WO2019151547A1 - Composition for preventing, improving or treating degenerative neurological disease comprising fraction of a quilaria agallocha roxburgh extract as active ingredient - Google Patents

Composition for preventing, improving or treating degenerative neurological disease comprising fraction of a quilaria agallocha roxburgh extract as active ingredient Download PDF

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Publication number
WO2019151547A1
WO2019151547A1 PCT/KR2018/001339 KR2018001339W WO2019151547A1 WO 2019151547 A1 WO2019151547 A1 WO 2019151547A1 KR 2018001339 W KR2018001339 W KR 2018001339W WO 2019151547 A1 WO2019151547 A1 WO 2019151547A1
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extract
fraction
disease
aroma
pharmaceutical composition
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PCT/KR2018/001339
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French (fr)
Korean (ko)
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손창규
이삼근
이진석
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대전대학교 산학협력단
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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
    • 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
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/83—Thymelaeaceae (Mezereum family), e.g. leatherwood or false ohelo
    • A61K36/835—Aquilaria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/322—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the nervous system or on mental function

Definitions

  • the present invention relates to a composition for the prevention, improvement or treatment of degenerative neurological diseases comprising a fraction of the aroma extract as an active ingredient.
  • Acupuncture is the wood that is hardened at the core part by depositing the resin of the incense tree. This has been prescribed to soothe the mind and calm down the stomach with the pharmacological effect of Suseunghwagang ( ⁇ ⁇ ⁇ ⁇ ) in oriental medicine, to warm the stomach and to maintain regular.
  • Alzheimer's disease is one of the socially important issues with a prevalence of 50 million people worldwide.
  • Degenerative brain disease including senile dementia, is a disease characterized by the degeneration of brain neurons.
  • the hippocampus is responsible for central functions such as brain cognition, persistence, and memory. Promotes disease progression
  • the hippocampus is known to have structural degeneration faster than that of other brains, and atrophy of the hippocampus adjacent to the lateral ventricle is also observed through PET PET or MRI magnetic resonance imaging of the elderly aged 65 or older. It is a very prominent pathological feature.
  • the main cause of the atrophy of the hippocampal region and the degeneration of brain neurons is considered to be high stress, especially in modern society.
  • Chronic stress is deeply involved in pathological conditions such as dysfunction of stressed endocrine system, immune system dysfunction and inflammation, imbalance of neurotransmitter system and oxidation-antioxidant system.
  • Excessive phosphorylation of the Tau protein or deposition of senile plaques, such as beta-amyloid peptides is further exacerbated by stress.
  • the secretion and delivery of related neurotransmitters must be balanced. This imbalance of homeostasis induces brain neuronal cell death and neuroglial dysfunction.
  • An unbalanced form of the representative brain neurotransmitter is an excessive increase in the excitatory neurotransmitter glutamate, where excessively increased glutamate concentrations in the synaptic gap are achieved through neuronal metabotropic receptors or ionotropic receptors. Induces intracellular calcium overload, which results in mitochondrial ROS overproduction and apoptotic signals dependent on caspase3 or calpain. In addition, it is induced by EAAT (excitatory amino acid transporter) of astrocytes and microglia cells adjacent to nerve cells, resulting in overactivity and eventually inflammatory effects such as IL-1 ⁇ , TNF- ⁇ , iNOS, and nitric oxide via NF- ⁇ B nucleic translocation.
  • EAAT excitatory amino acid transporter
  • neuronal excitatory toxicity and microglia hyperactivity due to excitatory neurotransmitter hypersecretion are very similar to those of deactivation of brain function, degenerative neuropathy and central nervous system disease caused by chronic stress.
  • the present inventors endeavored to develop a composition for improving, preventing or treating degenerative neurological diseases, which has fewer side effects on the human body than chemical synthetic medicines and includes an active ingredient derived from natural products that can be easily manufactured and consumed. Accordingly, the present invention was completed by objectively verifying the protective effect of hippocampal neurons, the histological effect of inhibiting histological oxidative damage, and the like of fractions of the aroma extracts through cell experiments.
  • the present invention is to provide a composition for the prevention, improvement or treatment of degenerative neurological diseases comprising a fraction of the aroma extract as an active ingredient.
  • the fragrance fraction according to the present invention has antioxidant activity, inhibits apoptosis of neurons, inhibits nerve stress and protects neurons, inhibits the production of reactive oxygen species (ROS) of neurons, extracellular Improvement of degenerative neuropathy due to its excellent action of reducing phosphorylation level of extracellular signal-regulated kinases (ERK), inhibition of nitric oxide (NO) production of neurons and DNA fragmentation inhibition of neurons. It can be usefully used for prevention or treatment.
  • the present invention for achieving the above object provides a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases comprising a fraction of the aroma extract as an active ingredient.
  • the aroma may be derived from Aquilaria genus plants.
  • the Aquilaria genus plants are Aquilaria cassiana (A. khasiana), Achillia apiculina (A. apiculina), Achilla blillonil (A. blillonil), A. baneonsis, A. brachyantha, A. cunmingiana, A. filaria, Achilla grandiflora. grandiflora, A. hilata, A. microcapa, A. rostrata, A. agallocha, Achilla malsensis ( A. malaccensis, A. sinensis or A. crassna.
  • the aroma extract is the extract of the first extract with aroma of water, C 1 to C 3 alcohol or a mixture thereof as a solvent, the second extraction using chloroform, ether or methylene chloride as a solvent Can be.
  • the fragrance extract may be an extract extracted by using aroma as a solvent of chloroform, ether or methylene chloride.
  • the aroma extract may be a methylene chloride extract of aroma.
  • the fraction may be a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases, characterized in that obtained by a method comprising the following steps:
  • step b) dissolving the filtrate obtained in step a) in an aqueous 100% (v / v) methanol solution to obtain a filtrate filtered;
  • step b) separating the filtrate obtained in step b) using distilled water and high performance liquid chromatography (HPLC) using acetonitrile or methanol solvent.
  • HPLC high performance liquid chromatography
  • step c) 10 ⁇ l of the filtrate obtained in step b) is passed through high-speed liquid liquid chromatography (HPLC) having a nonpolar column as a fixed phase, and measured at a wavelength of 280 nm.
  • HPLC high-speed liquid liquid chromatography
  • the nonpolar column may be a nonpolar column filled with octadecylsilane (C18), dodecylsilane (C12) or octasilane (C8).
  • the column may have a length of 3 to 30 cm, an inner diameter of 1.8 to 5 mm, and a diameter of particles packed in the column may be 1.5 to 5 ⁇ m.
  • the neurodegenerative disease may be induced by lipopolysaccharide (LPS).
  • LPS lipopolysaccharide
  • the degenerative neurological disease may be caused by glutamate.
  • the composition may have one or more of the following properties:
  • ROS reactive oxygen species
  • ERPs extracellular signal-regulated kinases
  • the composition may be one having antioxidant activity.
  • the degenerative neurological disease is Alzheimer's disease (Alzheimer's disease), Parkinson's disease (Parkinson's disease), Lou Gehrig disease (Lou Gehrig disease), Huntington's disease (Huntington's disease), Multiple sclerosis (Multiple sclerosis), Stroke, thrombosis, embolism, head trauma, cerebral circulatory metabolic disorder, cerebral coma or dementia.
  • Alzheimer's disease Alzheimer's disease
  • Parkinson's disease Parkinson's disease
  • Lou Gehrig disease Lou Gehrig disease
  • Huntington's disease Huntington's disease
  • Multiple sclerosis Multiple sclerosis
  • Stroke thrombosis
  • embolism embolism
  • head trauma cerebral circulatory metabolic disorder
  • cerebral coma or dementia dementia
  • the formulation of the composition may be an oral formulation selected from the group consisting of powders, granules, tablets, capsules, suspensions, emulsions and syrups.
  • the present invention also provides a food composition for the prevention or improvement of degenerative neurological diseases comprising a fraction of the aroma extract as an active ingredient.
  • Composition comprising a fraction of the aroma extract according to the present invention as an active ingredient, antioxidant activity, neuronal apoptosis (apoptosis) inhibitory effect, neuronal stress inhibition and neuronal cell protective action, neuronal reactive oxygen species (reactive oxygen species) : Inhibition of ROS production, reduction of phosphorylation level of extracellular signal-regulated kinases (ERK), inhibition of production of nitric oxide (NO) in neurons and inhibition of DNA fragmentation in neurons Indicates.
  • ERK extracellular signal-regulated kinases
  • NO nitric oxide
  • Alzheimer's disease Parkinson's disease, Lou Gehrig disease, Huntington's disease, Multiple sclerosis, stroke, thrombosis, and embolism It can be usefully used for the prevention or treatment of degenerative neurological diseases such as embolism, head trauma, cerebral circulatory metabolic disorder, brain coma or dementia.
  • Figure 1 is a process of searching for fractions showing the pharmaceutical efficacy of the aroma, it is a schematic of the process of extraction and separation of fractions according to hydrophobic or hydrophilic solvent.
  • Figure 2 shows the results of HPLC fingerprint analysis of the aroma methylene chloride extract.
  • FIG. 3 is a graph showing the protective effect against excitatory neurotransmitter (HT-22 cell) cell excitability killing induced by excitatory neurotransmitter (glutamate) of the solvent-specific aroma extract.
  • Figure 4 is a graph showing the protective effect against the excitatory neurotransmitter (HT-22 cell) cell excitability killing induced by excitatory neurotransmitter (glutamate) of any five fractions isolated from the aroma methylene chloride extract.
  • Figure 5a is a graph showing that the acupuncture E fraction according to an embodiment of the present invention inhibits apoptosis against cytotoxicity of mouse hippocampal neuronal cell line (HT-22 cell) induced by excitatory neurotransmitter (glutamate)
  • FIG. 5B shows that the Aroma E fraction according to one embodiment of the present invention inhibits extracellular leakage of Lactate dehydrogenase (LDH) in mouse hippocampal neuronal cell line (HT-22 cells) induced with excitatory neurotransmitter (glutamate). It is a graph.
  • LDH Lactate dehydrogenase
  • Figure 6a is a photograph showing the inhibitory effect of ROS generation of the fragrance E fraction in experiments in which ROS level was evaluated by DCFH-DA cytochemistry fluorescence technique in glutamate-induced HT-22 cells
  • Figure 6b is in the experiment
  • It is a graph showing the effect of inhibiting the ROS production of the fraction.
  • FIG. 7A shows that in the experiment evaluating the death of the mouse hippocampal neuronal cell line (HT-22 cell) induced by excitatory neurotransmitter (glutamate) of the fragrance E fraction by flow cytometry (FACs), the fragrance E fraction of the present invention was Annexin V / It is a graph showing that cell death is inhibited by inhibiting the PI signal, Figure 7b is a graph showing the quantified number of killed cells in the experiment.
  • FIG. 8A is an experiment evaluating DNA fragmentation level in glutamate-induced HT-22 cells by Hoechst33528 fluorescence microscopy.
  • FIG. 8A is a photograph showing that acupuncture E fraction inhibits neuronal cell death.
  • FIG. It is a graph quantifying the degree of fragmentation of cellular DNA.
  • FIG. 9 is a graph showing that acupuncture E fraction inhibits apoptosis by preventing phosphorylation of ERK in an experiment evaluating apoptosis in glutamate-induced HT-22 cells using Western blot technique.
  • FIG. 10 is a graph showing that aroma E fraction inhibits apoptosis by antioxidant activity in mouse hippocampal neuronal cell line (HT-22 cell) induced by Hydrogen peroxide (H 2 O 2 ).
  • FIG. 11 is a graph showing that the A-fraction E fraction inhibits the extracellular leakage of Lactate dehydrogenase (LDH) in mouse hippocampal neuronal cell line (HT-22 cell) induced with Hydrogen peroxide (H 2 O 2 ).
  • LDH Lactate dehydrogenase
  • Figure 12a is a graph showing that the aroma E fraction inhibits the increase of Lipopolysaccharide (LPS) -induced Nitric oxide (NOS) in mouse microglia neuronal cell lines (BV2 cells),
  • Figure 12b is a protein for the results shown in the experiment A graph showing quantitative values.
  • LPS Lipopolysaccharide
  • NOS Nitric oxide
  • the present inventors have investigated the brain hippocampal neurons treated with glutamate or lipopolysaccharide (LPS) in Ethylene acetate, methylene chloride, methanol, Experiments were carried out with aroma extract extracted using various organic solvents including ethanol. As a result, methylene chloride extract (hereinafter MC) was the most effective. Fingerprint analysis of the MC extract using HPLC confirmed the HPLC peaks of the five fractions. The five fractions (A, B, C, D, E) were separated using a Preperative LC instrument, and the inhibitory effect on neuronal excitatory toxicity was evaluated. As a result, a significant effect was observed in the E fraction. Therefore, it can be inferred that the E fraction plays a key role in pharmacological activity that improves the central nervous system disease of acupuncture.
  • LPS lipopolysaccharide
  • the present invention provides a composition for the prevention, improvement or treatment of degenerative neurological diseases comprising a fraction of the aroma extract as an active ingredient.
  • the aroma can be used that is derived from Aquilaria genus plants.
  • the plant of the genus Aquilaria is Achilla cassiana (A. khasiana), Achillia apiculina (A. apiculina), Achillia blillonil (A. blillonil), Achillia baneeonsis ( A. baneonsis, A. brachyantha, A. cunmingiana, A. filaria, A. filaria, A. grandiflora, Aquilia hill A. hilata, A. microcapa, A. rostrata, A. agallocha, A. malaccensis, Achilla Stems or roots, such as A. sinensis or A. crassna, preferably stems or roots on which resin is deposited can be used.
  • the aroma extract may be an extract of the aroma extract by using chloroform, ether or methylene chloride as a solvent, methylene chloride is preferred as the solvent.
  • the aroma extract may be prepared by extracting the aroma in accordance with a conventional method, such as cold water extraction, hot water extraction, organic solvent extraction or distillation, and then using the obtained extract as it is, or concentrated under reduced pressure or freeze-dried.
  • the fragrance extract means not only the substance obtained by extracting the aroma with the solvent, but also the substance obtained by the extract is fermented using a strain, or fractionally extracted with an organic solvent such as ether. It may mean.
  • the fragrance extract according to the present invention may be prepared as follows. First, about 1 to 80 times by weight, preferably 30 to 50 parts of chloroform, ether or methylene chloride, by weight of the fragrance or fragrance powder, which are selected and trimmed to an appropriate size, are used, for example, 5 to 100 Extraction is carried out by stirring extraction, boiling water extraction, cold extraction, reflux extraction or ultrasonic extraction for about 1 to 100 hours, preferably 10 to 40 hours at room temperature, preferably at room temperature. The obtained extract can be used as it is, or it can be filtered, concentrated or dried to obtain a fragrance extract. If necessary, the extract may be further fractionated with water or an organic solvent such as lower alcohol, ether or the like.
  • the aroma extract may be an extract obtained by extracting the first aroma using aroma, water, C 1 to C 3 alcohol or a mixture thereof as a solvent, a second extraction using chloroform, ether or methylene chloride as a solvent, the C 1 To C 3 alcohol is preferably ethanol, methanol or propanol. Most preferred as solvent of the first extraction of the invention is 20 to 50% (w / v) ethanol.
  • the fragrance extract may be prepared as follows. First, by using a 1 to 4 times the amount of water or a lower alcohol having 1 to 4 carbon atoms, such as ethanol, methanol, or a mixed solvent of the fragrance or aroma powder that has been selected and trimmed to an appropriate size, For example, primary extraction is performed at 5 to 100 ° C. for about 1 to 100 hours by stirring extraction, hot water extraction, cold extraction, reflux extraction, or ultrasonic extraction. The obtained primary extract may be used as it is, or it may be used by filtration, concentration or drying, and lyophilized to use in powder form.
  • a 1 to 4 times the amount of water or a lower alcohol having 1 to 4 carbon atoms such as ethanol, methanol, or a mixed solvent of the fragrance or aroma powder that has been selected and trimmed to an appropriate size.
  • primary extraction is performed at 5 to 100 ° C. for about 1 to 100 hours by stirring extraction, hot water extraction, cold extraction, reflux extraction, or ultrasonic extraction.
  • the obtained primary extract may be used as it is
  • the primary extract When the primary extract is in powder form, the primary extract may be used in an amount of about 5 to 400 times by weight, preferably 150 to 250 times, by weight of chloroform, ether, or methylene chloride, for example, 5 to 400 times. Extraction is carried out at 100 DEG C, preferably at room temperature for about 1 to 100 hours, preferably 10 to 40 hours, by stirring extraction, hot water extraction, cold extraction, reflux extraction or ultrasonic extraction. The obtained extract can be used as it is, or it can be filtered, concentrated or dried to obtain a fragrance extract.
  • Fragrance extract used in the present invention may be prepared in a powder state by an additional process such as vacuum distillation and freeze drying or spray drying.
  • the fraction of the fragrance extract can be obtained by a method comprising the following steps:
  • step b) dissolving the filtrate obtained in step a) in an aqueous 100% (v / v) methanol solution to obtain a filtrate filtered;
  • step b) separating the filtrate obtained in step b) using distilled water and high performance liquid chromatography (HPLC) using acetonitrile or methanol solvent.
  • HPLC high performance liquid chromatography
  • step c) 10 ⁇ l of the filtrate obtained in step b) is passed through a high performance liquid chromatography (HPLC) having a nonpolar column as a stationary phase and measured at a wavelength of 280 nm.
  • HPLC high performance liquid chromatography
  • the coefficient of variation is 0.024%.
  • a fraction having a peak can be separated.
  • the nonpolar column may be a nonpolar column filled with octadecylsilane (C18), dodecylsilane (C12) or octasilane (C8), preferably octadecylsilane (C18).
  • the column has a length of 3 to 30cm, an inner diameter of 1.8 to 5mm, the diameter of the particles packed in the column may be 1.5 to 5 ⁇ m.
  • step c) distilled water and acetonitrile are used as the mobile phase of the column, but 0 to 0.01 minutes at 30% (v / v) acetonitrile, 0.01 to 10 minutes at 0.01 Gradient of -40% (v / v) acetonitrile, gradient of 40-50% (v / v) acetonitrile at 10-15 minutes, gradient of 50-60% (v / v) acetonitrile at 15-30 minutes , 100% (v / v) acetonitrile at 30-40 minutes, 100% (v / v) acetonitrile at 40-50 minutes, the flow rate may be 1 mL / min.
  • the term 'comprising as an active ingredient' means including an amount sufficient to achieve the efficacy or activity of the fragrance fraction.
  • composition of the present invention comprises a fraction separated from the extract of aroma of natural plant material as an active ingredient, there is no side effect to the human body even when excessively administered.
  • GSH glutathione peroxidase
  • GSH is a protein that combines three amino acids, glycine, glutamate and cysteine, and removes free radicals and various toxic substances from liver and cells to prevent cell damage. Plays a role in enhancing immunity [Shin AY, et al., J. Neurosci.
  • Glutamate among the amino acids that make up GSH acts as a central excitatory neurotransmitter, which accounts for 15-20% of the central nervous system synapse, but excessive accumulation in the brain decreases the function of the antioxidant system in the body Imbalance of the system leads to excessive excitement of the nerve, resulting in excitotoxicity [Kim MH, et al., J. Neurosci. 2008, 28, 10852-10863; Penugonda S., et al., Toxicol. Appl. Pharm.
  • the fragrance fraction according to the present invention can be usefully used for the prevention, improvement or treatment of neurodegenerative diseases caused by glutamate.
  • fragrance fraction according to the present invention serves to protect neurons.
  • the fragrance fraction serves to protect neurons by inhibiting the death of neurons.
  • the protective action of the neurons is usually the protective action of brain neurons.
  • Brain neuron protection refers to the action of reducing or ameliorating damage to, or protecting or repairing, nerve cells or tissues caused by metabolic causes, toxic causes, neurotoxic causes and physical and chemical causes. do.
  • the fragrance fraction of the present invention acts to protect neurons from the excitatory toxicity of brain neurons induced by glutamate.
  • the term 'brain nerve cell' refers to neurons, glial cells (microglia, astrocytes), nerve support cells, glia and Schumann cells, etc. that make up the brain, hippocampus, brainstem and spinal cord.
  • Brain nerve cell protection by the composition of the present invention can be achieved through a variety of mechanisms, for example, can be achieved by inhibiting cell death of nerve cells.
  • the aroma fraction of the present invention as shown in the following experimental examples, antioxidant activity, neuronal apoptosis (apoptosis) inhibitory action, neuronal stress inhibition and neuronal cell protective action, neuronal reactive oxygen species (reactive oxygen species) : Inhibits ROS production, decreases phosphorylation levels of extracellular signal-regulated kinases (ERKs), inhibits the production of nitric oxide (NO) in neurons, and inhibits DNA fragmentation in neurons The above characteristics are shown.
  • ERKs extracellular signal-regulated kinases
  • NO nitric oxide
  • the fragrance fraction of the present invention has the effect of inhibiting the activated microglia or the production of pro-inflammatory substances secreted by the activated microglia.
  • Microglia the glial cells, account for 10-12% of the central nervous system glial cells and were first reported in 1932 by the morphological studies and tissue staining methods of Rio-Hortega. Microglia, which act on the degeneration of neurons and foreign substances in tissues, play an important role in transporting, destroying, removing, and cleaning pathological metabolites. It is considered a macrophage.
  • glial cells astrocytes, oligodendrocytes
  • astrocytes oligodendrocytes
  • macrophage-like functions such as phagocytosis.
  • monocytes which are thought to be precursors of these cells, during development.
  • monocytes which are thought to be precursors of these cells, during development.
  • the primary defense against microbial infections and trauma in the central nervous system is that At this time, they move to the site of infection or through local proliferation to devour infected microorganisms and digest the remnants of damaged neurons.
  • Microglial cells perform their intrinsic function of self-defense in the central nervous system, but are produced for defensive purposes such as tumor necrosis factor (TNF), interleukin (IL) -1 ⁇ , reactive oxygen (ROS) or nitrogen compounds. Excessive secretion of inflammatory mediators or prolonged activation of the cells themselves can lead to serious side effects of nerve tissue damage.
  • TNF tumor necrosis factor
  • IL interleukin
  • ROS reactive oxygen
  • microglia as mediators of inflammatory and degenerative diseases.
  • Alzheimer's and Parkinson's as well as traumatic and ischemic neuronal damage
  • Microglia as mediators of inflammatory and degenerative diseases.
  • the neuroinflammatory response represented by the activation of microglia plays an important pathological role in various nerve tissue damages, and ultimately, it is possible to seek ways to inhibit nerve tissue damage by inhibiting the inflammatory activation of the microglia. Therefore, it will be possible to develop clinically applicable preventive and therapeutic agents for neurodegenerative diseases.
  • composition of the present invention can be usefully used for the prevention, improvement or treatment of degenerative neurological diseases.
  • the degenerative neurological disease is Alzheimer's disease, Alzheimer's disease, Parkinson's disease, Lou Gehrig disease, Huntington's disease, Multiple sclerosis, Multiple sclerosis, Stroke, thrombosis, embolism, head trauma, cerebral circulatory metabolic disorder, brain coma or dementia, but are not limited thereto.
  • the neurodegenerative disease may be induced by lipopolysaccharide (LPS) or may be induced by glutamate.
  • LPS lipopolysaccharide
  • the present invention provides a pharmaceutical composition for preventing or treating neurodegenerative diseases.
  • the pharmaceutical composition for preventing or treating degenerative neurological diseases of the present invention may include the fraction in an amount of 0.02 to 80% by weight, preferably 0.02 to 50% by weight based on the total weight of the pharmaceutical composition.
  • compositions of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.
  • compositions according to the invention may be used in the form of their pharmaceutically acceptable salts, or may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.
  • compositions according to the invention can be used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., respectively, according to conventional methods.
  • sterile injectable solutions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., respectively, according to conventional methods.
  • Carriers, excipients and diluents that may be included in the pharmaceutical compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzo mixture, and the like.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in the pharmaceutical composition, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. It can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups. In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, suspensions, emulsions, lyophilized preparations and preservatives Etc. may be included.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
  • non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used.
  • base of the suppository utopsol, macrogol, tween 61, cacao butter, lauzin paper, glycerogelatin and the like can be used.
  • Preferred dosages of the compositions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art.
  • the fraction of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. Most preferably from 50 to 100 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.
  • composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections.
  • the present invention also provides a nutraceutical composition for the prevention or improvement of degenerative neurological diseases comprising aroma fraction as an active ingredient.
  • the health functional food composition is not particularly limited as long as it can be ingested to prevent or improve degenerative neurological diseases.
  • the fraction When the composition of the present invention is used as a food additive, the fraction may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method.
  • the mixed amount of the active ingredient can be suitably determined according to the purpose of use (prevention, health or therapeutic treatment).
  • the fractions of the invention are added in an amount of up to 15 parts by weight, preferably up to 10 parts by weight relative to the raw materials.
  • the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. .
  • the kind of food there is no particular limitation on the kind of food.
  • examples of the food to which the fraction may be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, and the like means all of the health food in the conventional sense.
  • composition of the present invention When the composition of the present invention is used as a health beverage, various flavors or natural carbohydrates and the like may be contained as additional ingredients, as in general beverages.
  • the above-mentioned natural carbohydrates are sugars such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclotensin, xylitol, sorbitol and erythritol.
  • sweetening agent natural sweetening agents such as tautin and stevia extract, and synthetic sweetening agents such as saccharin and aspartame can be used.
  • the ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 g of the composition of the present invention.
  • the composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, And carbonating agents used in carbonated drinks.
  • the composition of the present invention may contain a natural fruit juice, a pulp for producing a vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical, but the composition of the present invention is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight.
  • Aquilariae Lignum used powdered aromas obtained from an herbal pharmaceutical company (Dae Han Bio Pharm Inc., Gyeonggi-do, Korea), which was approved by the Ministry of Food and Drug Safety.
  • the column flow rate was 1 mL / min, and the mobile phase conditions were carried out using (A) distilled water and (B) acetonitrile as follows: 0 to 0.01 min, 30% B (isosolvent eluting); 0.01 to 10 min, 0.01 to 40% B (linear gradient elution); 10 to 15 min, 40 to 50% B (linear gradient elution); 15 to 30 min, 50 to 60% B (linear gradient elution); 30 to 40 min, 100% B (linear gradient elution); and 40 to 50 min (isosolvent elution). This was detected by a UV detector.
  • the mouse hippocampal cell line (HT-22 cell) was used to evaluate the medium was used DMEM containing 10% (v / v) fetal bovine serum (FBS), 1% (v / v) penicillin, the culture was 37 °C , 5% CO 2, 95% humidified air incubator was used. Cytotoxicity was measured by modifying the method of Rochem et al. WST-8 and LDH to run the test in the safe concentration range of the sample. 4 x 10 3 cells / well were dispensed into 96 wells plate and incubated for 12 hours.
  • MC methylene chloride
  • EA ethyl acetate
  • Rest rest extracts of aroma
  • Figure 3 is a graph showing the protective effect of the excitatory neurotransmitter (glutamate) induced mouse hippocampal neuronal cell line (HT-22 cell) cell excitability killing of the solvent-specific extracts.
  • MC methylene chloride
  • the mouse hippocampal cell line (HT-22 cell) was used to evaluate the medium was used DMEM containing 10% (v / v) fetal bovine serum (FBS), 1% (v / v) penicillin, the culture was 37 °C , 5% CO 2, 95% humidified air incubator was used. Cytotoxicity was measured by modifying the method of Rochem et al. WST-8 and LDH to run the test in the safe concentration range of the sample. 4 x 10 3 cells / well were dispensed into 96 wells plate and incubated for 12 hours. The A-E fractions were then dispensed at a final concentration of 10 ⁇ g / ml and cultured for 2 hours.
  • the final concentration of glutamate was treated with 20 mM to induce apoptosis after 20 hours.
  • 20 ⁇ L of WST-8 solution was added and incubated for 1 hour and 30 minutes at 37 ° C, and then measured by absorbance at 450 nm of UV spectrometer.
  • the reaction was carried out at 37 ° C. for 15 minutes using a culture supernatant and measured at 440 nm absorbance. The results are shown in FIG. 4.
  • Figure 4 is a graph showing the protective effect against the excitatory neurotransmitter (HT-22 cell) cell excitability killing induced by excitatory neurotransmitter (glutamate) of any five fractions isolated from the aroma methylene chloride extract.
  • the A-fraction E fraction was most effective in inhibiting apoptosis on neurocytotoxicity.
  • the mouse hippocampal cell line (HT-22 cell) was used to evaluate the medium was used DMEM containing 10% (v / v) fetal bovine serum (FBS), 1% (v / v) penicillin, the culture was 37 °C , 5% CO 2, 95% humidified air incubator was used. Cytotoxicity was measured by modifying the method of Rochem et al. WST-8 and LDH to run the test in the safe concentration range of the sample. 4 x 10 3 cells / well were dispensed into 96 wells plate and incubated for 12 hours. Aroma E fractions were then dispensed at a final concentration of 1.25, 2.5, 5 ⁇ g / ml and cultured for 2 hours.
  • Figure 5a is a graph showing that the acupuncture E fraction according to an embodiment of the present invention inhibits apoptosis against cytotoxicity of mouse hippocampal neuronal cell line (HT-22 cell) induced by excitatory neurotransmitter (glutamate)
  • FIG. 5B shows that the Aroma E fraction according to one embodiment of the present invention inhibits extracellular leakage of Lactate dehydrogenase (LDH) in mouse hippocampal neuronal cell line (HT-22 cells) induced with excitatory neurotransmitter (glutamate). It is a graph.
  • LDH Lactate dehydrogenase
  • HT-22 cells were injected into 6 well plates at 4 ⁇ 10 3 cells / well and incubated for 12 hours.
  • Cellular excitatory toxicity was induced by treatment with glutamate 20 mM after 2 hours treatment with E fraction at 5 ⁇ g / mL.
  • the DCFH-DA reagent was treated with 10 ⁇ M and reacted at 37 ° C. for 30 minutes, and then ROS levels were observed under a fluorescence microscope. The results are shown in FIG.
  • Figure 6a is a photograph showing the inhibitory effect of ROS generation of the fragrance E fraction in experiments in which ROS level was evaluated by DCFH-DA cytochemistry fluorescence technique in glutamate-induced HT-22 cells
  • Figure 6b is in the experiment
  • It is a graph showing the effect of inhibiting the ROS production of the fraction.
  • HT-22 cells were aliquoted at 100 x Dish at 5 X 10 4 cells / mL and incubated for 12 hours. Cellular excitatory toxicity was induced by treatment with glutamate 20 mM after 2 hours treatment with E fraction at 5 ⁇ g / mL. After incubation for 10 hours, the cells are collected in a centrifuge (1300rpm, 3min), dissolved by adding 500 ⁇ L of 1X Annexin V binding buffer and stored on ice. Treatment with anneal V 1 ⁇ L and PI 0.5 ⁇ L confirmed the death of HT-22 cells using flow cytometry (FACs). The results are shown in FIG.
  • FIG. 7A shows that in the experiment evaluating the death of the mouse hippocampal neuronal cell line (HT-22 cell) induced by excitatory neurotransmitter (glutamate) of the fragrance E fraction by flow cytometry (FACs), the fragrance E fraction of the present invention was Annexin V / It is a graph showing that cell death is inhibited by inhibiting the PI signal, Figure 7b is a graph showing the quantified number of killed cells in the experiment.
  • HT-22 cells were injected into 6 well plates at 4 ⁇ 10 3 cells / well and incubated for 12 hours.
  • Cellular excitatory toxicity was induced by treatment with glutamate 20 mM after 2 hours treatment with E fraction at 5 ⁇ g / mL.
  • E fraction at 5 ⁇ g / mL.
  • the cells were fixed with 4% paraformaldehyde and treated with Hoechst33528 reagent at a concentration of 10 ⁇ M and PI 10 ⁇ g / mL to confirm HT-22 cell DNA fragmentation. The results are shown in FIG.
  • FIG. 8A is an experiment evaluating DNA fragmentation level in glutamate-induced HT-22 cells by Hoechst33528 fluorescence microscopy.
  • FIG. 8A is a photograph showing that acupuncture E fraction inhibits neuronal cell death.
  • FIG. It is a graph quantifying the degree of fragmentation of cellular DNA.
  • HT-22 cells were aliquoted at 100 x Dish at 5 X 10 4 cells / mL and incubated for 12 hours.
  • Aromatosis E fractions were treated for 2 hours at a concentration of 5 ⁇ g / mL and cell excitability was induced by glutamate 20 mM treatment.
  • protein expression and phosphorylation of ERK were evaluated using Western blot technique, and the results are shown in FIG. 9.
  • FIG. 9 is a graph showing that acupuncture E fraction inhibits apoptosis by preventing phosphorylation of ERK in an experiment evaluating apoptosis in glutamate-induced HT-22 cells using Western blot technique.
  • the mouse hippocampal cell line (HT-22 cell) was used to evaluate the medium was used DMEM containing 10% (v / v) fetal bovine serum (FBS), 1% (v / v) penicillin, the culture was 37 °C , 5% CO 2, 95% humidified air incubator was used. Cytotoxicity was measured by modifying the method of Rochem et al. WST-8 and LDH to run the test in the safe concentration range of the sample. 4 x 10 3 cells / well were dispensed into 96 wells plate and incubated for 12 hours. Aroma E fractions were then dispensed at a final concentration of 5 ⁇ g / ml and cultured for 2 hours.
  • the final concentration of glutamate 20mM was then treated to induce cell death after 20 hours.
  • 20 ⁇ L of WST-8 solution was added and incubated for 1 hour and 30 minutes at 37 ° C, and then measured by absorbance at 450 nm of UV spectrometer.
  • the culture supernatant was reacted with a reagent at 37 ° C. for 15 minutes and measured at 440 nm absorbance. The results are shown in FIGS. 10 and 11.
  • FIG. 10 is a graph showing that aroma E fraction inhibits apoptosis by antioxidant activity in mouse hippocampal neuronal cell line (HT-22 cell) induced by Hydrogen peroxide (H 2 O 2 ).
  • FIG. 11 is a graph showing that the A-fraction E fraction inhibits the extracellular leakage of Lactate dehydrogenase (LDH) in mouse hippocampal neuronal cell line (HT-22 cell) induced with Hydrogen peroxide (H 2 O 2 ).
  • LDH Lactate dehydrogenase
  • the mouse microglia (BV2 cell) was used to evaluate the medium.
  • DMEM containing 10% (v / v) fetal bovine serum (FBS), 1% (v / v) penicillin, and DMEM were used.
  • NO assay was measured using griess reagent, and corrected by protein quantitative value measured by Bradford reagent to quantify the concentration of the protein per mg. 4 x 10 4 cells / well were dispensed into 96 wells plates and incubated for 12 hours. The E fractions were then dispensed at a final concentration of 5 ⁇ g / ml and cultured for 2 hours.
  • the final concentration of LPS was treated with 1 ⁇ g / mL to induce hyperactivity of the microglia after 24 hours.
  • the culture supernatant was reacted with griess reagent for 30 minutes at 37 ° C. and measured with 540 nm absorbance. The results are shown in FIG.
  • Figure 12a is a graph showing that the aroma E fraction inhibits the increase of Lipopolysaccharide (LPS) -induced Nitric oxide (NOS) in mouse microglia neuronal cell lines (BV2 cells),
  • Figure 12b is a protein for the results shown in the experiment A graph showing quantitative values.
  • LPS Lipopolysaccharide
  • NOS Nitric oxide
  • the aroma fraction of the present invention the anti-oxidant action, the inhibitory action of apoptosis (apoptosis) of nerve cells, neuronal stress inhibition and neuronal cell protective action, neuronal reactive oxygen species (ROS) Inhibits production, decreases the phosphorylation level of extracellular signal-regulated kinases (ERK), inhibits the production of nitric oxide (NO) in neurons, and inhibits DNA fragmentation in neurons.
  • ROS neuronal reactive oxygen species
  • the fragrance fraction according to the present invention inhibits excitatory toxicity of hippocampal neurons, inhibits apoptosis due to brain oxidative damage, and lowers the inflammatory response of microglia, and thus has excellent neuronal protective effects. Excellent effects are expected for prevention or treatment.
  • composition comprising the fragrance fraction of the present invention as an active ingredient may be usefully used for the prevention, improvement or treatment of degenerative neurological diseases, especially central nervous system diseases caused by neuronal damage and death.

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Abstract

The present invention relates to a composition for preventing, improving or treating degenerative neurological diseases comprising a fraction of Aquilaria agallocha Roxburgh extract as an active ingredient. The composition comprising a fraction of Aquilaria agallocha Roxburgh extract as an active ingredient according to the present invention exhibits antioxidant activity, inhibition of apoptosis of neuronal cells, inhibition of cranial nerve stress, neuronal cell protection, inhibition of the production of reactive oxygen species (ROS) in neuronal cells, decrease in phosphorylation level of extracellular signal-regulated kinases (ERK), inhibition of the production of nitric oxide (NO) in neuronal cells, and inhibition of DNA segmentation of neuronal cells. Accordingly, the composition has an effect on degenerative neurological diseases, such as Alzheimer's disease, Parkinson's disease, Lou Gehrig disease, Huntington's disease, multiple sclerosis, stroke, thrombosis, embolism, head trauma, metabolic disorder of cerebral circulation, brain functional coma or dementia, etc.

Description

침향 추출물의 분획물을 유효성분으로 포함하는 퇴행성 신경질환의 예방, 개선 또는 치료용 조성물A composition for the prevention, improvement or treatment of degenerative neurological diseases comprising fractions of the aroma extract as an active ingredient
본 발명은 침향 추출물의 분획물을 유효성분으로 포함하는 퇴행성 신경질환의 예방, 개선 또는 치료용 조성물에 관한 것이다. The present invention relates to a composition for the prevention, improvement or treatment of degenerative neurological diseases comprising a fraction of the aroma extract as an active ingredient.
침향은 침향나무의 수지가 침착된 것으로 심재부위에서 굳어 만들어진 목재이다. 이는 과거 한의학에서 수승화강(水昇火降)하는 약리적 효능으로 정신을 맑게 해주고 화를 가라앉힘으로써 진정 작용을 하며 위를 따뜻하게 하고 정기를 보하기 위해 처방되어져 왔다. Acupuncture is the wood that is hardened at the core part by depositing the resin of the incense tree. This has been prescribed to soothe the mind and calm down the stomach with the pharmacological effect of Suseunghwagang (水 昇 火 降) in oriental medicine, to warm the stomach and to maintain regular.
한편, 인구 고령화와 고도의 산업발전 및 식습관의 서구화에 따라 뇌졸중, 심/뇌혈관성 질환 그리고 알츠하이머성 치매와 같은 퇴행성 뇌 질환의 발병률이 매년 급격히 증가하고 있다. 특히 알츠하이머 병 (Alzheimer's disease)은 전 세계 5천만 명의 유병률을 나타낼 정도로 사회의학적으로 중요한 이슈 중 하나이다. On the other hand, with the aging of the population, high industrial development, and westernization of eating habits, the incidence of degenerative brain diseases such as stroke, cardio / cerebrovascular disease, and Alzheimer's dementia increases rapidly every year. Alzheimer's disease, in particular, is one of the socially important issues with a prevalence of 50 million people worldwide.
노인성 치매를 비롯한 퇴행성 뇌 질환은 뇌 신경세포의 퇴화가 주된 특징으로 나타나는 질병인데, 특히 해마는 뇌 인지력, 지남력, 기억력과 같은 중추기능을 담당하는 영역으로 이의 신경세포 사멸은 상기 기능의 저하 및 관련 질병의 진행을 촉진시킨다. 또한 이 해마영역은 타 뇌의 영역에 비해 구조적 퇴화가 빠르게 진행되는 것으로 잘 알려져 있으며, 65세 이상 노인의 뇌 PET 단층촬영이나 MRI 자기공명장치 촬영을 통해서도 측뇌실 (lateral ventricle)에 인접한 해마의 위축은 매우 현저한 병리적 특징이다.Degenerative brain disease, including senile dementia, is a disease characterized by the degeneration of brain neurons. In particular, the hippocampus is responsible for central functions such as brain cognition, persistence, and memory. Promotes disease progression The hippocampus is known to have structural degeneration faster than that of other brains, and atrophy of the hippocampus adjacent to the lateral ventricle is also observed through PET PET or MRI magnetic resonance imaging of the elderly aged 65 or older. It is a very prominent pathological feature.
이 해마영역의 위축이나 뇌 신경세포의 퇴화를 일으키는 주된 원인은 특히 현대사회에 이르러 발생빈도가 높은 스트레스로 여겨진다. 만성 스트레스는 스트레스성 내분비계의 기능이상, 면역계의 기능저하 및 염증반응, 신경전달물질계 및 산화-항산화 체계의 불균형 등의 병리적 상태에 깊게 관여한다. 또한 Tau protein의 과도한 인산화나 beta-amyloid peptide와 같은 노인반 (senile plaque)의 침착이 스트레스에 의해 더욱 악화된다. 특히 뇌의 발달이나 중추적 기능이 작용하기 위해서는 관련 신경전달물질의 분비 및 전달이 균형을 이루어야 하는데, 이 항상성의 불균형이 뇌 신경세포의 사멸이나 신경아교세포의 기능장애를 유도한다. The main cause of the atrophy of the hippocampal region and the degeneration of brain neurons is considered to be high stress, especially in modern society. Chronic stress is deeply involved in pathological conditions such as dysfunction of stressed endocrine system, immune system dysfunction and inflammation, imbalance of neurotransmitter system and oxidation-antioxidant system. Excessive phosphorylation of the Tau protein or deposition of senile plaques, such as beta-amyloid peptides, is further exacerbated by stress. In particular, in order for brain development or central function to function, the secretion and delivery of related neurotransmitters must be balanced. This imbalance of homeostasis induces brain neuronal cell death and neuroglial dysfunction.
대표적 뇌 신경전달물질의 불균형 형태는 흥분성 신경전달물질인 glutamate의 과도한 증가인데, 시냅스 간극에서 과도하게 증가된 glutamate의 농도는 신경세포의 대사성 수용기 (metabotropic receptor) 또는 이온성 수용기 (ionotropic receptor)를 통해 세포 내 calcium overload를 유도하고, 이는 mitochondrial ROS 과생성 및 caspase3 또는 calpain 의존적으로 apoptotic signal을 나타낸다. 더불어 신경세포에 인접한 astrocyte와 microglia cell의 EAAT (excitatory amino acid transporter)에 의해 유입되어 과활성을 일으키고, 결국 NF-κB nucleic translocation을 경로로 IL-1β, TNF-α, iNOS, nitric oxide와 같은 염증성 인자를 분비하여 신경세포의 사멸에 기여한다. 이와 같이 흥분성 신경전달물질 과분비에 따른 신경세포 흥분독성과 미세아교세포의 과활성은 만성 스트레스로 기인하는 뇌 기능의 활성저하, 퇴행성 신경질환 및 중추신경계 질환의 발병 형태와 매우 유사하다. An unbalanced form of the representative brain neurotransmitter is an excessive increase in the excitatory neurotransmitter glutamate, where excessively increased glutamate concentrations in the synaptic gap are achieved through neuronal metabotropic receptors or ionotropic receptors. Induces intracellular calcium overload, which results in mitochondrial ROS overproduction and apoptotic signals dependent on caspase3 or calpain. In addition, it is induced by EAAT (excitatory amino acid transporter) of astrocytes and microglia cells adjacent to nerve cells, resulting in overactivity and eventually inflammatory effects such as IL-1β, TNF-α, iNOS, and nitric oxide via NF-κB nucleic translocation. Secrete factors to contribute to neuronal cell death. As such, neuronal excitatory toxicity and microglia hyperactivity due to excitatory neurotransmitter hypersecretion are very similar to those of deactivation of brain function, degenerative neuropathy and central nervous system disease caused by chronic stress.
최근, 침향 추출물의 골 대사성 질환에 대한 치료 효과에 대해서는 보고된 바 있으나, 신경 퇴행성 질환에 대한 개선, 예방 또는 치료 효과는 보고된 바가 없다. Recently, there has been a report on the therapeutic effect of aroma extracts on bone metabolic diseases, but there has been no report on the improvement, prevention or treatment of neurodegenerative diseases.
본 발명자들은 화학적 합성 의약보다 인체에 대한 부작용이 적고, 용이하게 제조 및 섭취할 수 있는 천연물 유래의 유효성분을 포함하는 퇴행성 신경질환의 개선, 예방 또는 치료용 조성물을 개발하고자 노력하였다. 이에, 세포실험을 통해 침향 추출물의 분획물의 해마 신경세포 보호효과, 해마의 조직학적 산화 손상 억제효과 효과 등을 객관적으로 검증함으로써 본 발명을 완성하였다. The present inventors endeavored to develop a composition for improving, preventing or treating degenerative neurological diseases, which has fewer side effects on the human body than chemical synthetic medicines and includes an active ingredient derived from natural products that can be easily manufactured and consumed. Accordingly, the present invention was completed by objectively verifying the protective effect of hippocampal neurons, the histological effect of inhibiting histological oxidative damage, and the like of fractions of the aroma extracts through cell experiments.
본 발명은 침향 추출물의 분획물을 유효성분으로 포함하는 퇴행성 신경질환의 예방, 개선 또는 치료용 조성물을 제공하기 위한 것이다. 본 발명에 따른 침향 분획물은 항산화 작용, 신경세포의 아팝토시스(apoptosis) 억제 작용, 뇌신경 스트레스 저해 및 신경세포 보호 작용, 신경세포의 활성산소종(reactive oxygen species:ROS) 생성 억제 작용, 세포외 신호조절인산화효소(ERK; extracellular signal-regulated kinases)의 인산화 수준 감소 작용, 신경세포의 일산화질소(NO)의 생성 억제 작용 및 신경세포의 DNA 분절 저해 작용 등이 우수하므로, 퇴행성 신경질환의 개선, 예방 또는 치료를 위해 유용하게 이용될 수 있다. The present invention is to provide a composition for the prevention, improvement or treatment of degenerative neurological diseases comprising a fraction of the aroma extract as an active ingredient. The fragrance fraction according to the present invention has antioxidant activity, inhibits apoptosis of neurons, inhibits nerve stress and protects neurons, inhibits the production of reactive oxygen species (ROS) of neurons, extracellular Improvement of degenerative neuropathy due to its excellent action of reducing phosphorylation level of extracellular signal-regulated kinases (ERK), inhibition of nitric oxide (NO) production of neurons and DNA fragmentation inhibition of neurons. It can be usefully used for prevention or treatment.
상기한 목적을 달성하기 위한 본 발명은 침향 추출물의 분획물을 유효성분으로 포함하는 퇴행성 신경질환의 예방 또는 치료용 약학 조성물을 제공한다.The present invention for achieving the above object provides a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases comprising a fraction of the aroma extract as an active ingredient.
본 발명의 일 실시예에 의하면, 상기 침향은 아킬라리아(Aquilaria) 속 식물로부터 유래된 것일 수 있다.According to one embodiment of the present invention, the aroma may be derived from Aquilaria genus plants.
본 발명의 일 실시예에 의하면, 상기 아킬라리아(Aquilaria) 속 식물은 아킬라리아 카시아나(A. khasiana), 아킬라리아 아피쿠리나(A. apiculina), 아킬라리아 블리로닐(A. blillonil), 아킬라리아 바네온시스(A. baneonsis), 아킬라리아 브라키안타(A. brachyantha), 아킬라리아 컨밍이아나(A. cunmingiana), 아킬라리아 필라리아(A. filaria), 아킬라리아 그란디플로라(A. grandiflora), 아킬라리아 힐라타(A. hilata), 아킬라리아 마이크로카파(A. microcapa), 아킬라리아 로스트라타(A. rostrata), 아킬라리아 아갈로차(A. agallocha), 아킬라리아 말라센시스(A. malaccensis), 아킬라리아 시넨시스(A. sinensis) 또는 아킬라리아 크라사나(A. crassna)인 것일 수 있다.According to an embodiment of the present invention, the Aquilaria genus plants are Aquilaria cassiana (A. khasiana), Achillia apiculina (A. apiculina), Achilla blillonil (A. blillonil), A. baneonsis, A. brachyantha, A. cunmingiana, A. filaria, Achilla grandiflora. grandiflora, A. hilata, A. microcapa, A. rostrata, A. agallocha, Achilla malsensis ( A. malaccensis, A. sinensis or A. crassna.
본 발명의 일 실시예에 의하면, 상기 침향 추출물은 침향을 물, C1 내지 C3 알코올 또는 이들의 혼합물을 용매로 하여 1차 추출한 추출물을 클로로포름, 에테르 또는 메틸렌클로라이드를 용매로 하여 2차 추출한 추출물일 수 있다.According to an embodiment of the present invention, the aroma extract is the extract of the first extract with aroma of water, C 1 to C 3 alcohol or a mixture thereof as a solvent, the second extraction using chloroform, ether or methylene chloride as a solvent Can be.
본 발명의 일 실시예에 의하면, 상기 침향 추출물은 침향을 클로로포름, 에테르 또는 메틸렌클로라이드를 용매로 하여 추출한 추출물일 수 있다.According to an embodiment of the present invention, the fragrance extract may be an extract extracted by using aroma as a solvent of chloroform, ether or methylene chloride.
본 발명의 일 실시예에 의하면, 상기 침향 추출물은 침향의 메틸렌클로라이드 추출물일 수 있다.According to an embodiment of the present invention, the aroma extract may be a methylene chloride extract of aroma.
본 발명의 일 실시예에 의하면, 상기 분획물은 다음 단계를 포함하는 방법에 의해 얻어진 것을 특징으로 하는 퇴행성 신경질환의 예방 또는 치료용 약학 조성물일 수 있다:According to one embodiment of the invention, the fraction may be a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases, characterized in that obtained by a method comprising the following steps:
a) 침향 또는 침향 에탄올 추출물을 메틸렌클로라이드에 용해시킨 후 여과한 여과액을 수득하는 단계;a) dissolving incense or incense ethanol extract in methylene chloride to obtain a filtrate which is filtered;
b) 상기 a) 단계에서 수득한 여과액을 100%(v/v) 메탄올 수용액에 용해시킨 후 여과한 여과액을 수득하는 단계; 및b) dissolving the filtrate obtained in step a) in an aqueous 100% (v / v) methanol solution to obtain a filtrate filtered; And
c) 상기 b) 단계에서 수득한 여과액을 증류수, 및 아세토니트릴 또는 메탄올 용매를 사용한 고속 액체 크로마토그래피(HPLC)를 이용하여 분리하는 단계.c) separating the filtrate obtained in step b) using distilled water and high performance liquid chromatography (HPLC) using acetonitrile or methanol solvent.
본 발명의 일 실시예에 의하면, 상기 c) 단계에서는, 상기 b) 단계에서 수득한 10 ㎕의 여과액을 고정상으로서 비극성 컬럼을 갖는 고속 액체 액체크로마토그래피(HPLC)에 통과시켜 280 nm 파장에서 측정한 결과, 변동계수 0.024%로, 머무름 시간(retention time) 28.52분에 피크를 가지는 분획물(E 분획물)을 분리하였으며, According to an embodiment of the present invention, in step c), 10 μl of the filtrate obtained in step b) is passed through high-speed liquid liquid chromatography (HPLC) having a nonpolar column as a fixed phase, and measured at a wavelength of 280 nm. As a result, a fraction (E fraction) having a peak at 28.52 minutes of retention time was isolated with a coefficient of variation of 0.024%.
상기 컬럼의 이동상은, 용매로 증류수와 아세토니트릴을 사용하되, 0~0.01 분에서 30%(v/v) 아세토니트릴, 0.01-10 분에서 0.01-40%(v/v) 아세토니트릴의 구배, 10-15 분에서 40-50%(v/v) 아세토니트릴의 구배, 15-30 분에서 50-60%(v/v) 아세토니트릴의 구배, 30-40 분에서 100%(v/v) 아세토니트릴, 40-50 분에서 100%(v/v) 아세토니트릴을 사용하고, 유속을 1 mL/min으로 한 것일 수 있다.In the mobile phase of the column, using distilled water and acetonitrile as a solvent, a gradient of 30% (v / v) acetonitrile at 0 ~ 0.01 minutes, 0.01-40% (v / v) acetonitrile at 0.01-10 minutes, Gradient of 40-50% (v / v) acetonitrile at 10-15 min, gradient of 50-60% (v / v) acetonitrile at 15-30 min, 100% (v / v) at 30-40 min Acetonitrile, 100% (v / v) acetonitrile at 40-50 minutes may be used and the flow rate is 1 mL / min.
본 발명의 일 실시예에 의하면, 상기 비극성 컬럼은 옥타데실실란(C18), 도데실실란(C12) 또는 옥타실란(C8)이 충전된 비극성 컬럼일 수 있다.According to one embodiment of the present invention, the nonpolar column may be a nonpolar column filled with octadecylsilane (C18), dodecylsilane (C12) or octasilane (C8).
본 발명의 일 실시예에 의하면, 상기 컬럼은 길이가 3 내지 30cm, 내경이 1.8 내지 5mm이고, 상기 컬럼에 충전된 입자의 직경이 1.5 내지 5㎛일 수 있다.According to an embodiment of the present invention, the column may have a length of 3 to 30 cm, an inner diameter of 1.8 to 5 mm, and a diameter of particles packed in the column may be 1.5 to 5 μm.
본 발명의 일 실시예에 의하면, 상기 퇴행성 신경질환은 지질다당류(lipopolysaccharide, LPS)에 의하여 유도되는 것일 수 있다.According to one embodiment of the present invention, the neurodegenerative disease may be induced by lipopolysaccharide (LPS).
본 발명의 일 실시예에 의하면, 상기 퇴행성 신경질환은 글루타메이트에 의해 유발된 것일 수 있다.According to one embodiment of the invention, the degenerative neurological disease may be caused by glutamate.
본 발명의 일 실시예에 의하면, 상기 조성물은 하기 특성 중 하나 이상을 가질 수 있다:According to one embodiment of the invention, the composition may have one or more of the following properties:
1) 신경세포의 아팝토시스(apoptosis)를 억제;1) inhibit apoptosis of neurons;
2) 신경세포의 활성산소종(reactive oxygen species:ROS) 생성 억제;2) inhibiting the production of reactive oxygen species (ROS) of neurons;
3) 세포외 신호조절인산화효소(ERK; extracellular signal-regulated kinases)의 인산화 수준의 감소; 3) reduction of phosphorylation levels of extracellular signal-regulated kinases (ERKs);
4) 신경세포의 일산화질소(NO)의 생성 억제; 및4) inhibition of production of nitric oxide (NO) in neurons; And
5) 신경세포의 DNA 분절 저해.5) Inhibition of DNA segmentation of neurons.
본 발명의 일 실시예에 의하면, 상기 조성물은 항산화 활성을 갖는 것일 수 있다.According to an embodiment of the present invention, the composition may be one having antioxidant activity.
본 발명의 일 실시예에 의하면, 상기 퇴행성 신경질환은 알츠하이머병(Alzheimer's disease), 파킨슨병(Parkinson's disease), 루게릭병(Lou Gehrig disease), 헌팅톤병(Huntington's disease), 다발성 경화증(Multiple sclerosis), 뇌졸중(stroke), 혈전증(thrombosis), 색전증(embolism), 두부손상(head trauma), 뇌순환대사장애, 뇌 기능혼수 또는 치매(dementia)일 수 있다.According to one embodiment of the invention, the degenerative neurological disease is Alzheimer's disease (Alzheimer's disease), Parkinson's disease (Parkinson's disease), Lou Gehrig disease (Lou Gehrig disease), Huntington's disease (Huntington's disease), Multiple sclerosis (Multiple sclerosis), Stroke, thrombosis, embolism, head trauma, cerebral circulatory metabolic disorder, cerebral coma or dementia.
본 발명의 일 실시예에 의하면, 상기 조성물의 제형이 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼 및 시럽으로 이루어진 군으로부터 선택되는 경구용 제형일 수 있다.According to one embodiment of the invention, the formulation of the composition may be an oral formulation selected from the group consisting of powders, granules, tablets, capsules, suspensions, emulsions and syrups.
또한, 본 발명은 침향 추출물의 분획물을 유효성분으로 포함하는 퇴행성 신경질환의 예방 또는 개선용 식품 조성물을 제공한다.The present invention also provides a food composition for the prevention or improvement of degenerative neurological diseases comprising a fraction of the aroma extract as an active ingredient.
본 발명에 따른 침향 추출물의 분획물을 유효성분으로 포함하는 조성물은 항산화 작용, 신경세포의 아팝토시스(apoptosis) 억제 작용, 뇌신경 스트레스 저해 및 신경세포 보호 작용, 신경세포의 활성산소종(reactive oxygen species:ROS) 생성 억제 작용, 세포외 신호조절인산화효소(ERK; extracellular signal-regulated kinases)의 인산화 수준 감소 작용, 신경세포의 일산화질소(NO)의 생성 억제 작용 및 신경세포의 DNA 분절 저해 작용 등을 나타낸다. 이에 따라, 알츠하이머병(Alzheimer's disease), 파킨슨병(Parkinson's disease), 루게릭병(Lou Gehrig disease), 헌팅톤병(Huntington's disease), 다발성 경화증(Multiple sclerosis), 뇌졸중(stroke), 혈전증(thrombosis), 색전증(embolism), 두부손상(head trauma), 뇌순환대사장애, 뇌 기능혼수 또는 치매(dementia) 등의 퇴행성 신경질환의 예방 또는 치료에 유용하게 사용할 수 있다.Composition comprising a fraction of the aroma extract according to the present invention as an active ingredient, antioxidant activity, neuronal apoptosis (apoptosis) inhibitory effect, neuronal stress inhibition and neuronal cell protective action, neuronal reactive oxygen species (reactive oxygen species) : Inhibition of ROS production, reduction of phosphorylation level of extracellular signal-regulated kinases (ERK), inhibition of production of nitric oxide (NO) in neurons and inhibition of DNA fragmentation in neurons Indicates. As a result, Alzheimer's disease, Parkinson's disease, Lou Gehrig disease, Huntington's disease, Multiple sclerosis, stroke, thrombosis, and embolism It can be usefully used for the prevention or treatment of degenerative neurological diseases such as embolism, head trauma, cerebral circulatory metabolic disorder, brain coma or dementia.
도 1은 침향의 약학적 유효성을 나타내는 분획물을 탐색하는 과정으로, 소수성 또는 친수성 용매에 따른 추출 및 분획물을 분리하는 과정을 모식화한 것이다.Figure 1 is a process of searching for fractions showing the pharmaceutical efficacy of the aroma, it is a schematic of the process of extraction and separation of fractions according to hydrophobic or hydrophilic solvent.
도 2는 침향 메틸렌클로라이드 추출물의 HPLC 지문분석 결과이다.Figure 2 shows the results of HPLC fingerprint analysis of the aroma methylene chloride extract.
도 3은 용매별 침향 추출물의 흥분성 신경전달물질 (glutamate)로 유도된 마우스 해마 신경세포주 (HT-22 cell) 세포흥분독성 사멸에 대한 보호효과를 나타내는 그래프이다.3 is a graph showing the protective effect against excitatory neurotransmitter (HT-22 cell) cell excitability killing induced by excitatory neurotransmitter (glutamate) of the solvent-specific aroma extract.
도 4는 침향 메틸렌클로라이드 추출물에서 분리한 임의의 5가지 분획물의 흥분성 신경전달물질(glutamate)로 유도된 마우스 해마신경세포주 (HT-22 cell) 세포흥분독성 사멸에 대한 보호효과를 나타내는 그래프이다. Figure 4 is a graph showing the protective effect against the excitatory neurotransmitter (HT-22 cell) cell excitability killing induced by excitatory neurotransmitter (glutamate) of any five fractions isolated from the aroma methylene chloride extract.
도 5a은 본 발명의 일 실시예에 의한 침향 E 분획물이 흥분성 신경전달물질(glutamate)로 유도된 마우스 해마 신경세포주(HT-22 cell)의 세포흥분독성에 대하여 세포사멸을 억제하는 것을 나타내는 그래프이고, 도 5b는 본 발명의 일 실시예에 의한 침향 E 분획물이 흥분성 신경전달물질(glutamate)로 유도된 마우스 해마 신경세포주(HT-22 cell)에서 Lactate dehydrogenase(LDH)의 세포 외 유출을 억제하는 것을 나타내는 그래프이다. Figure 5a is a graph showing that the acupuncture E fraction according to an embodiment of the present invention inhibits apoptosis against cytotoxicity of mouse hippocampal neuronal cell line (HT-22 cell) induced by excitatory neurotransmitter (glutamate) FIG. 5B shows that the Aroma E fraction according to one embodiment of the present invention inhibits extracellular leakage of Lactate dehydrogenase (LDH) in mouse hippocampal neuronal cell line (HT-22 cells) induced with excitatory neurotransmitter (glutamate). It is a graph.
도 6a은 glutamate로 유도된 HT-22 cell에서 ROS 수준을 DCFH-DA 세포화학형광기법으로 평가한 실험에서, 침향 E 분획물의 ROS 생성 억제효능을 나타내는 사진이고, 도 6b는 상기 실험에서, 침향 E 분획물의 ROS 생성 억제 효능을 나타내는 그래프이다. Figure 6a is a photograph showing the inhibitory effect of ROS generation of the fragrance E fraction in experiments in which ROS level was evaluated by DCFH-DA cytochemistry fluorescence technique in glutamate-induced HT-22 cells, Figure 6b is in the experiment, It is a graph showing the effect of inhibiting the ROS production of the fraction.
도 7a는 침향 E 분획물의 흥분성 신경전달물질(glutamate)로 유도된 마우스 해마 신경세포주(HT-22 cell) 사멸을 유세포 분석기(FACs)로 평가한 실험에서, 본 발명의 침향 E 분획물이 Annexin V/PI 신호를 억제함에 따라 세포 사멸이 억제되는 것을 나타내는 그래프이고, 도 7b는 상기 실험에서, 사멸된 세포의 수를 정량화하여 나타내는 그래프이다. FIG. 7A shows that in the experiment evaluating the death of the mouse hippocampal neuronal cell line (HT-22 cell) induced by excitatory neurotransmitter (glutamate) of the fragrance E fraction by flow cytometry (FACs), the fragrance E fraction of the present invention was Annexin V / It is a graph showing that cell death is inhibited by inhibiting the PI signal, Figure 7b is a graph showing the quantified number of killed cells in the experiment.
도 8a는 glutamate로 유도된 HT-22 cell에서의 DNA fragmentation 수준을 Hoechst33528 형광 현미경으로 평가한 실험으로, 침향 E 분획물이 신경세포의 사멸을 억제한다는 것을 나타내는 사진이고, 도 8b는 상기 실험에서, 신경세포 DNA의 분절(fragmentation) 정도를 정량화하여 나타내는 그래프이다. FIG. 8A is an experiment evaluating DNA fragmentation level in glutamate-induced HT-22 cells by Hoechst33528 fluorescence microscopy. FIG. 8A is a photograph showing that acupuncture E fraction inhibits neuronal cell death. FIG. It is a graph quantifying the degree of fragmentation of cellular DNA.
도 9는 glutamate로 유도된 HT-22 cell에서의 세포사멸기전을 웨스턴블랏 기법을 이용하여 평가한 실험에서, 침향 E 분획물이 ERK의 인산화를 방지하여 세포사멸을 억제한다는 것을 나타내는 그래프이다. 9 is a graph showing that acupuncture E fraction inhibits apoptosis by preventing phosphorylation of ERK in an experiment evaluating apoptosis in glutamate-induced HT-22 cells using Western blot technique.
도 10은 침향 E 분획물이 Hydrogen peroxide(H2O2)로 유도된 마우스 해마 신경세포주(HT-22 cell)에서 항산화 작용을 하여 세포사멸을 억제한다는 것을 나타내는 그래프이다. FIG. 10 is a graph showing that aroma E fraction inhibits apoptosis by antioxidant activity in mouse hippocampal neuronal cell line (HT-22 cell) induced by Hydrogen peroxide (H 2 O 2 ).
도 11은 침향 E 분획물이 Hydrogen peroxide(H2O2)로 유도된 마우스 해마 신경세포주(HT-22 cell)에서 Lactate dehydrogenase(LDH)의 세포 외 유출을 억제하는 것을 나타내는 그래프이다. FIG. 11 is a graph showing that the A-fraction E fraction inhibits the extracellular leakage of Lactate dehydrogenase (LDH) in mouse hippocampal neuronal cell line (HT-22 cell) induced with Hydrogen peroxide (H 2 O 2 ).
도 12a은 침향 E 분획물이 마우스 미세아교신경세포주(BV2 cell)에서 Lipopolysaccharide(LPS)로 유도된 Nitric oxide(NO)의 증가를 억제한다는 것을 나타내는 그래프이고, 도 12b는 상기 실험에서 나타낸 결과에 대한 단백질 정량값을 나타낸 그래프이다. Figure 12a is a graph showing that the aroma E fraction inhibits the increase of Lipopolysaccharide (LPS) -induced Nitric oxide (NOS) in mouse microglia neuronal cell lines (BV2 cells), Figure 12b is a protein for the results shown in the experiment A graph showing quantitative values.
이하, 본 발명을 상세하게 설명한다. EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.
본 발명자들은 침향의 퇴행성 신경질환에 대한 개선, 예방 또는 치료 효과를 나타내는 주요 약리활성 성분을 동정하고자, 글루타메이트 또는 지질다당류(lipopolysaccharide, LPS) 처리한 뇌 해마 신경세포를 Ethylene acetate, methylene chloride, methanol, ethanol 등을 비롯한 각종 유기용매를 이용하여 추출한 침향 추출물로 실험하였다. 그 결과, methylene chloride 추출물(이하 MC)이 가장 유효하였다. 상기 MC 추출물을 HPLC를 이용하여 지문분석한 결과, 5가지의 분획물의 HPLC peak를 확인하였다. Preperative LC 기기를 이용하여 상기 5가지의 분획물(A, B, C, D, E)을 분리한 뒤 신경세포 흥분독성에 대한 억제효과를 평가한 결과, E 분획물에서 현저한 유효성이 관찰되었다. 따라서 침향의 뇌 중추신경계 질환을 개선하는 약리활성은 E 분획물이 핵심적인 역할을 하는 것으로 유추할 수 있다. In order to identify the major pharmacologically active ingredients exhibiting amelioration, prophylactic or therapeutic effects of acupuncture degenerative neurological diseases, the present inventors have investigated the brain hippocampal neurons treated with glutamate or lipopolysaccharide (LPS) in Ethylene acetate, methylene chloride, methanol, Experiments were carried out with aroma extract extracted using various organic solvents including ethanol. As a result, methylene chloride extract (hereinafter MC) was the most effective. Fingerprint analysis of the MC extract using HPLC confirmed the HPLC peaks of the five fractions. The five fractions (A, B, C, D, E) were separated using a Preperative LC instrument, and the inhibitory effect on neuronal excitatory toxicity was evaluated. As a result, a significant effect was observed in the E fraction. Therefore, it can be inferred that the E fraction plays a key role in pharmacological activity that improves the central nervous system disease of acupuncture.
본 발명의 일 구현예에 따르면, 본 발명은 침향 추출물의 분획물을 유효성분으로 포함하는 퇴행성 신경질환의 예방, 개선 또는 치료용 조성물을 제공한다.According to one embodiment of the invention, the present invention provides a composition for the prevention, improvement or treatment of degenerative neurological diseases comprising a fraction of the aroma extract as an active ingredient.
본 발명에서, 상기 침향은 아킬라리아(Aquilaria) 속 식물로부터 유래된 것을 사용할 수 있다. 구체적으로, 상기 아킬라리아(Aquilaria) 속 식물은 아킬라리아 카시아나(A. khasiana), 아킬라리아 아피쿠리나(A. apiculina), 아킬라리아 블리로닐(A. blillonil), 아킬라리아 바네온시스(A. baneonsis), 아킬라리아 브라키안타(A. brachyantha), 아킬라리아 컨밍이아나(A. cunmingiana), 아킬라리아 필라리아(A. filaria), 아킬라리아 그란디플로라(A. grandiflora), 아킬라리아 힐라타(A. hilata), 아킬라리아 마이크로카파(A. microcapa), 아킬라리아 로스트라타(A. rostrata), 아킬라리아 아갈로차(A. agallocha), 아킬라리아 말라센시스(A. malaccensis), 아킬라리아 시넨시스(A. sinensis) 또는 아킬라리아 크라사나(A. crassna) 등의 줄기 또는 뿌리, 바람직하게는 수지가 침착된 줄기 또는 뿌리를 사용할 수 있다.In the present invention, the aroma can be used that is derived from Aquilaria genus plants. Specifically, the plant of the genus Aquilaria is Achilla cassiana (A. khasiana), Achillia apiculina (A. apiculina), Achillia blillonil (A. blillonil), Achillia baneeonsis ( A. baneonsis, A. brachyantha, A. cunmingiana, A. filaria, A. filaria, A. grandiflora, Aquilia hill A. hilata, A. microcapa, A. rostrata, A. agallocha, A. malaccensis, Achilla Stems or roots, such as A. sinensis or A. crassna, preferably stems or roots on which resin is deposited can be used.
또한, 상기 침향 추출물은 침향을 클로로포름, 에테르 또는 메틸렌클로라이드를 용매로 하여 추출한 추출물일 수 있고, 상기 용매로 바람직한 것은 메틸렌클로라이드이다.In addition, the aroma extract may be an extract of the aroma extract by using chloroform, ether or methylene chloride as a solvent, methylene chloride is preferred as the solvent.
상기 침향 추출물은 통상의 방법에 따라, 침향을 냉수 추출, 열수 추출, 유기용매 추출 또는 증류 등의 방법으로 추출한 다음, 수득한 추출액을 그대로 사용하거나, 감압 농축 또는 동결 건조함으로써 제조할 수 있다. 본 발명에서 침향 추출물은 침향을 상기 용매로 추출하여 수득한 물질 그 자체를 의미할 뿐만 아니라, 상기 추출하여 수득된 물질을 균주를 사용하여 발효시키거나, 에테르 등의 유기 용매로 더욱 분별 추출한 것 등을 의미할 수 있다. The aroma extract may be prepared by extracting the aroma in accordance with a conventional method, such as cold water extraction, hot water extraction, organic solvent extraction or distillation, and then using the obtained extract as it is, or concentrated under reduced pressure or freeze-dried. In the present invention, the fragrance extract means not only the substance obtained by extracting the aroma with the solvent, but also the substance obtained by the extract is fermented using a strain, or fractionally extracted with an organic solvent such as ether. It may mean.
구체적으로, 본 발명에 따른 침향 추출물은 다음과 같이 제조될 수 있다. 먼저, 선별하여 적절한 크기로 다듬어진 침향 또는 침향 분말을 중량 기준으로 약 1 내지 80배 분량, 바람직하게는 30 내지 50 분량의 클로로포름, 에테르 또는 메틸렌클로라이드를 용매로 사용하여, 예를 들어 5 내지 100 ℃, 바람직하게는 상온에서 약 1 내지 100 시간, 바람직하게는 10 내지 40 시간 동안 교반 추출, 열탕 추출, 냉침 추출, 환류 추출 또는 초음파 추출 등의 방법으로 추출한다. 수득한 추출액은 그대로 사용하거나, 이를 여과, 농축 또는 건조하여 침향 추출물을 얻을 수 있다. 필요한 경우, 상기 추출물을 물 또는 저급 알콜, 에테르 등의 유기 용매로 더 분획하여 사용할 수 있다. Specifically, the fragrance extract according to the present invention may be prepared as follows. First, about 1 to 80 times by weight, preferably 30 to 50 parts of chloroform, ether or methylene chloride, by weight of the fragrance or fragrance powder, which are selected and trimmed to an appropriate size, are used, for example, 5 to 100 Extraction is carried out by stirring extraction, boiling water extraction, cold extraction, reflux extraction or ultrasonic extraction for about 1 to 100 hours, preferably 10 to 40 hours at room temperature, preferably at room temperature. The obtained extract can be used as it is, or it can be filtered, concentrated or dried to obtain a fragrance extract. If necessary, the extract may be further fractionated with water or an organic solvent such as lower alcohol, ether or the like.
또한, 상기 침향 추출물은 침향을 물, C1 내지 C3 알코올 또는 이들의 혼합물을 용매로 하여 1차 추출한 추출물을 클로로포름, 에테르 또는 메틸렌클로라이드를 용매로 하여 2차 추출한 추출물일 수 있으며, 상기 C1 내지 C3 알코올은 에탄올, 메탄올 또는 프로판올인 것이 바람직하다. 본 발명의 1차 추출의 용매로서 가장 바람직한 것은 20 내지 50 %(w/v)의 에탄올이다. In addition, the aroma extract may be an extract obtained by extracting the first aroma using aroma, water, C 1 to C 3 alcohol or a mixture thereof as a solvent, a second extraction using chloroform, ether or methylene chloride as a solvent, the C 1 To C 3 alcohol is preferably ethanol, methanol or propanol. Most preferred as solvent of the first extraction of the invention is 20 to 50% (w / v) ethanol.
구체적으로, 상기 침향 추출물은 다음과 같이 제조될 수 있다. 먼저, 선별하여 적절한 크기로 다듬어진 침향 또는 침향 분말을 중량 기준으로 약 1 내지 50배 분량의 물, 에탄올, 메탄올 등과 같은 탄소수 1 내지 4의 저급 알코올, 또는 이들의 혼합 용매를 사용하여, 예를 들어 5 내지 100 ℃에서 약 1 내지 100 시간 동안 교반 추출, 열탕 추출, 냉침 추출, 환류 추출 또는 초음파 추출 등의 방법으로 1차 추출한다. 수득한 1차 추출액은 그대로 사용하거나, 이를 여과, 농축 또는 건조하여 사용할 수 있고, 동결건조하여 분말 형태로 이용하는 것이 바람직하다. 상기 1차 추출물이 분말 형태인 경우에는 1차 추출물을 중량 기준으로 약 5 내지 400 배 분량, 바람직하게는 150 내지 250 배 분량의 클로로포름, 에테르 또는 메틸렌클로라이드를 용매로 사용하여, 예를 들어 5 내지 100 ℃, 바람직하게는 상온에서 약 1 내지 100 시간, 바람직하게는 10 내지 40 시간 동안 교반 추출, 열탕 추출, 냉침 추출, 환류 추출 또는 초음파 추출 등의 방법으로 추출한다. 수득한 추출액은 그대로 사용하거나, 이를 여과, 농축 또는 건조하여 침향 추출물을 얻을 수 있다. Specifically, the fragrance extract may be prepared as follows. First, by using a 1 to 4 times the amount of water or a lower alcohol having 1 to 4 carbon atoms, such as ethanol, methanol, or a mixed solvent of the fragrance or aroma powder that has been selected and trimmed to an appropriate size, For example, primary extraction is performed at 5 to 100 ° C. for about 1 to 100 hours by stirring extraction, hot water extraction, cold extraction, reflux extraction, or ultrasonic extraction. The obtained primary extract may be used as it is, or it may be used by filtration, concentration or drying, and lyophilized to use in powder form. When the primary extract is in powder form, the primary extract may be used in an amount of about 5 to 400 times by weight, preferably 150 to 250 times, by weight of chloroform, ether, or methylene chloride, for example, 5 to 400 times. Extraction is carried out at 100 DEG C, preferably at room temperature for about 1 to 100 hours, preferably 10 to 40 hours, by stirring extraction, hot water extraction, cold extraction, reflux extraction or ultrasonic extraction. The obtained extract can be used as it is, or it can be filtered, concentrated or dried to obtain a fragrance extract.
본 발명에서 이용되는 침향 추출물은 감압 증류 및 동결 건조 또는 분무 건조 등과 같은 추가적인 과정에 의해 분말 상태로 제조될 수 있다.Fragrance extract used in the present invention may be prepared in a powder state by an additional process such as vacuum distillation and freeze drying or spray drying.
본 발명에서 상기 침향 추출물의 분획물은 다음 단계를 포함하는 방법에 의해 얻어질 수 있다:In the present invention, the fraction of the fragrance extract can be obtained by a method comprising the following steps:
a) 침향 또는 침향 에탄올 추출물을 메틸렌클로라이드에 용해시킨 후 여과한 여과액을 수득하는 단계;a) dissolving incense or incense ethanol extract in methylene chloride to obtain a filtrate which is filtered;
b) 상기 a) 단계에서 수득한 여과액을 100%(v/v) 메탄올 수용액에 용해시킨 후 여과한 여과액을 수득하는 단계; 및b) dissolving the filtrate obtained in step a) in an aqueous 100% (v / v) methanol solution to obtain a filtrate filtered; And
c) 상기 b) 단계에서 수득한 여과액을 증류수, 및 아세토니트릴 또는 메탄올 용매를 사용한 고속 액체 크로마토그래피(HPLC)를 이용하여 분리하는 단계.c) separating the filtrate obtained in step b) using distilled water and high performance liquid chromatography (HPLC) using acetonitrile or methanol solvent.
상기 c) 단계에서는, 상기 b) 단계에서 수득한 10 ㎕의 여과액을 고정상으로서 비극성 컬럼을 갖는 고속 액체 액체크로마토그래피(HPLC)에 통과시켜 280 nm 파장에서 측정한 결과, 변동계수 0.024%로, 머무름 시간(retention time) 28.52분에 피크를 가지는 분획물을 분리할 수 있다.In step c), 10 μl of the filtrate obtained in step b) is passed through a high performance liquid chromatography (HPLC) having a nonpolar column as a stationary phase and measured at a wavelength of 280 nm. The coefficient of variation is 0.024%. At 28.52 minutes of retention time, a fraction having a peak can be separated.
상기 비극성 컬럼은 옥타데실실란(C18), 도데실실란(C12) 또는 옥타실란(C8)이 충전된 비극성 컬럼일 수 있고, 바람직하게는 옥타데실실란(C18)일 수 있다.The nonpolar column may be a nonpolar column filled with octadecylsilane (C18), dodecylsilane (C12) or octasilane (C8), preferably octadecylsilane (C18).
또한, 상기 컬럼은 길이가 3 내지 30cm, 내경이 1.8 내지 5mm이고, 상기 컬럼에 충전된 입자의 직경이 1.5 내지 5㎛일 수 있다.In addition, the column has a length of 3 to 30cm, an inner diameter of 1.8 to 5mm, the diameter of the particles packed in the column may be 1.5 to 5㎛.
또한, 본 발명의 바람직한 일 실시예에서 상기 c) 단계에서는, 상기 컬럼의 이동상으로 증류수와 아세토니트릴을 사용하되, 0~0.01 분에서 30%(v/v) 아세토니트릴, 0.01-10 분에서 0.01-40%(v/v) 아세토니트릴의 구배, 10-15 분에서 40-50%(v/v) 아세토니트릴의 구배, 15-30 분에서 50-60%(v/v) 아세토니트릴의 구배, 30-40 분에서 100%(v/v) 아세토니트릴, 40-50 분에서 100%(v/v) 아세토니트릴을 사용하고, 유속을 1 mL/min으로 한 것일 수 있다. In addition, in the preferred embodiment of the present invention, in step c), distilled water and acetonitrile are used as the mobile phase of the column, but 0 to 0.01 minutes at 30% (v / v) acetonitrile, 0.01 to 10 minutes at 0.01 Gradient of -40% (v / v) acetonitrile, gradient of 40-50% (v / v) acetonitrile at 10-15 minutes, gradient of 50-60% (v / v) acetonitrile at 15-30 minutes , 100% (v / v) acetonitrile at 30-40 minutes, 100% (v / v) acetonitrile at 40-50 minutes, the flow rate may be 1 mL / min.
본 명세서에서 용어 '유효성분으로 포함하는'이란 침향 분획물의 효능 또는 활성을 달성하는데 충분한 양을 포함하는 것을 의미한다. As used herein, the term 'comprising as an active ingredient' means including an amount sufficient to achieve the efficacy or activity of the fragrance fraction.
본 발명의 조성물은 천연식물재료인 침향의 추출물로부터 분리한 분획물을 유효성분으로 포함하는 것이므로 과량 투여하여도 인체에 부작용이 없다.Since the composition of the present invention comprises a fraction separated from the extract of aroma of natural plant material as an active ingredient, there is no side effect to the human body even when excessively administered.
인체 내에는 SOD(superoxide dismutase), catalase, 글루타치온 과산화효소(glutathione peroxidase) 등의 항산화 효소와 글루타치온(glutathione), 요산(uric acid) 등의 여러 가지 비효소적 항산화 물질이 있어 활성산소의 축적이나 해로운 산화작용을 막아주게 된다. 그 중 글루타치온 과산화효소(glutathione peroxidase)는 GSH를 생산하여 우리 몸의 균형을 이루어주게 되는데 [Murphy T. H., et al., Neuron. 2, 1547-1558], 이때 GSH는 글라이신(glycine), 글루타메이트(glutamate) 및 시스테인(cysteine)의 세 가지 아미노산이 결합된 단백질로 간과 세포에서 활성산소 및 각종 독성물질을 제거하여 세포손상을 방지하고 면역력을 높이는 역할을 한다 [Shin A. Y., et al., J. Neurosci. 2006, 26, 10514-10523; Milatovic D., et al., Toxicol. Appl. Pharm. 2009, 240, 219-225]. 이러한 GSH를 이루고 있는 아미노산 중 글루타메이트(glutamate)는 중추신경계 시냅스의 15 ~ 20 %를 차지하는 중심적인 흥분성 신경전달물질로 작용하지만, 뇌 속의 과다한 축적은 체내의 항산화 시스템의 기능을 저하시킴으로 인해 산화계와 항산화계의 불균형을 초래하여 신경을 과도하게 흥분시킴으로 인해 흥분독성(excitotoxicity)을 야기하게 된다 [Kim M. H., et al., J. Neurosci. 2008, 28, 10852-10863; Penugonda S., et al., Toxicol. Appl. Pharm. 2006, 216, 197-205; Federico H., et al., J. Neurochem. 2007, 100, 736-746]. 흥분독성은 세포의 사멸을 일으키게 되어 알츠하이머병, 파킨슨병과 같은 여러 퇴행성 신경질환을 유발하게 된다 [Choi B. H., et al., J. Cell. Sci. 2005, 119, 1329-1340; Lim C. S., et al., Biol. Pharm. Bull. 2006, 29, 1212-1216]. There are many non-enzymatic antioxidants such as SOD (superoxide dismutase), catalase, glutathione peroxidase, glutathione, uric acid, etc. It prevents oxidation. Among them, glutathione peroxidase produces GSH and balances our body. [Murphy T. H., et al., Neuron. 2, 1547-1558], wherein GSH is a protein that combines three amino acids, glycine, glutamate and cysteine, and removes free radicals and various toxic substances from liver and cells to prevent cell damage. Plays a role in enhancing immunity [Shin AY, et al., J. Neurosci. 2006, 26, 10514-10523; Milatovic D., et al., Toxicol. Appl. Pharm. 2009, 240, 219-225. Glutamate (glutamate) among the amino acids that make up GSH acts as a central excitatory neurotransmitter, which accounts for 15-20% of the central nervous system synapse, but excessive accumulation in the brain decreases the function of the antioxidant system in the body Imbalance of the system leads to excessive excitement of the nerve, resulting in excitotoxicity [Kim MH, et al., J. Neurosci. 2008, 28, 10852-10863; Penugonda S., et al., Toxicol. Appl. Pharm. 2006, 216, 197-205; Federico H., et al., J. Neurochem. 2007, 100, 736-746. Excitotoxicity causes cell death, leading to several degenerative neurological diseases such as Alzheimer's disease and Parkinson's disease [Choi B. H., et al., J. Cell. Sci. 2005, 119, 1329-1340; Lim C. S., et al., Biol. Pharm. Bull. 2006, 29, 1212-1216.
본 발명에 따른 침향 분획물은 상기 글루타메이트에 의해 유발되는 퇴행성 신경질환의 예방, 개선 또는 치료용으로 유용하게 이용될 수 있다.The fragrance fraction according to the present invention can be usefully used for the prevention, improvement or treatment of neurodegenerative diseases caused by glutamate.
또한, 본 발명에 따른 침향 분획물은 신경세포의 보호 작용을 한다.In addition, the fragrance fraction according to the present invention serves to protect neurons.
본 발명의 일 구현예에 따르면, 상기 침향 분획물은 신경세포의 사멸을 억제하여 신경세포의 보호 작용을 한다. 상기 신경세포의 보호 작용은 대개 뇌 신경세포의 보호 작용이다. 뇌 신경세포 보호란, 대사성 원인, 독성 원인, 신경 독성원인 및 물리, 화학적 원인 등에 의해 초래되는 신경 세포 또는 신경 조직의 손상을 경감 또는 개선하거나 이러한 손상을 입은 신경 세포를 보호 또는 회복하는 작용을 의미한다. 특히, 본 발명의 침향 분획물은 글루타메이트로 유발된 뇌 신경세포의 흥분독성으로부터 신경세포를 보호하는 작용을 한다.According to one embodiment of the present invention, the fragrance fraction serves to protect neurons by inhibiting the death of neurons. The protective action of the neurons is usually the protective action of brain neurons. Brain neuron protection refers to the action of reducing or ameliorating damage to, or protecting or repairing, nerve cells or tissues caused by metabolic causes, toxic causes, neurotoxic causes and physical and chemical causes. do. In particular, the fragrance fraction of the present invention acts to protect neurons from the excitatory toxicity of brain neurons induced by glutamate.
본 명세서에서, 용어 '뇌 신경 세포'는 뇌, 해마, 뇌간 및 척수를 구성하는 뉴런, 신경 교세포(미세아교세포, 성상교세포), 신경지지 세포, 글리아 및 슈만 세포 등을 의미한다. 이러한 본 발명의 조성물에 의한 뇌 신경 세포 보호는 다양한 기전을 통하여 이루어질 수 있으며, 예컨대, 신경 세포의 세포 사멸을 억제하여 이루어질 수 있다. As used herein, the term 'brain nerve cell' refers to neurons, glial cells (microglia, astrocytes), nerve support cells, glia and Schumann cells, etc. that make up the brain, hippocampus, brainstem and spinal cord. Brain nerve cell protection by the composition of the present invention can be achieved through a variety of mechanisms, for example, can be achieved by inhibiting cell death of nerve cells.
또한, 본 발명의 침향 분획물은 하기 실험예에 나타낸 바와 같이, 항산화 작용, 신경세포의 아팝토시스(apoptosis) 억제 작용, 뇌신경 스트레스 저해 및 신경세포 보호 작용, 신경세포의 활성산소종(reactive oxygen species:ROS) 생성 억제 작용, 세포외 신호조절인산화효소(ERK; extracellular signal-regulated kinases)의 인산화 수준 감소 작용, 신경세포의 일산화질소(NO)의 생성 억제 작용 및 신경세포의 DNA 분절 저해 작용 중에서 하나 이상의 특성을 나타낸다. In addition, the aroma fraction of the present invention, as shown in the following experimental examples, antioxidant activity, neuronal apoptosis (apoptosis) inhibitory action, neuronal stress inhibition and neuronal cell protective action, neuronal reactive oxygen species (reactive oxygen species) : Inhibits ROS production, decreases phosphorylation levels of extracellular signal-regulated kinases (ERKs), inhibits the production of nitric oxide (NO) in neurons, and inhibits DNA fragmentation in neurons The above characteristics are shown.
또한, 본 발명의 침향 분획물은 활성화된 미세아교세포(microglia)를 억제하거나 활성화된 미세아교세포가 분비하는 염증유발 물질들의 생성을 억제하는 효과가 있다.In addition, the fragrance fraction of the present invention has the effect of inhibiting the activated microglia or the production of pro-inflammatory substances secreted by the activated microglia.
신경교세포 중 미세아교세포(microglia)는 중추신경계 신경교세포의 10-12%를 차지하는 세포로서 1932년 리오 오르테가(Rio-Hortega)의 형태학적인 연구와 조직염색 방법에 의해 처음으로 보고되었다. 미세아교세포(microglia)는 조직내에서 변성된 뉴런(neuron)과 이물질 등을 잡아먹는 작용을 하여 물질의 운반과 파괴, 제거 및 병적 대사 물질의 청소 등중요한 역할을 하여 일반적으로 중추신경계에 존재하는 대식세포(macrophage)로 여겨진다.Microglia, the glial cells, account for 10-12% of the central nervous system glial cells and were first reported in 1932 by the morphological studies and tissue staining methods of Rio-Hortega. Microglia, which act on the degeneration of neurons and foreign substances in tissues, play an important role in transporting, destroying, removing, and cleaning pathological metabolites. It is considered a macrophage.
또한, 중추신경계를 구성하는 신경세포(neuron)와 다른 신경교세포들(astrocytes, oligodendrocytes)과 구별되는 특징적인 세포표면 항원을 발현하고, 탐식작용 등 대식세포와 유사한 기능을 수행하는 것으로 알려져 있다. 이러한 미세아교세포는 발생과정 중에 이들세포의 전구 세포로 여겨지는 단핵구세포(monocyte)들이 중추신경계에 들어와 분화한 것으로 알려져 있는데, 중추신경계내에서 미생물 감염이나 외상이 있을 경우 이에 대한 일차적인 방어 작용을 수행하며, 이때 감염 장소로 이동하거나 국소적인 증식을 통해 감염미생물을 탐식하고 손상된 신경세포들의 잔유 물질들을 소화하게 된다.In addition, it is known to express characteristic cell surface antigens that are distinguished from neurons and other glial cells (astrocytes, oligodendrocytes) constituting the central nervous system, and perform macrophage-like functions such as phagocytosis. These microglia are known to have differentiated into the central nervous system by monocytes, which are thought to be precursors of these cells, during development. The primary defense against microbial infections and trauma in the central nervous system is that At this time, they move to the site of infection or through local proliferation to devour infected microorganisms and digest the remnants of damaged neurons.
미세아교세포는 중추신경계에서 자기방어라는 본래의 기능을 수행하지만, 방어목적으로 생산된 TNF(tumor necrosis factor)-α, 인터류킨(interleukin)(IL)-1β, 반응성 산소 (ROS) 혹은 질소 화합물 등의 염증 유발 물질들이 과다하게 분비되거나 세포 자체가 활성화된 상태로 오래 지속될 경우 신경 조직 손상이라는 심각한 부작용을 초래하게 된다.Microglial cells perform their intrinsic function of self-defense in the central nervous system, but are produced for defensive purposes such as tumor necrosis factor (TNF), interleukin (IL) -1β, reactive oxygen (ROS) or nitrogen compounds. Excessive secretion of inflammatory mediators or prolonged activation of the cells themselves can lead to serious side effects of nerve tissue damage.
최근, 알츠하이머(Alzheimer), 파킨슨병(Parkinson) 등의 퇴행성 신경질환뿐 아니라 외상 및 허혈 상태에 따른 신경세포 손상에도 이러한 미세아교세포의 과민 활성화가 관련되어있다는 연구 보고가 나오고 있으며[Microglia as mediators of inflammatory and degenerative diseases. Gonzalez-Scarano, F and Baltuch, G.Recently, research reports suggest that hyperglia activation of microglia is involved in neurodegenerative diseases such as Alzheimer's and Parkinson's as well as traumatic and ischemic neuronal damage [Microglia as mediators of inflammatory and degenerative diseases. Gonzalez-Scarano, F and Baltuch, G.
Annu. Rev. Neurosci. 22:219-40 (1999)], 이들 활성화된 미세아교세포를 억제하거나 활성화된 미세아교세포가 분비하는 염증유발 물질들의 작용을 막는 치료법이 개발되고 있다.Annu. Rev. Neurosci. 22: 219-40 (1999)], therapeutics are being developed that inhibit these activated microglia or prevent the action of proinflammatory substances secreted by the activated microglia.
즉, 미세아교세포의 활성화로 대변되는 신경염증반응이 다양한 신경조직손상에 있어서 중요한 병리적 역할을 수행하며, 궁극적으로 이러한 미세아교세포의 염증 활성화를 저해함으로써 신경조직손상 억제방안을 모색할 수 있고, 임상적으로 적용할 수 있는 퇴행성 신경질환 예방 및 치료제의 개발이 가능할 것이다.That is, the neuroinflammatory response represented by the activation of microglia plays an important pathological role in various nerve tissue damages, and ultimately, it is possible to seek ways to inhibit nerve tissue damage by inhibiting the inflammatory activation of the microglia. Therefore, it will be possible to develop clinically applicable preventive and therapeutic agents for neurodegenerative diseases.
따라서, 본 발명의 조성물은 퇴행성 신경질환의 예방, 개선 또는 치료에 유용하게 이용될 수 있다.Therefore, the composition of the present invention can be usefully used for the prevention, improvement or treatment of degenerative neurological diseases.
본 발명의 일 구현예에 따르면, 상기 퇴행성 신경질환은 알츠하이머병(Alzheimer's disease), 파킨슨병(Parkinson's disease), 루게릭병(Lou Gehrig disease), 헌팅톤병(Huntington's disease), 다발성 경화증(Multiple sclerosis), 뇌졸중(stroke), 혈전증(thrombosis), 색전증(embolism), 두부손상(head trauma), 뇌순환대사장애, 뇌 기능혼수 또는 치매(dementia) 등이 있으나, 이에 제한되지 않는다.According to one embodiment of the present invention, the degenerative neurological disease is Alzheimer's disease, Alzheimer's disease, Parkinson's disease, Lou Gehrig disease, Huntington's disease, Multiple sclerosis, Multiple sclerosis, Stroke, thrombosis, embolism, head trauma, cerebral circulatory metabolic disorder, brain coma or dementia, but are not limited thereto.
본 발명의 명세서에서, 상기 퇴행성 신경질환은 지질다당류(lipopolysaccharide, LPS)에 의하여 유도된 것이거나, 글루타메이트에 의해 유발된 것일 수 있다.In the specification of the present invention, the neurodegenerative disease may be induced by lipopolysaccharide (LPS) or may be induced by glutamate.
본 발명의 일 구현예에 따르면, 본 발명은 퇴행성 신경질환의 예방 또는 치료용 약학 조성물을 제공한다.According to one embodiment of the present invention, the present invention provides a pharmaceutical composition for preventing or treating neurodegenerative diseases.
본 발명의 퇴행성 신경질환의 예방 또는 치료용 약학 조성물은 약학 조성물 총 중량에 대하여 상기 분획물을 0.02 내지 80 중량%, 바람직하게는 0.02 내지 50 중량%로 포함할 수 있다.The pharmaceutical composition for preventing or treating degenerative neurological diseases of the present invention may include the fraction in an amount of 0.02 to 80% by weight, preferably 0.02 to 50% by weight based on the total weight of the pharmaceutical composition.
본 발명의 약학 조성물은 약학적 조성물의 제조에 통상적으로 사용되는 적절한 담체, 부형제 및 희석제를 더 포함할 수도 있다.The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.
본 발명에 따른 조성물의 약학적 투여 형태는 이들의 약학적 허용 가능한 염의 형태로도 사용될 수 있고, 또한 단독으로 또는 타 약학적 활성 화합물과 결합 뿐만 아니라 적당한 집합으로 사용될 수 있다.The pharmaceutical dosage forms of the compositions according to the invention may be used in the form of their pharmaceutically acceptable salts, or may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.
본 발명에 따른 약학 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 본 발명의 약학 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조 혼합물 등을 들 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 약학 조성물에 적어도 하나 이상의 부형제, 예를 들면전분, 칼슘카보네이트, 수크로스 또는 락토오스, 젤라틴 등을 섞어 제조될 수 있다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 현탁제, 유제, 동결건조 제제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 오일, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔, 마크로골, 트윈 61, 카카오지, 라우진지, 글리세로제라틴 등이 사용될 수 있다.The pharmaceutical compositions according to the invention can be used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., respectively, according to conventional methods. Can be. Carriers, excipients and diluents that may be included in the pharmaceutical compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzo mixture, and the like. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in the pharmaceutical composition, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. It can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups. In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, suspensions, emulsions, lyophilized preparations and preservatives Etc. may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, utopsol, macrogol, tween 61, cacao butter, lauzin paper, glycerogelatin and the like can be used.
본 발명의 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물 형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. Preferred dosages of the compositions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art.
그러나, 바람직한 효과를 위해서, 본 발명의 분획물은 1일 0.0001 내지 100 mg/kg으로, 바람직하게는 0.001 내지 100 mg/kg으로 투여하는 것이 좋다. 가장 바람직하게는 50 내지 100 mg/kg으로 투여하는 것이다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.However, for the desired effect, the fraction of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. Most preferably from 50 to 100 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.
본 발명의 조성물은 쥐, 생쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면 경구, 직장 또는 정맥, 근육, 피하, 자궁내 경막 또는 뇌혈관 내 주사에 의해 투여될 수 있다.The composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections.
본 발명은 또한, 침향 분획물을 유효성분으로 포함하는 퇴행성 신경질환의 예방 또는 개선용 건강기능식품 조성물을 제공한다.The present invention also provides a nutraceutical composition for the prevention or improvement of degenerative neurological diseases comprising aroma fraction as an active ingredient.
상기 건강기능식품 조성물은 퇴행성 신경질환을 예방 또는 개선하기 위해 섭취할 수 있는 것이면 특별히 제한되지 않는다.The health functional food composition is not particularly limited as long as it can be ingested to prevent or improve degenerative neurological diseases.
본 발명의 상기 조성물을 식품 첨가물로 사용하는 경우, 상기 분획물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합양은 그의 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조시에 본 발명의 분획물은 원료에 대하여 15 중량부 이하, 바람직하게는 10 중량부 이하의 양으로 첨가된다. 그러나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.When the composition of the present invention is used as a food additive, the fraction may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient can be suitably determined according to the purpose of use (prevention, health or therapeutic treatment). Generally, in the preparation of food or beverages, the fractions of the invention are added in an amount of up to 15 parts by weight, preferably up to 10 parts by weight relative to the raw materials. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. .
상기 식품의 종류에는 특별한 제한은 없다. 상기 분획물을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농 제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 의미한다.There is no particular limitation on the kind of food. Examples of the food to which the fraction may be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, and the like means all of the health food in the conventional sense.
본 발명의 조성물을 건강음료로 사용할 경우, 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토스, 슈크로스와 같은 디사카라이드, 덱스트린, 사이클로텐스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100g 당 일반적으로 약 0.01~0.04g, 바람직하게는 약 0.02~0.03g 이다.When the composition of the present invention is used as a health beverage, various flavors or natural carbohydrates and the like may be contained as additional ingredients, as in general beverages. The above-mentioned natural carbohydrates are sugars such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclotensin, xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, and synthetic sweetening agents such as saccharin and aspartame can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 g of the composition of the present invention.
상기 외에 본 발명의 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 박에 본 발명의 조성물은 천연 과일쥬스, 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 혼합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 조성물은 100 중량부당 0.01~0.1 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, And carbonating agents used in carbonated drinks. In the foil, the composition of the present invention may contain a natural fruit juice, a pulp for producing a vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical, but the composition of the present invention is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight.
이하, 본 발명을 실시예를 통하여 상세히 설명한다. 그러나 이들 실시예는 본 발명을 예시하기 위한 것으로, 본 발명의 권리범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않으며, 본 발명의 사상과 범위 내에서 여러 가지 변형 또는 수정할 수 있음은 당업자에게 자명한 것이다.Hereinafter, the present invention will be described in detail through examples. However, these examples are intended to illustrate the present invention, and the scope of the present invention is not to be construed as limited by these embodiments, and various modifications or changes can be made within the spirit and scope of the present invention to those skilled in the art. It is self-evident.
실시예 Example
실시예 1. 침향 분획물 분리Example 1 Isolation of Aroma Fraction
1-1. 시료조제1-1. Sample Preparation
침향(Aquilariae Lignum)은 식품의약품안전처의 승인을 받은 herbal pharmaceutical company(Dae Han Bio Pharm Inc., Gyeonggi-do, Korea)로부터 수득한 분말상 침향을 사용하였다. Aquilariae Lignum used powdered aromas obtained from an herbal pharmaceutical company (Dae Han Bio Pharm Inc., Gyeonggi-do, Korea), which was approved by the Ministry of Food and Drug Safety.
먼저, 분말 침향 10g을 30% 에탄올 200mL로 실온(25℃)에서 72시간 동안 교반추출 하였다. 교반 후 원심분리기(5000rpm 30min)에서 침전물을 가라앉힌 후 상층액은 300 메쉬의 필터페이퍼(fillter paper: Advantac, Tokyo Roshi Kaisah, Tokyo, Japan) 50mm로 여과하고, 여과된 액체(fillterate)는 순환식 증발기에서 농축한 후 동결건조 하였다. 최종 추출물의 수율은 6.42% (W/W) 이었으며, 이후 사용을 위해 -80℃에 보관하였다.First, 10g of the powdered aroma was extracted with 200mL of 30% ethanol at room temperature (25 ° C) for 72 hours. After stirring, the precipitate was allowed to settle in a centrifuge (5000 rpm 30 min), and the supernatant was filtered with 300 mm of filter paper (Advantac, Tokyo Roshi Kaisah, Tokyo, Japan), and the filtered liquid was circulated. It was concentrated on an evaporator and then lyophilized. The yield of the final extract was 6.42% (W / W) and stored at −80 ° C. for later use.
1-2. 침향의 methylene chloride 추출물로부터 분획물의 분리(1-2. Fractionation of Fraction from Methylene Chloride Extract of Aroma Aquilariae LignumAquilariae lignum methylene chloride methylene chloride extract & separation of fraction layer)extract & separation of fraction layer)
상기 1-1에서 제조한 침향 추출물 분말 5g을 200g의 메틸렌클로라이드(methylene chloride)와 혼합한 후 상온에서 24시간 동안 교반하여 용매 추출하였다. 상기 침향의 메틸렌클로라이드 추출물을 0.2 μm의 나일론 필터로 여과하고, 메틸렌클로라이드로 2번 세척한 뒤 여과액을 수집하였다. 수집한 여과액을 다시 100mL의 증류수에 용해시킨 후, 수용액층은 제거하고, 메틸렌클로라이드층은 황산나트륨으로 건조시켰다. 회전농축기로 휘발성분을 제거하여 황색의 농축액(메틸렌클로라이드 추출물) 0.62g을 획득하였다. 5 g of the aroma extract powder prepared in 1-1 was mixed with 200 g of methylene chloride, and then stirred at room temperature for 24 hours to extract a solvent. The methylene chloride extract of the aroma was filtered through a 0.2 μm nylon filter, washed twice with methylene chloride, and the filtrate was collected. The collected filtrate was dissolved in 100 mL of distilled water again, the aqueous layer was removed, and the methylene chloride layer was dried over sodium sulfate. Volatile components were removed using a rotary concentrator to obtain 0.62 g of a yellow concentrate (methylene chloride extract).
상기 메틸렌클로라이드 추출물 1mg을 1mL의 100% 메탄올에 용해시킨 뒤 0.2 μm의 나일론 필터로 여과하였다. 그리고 10μL의 여과액을 C18 컬럼을 이용한 HPLC 분석기기에 주입하여 지문분석을 실시하고, 그 결과를 도 2에 나타내었다. 1 mg of the methylene chloride extract was dissolved in 1 mL of 100% methanol and filtered through a 0.2 μm nylon filter. 10 μL of the filtrate was injected into an HPLC analyzer using a C18 column to conduct fingerprint analysis, and the results are shown in FIG. 2.
이때, 컬럼 유동률은 1mL/min이고, 이동상 조건은 (A) 증류수와 (B) 아세토니트릴을 이용하여 다음과 같이 실시하였다: 0 to 0.01 min, 30 % B (등용매 용리); 0.01 to 10 min, 0.01 to 40 % B (선형 기울기 용리); 10 to 15 min, 40 to 50 % B (선형 기울기 용리); 15 to 30 min, 50 to 60 % B (선형 기울기 용리); 30 to 40 min, 100 % B (선형 기울기 용리); and 40 to 50 min (등용매 용리). 이것을 UV 검출기로 검출하였다. 그 결과 총 280 nm UV 파장에서 5개의 메인 피크가 변동계수 0.024%로, 머무름 시간(retention time) 8.13 분(A 분획물), 13.80 분(B 분획물), 17.70 분(C 분획물), 20.92 분(D 분획물) 및 28.52 분(E 분획물)에 검출되었다. At this time, the column flow rate was 1 mL / min, and the mobile phase conditions were carried out using (A) distilled water and (B) acetonitrile as follows: 0 to 0.01 min, 30% B (isosolvent eluting); 0.01 to 10 min, 0.01 to 40% B (linear gradient elution); 10 to 15 min, 40 to 50% B (linear gradient elution); 15 to 30 min, 50 to 60% B (linear gradient elution); 30 to 40 min, 100% B (linear gradient elution); and 40 to 50 min (isosolvent elution). This was detected by a UV detector. As a result, the five main peaks at a total 280 nm UV wavelength with a coefficient of variation of 0.024%, retention time 8.13 minutes (A fraction), 13.80 minutes (B fraction), 17.70 minutes (C fraction), 20.92 minutes (D Fractions) and 28.52 minutes (E fraction).
또한, 상기 메틸렌클로라이드 추출물의 활성성분을 동정하기 위한 단계로 먼저 분취 액체크로마토그래피(preparative LC(이하 prep. LC))를 이용하여, 상기 주요 5개(A, B, C, D 및 E 분획물)의 HPLC 피크들 중 가장 유효성이 뛰어난 E 분획물을 분리하고자 하였다.In addition, the first five (A, B, C, D and E fractions) using preparative LC (prep. LC) as a step for identifying the active ingredient of the methylene chloride extract We tried to separate the most effective E fractions of the HPLC peaks of.
이를 위하여, 먼저 75mg의 메틸렌클로라이드 추출물을 1.5mL의 메탄올에 용해시켜 0.2 μm의 나일론 필터로 여과하였다. 그리고 상기 여과액 2000μL를 prep. LC 기기에 주입하였다. 이때, 컬럼 유동률은 30 mL/min으로 하고, 이동상 조건은 (A) 증류수와 (B) 메탄올을 이용하여 다음과 같이 실시하였다: 0 to 0.01 min, 50 % B (등용매 용리); 0.01 to 10 min, 0.01 to 55 % B (선형 기울기 용리); 10 to 15 min, 55 to 65 % B (선형 기울기 용리); 15 to 30 min, 65 to 75 % B (선형 기울기 용리); 30 to 40 min, 100 % B (선형 기울기 용리); and 40 to 50 min (등용매 용리). 이것은 광다이오드 어레이 검출기에서 190-700nm의 파장으로 검출하였다. 그리고, 회전 농축기로 휘발성분을 제거한 뒤 황색의 E 분획물 2.2mg을 획득하였다. For this purpose, 75 mg of methylene chloride extract was first dissolved in 1.5 mL of methanol and filtered through a 0.2 μm nylon filter. And prep. 2000 μL of the filtrate. Injection into the LC instrument. At this time, the column flow rate was 30 mL / min, and the mobile phase conditions were carried out using (A) distilled water and (B) methanol as follows: 0 to 0.01 min, 50% B (isosolvent eluting); 0.01 to 10 min, 0.01 to 55% B (linear gradient elution); 10 to 15 min, 55 to 65% B (linear gradient elution); 15 to 30 min, 65 to 75% B (linear gradient elution); 30 to 40 min, 100% B (linear gradient elution); and 40 to 50 min (isosolvent elution). This was detected at a wavelength of 190-700 nm with a photodiode array detector. Then, 2.2 mg of a yellow E fraction was obtained after removing the volatile component using a rotary concentrator.
실험예 Experimental Example
실험예 1. 용매별 침향 추출물의 세포흥분독성에 대한 세포사멸 억제효능Experimental Example 1. Inhibitory effect on apoptosis of aroma extracts by solvent
마우스 해마 세포주(HT-22 cell)을 이용하여 평가하였으며 배지는 10% (v/v) fetal bovine serum (FBS), 1% (v/v) penicillin을 함유한 DMEM을 사용하였고, 배양은 37℃, 5% CO2, 95% humid air로 조절된 배양기를 사용하였다. 시료의 안전성 농도 범위에서 시험을 실행하기 위해 세포독성은 Rochem 등의 방법을 변형하여 WST-8과 LDH를 측정하였다. 96 wells plate에 4 X 103 cells/well의 세포를 분주한 후 12 시간동안 배양하였다. 이후 침향의 메틸렌클로라이드(MC), 에틸아세테이트(EA), 레스트(Rest) 추출물 각각을 25㎍/㎖의 최종농도로 분주하고 재배양을 2시간 동안 하였다. 이후 최종농도 글루타메이트 20mM를 처리하여 20 시간 후 세포사멸을 유도하였다. 각 용매추출물의 세포사멸 억제효과를 평가하기 위해 WST-8 용액 20μL을 첨가하고 37℃에서 1시간 30분 배양한 뒤, UV spectrometer 450nm의 흡광도로 측정하였으며, 그 결과를 도 3에 나타내었다.The mouse hippocampal cell line (HT-22 cell) was used to evaluate the medium was used DMEM containing 10% (v / v) fetal bovine serum (FBS), 1% (v / v) penicillin, the culture was 37 ℃ , 5% CO 2, 95% humidified air incubator was used. Cytotoxicity was measured by modifying the method of Rochem et al. WST-8 and LDH to run the test in the safe concentration range of the sample. 4 x 10 3 cells / well were dispensed into 96 wells plate and incubated for 12 hours. Afterwards, methylene chloride (MC), ethyl acetate (EA), and rest (Rest) extracts of aroma were dispensed at a final concentration of 25 µg / ml and cultured for 2 hours. Thereafter, the final concentration of glutamate was treated with 20 mM to induce apoptosis after 20 hours. In order to evaluate the apoptosis inhibitory effect of each solvent extract was added 20μL of WST-8 solution and incubated for 1 hour 30 minutes at 37 ℃, was measured by absorbance of 450nm UV spectrometer, the results are shown in FIG.
도 3은 용매별 추출물의 흥분성 신경전달물질 (glutamate)로 유도된 마우스 해마 신경세포주 (HT-22 cell) 세포흥분독성 사멸에 대한 보호효과를 나타내는 그래프이다.Figure 3 is a graph showing the protective effect of the excitatory neurotransmitter (glutamate) induced mouse hippocampal neuronal cell line (HT-22 cell) cell excitability killing of the solvent-specific extracts.
도 3을 살펴보면, 메틸렌클로라이드(MC) 추출물이 신경세포독성에 의한 세포사멸(아팝토시스) 억제 효과가 가장 뛰어난 것을 알 수 있다. Looking at Figure 3, it can be seen that methylene chloride (MC) extract is the most excellent effect of inhibiting apoptosis (apoptosis) by neurocytotoxicity.
실험예 2. 침향 A~E 분획물의 세포흥분독성에 대한 세포사멸 억제효능Experimental Example 2. Inhibitory effect of apoptosis on cell excitability of Aroma A ~ E fraction
마우스 해마 세포주(HT-22 cell)을 이용하여 평가하였으며 배지는 10% (v/v) fetal bovine serum (FBS), 1% (v/v) penicillin을 함유한 DMEM을 사용하였고, 배양은 37℃, 5% CO2, 95% humid air로 조절된 배양기를 사용하였다. 시료의 안전성 농도 범위에서 시험을 실행하기 위해 세포독성은 Rochem 등의 방법을 변형하여 WST-8과 LDH를 측정하였다. 96 wells plate에 4 X 103 cells/well의 세포를 분주한 후 12 시간동안 배양하였다. 이후 A~E 분획물을 각각 10 ㎍/㎖의 최종농도로 분주하고 재배양을 2시간 동안 하였다. 이후 최종농도 글루타메이트 20mM를 처리하여 20 시간 후 세포사멸을 유도하였다. 각 분획물의 세포사멸 억제효과를 평가하기 위해 WST-8 용액 20μL을 첨가하고 37℃에서 1시간 30분 배양한 뒤, UV spectrometer 450nm의 흡광도로 측정하였다. 또한 세포사멸시 유리되는 LDH의 활성을 측정하기 위해 배양 상층액을 이용하여 시약과 37℃에서 15분간 반응하고 440nm 흡광도로 측정하였으며, 그 결과를 도 4에 나타내었다. The mouse hippocampal cell line (HT-22 cell) was used to evaluate the medium was used DMEM containing 10% (v / v) fetal bovine serum (FBS), 1% (v / v) penicillin, the culture was 37 ℃ , 5% CO 2, 95% humidified air incubator was used. Cytotoxicity was measured by modifying the method of Rochem et al. WST-8 and LDH to run the test in the safe concentration range of the sample. 4 x 10 3 cells / well were dispensed into 96 wells plate and incubated for 12 hours. The A-E fractions were then dispensed at a final concentration of 10 μg / ml and cultured for 2 hours. Thereafter, the final concentration of glutamate was treated with 20 mM to induce apoptosis after 20 hours. In order to evaluate the effect of inhibiting apoptosis of each fraction, 20μL of WST-8 solution was added and incubated for 1 hour and 30 minutes at 37 ° C, and then measured by absorbance at 450 nm of UV spectrometer. In addition, in order to measure the activity of LDH liberated upon cell death, the reaction was carried out at 37 ° C. for 15 minutes using a culture supernatant and measured at 440 nm absorbance. The results are shown in FIG. 4.
도 4는 침향 메틸렌클로라이드 추출물에서 분리한 임의의 5가지 분획물의 흥분성 신경전달물질(glutamate)로 유도된 마우스 해마신경세포주 (HT-22 cell) 세포흥분독성 사멸에 대한 보호효과를 나타내는 그래프이다. Figure 4 is a graph showing the protective effect against the excitatory neurotransmitter (HT-22 cell) cell excitability killing induced by excitatory neurotransmitter (glutamate) of any five fractions isolated from the aroma methylene chloride extract.
상기 도 4를 보면, 침향 E 분획물이 신경세포독성에 대한 세포사멸(아팝토시스) 억제 효과가 가장 뛰어났다. Referring to FIG. 4, the A-fraction E fraction was most effective in inhibiting apoptosis on neurocytotoxicity.
실험예 3. 침향 E 분획물의 세포흥분독성에 대한 농도별 세포사멸 억제효능Experimental Example 3. Inhibitory effect of apoptosis E fraction on cell excitability by concentration
마우스 해마 세포주 (HT-22 cell)을 이용하여 평가하였으며 배지는 10% (v/v) fetal bovine serum (FBS), 1% (v/v) penicillin, 함유한 DMEM을 사용하였고, 배양은 37℃, 5% CO2, 95% humid air로 조절된 배양기를 사용하였다. 시료의 안전성 농도 범위에서 시험을 실행하기 위해 세포독성은 Rochem 등의 방법을 변형하여 WST-8과 LDH를 측정하였다. 96 wells plate에 4 X 103 cells/well의 세포를 분주한 후 12 시간동안 배양하였다. 이후 침향 E 분획물을 1.25, 2.5, 5㎍/㎖의 최종농도로 분주하고 재배양을 2시간 동안 하였다. 이후 최종농도 Glutamate 20mM를 처리하여 20 시간 후 세포사멸을 유도하였다. E 분획물의 세포사멸 억제효과를 평가하기 위해 WST-8 용액 20μL을 첨가하고 37℃에서 1시간 30분 배양한 뒤, UV spectrometer 450nm의 흡광도로 측정하였다. 또한 세포사멸시 유리되는 LDH의 활성을 측정하기 위해 배양 상층액을 이용하여 시약과 37℃에서 15분간 반응하고 440nm 흡광도로 측정하였으며, 그 결과를 도 5에 나타내었다. The mouse hippocampal cell line (HT-22 cell) was used to evaluate the medium was used DMEM containing 10% (v / v) fetal bovine serum (FBS), 1% (v / v) penicillin, the culture was 37 ℃ , 5% CO 2, 95% humidified air incubator was used. Cytotoxicity was measured by modifying the method of Rochem et al. WST-8 and LDH to run the test in the safe concentration range of the sample. 4 x 10 3 cells / well were dispensed into 96 wells plate and incubated for 12 hours. Aroma E fractions were then dispensed at a final concentration of 1.25, 2.5, 5 μg / ml and cultured for 2 hours. After treatment with a final concentration of Glutamate 20mM induced apoptosis after 20 hours. In order to evaluate the apoptosis inhibitory effect of E fraction, 20μL of WST-8 solution was added and incubated for 1 hour and 30 minutes at 37 ° C, and then measured by absorbance at 450 nm of UV spectrometer. In addition, in order to measure the activity of LDH released during cell death, the reaction was carried out at 37 ° C. for 15 minutes using a culture supernatant, and measured by absorbance at 440 nm. The results are shown in FIG. 5.
도 5a은 본 발명의 일 실시예에 의한 침향 E 분획물이 흥분성 신경전달물질(glutamate)로 유도된 마우스 해마 신경세포주(HT-22 cell)의 세포흥분독성에 대하여 세포사멸을 억제하는 것을 나타내는 그래프이고, 도 5b는 본 발명의 일 실시예에 의한 침향 E 분획물이 흥분성 신경전달물질(glutamate)로 유도된 마우스 해마 신경세포주(HT-22 cell)에서 Lactate dehydrogenase(LDH)의 세포 외 유출을 억제하는 것을 나타내는 그래프이다. Figure 5a is a graph showing that the acupuncture E fraction according to an embodiment of the present invention inhibits apoptosis against cytotoxicity of mouse hippocampal neuronal cell line (HT-22 cell) induced by excitatory neurotransmitter (glutamate) FIG. 5B shows that the Aroma E fraction according to one embodiment of the present invention inhibits extracellular leakage of Lactate dehydrogenase (LDH) in mouse hippocampal neuronal cell line (HT-22 cells) induced with excitatory neurotransmitter (glutamate). It is a graph.
실험예 4. 침향 E 분획물의 세포흥분독성에 의한 ROS 생성 억제 효능Experimental Example 4. Inhibitory effect of ROS generation by cytotoxicity of Aroma E fraction
HT-22 세포를 4 X 103 cells/well으로 6 well plate에 분주하여 12시간 동안 배양하였다. E 분획물 5μg/mL 농도로 2시간 처리한 후 세포흥분독성을 glutamate 20mM 처리하여 유도하였다. 6시간 배양 후, DCFH-DA 시약을 10μM 처리하고 37℃에서 30분간 반응한 뒤 ROS 수준을 형광현미경으로 관찰하였다. 그 결과는 도 6에 나타내었다. HT-22 cells were injected into 6 well plates at 4 × 10 3 cells / well and incubated for 12 hours. Cellular excitatory toxicity was induced by treatment with glutamate 20 mM after 2 hours treatment with E fraction at 5 μg / mL. After 6 hours of incubation, the DCFH-DA reagent was treated with 10 μM and reacted at 37 ° C. for 30 minutes, and then ROS levels were observed under a fluorescence microscope. The results are shown in FIG.
도 6a은 glutamate로 유도된 HT-22 cell에서 ROS 수준을 DCFH-DA 세포화학형광기법으로 평가한 실험에서, 침향 E 분획물의 ROS 생성 억제효능을 나타내는 사진이고, 도 6b는 상기 실험에서, 침향 E 분획물의 ROS 생성 억제 효능을 나타내는 그래프이다. Figure 6a is a photograph showing the inhibitory effect of ROS generation of the fragrance E fraction in experiments in which ROS level was evaluated by DCFH-DA cytochemistry fluorescence technique in glutamate-induced HT-22 cells, Figure 6b is in the experiment, It is a graph showing the effect of inhibiting the ROS production of the fraction.
실험예 5. 침향 E 분획물의 세포흥분독성에 의한 세포 사멸(apoptosis) 억제 효능Experimental Example 5. Inhibition of Apoptosis by Cytotoxicity of Aroma E Fraction
HT-22 세포를 5 X 104 cells/mL으로 100Φ Dish에 분주하여 12시간 동안 배양하였다. E 분획물 5μg/mL 농도로 2시간 처리한 후 세포흥분독성을 glutamate 20mM 처리하여 유도하였다. 10시간 배양 후, 원심분리기 (1300rpm, 3min)에서 cell을 모은 후 1X Annexin V binding buffer 500μL를 첨가하여 녹이고 얼음에 보관한다. Annexin V 1μL 와 PI 0.5μL 처리하여 유세포 분석기(FACs)를 이용해 HT-22 세포의 사멸을 확인하였다. 그 결과는 도 7에 나타내었다. HT-22 cells were aliquoted at 100 x Dish at 5 X 10 4 cells / mL and incubated for 12 hours. Cellular excitatory toxicity was induced by treatment with glutamate 20 mM after 2 hours treatment with E fraction at 5 μg / mL. After incubation for 10 hours, the cells are collected in a centrifuge (1300rpm, 3min), dissolved by adding 500μL of 1X Annexin V binding buffer and stored on ice. Treatment with anneal V 1μL and PI 0.5μL confirmed the death of HT-22 cells using flow cytometry (FACs). The results are shown in FIG.
도 7a는 침향 E 분획물의 흥분성 신경전달물질(glutamate)로 유도된 마우스 해마 신경세포주(HT-22 cell) 사멸을 유세포 분석기(FACs)로 평가한 실험에서, 본 발명의 침향 E 분획물이 Annexin V/PI 신호를 억제함에 따라 세포 사멸이 억제되는 것을 나타내는 그래프이고, 도 7b는 상기 실험에서, 사멸된 세포의 수를 정량화하여 나타내는 그래프이다. FIG. 7A shows that in the experiment evaluating the death of the mouse hippocampal neuronal cell line (HT-22 cell) induced by excitatory neurotransmitter (glutamate) of the fragrance E fraction by flow cytometry (FACs), the fragrance E fraction of the present invention was Annexin V / It is a graph showing that cell death is inhibited by inhibiting the PI signal, Figure 7b is a graph showing the quantified number of killed cells in the experiment.
실험예 6. 침향 E 분획물의 세포흥분독성에 의한 DNA fragmentation 억제 효능Experimental Example 6. Inhibitory effect of DNA fragmentation on cell excitability of Aroma F fraction
HT-22 세포를 4 X 103 cells/well으로 6 well plate에 분주하여 12시간 동안 배양하였다. E 분획물 5μg/mL 농도로 2시간 처리한 후 세포흥분독성을 glutamate 20mM 처리하여 유도하였다. 8시간 배양 후, 4% paraformaldehyde로 세포고정 후 Hoechst33528 시약을 10μM, PI 10μg/mL 농도로 처리하여 HT-22 세포 DNA fragmentation을 확인하였다. 그 결과를 도 8에 나타내었다. HT-22 cells were injected into 6 well plates at 4 × 10 3 cells / well and incubated for 12 hours. Cellular excitatory toxicity was induced by treatment with glutamate 20 mM after 2 hours treatment with E fraction at 5 μg / mL. After 8 hours of incubation, the cells were fixed with 4% paraformaldehyde and treated with Hoechst33528 reagent at a concentration of 10 μM and PI 10 μg / mL to confirm HT-22 cell DNA fragmentation. The results are shown in FIG.
도 8a는 glutamate로 유도된 HT-22 cell에서의 DNA fragmentation 수준을 Hoechst33528 형광 현미경으로 평가한 실험으로, 침향 E 분획물이 신경세포의 사멸을 억제한다는 것을 나타내는 사진이고, 도 8b는 상기 실험에서, 신경세포 DNA의 분절(fragmentation) 정도를 정량화하여 나타내는 그래프이다. FIG. 8A is an experiment evaluating DNA fragmentation level in glutamate-induced HT-22 cells by Hoechst33528 fluorescence microscopy. FIG. 8A is a photograph showing that acupuncture E fraction inhibits neuronal cell death. FIG. It is a graph quantifying the degree of fragmentation of cellular DNA.
실험예 7. 침향 E 분획물의 세포흥분독성에 의한 세포 내 세포사멸기전Experimental Example 7 Intracellular Apoptosis Mechanism by Cytotoxicity of Aroma E Fraction
HT-22 세포를 5 X 104 cells/mL으로 100Φ Dish에 분주하여 12시간 동안 배양하였다. 침향 E 분획물을 5μg/mL 농도로 2시간 처리한 후 세포흥분독성을 glutamate 20mM 처리하여 유도하였다. 상기 세포를 8 시간 배양 후, ERK의 단백질 발현과 인산화 정도를 웨스턴 블랏 기법을 이용하여 평가하였고, 그 결과를 도 9에 나타내었다. HT-22 cells were aliquoted at 100 x Dish at 5 X 10 4 cells / mL and incubated for 12 hours. Aromatosis E fractions were treated for 2 hours at a concentration of 5 μg / mL and cell excitability was induced by glutamate 20 mM treatment. After culturing the cells for 8 hours, protein expression and phosphorylation of ERK were evaluated using Western blot technique, and the results are shown in FIG. 9.
도 9는 glutamate로 유도된 HT-22 cell에서의 세포사멸기전을 웨스턴블랏 기법을 이용하여 평가한 실험에서, 침향 E 분획물이 ERK의 인산화를 방지하여 세포사멸을 억제한다는 것을 나타내는 그래프이다. 9 is a graph showing that acupuncture E fraction inhibits apoptosis by preventing phosphorylation of ERK in an experiment evaluating apoptosis in glutamate-induced HT-22 cells using Western blot technique.
실험예 8. 침향 E 분획물의 신경세포 산화 손상에 대한 생존률Experimental Example 8. Survival Rate for Neuronal Oxidative Damage of Aroma E Fraction
마우스 해마 세포주 (HT-22 cell)을 이용하여 평가하였으며 배지는 10% (v/v) fetal bovine serum (FBS), 1% (v/v) penicillin, 함유한 DMEM을 사용하였고, 배양은 37℃, 5% CO2, 95% humid air로 조절된 배양기를 사용하였다. 시료의 안전성 농도 범위에서 시험을 실행하기 위해 세포독성은 Rochem 등의 방법을 변형하여 WST-8과 LDH를 측정하였다. 96 wells plate에 4 X 103 cells/well의 세포를 분주한 후 12 시간동안 배양하였다. 이후 침향 E 분획물을 5㎍/㎖의 최종농도로 분주하고 재배양을 2시간 동안 하였다. 이후 최종농도 glutamate 20mM를 처리하여 20 시간 후 세포사멸을 유도하였다. E 분획물의 세포사멸 억제효과를 평가하기 위해 WST-8 용액 20μL을 첨가하고 37℃에서 1시간 30분 배양한 뒤, UV spectrometer 450nm의 흡광도로 측정하였다. 또한 세포사멸 시 유리되는 LDH의 활성을 측정하기 위해 배양 상층액을 이용하여 시약과 37℃에서 15분간 반응하고 440nm 흡광도로 측정하였다. 그 결과는 도 10 및 11에 나타내었다. The mouse hippocampal cell line (HT-22 cell) was used to evaluate the medium was used DMEM containing 10% (v / v) fetal bovine serum (FBS), 1% (v / v) penicillin, the culture was 37 ℃ , 5% CO 2, 95% humidified air incubator was used. Cytotoxicity was measured by modifying the method of Rochem et al. WST-8 and LDH to run the test in the safe concentration range of the sample. 4 x 10 3 cells / well were dispensed into 96 wells plate and incubated for 12 hours. Aroma E fractions were then dispensed at a final concentration of 5 μg / ml and cultured for 2 hours. The final concentration of glutamate 20mM was then treated to induce cell death after 20 hours. In order to evaluate the apoptosis inhibitory effect of E fraction, 20μL of WST-8 solution was added and incubated for 1 hour and 30 minutes at 37 ° C, and then measured by absorbance at 450 nm of UV spectrometer. In addition, in order to measure the activity of LDH released during apoptosis, the culture supernatant was reacted with a reagent at 37 ° C. for 15 minutes and measured at 440 nm absorbance. The results are shown in FIGS. 10 and 11.
도 10은 침향 E 분획물이 Hydrogen peroxide(H2O2)로 유도된 마우스 해마 신경세포주(HT-22 cell)에서 항산화 작용을 하여 세포사멸을 억제한다는 것을 나타내는 그래프이다. FIG. 10 is a graph showing that aroma E fraction inhibits apoptosis by antioxidant activity in mouse hippocampal neuronal cell line (HT-22 cell) induced by Hydrogen peroxide (H 2 O 2 ).
도 11은 침향 E 분획물이 Hydrogen peroxide(H2O2)로 유도된 마우스 해마 신경세포주(HT-22 cell)에서 Lactate dehydrogenase(LDH)의 세포 외 유출을 억제하는 것을 나타내는 그래프이다. FIG. 11 is a graph showing that the A-fraction E fraction inhibits the extracellular leakage of Lactate dehydrogenase (LDH) in mouse hippocampal neuronal cell line (HT-22 cell) induced with Hydrogen peroxide (H 2 O 2 ).
실험예 9. 침향 E 분획물의 신경세포 염증 반응에 대한 NO 생성 억제Experimental Example 9. Inhibition of NO Production in Neuronal Inflammatory Response of Aroma E Fraction
마우스 미세아교세포주 (BV2 cell)을 이용하여 평가하였으며 배지는 10% (v/v) fetal bovine serum (FBS), 1% (v/v) penicillin, 함유한 DMEM을 사용하였고, 배양은 37℃, 5% CO2, 95% humid air로 조절된 배양기를 사용하였다. NO assay는 griess 시약을 이용하여 측정하였고, 이의 단백질 1mg당 농도를 정량하기 위해 Bradford 시약을 이용하여 측정한 단백질 정량값으로 보정하였다. 96 wells plate에 4 × 104 cells/well의 세포를 분주한 후 12 시간동안 배양하였다. 이후 E 분획물을 5㎍/㎖의 최종농도로 분주하고 재배양을 2시간 동안 하였다. 이후 최종농도 LPS 1㎍/mL을 처리하여 24 시간 후 미세아교세포의 과활성을 유도하였다. 과활성 시 유리되는 NO의 농도를 측정하기 위해 배양 상층액을 griess 시약과 37℃에서 30분간 반응하고 540nm 흡광도로 측정하였다. 그 결과는 도 12에 나타내었다. The mouse microglia (BV2 cell) was used to evaluate the medium. DMEM containing 10% (v / v) fetal bovine serum (FBS), 1% (v / v) penicillin, and DMEM were used. An incubator controlled with 5% CO 2, 95% humid air was used. NO assay was measured using griess reagent, and corrected by protein quantitative value measured by Bradford reagent to quantify the concentration of the protein per mg. 4 x 10 4 cells / well were dispensed into 96 wells plates and incubated for 12 hours. The E fractions were then dispensed at a final concentration of 5 μg / ml and cultured for 2 hours. Thereafter, the final concentration of LPS was treated with 1 µg / mL to induce hyperactivity of the microglia after 24 hours. In order to measure the concentration of NO released during overactivity, the culture supernatant was reacted with griess reagent for 30 minutes at 37 ° C. and measured with 540 nm absorbance. The results are shown in FIG.
도 12a은 침향 E 분획물이 마우스 미세아교신경세포주(BV2 cell)에서 Lipopolysaccharide(LPS)로 유도된 Nitric oxide(NO)의 증가를 억제한다는 것을 나타내는 그래프이고, 도 12b는 상기 실험에서 나타낸 결과에 대한 단백질 정량값을 나타낸 그래프이다. Figure 12a is a graph showing that the aroma E fraction inhibits the increase of Lipopolysaccharide (LPS) -induced Nitric oxide (NOS) in mouse microglia neuronal cell lines (BV2 cells), Figure 12b is a protein for the results shown in the experiment A graph showing quantitative values.
상기 실험예들을 통해, 본 발명에 따른 침향 분획물이 항산화 작용, 신경세포의 아팝토시스(apoptosis) 억제 작용, 뇌신경 스트레스 저해 및 신경세포 보호 작용, 신경세포의 활성산소종(reactive oxygen species:ROS) 생성 억제 작용, 세포외 신호조절인산화효소(ERK; extracellular signal-regulated kinases)의 인산화 수준 감소 작용, 신경세포의 일산화질소(NO)의 생성 억제 작용 및 신경세포의 DNA 분절 저해 작용 등을 나타내는 것을 구체적으로 확인할 수 있다. Through the above experimental examples, the aroma fraction of the present invention, the anti-oxidant action, the inhibitory action of apoptosis (apoptosis) of nerve cells, neuronal stress inhibition and neuronal cell protective action, neuronal reactive oxygen species (ROS) Inhibits production, decreases the phosphorylation level of extracellular signal-regulated kinases (ERK), inhibits the production of nitric oxide (NO) in neurons, and inhibits DNA fragmentation in neurons. You can check with
비록 본 발명이 상기에 언급된 바람직한 실시예로서 설명되었으나, 발명의 요지와 범위로부터 벗어남이 없이 다양한 수정이나 변형을 하는 것이 가능하다. 또한, 첨부된 특허청구범위는 본 발명의 요지에 속하는 이러한 수정이나 변형을 포함한다.Although the present invention has been described as the preferred embodiment mentioned above, it is possible to make various modifications or variations without departing from the spirit and scope of the invention. Also, the appended claims cover such modifications and variations as fall within the spirit of the invention.
본 발명에 따른 침향 분획물은 해마 신경세포의 흥분독성을 억제하고, 뇌 산화 손상에 의한 세포사멸을 억제하며, 미세아교세포의 염증반응을 저하시키는 등 신경세포 보호 효과가 탁월하므로, 퇴행성 신경질환의 예방 또는 치료에 우수한 효과가 기대된다.The fragrance fraction according to the present invention inhibits excitatory toxicity of hippocampal neurons, inhibits apoptosis due to brain oxidative damage, and lowers the inflammatory response of microglia, and thus has excellent neuronal protective effects. Excellent effects are expected for prevention or treatment.
이에 따라, 본 발명의 침향 분획물을 유효성분으로 포함하는 조성물은 신경세포 손상 및 사멸에 따른 퇴행성 신경 질환, 특히 중추신경계 질환의 예방, 개선 또는 치료에 유용하게 이용될 수 있다.Accordingly, the composition comprising the fragrance fraction of the present invention as an active ingredient may be usefully used for the prevention, improvement or treatment of degenerative neurological diseases, especially central nervous system diseases caused by neuronal damage and death.

Claims (17)

  1. 침향 추출물의 분획물을 유효성분으로 포함하는 퇴행성 신경질환의 예방 또는 치료용 약학 조성물.Pharmaceutical composition for the prevention or treatment of degenerative neurological diseases comprising a fraction of the aroma extract as an active ingredient.
  2. 제1항에 있어서,The method of claim 1,
    상기 침향은 아킬라리아(Aquilaria) 속 식물로부터 유래된 것임을 특징으로 하는 퇴행성 신경질환의 예방 또는 치료용 약학 조성물.The aroma is a pharmaceutical composition for the prevention or treatment of degenerative neurological disease, characterized in that derived from Aquilaria ( Aquilaria ) plants.
  3. 제2항에 있어서,The method of claim 2,
    상기 아킬라리아(Aquilaria) 속 식물은 아킬라리아 카시아나(A. khasiana), 아킬라리아 아피쿠리나(A. apiculina), 아킬라리아 블리로닐(A. blillonil), 아킬라리아 바네온시스(A. baneonsis), 아킬라리아 브라키안타(A. brachyantha), 아킬라리아 컨밍이아나(A. cunmingiana), 아킬라리아 필라리아(A. filaria), 아킬라리아 그란디플로라(A. grandiflora), 아킬라리아 힐라타(A. hilata), 아킬라리아 마이크로카파(A. microcapa), 아킬라리아 로스트라타(A. rostrata), 아킬라리아 아갈로차(A. agallocha), 아킬라리아 말라센시스(A. malaccensis), 아킬라리아 시넨시스(A. sinensis) 또는 아킬라리아 크라사나(A. crassna)인 것을 특징으로 하는 퇴행성 신경질환의 예방 또는 치료용 약학 조성물.The plant of the genus Aquilaria is A. khasiana, A. apiculina, A. blillonil, A. baneonsis ), A. brachyantha, A. cunmingiana, A. filaria, A. grandiflora, Aquila hilara (A) hilata, A. microcapa, A. rostrata, A. agallocha, A. malaccensis, Achilla sinensis A pharmaceutical composition for the prevention or treatment of degenerative neurological diseases, characterized in that (A. sinensis) or A. crassna (A. crassna).
  4. 제1항에 있어서,The method of claim 1,
    상기 침향 추출물이 침향을 클로로포름, 에테르 또는 메틸렌클로라이드를 용매로 하여 추출한 추출물인 것을 특징으로 하는 퇴행성 신경질환의 예방 또는 치료용 약학 조성물.The aroma extract is a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases, characterized in that the extract extracted with aroma chloroform, ether or methylene chloride as a solvent.
  5. 제1항에 있어서,The method of claim 1,
    상기 침향 추출물이 침향의 메틸렌클로라이드 추출물인 것을 특징으로 하는 퇴행성 신경질환의 예방 또는 치료용 약학 조성물.A pharmaceutical composition for the prevention or treatment of degenerative neurological diseases, characterized in that the aroma extract is methylene chloride extract of aroma.
  6. 제1항에 있어서,The method of claim 1,
    상기 침향 추출물이 침향을 물, C1 내지 C3 알코올 또는 이들의 혼합물을 용매로 하여 1차 추출한 추출물을 클로로포름, 에테르 또는 메틸렌클로라이드를 용매로 하여 2차 추출한 추출물인 것을 특징으로 하는 퇴행성 신경질환의 예방 또는 치료용 약학 조성물.The extract of the aroma is degenerative neurological disease, characterized in that the extract of the first extract with aroma of water, C 1 to C 3 alcohol or a mixture thereof as a solvent, the second extract using chloroform, ether or methylene chloride as a solvent. Prophylactic or therapeutic pharmaceutical composition.
  7. 제1항에 있어서, 상기 분획물은 다음 단계를 포함하는 방법에 의해 얻어진 것을 특징으로 하는 퇴행성 신경질환의 예방 또는 치료용 약학 조성물:The pharmaceutical composition for preventing or treating degenerative neurological disease according to claim 1, wherein the fraction is obtained by a method comprising the following steps:
    a) 침향 또는 침향 에탄올 추출물을 메틸렌클로라이드에 용해시킨 후 여과한 여과액을 수득하는 단계;a) dissolving incense or incense ethanol extract in methylene chloride to obtain a filtrate which is filtered;
    b) 상기 a) 단계에서 수득한 여과액을 100%(v/v) 메탄올에 용해시킨 후 여과한 여과액을 수득하는 단계; 및b) dissolving the filtrate obtained in step a) in 100% (v / v) methanol to obtain a filtrate which is filtered; And
    c) 상기 b) 단계에서 수득한 여과액을 증류수, 및 아세토니트릴 또는 메탄올 용매를 사용한 고속 액체 크로마토그래피(HPLC)를 이용하여 분리하는 단계.c) separating the filtrate obtained in step b) using distilled water and high performance liquid chromatography (HPLC) using acetonitrile or methanol solvent.
  8. 제7항에 있어서, 상기 c) 단계에서는The method of claim 7, wherein in step c)
    상기 b) 단계에서 수득한 10 ㎕의 여과액을 고정상으로서 비극성 컬럼을 갖는 고속 액체 액체크로마토그래피(HPLC)에 통과시켜 280 nm 파장에서 측정한 결과, 변동계수 0.024%로, 머무름 시간(retention time) 28.52분에 피크를 나타내는 분획물을 분리하였으며, 10 μl of the filtrate obtained in step b) was passed through a high-performance liquid chromatography (HPLC) having a nonpolar column as a stationary phase and measured at a wavelength of 280 nm. As a result, the retention time was 0.024%. Fractions showing peaks were separated at 28.52 minutes,
    상기 컬럼의 이동상은, 증류수와 아세토니트릴을 사용하되, 0~0.01 분에서 30%(v/v) 아세토니트릴, 0.01-10 분에서 0.01-40%(v/v) 아세토니트릴의 구배, 10-15 분에서 40-50%(v/v) 아세토니트릴의 구배, 15-30 분에서 50-60%(v/v) 아세토니트릴의 구배, 30-40 분에서 100%(v/v) 아세토니트릴, 40-50 분에서 100%(v/v) 아세토니트릴을 사용하고, 유속을 1 mL/min으로 한 것을 특징으로 하는 퇴행성 신경질환의 예방 또는 치료용 약학 조성물.For the mobile phase of the column, distilled water and acetonitrile were used, but a gradient of 30% (v / v) acetonitrile at 0-0.01 min, 0.01-40% (v / v) acetonitrile at 0.01-10 min, 10- Gradient of 40-50% (v / v) acetonitrile at 15 minutes, gradient of 50-60% (v / v) acetonitrile at 15-30 minutes, 100% (v / v) acetonitrile at 30-40 minutes , 100% (v / v) acetonitrile at 40-50 minutes, the flow rate of 1 mL / min pharmaceutical composition for the prevention or treatment of neurodegenerative diseases.
  9. 제8항에 있어서,The method of claim 8,
    상기 비극성 컬럼은 옥타데실실란(C18), 도데실실란(C12) 또는 옥타실란(C8)이 충전된 비극성 컬럼인 것을 특징으로 하는 퇴행성 신경질환의 예방 또는 치료용 약학 조성물.The non-polar column is a pharmaceutical composition for preventing or treating neurodegenerative diseases, characterized in that the non-polar column filled with octadecyl (C18), dodecylsilane (C12) or octasilane (C8).
  10. 제8항에 있어서,The method of claim 8,
    상기 컬럼은 길이가 3 내지 30cm, 내경이 1.8 내지 5mm이고, The column has a length of 3 to 30 cm, an inner diameter of 1.8 to 5 mm,
    상기 컬럼에 충전된 입자의 직경이 1.5 내지 5㎛인 것을 특징으로 하는 퇴행성 신경질환의 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating neurodegenerative diseases, characterized in that the diameter of the particles packed in the column is 1.5 to 5㎛.
  11. 제1항에 있어서, 상기 분획물이 하기 특성 중 하나 이상을 가지는 것을 특징으로 하는 퇴행성 신경질환의 예방 또는 치료용 약학 조성물:The pharmaceutical composition for preventing or treating neurodegenerative disease according to claim 1, wherein the fraction has one or more of the following characteristics:
    1) 신경세포의 아팝토시스(apoptosis)를 억제;1) inhibit apoptosis of neurons;
    2) 신경세포의 활성산소종(reactive oxygen species:ROS) 생성 억제;2) inhibiting the production of reactive oxygen species (ROS) of neurons;
    3) 세포외 신호조절인산화효소(ERK; extracellular signal-regulated kinases)의 인산화 수준의 감소; 3) reduction of phosphorylation levels of extracellular signal-regulated kinases (ERKs);
    4) 신경세포의 일산화질소(NO)의 생성 억제; 및4) inhibition of production of nitric oxide (NO) in neurons; And
    5) 신경세포의 DNA 분절 저해.5) Inhibition of DNA segmentation of neurons.
  12. 제1항에 있어서,The method of claim 1,
    상기 분획물은 항산화 활성을 갖는 것을 특징으로 하는 퇴행성 신경질환의 예방 또는 치료용 약학 조성물.The fraction is a pharmaceutical composition for the prevention or treatment of degenerative neurological disease, characterized in that it has an antioxidant activity.
  13. 제1항에 있어서,The method of claim 1,
    상기 퇴행성 신경질환은 알츠하이머병(Alzheimer's disease), 파킨슨병(Parkinson's disease), 루게릭병(Lou Gehrig disease), 헌팅톤병(Huntington's disease), 다발성 경화증(Multiple sclerosis), 뇌졸중(stroke), 혈전증(thrombosis), 색전증(embolism), 두부손상(head trauma), 뇌순환대사장애, 뇌 기능혼수 또는 치매(dementia)인 것을 특징으로 하는 퇴행성 신경질환의 예방 또는 치료용 조성물.The neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Lou Gehrig disease, Huntington's disease, Multiple sclerosis, stroke, thrombosis , Embolism, head trauma (head trauma), cerebral circulatory metabolic disorders, brain function coma or dementia (dementia) composition for the prevention or treatment of neurodegenerative diseases.
  14. 제1항에 있어서,The method of claim 1,
    상기 퇴행성 신경질환은 지질다당류(lipopolysaccharide, LPS)에 의하여 유도되는 것을 특징으로 하는 퇴행성 신경질환의 예방 또는 치료용 약학 조성물.The degenerative neurological disease is a pharmaceutical composition for the prevention or treatment of degenerative neurological disease, characterized in that it is induced by lipopolysaccharide (LPS).
  15. 제1항에 있어서, The method of claim 1,
    상기 퇴행성 신경질환은 글루타메이트에 의해 유발된 것을 특징으로 하는 퇴행성 신경질환의 예방 또는 치료용 약학 조성물.The neurodegenerative disease is a pharmaceutical composition for preventing or treating neurodegenerative disease, characterized in that caused by glutamate.
  16. 제 1항에 있어서,The method of claim 1,
    상기 조성물의 제형이 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼 및 시럽으로 이루어진 군으로부터 선택되는 경구용 제형인 것을 특징으로 하는 퇴행성 신경질환의 예방 또는 치료용 약학 조성물.The pharmaceutical composition for the prevention or treatment of degenerative neurological diseases, characterized in that the formulation of the composition is an oral formulation selected from the group consisting of powders, granules, tablets, capsules, suspensions, emulsions and syrups.
  17. 침향 추출물의 분획물을 유효성분으로 포함하는 퇴행성 신경질환의 예방 또는 개선용 건강기능식품 조성물.Health functional food composition for the prevention or improvement of degenerative neurological disease comprising a fraction of the aroma extract as an active ingredient.
PCT/KR2018/001339 2018-01-30 2018-01-31 Composition for preventing, improving or treating degenerative neurological disease comprising fraction of a quilaria agallocha roxburgh extract as active ingredient WO2019151547A1 (en)

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