHP (300μM)과 트레할로스 (100 μM)를 24시간 동안 동시 처리하였다. 양성 대조군으로 NAC (1.0 mM)을 사용하였다. 세포 생존율을 확인한 결과, tBHP 처리군은 정상군에 비해 약 50% 정도 세포 생존율을 보였고 트레할로스 (18)에서도 세포 보호 효과를 확인하였다(b, c).As shown in Figure 5, to confirm the intracellular toxicity of trehalose, human retinal epithelial cell line (ARPE-19) cells were cultured with 100 μM for 24 hours and cell viability was checked, and no cytotoxicity was observed at 100 μM. (a). Based on the above results, tBHP (tert-Butyl hydroperoxide) was used to apply oxidative stress to cells, and tBHP (300 μM) and trehalose (100 μM) were simultaneously treated for 24 hours to determine whether there was a cytoprotective effect. NAC (1.0 mM) was used as a positive control. As a result of checking the cell viability, the tBHP-treated group showed a cell survival rate of about 50% compared to the normal group, and a cytoprotective effect was also confirmed with trehalose (18) (b, c).
도 6은 산화적 스트레스에 의한 ARPE-19 세포 손상에서 저농도 트레할로스(50 μM)의 세포보효 효과를 확인한 그래프이다. 도 6은 산화적 스트레스 유도된 세포에서 트레할로스(50 μM, 18번)의 세포생존율을 평가한 것이고(#, P<0.05 vs. CTR; * P<0.05 vs. tBHT), 도 7은 산화적 스트레스가 유도된 세포에서 트레할로스(JK-18)의 세포생존율을 평가한 것이다(#, P<0.05 vs. CTR; * P<0.05 vs. tBHT). 도 6에서 18번 및 JK-18이 트레할로스를 의미한다.Figure 6 is a graph confirming the cytoprotective effect of low concentration trehalose (50 μM) on ARPE-19 cell damage caused by oxidative stress. Figure 6 evaluates the cell viability of trehalose (50 μM, No. 18) in oxidative stress-induced cells (#, P<0.05 vs. CTR; * P<0.05 vs. tBHT), and Figure 7 shows oxidative stress Cell survival rate of trehalose (JK-18) was evaluated in induced cells (#, P<0.05 vs. CTR; * P<0.05 vs. tBHT). In Figure 6, numbers 18 and JK-18 refer to trehalose.
도 6에 나타난 바와 같이, 정상군에 비해 유의적으로 감소한 tBHP 군에서 트레할로스를 1, 10, 50, 100 μM에서 확인한 결과, 50, 100 μM에서 모두 tBHP 군 보다 유의적으로 세포 생존율을 회복시키는 것을 확인하였다.As shown in Figure 6, as a result of confirming trehalose at 1, 10, 50, and 100 μM in the tBHP group, which significantly decreased compared to the normal group, it was found that both 50 and 100 μM significantly restored cell viability compared to the tBHP group. Confirmed.
도 7은 산화적 스트레스에 의해 손상된 미토콘드리아 기능에 대한 트레할로스의 보호 효과를 확인한 그래프이다. 구체적으로, (a) 트레할로스의 산소 소모 변화(OCR, Oxygen Consumption Rate) 평가, (b) 비미토콘드리아 산소 소비량(#, P<0.05 vs. CTR; * P<0.05 vs. tBHT), (c) 기초 산소 소비량(#, P<0.05 vs. CTR; * P<0.05 vs. tBHT), (d) 최대 호흡량(#, P<0.05 vs. CTR; * P<0.05 vs. tBHT), (e) ATP 생산(#, P<0.05 vs. CTR; * P<0.05 vs. tBHT) 및 (f) 유출된 전자량(#, P<0.05 vs. CTR; * P<0.05 vs. tBHT)을 나타낸다. 도 7에서 JK-18이 트레할로스를 의미한다.Figure 7 is a graph confirming the protective effect of trehalose on mitochondrial function damaged by oxidative stress. Specifically, (a) assessment of oxygen consumption rate (OCR) changes in trehalose, (b) non-mitochondrial oxygen consumption (#, P<0.05 vs. CTR; * P<0.05 vs. tBHT), (c) basal Oxygen consumption (#, P<0.05 vs. CTR; * P<0.05 vs. tBHT), (d) maximal respiratory rate (#, P<0.05 vs. CTR; * P<0.05 vs. tBHT), (e) ATP production (#, P<0.05 vs. CTR; * P<0.05 vs. tBHT) and (f) amount of electrons leaked (#, P<0.05 vs. CTR; * P<0.05 vs. tBHT). In Figure 7, JK-18 refers to trehalose.
도 7에 나타난 바와 같이, 미토콘드리아의 기능은 발생하는 호흡량을 분석하여 확인하였다. 산화적 스트레스를 자극한 대조군의 기저호흡 구간은 46.92±1.6 pmole/min으로 나타났으며 (a, c), 트레할로스 처리군에서는 67.63±0.96 pmole/min으로 유의적으로 회복되었다. 산화적 스트레스를 자극한 대조군에서 oligomycin 투여 후 24.34±8.3 pmole/min로 낮아졌고, FCCP 투여 후 최대 호흡 구간은 70.46±6.26 pmole/min (d)까지 낮아졌다. 반면, 트레할로스 및 정상군은 각각 최대 호흡구간 (maximal respiration)에서 정상 대조군 131.24±2.49 pmole/min이고, 트레할로스(JK-18)는 93.19±5.7 pmole/min까지 최대 호흡이 유의적으로 증가하였다. 그리고 기저 호흡 구간 및 ATP 생산구간 (e) 은 산화적 스트레스에 의해 감소되었으나 (26.59±5.11 pmole/min) 트레할로스에 의해 회복이 되는 것을 확인하였다(JK-18, 47.12±1.2 pmole/min). 정상적인 미토콘드리아 대사과정에서 일부의 전자는 유출되어 산화를 완전히 환원시키지 못해서 활성산소를 많이 만들게 된다. 산화적 스트레스에 의해 증가된 proton leak는 트레할로스에 의해 유의적인 감소를 나타냈다 (f). As shown in Figure 7, the function of mitochondria was confirmed by analyzing the amount of respiration that occurred. The basal respiratory interval in the control group stimulated with oxidative stress was found to be 46.92 ± 1.6 pmole/min (a, c), and in the trehalose-treated group, it was significantly recovered to 67.63 ± 0.96 pmole/min. In the control group in which oxidative stress was stimulated, after oligomycin administration, the maximum respiratory interval was lowered to 24.34 ± 8.3 pmole/min, and after FCCP administration, the maximum respiratory interval was lowered to 70.46 ± 6.26 pmole/min (d). On the other hand, the maximum respiration of the trehalose and normal groups was 131.24 ± 2.49 pmole/min for the normal control group, and the maximum respiration for trehalose (JK-18) was significantly increased to 93.19 ± 5.7 pmole/min. Additionally, the basal respiration section and ATP production section (e) were decreased by oxidative stress (26.59±5.11 pmole/min), but recovered by trehalose (JK-18, 47.12±1.2 pmole/min). During normal mitochondrial metabolism, some electrons are leaked out and oxidation cannot be completely reduced, creating a lot of active oxygen. Proton leak increased by oxidative stress was significantly reduced by trehalose (f).
도 8은 산화적 스트레스에 의해 손상된 ARPE-19 세포의 ROS (reactive oxygzen species) 생산과 미토콘드리아 손상에 대한 트레할로스의 효능 평가, 미토콘드리아 염색 및 공초점현미경 이미지이다. 구체적으로, (a) DCFDA 형광량 측정(#, P<0.05 vs. CTR; * P<0.05 vs. tBHT) 및 (b) Mitotracker 평균발현량 측정(#, P<0.05 vs. CTR; * P<0.05 vs. tBHT) 결과를 나타낸다. 도 8에서 JK-18이 트레할로스를 의미한다.Figure 8 shows evaluation of the efficacy of trehalose on ROS (reactive oxygen species) production and mitochondrial damage in ARPE-19 cells damaged by oxidative stress, mitochondrial staining, and confocal microscopy images. Specifically, (a) DCFDA fluorescence measurement (#, P<0.05 vs. CTR; * P<0.05 vs. tBHT) and (b) Mitotracker average expression measurement (#, P<0.05 vs. CTR; * P< 0.05 vs. tBHT) results are shown. In Figure 8, JK-18 refers to trehalose.
도 8에 나타난 바와 같이, 산화적 스트레스에 의한 미토콘드리아 손상에서 트레할로스의 효과를 정성적, 정량적으로 확인하였다. 세포내 산화적 스트레스 (oxidative stress)를 확인하고자 mitotracker와 DCFDA(2,7-dichlorodihydrofluoroscein diacetate)를 사용하여 세포를 염색한 후 공초점 현미경으로 관찰하였다. NAC은 양성 대조군으로 사용되었다. tBHP에 의해 세포내 증가된 ROS의 세기(intensity)는 정상군 (372.33±191.54 IU) 과 비교하였을 때 약 6.6 배 증가하였고 (2453±259.05 IU), 반면 트레할로스에서는 ROS 세기가 유의적으로 감소함을 확인하였다 (JK-18, 1438±274.32 IU) (a). 동시에 미토콘드리아의 세기(intensity)를 mitotracker로 확인한 결과, 정상군 (2884.67±903.55 IU) 대비 tBHP에 의해 감소된 mitotracker의 세기 (1472.33±224.46 IU)는 트레할로스에 의해 미토콘드리아 세기가 유의적으로 회복됨을 정성적, 정량적으로 확인하였다 (JK-18, 2591.33±343.43 IU).As shown in Figure 8, the effect of trehalose on mitochondrial damage caused by oxidative stress was confirmed qualitatively and quantitatively. To confirm intracellular oxidative stress, cells were stained using mitotracker and DCFDA (2,7-dichlorodihydrofluoroscein diacetate) and observed under a confocal microscope. NAC was used as a positive control. The intensity of intracellular ROS increased by tBHP increased approximately 6.6 times (2453±259.05 IU) compared to the normal group (372.33±191.54 IU), whereas the intensity of ROS significantly decreased with tBHP. Confirmed (JK-18, 1438±274.32 IU) (a). At the same time, as a result of checking the intensity of mitochondria with mitotracker, the intensity of mitotracker decreased by tBHP (1472.33 ± 224.46 IU) compared to the normal group (2884.67 ± 903.55 IU), qualitatively showing that the mitochondrial intensity was significantly restored by trehalose. , confirmed quantitatively (JK-18, 2591.33±343.43 IU).
다양한 산화적 스트레스에 의한 세포 손상과 연계된 ROS의 생성에는 세포내 특정 신호계가 관여할 수 있지만, 전자 전달계 교란에 따른 미토콘드리아의 기능 손상이 주요 요인으로 작용한다. 따라서 상기 실험예와 같이 tBHP가 처리된 ARPE-19세포에서 세포 독성 유발 및 ROS 생성 증가가 미토콘드리아 손상과 연관되었는지를 확인하였고, 트레할로스가 이에 미치는 영향을 평가하였다. Although a specific intracellular signaling system may be involved in the generation of ROS linked to cell damage caused by various oxidative stresses, mitochondrial function damage due to disturbance of the electron transport system acts as a major factor. Therefore, as in the above experimental example, it was confirmed whether cytotoxicity and increased ROS production were associated with mitochondrial damage in ARPE-19 cells treated with tBHP, and the effect of trehalose on this was evaluated.
평가 결과, tBHP 의한 ARPE-19 세포 손상은 미토콘드리아의 기능 손상을 동반하였으며, 이는 산화적 스트레스에 의한 ROS 생성과 연관성이 있음을 알 수 있다. 또한, 트레할로스는 산화적 스트레스, 미토콘드리아 손상 및 DNA 손상에 대한 억제 효과 및 보호 효과가 있음이 확인되었기 때문에, 이와 연관된 망막 질환(습식 노인황반변성, 색소성 망막염, 당뇨병성 망막병증, 미숙아 망막병증, 녹내장 등)의 예방, 개선 또는 치료 용도로 사용하기 적합함을 알 수 있다.As a result of the evaluation, it was found that ARPE-19 cell damage caused by tBHP was accompanied by impaired mitochondrial function, which was related to ROS generation due to oxidative stress. In addition, since trehalose has been confirmed to have an inhibitory and protective effect against oxidative stress, mitochondrial damage, and DNA damage, it has been shown to be effective in treating retinal diseases associated with them (wet age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, retinopathy of prematurity, It can be seen that it is suitable for use in the prevention, improvement or treatment of (glaucoma, etc.).
이상에서 본 발명의 실시예를 중심으로 설명하였으나 이는 단지 예시일 뿐 본 발명을 한정하는 것이 아니며, 본 발명이 속하는 분야에서 통상의 지식을 가진 자라면 본 발명의 실시예의 본질적인 특성을 벗어나지 않는 범위에서 이상에 예시되지 않은 여러 가지의 변형과 응용이 가능함을 알 수 있을 것이다. 예를 들어, 본 발명의 실시예에 구체적으로 나타난 각 구성 요소는 변형하여 실시할 수 있다. 그리고 이러한 변형과 응용에 관계된 차이점들은 첨부된 청구 범위에서 규정하는 본 발명의 범위에 포함되는 것으로 해석되어야 할 것이다.Although the description has been made above with a focus on embodiments of the present invention, this is merely an example and does not limit the present invention, and those skilled in the art will be able to understand the present invention without departing from the essential characteristics of the embodiments of the present invention. It will be apparent that various modifications and applications not exemplified above are possible. For example, each component specifically shown in the embodiments of the present invention can be modified and implemented. And these variations and differences in application should be construed as being included in the scope of the present invention as defined in the appended claims.

Claims (4)

  1. 트레할로스(trehalose)를 포함하는 Contains trehalose
    망막 질환 또는 퇴행성 뇌 질환의 예방 또는 개선용 건강식품 조성물.Health food composition for preventing or improving retinal disease or degenerative brain disease.
  2. 청구항 1에 있어서,In claim 1,
    상기 망막 질환은 노인황반변성(AMD), 색소성 망막염(RP), 당뇨병성 망막병증(DR), 미숙아 망막병증(ROP) 및 녹내장(glaucoma)으로 이루어진 군에서 선택되는 하나 이상을 포함하고, The retinal disease includes one or more selected from the group consisting of age-related macular degeneration (AMD), retinitis pigmentosa (RP), diabetic retinopathy (DR), retinopathy of prematurity (ROP), and glaucoma,
    상기 퇴행성 뇌 질환은 알츠하이머병(Alzheimer's disease), 파킨슨병(Parkinson's disease), 헌팅턴병(huntington's disease), 다발성경화증(multiple sclerosis),　경도인지장애(mild cognitive impairment), 대뇌　아밀로이드　맥관병증, 아밀로이드성 뇌졸증(stroke), 전신성　아밀로이드병, 더취(Dutch)형　아밀로이드증, 니만-픽병(니만-피크 병(Niemann­Pick disease), 근위축성 측삭 경화증 (amyotrophic lateral sclerosis), 척수소뇌성 운동실조증(Spinocerebellar Atrophy), 뚜렛 증후군(Tourette's Syndrome), 프리드리히 보행실조(Friedrich's Ataxia), 마차도-조셉 병(Machado-Joseph's disease), 루이 소체 치매(Lewy Body Dementia), 근육긴장이상(Dystonia) 및 진행성 핵상 마비(Progressive Supranuclear Palsy)로 이루어진 군에서 선택되는 하나 이상을 포함하는The degenerative brain diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, mild cognitive impairment, cerebral amyloid angiopathy, amyloid stroke ( stroke), systemic amyloid disease, Dutch amyloidosis, Niemann-Pick disease, amyotrophic lateral sclerosis, Spinocerebellar Atrophy, Tourette syndrome ( A group consisting of Tourette's Syndrome, Friedrich's Ataxia, Machado-Joseph's disease, Lewy Body Dementia, Dystonia, and Progressive Supranuclear Palsy. containing one or more selected from
    망막 질환 또는 퇴행성 뇌 질환의 예방 또는 개선용 건강식품 조성물.Health food composition for preventing or improving retinal disease or degenerative brain disease.
  3. 청구항 2에 있어서,In claim 2,
    상기 망막 질환은 노인황반변성이고, The retinal disease is age-related macular degeneration,
    상기 퇴행성 뇌 질환은 알츠하이머병인The degenerative brain disease is Alzheimer's disease
    망막 질환 또는 퇴행성 뇌 질환의 예방 또는 개선용 건강식품 조성물.Health food composition for preventing or improving retinal disease or degenerative brain disease.
  4. 청구항 1에 있어서,In claim 1,
    과일농축액, 사과산, 알로에베라분말, 사탕수수분말, 비타민, 젖산칼슘, 증점제, 수크랄로스 및 정제수로 이루어진 군에서 선택되는 하나 이상을 더 포함하는It further contains one or more selected from the group consisting of fruit concentrate, malic acid, aloe vera powder, sugarcane powder, vitamins, calcium lactate, thickener, sucralose and purified water.
    망막 질환 또는 퇴행성 뇌 질환의 예방 또는 개선용 건강식품 조성물.Health food composition for preventing or improving retinal disease or degenerative brain disease.
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