Composition for protecting nerve cells containing Asyneuma japonicum (Miq.) Briq. extract as an active ingredient

Abstract

The present invention relates to a neuroprotective composition comprising an extract of *Adenophora triphylla* as an active ingredient, which can exhibit a neuroprotective effect by inhibiting the death of neurons induced by oxidative stress. In addition, the present invention provides a functional food composition for the prevention of degenerative brain diseases and a pharmaceutical composition for the prevention and treatment of degenerative brain diseases, comprising the above-mentioned neuroprotective composition.

Description

Technology Field

[0001] The present invention relates to a neuroprotective composition comprising an extract of *Adenophora triphylla* as an active ingredient. Background Technology

[0002] Recently, along with improvements in economic standards and advancements in medicine and life sciences, the elderly population is rapidly increasing worldwide. According to data released by Statistics Korea, as of 2005, the proportion of the elderly population aged 65 or older in the total population reached 9.1%, while the proportion of the youth population aged 14 or younger has been gradually decreasing; consequently, Korea is assessed to have already entered an aging society. Furthermore, it is projected that the proportion of the elderly population aged 65 or older will exceed 14% of the total population in 2019 and approach 20% in 2020, making Korea the second most aging nation after Japan.

[0003] Along with the aging of the population, the incidence and prevalence of various age-related diseases are increasing. In particular, degenerative brain diseases show a high rate, ranking first in incidence among diseases of the elderly.

[0004] Neurodegenerative diseases are conditions caused by damage to brain nerve cells, and there is currently no fundamental cure due to a lack of specific understanding regarding the mechanisms of onset or progression. To date, various factors such as genetic factors, oxidative stress, excessive inflammatory responses, and the accumulation of abnormal peptides have been identified as causes of neurodegenerative diseases through related research.

[0005] Representative degenerative brain diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease.

[0006] First, Alzheimer's disease is a degenerative brain disease in which neurons in the cerebral cortex die, causing the gyri of the frontal and temporal lobes to atrophy or shrink. It is one of the causes of dementia that occurs as brain tissue loses function during the aging process. It initially begins with forgetfulness, and in the late stages, personality disintegration and psychotic symptoms appear. Although the exact cause has not yet been identified, it is suspected to be due to genetics, structural changes in the nervous system, inflammatory mediators, the cholinergic nervous system, and abnormalities in other neurotransmitters.

[0007] Parkinson's disease is a degenerative neurological disorder characterized by tremors, stiffness, and loss of balance in the arms, legs, or the entire body. It occurs due to problems with brain nerve cells, resulting in abnormal secretion of the neurotransmitter dopamine and the gradual destruction of specific nerve cells in the brain.

[0008] The typical symptoms of Parkinson's disease are movement disorders characterized by tremors, muscle rigidity, and slowed body movements. Because the disease progresses slowly, it is difficult to determine exactly when it began; by the time symptoms are discovered and the disease is suspected, nerve cells in the substantia nigra of the brain have often already undergone significant degeneration. In addition, facial expressions may become rigid, strides may shorten, and depression or bowel and bladder dysfunction may occur.

[0009] Huntington's disease is caused by the degeneration of neurons in the caudate nucleus and putamen of the brain. Huntington's disease is accompanied by uncontrollable body movements, particularly spasmodic movements of the arms and legs. As the disease progresses, it can lead to cognitive and emotional disorders, and ultimately results in death within approximately 10 to 15 years of the onset of symptoms.

[0010] Amyotrophic Lateral Sclerosis (ALS), better known as Lou Gehrig's disease, is a disease characterized by the selective death of motor neurons, appearing to be marked by the gradual destruction of both upper motor neurons in the cerebral cortex and lower motor neurons in the brainstem and spinal cord. Clinical symptoms begin with the slow-progressing weakness and atrophy of the limbs, and as the disease progresses, it eventually leads to death within a few years due to respiratory muscle paralysis.

[0011] The Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis described above are all caused by damage to brain neurons, and oxidative stress involving radical formation is known to be a major cause of such damage.

[0012] Oxidative stress refers to damage to cells or tissues caused by toxic free radicals. Brain neurons, in particular, are sensitive to oxidative stress for the following reasons.

[0013] The process by which the brain obtains energy by oxidizing sugar is a highly aerobic metabolic process that consumes a large amount of oxygen. Although the brain accounts for only about 2% of body weight, it consumes enough oxygen to use 20% of total oxygen consumption. Consequently, brain nerve cells are damaged by the large amount of reactive oxygen species generated along with this consumption.

[0014] In addition, brain neurons contain high concentrations of easily oxidized polyunsaturated fatty acids, and metal ions (Fe) used as catalysts during radical formation. 2+ , Cu 2+ It is rich in ) etc., and is more vulnerable to oxidative stress because it lacks antioxidant enzymes such as catalase compared to other tissues, so its defense mechanisms against oxidative stress are insufficient.

[0015] Furthermore, another cause of brain nerve cell damage is damage caused by inflammatory responses. Inflammation is one of the pathological phenomena observed in most degenerative brain diseases, such as Alzheimer's and Parkinson's disease, and it is hypothesized that neuronal death is accelerated by inflammatory mediators, such as cytokines and oxidative substances, produced by immune cells during the inflammatory response.

[0016] Therefore, there is a need to develop a composition capable of providing a protective effect on brain neurons and suppressing inflammatory responses caused by oxidative stress or various other factors. Prior art literature

[0017] (Patent Document 0001) KR 10-2023-0000748 A The problem to be solved

[0018] The objective of the present invention is to provide a neuroprotective composition comprising an extract of *Adenophora triphylla* as an active ingredient.

[0019] Another objective of the present invention is to provide a composition containing *Azalea extract* as an active ingredient that can exhibit a neuroprotective effect by inhibiting the death of nerve cells induced by oxidative stress.

[0020] Another objective of the present invention is to provide a functional food composition for the prevention of degenerative brain diseases and a pharmaceutical composition for the prevention and treatment of degenerative brain diseases, comprising the above-mentioned neuroprotective composition. means of solving the problem

[0021] To achieve the above objective, the present invention relates to a neuroprotective composition comprising an extract of *Adenophora triphylla* as an active ingredient.

[0022] In addition, the above extract can be extracted with a solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, and mixed solvents thereof.

[0023] In addition, the above composition may exhibit a neuroprotective effect by inhibiting the death of neurons induced by oxidative stress.

[0024] In addition, the above composition may exhibit a preventive or therapeutic effect for degenerative brain diseases.

[0025] In addition, the above-mentioned degenerative brain disease may be one or more selected from the group consisting of stroke, paralysis, memory loss, memory impairment, dementia, amnesia, Parkinson's disease, Alzheimer's disease, Pick's disease, and Creutzfeld-Jakob disease.

[0026] A functional food composition for the prevention of degenerative brain diseases according to another embodiment of the present invention may include the neuroprotective composition.

[0027] A pharmaceutical composition for the prevention and treatment of degenerative brain diseases according to another embodiment of the present invention may include the neuroprotective composition. Effects of the invention

[0028] The present invention includes an extract of *Adenophora triphylla* as an active ingredient and can exhibit a neuroprotective effect by inhibiting the death of nerve cells induced by oxidative stress.

[0029] In addition, the present invention provides a functional food composition for the prevention of degenerative brain diseases and a pharmaceutical composition for the prevention and treatment of degenerative brain diseases, comprising the above-mentioned neuroprotective composition. Brief explanation of the drawing

[0030] Figure 1 is the result of an evaluation test of the neuroprotective ability of a composition according to one embodiment of the present invention. Specific details for implementing the invention

[0031] Hereinafter, embodiments of the present invention are described in detail so that those skilled in the art can easily implement the invention. However, the present invention may be embodied in various different forms and is not limited to the embodiments described herein.

[0032] For the treatment of dementia by drug therapy, acetylcholinesterase inhibitors such as Tacrine or Aricept, which inhibit the breakdown of acetylcholine in the brain to maintain the concentration of acetylcholine at a certain level, are mainly used.

[0033] Although the efficacy of the above-mentioned drug is recognized to some extent, it has been pointed out that long-term high-dose administration causes significant side effects, such as muscle pain, nausea, vomiting, diarrhea, and loss of appetite due to gastrointestinal disorders, as well as liver toxicity. Accordingly, an alternative method has been proposed to promote the synthesis of acetylcholine by administering acetylcholine precursors such as choline.

[0034] However, these substances have the disadvantage of not being sufficiently effective because they cannot cross the blood-brain barrier. Consequently, attention is being focused on compositions derived from food or herbal medicines that enable safe prevention and treatment without side effects.

[0035] Accordingly, a composition for preventing or improving cognitive impairment has been developed using balloon flower extract, strawberries, and Ecklonia cava, but it has not been sold as an actual product.

[0036] Accordingly, the present invention aims to provide a herbal medicine-derived composition with excellent preventive and therapeutic effects without side effects, as described above.

[0037] Specifically, the neuroprotective composition of the present invention may include an extract of *Adenophora triphylla* as an active ingredient.

[0038] The aforementioned *Phyteuma japonicum* is a perennial herb found primarily in low-lying areas of mountain valleys throughout Korea. It grows to a height of 50 to 100 cm. Hairs are scattered throughout the plant, and the stem has ridges. The leaves are alternate, oblong with pointed tips, measuring 5 to 12 cm in length and 2.5 to 4 cm in width. The leaves at the base have short petioles, which disappear as they ascend; the margins are serrated, and the surface is covered with a few hairs. The flowers are purple, arranged in a raceme that branches from the base, and are hairless; the calyx is tubular. The corolla is also deeply divided into five lobes, each 1 mm wide and 1 to 1.2 cm long, curling backward. The filaments have hairs on the lower margins, and the style is 1 to 1.2 cm long with a three-lobed tip. The fruit is a capsule, flat and round in shape, with distinct longitudinal veins.

[0039] The present invention is characterized by providing a neuroprotective composition using an extract of active ingredients obtained from the aforementioned *Yongaja*. The *Yongaja* extract may exhibit a neuroprotective effect by inhibiting the death of neurons induced by oxidative stress.

[0040] The above extract can be extracted with a solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, and mixed solvents thereof.

[0041] The above extract may be an extract obtained by extracting with a solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, and mixed solvents thereof.

[0042] The above extract can be eluted using a polar solvent such as water, ethanol, methanol, or other alcohols having 1 to 6 carbon atoms, or a mixed solvent having a mixing ratio of 1:0.1 to 1:10 of alcohol and water, and more specifically, water can be used as the extraction solvent. At this time, the extraction temperature may be 10°C to 100°C, preferably at room temperature.

[0043] The above extraction solvent may be used in an amount of 2 to 50 times the weight of the sample, preferably 2 to 20 times. For extraction, the sample may be left in the extraction solvent for leaching for 1 to 72 hours, and specifically for 1 to 24 hours.

[0044] The extract obtained using the above organic solvent may further include a step of fractionating using an organic solvent, and the method for preparing the extract may be a conventional extraction method in the art, such as ultrasonic extraction, leaching, and reflux extraction.

[0045] The above reflux extraction method is based on 10 to 30 g of crushed natural product, 1 to 3 hours of reflux, and 50 to 100% of alcohol or water with 1 to 6 carbon atoms, based on 100 mL of alcohol with 1 to 6 carbon atoms. More specifically, based on 100 mL of alcohol or water with 1 to 6 carbon atoms, 10 to 20 g of crushed natural product, 1 to 2 hours of reflux, and 70 to 90% of alcohol or water with 1 to 4 carbon atoms.

[0046] The above leaching method is carried out at 15 to 30°C for 24 to 72 hours, and water or 50 to 100% alcohol having 1 to 6 carbon atoms is used as the extraction solvent. More specifically, it is carried out at 20 to 25°C for 30 to 54 hours, and the extraction solvent is water or 70 to 80% alcohol having 1 to 6 carbon atoms.

[0047] The above ultrasonic extraction method is carried out at 30 to 50°C for 0.5 to 2.5 hours, and the extraction solvent is water or 50 to 100% alcohol having 1 to 6 carbon atoms. Specifically, the extraction is carried out at 40 to 50°C for 1 to 2.5 hours, and the extraction solvent is water or 70 to 80% alcohol having 1 to 6 carbon atoms.

[0048] The above extraction solvent may be used in an amount of 2 to 50 times the weight of the sample, more specifically 2 to 20 times. For extraction, the sample may be left in the extraction solvent for 1 to 72 hours, more specifically 24 to 48 hours.

[0049] It may be, but is not limited to, a product obtained by concentrating a filtered extract under reduced pressure using a vacuum rotary concentrator, and includes all extracts, diluted or concentrated extracts, dried products obtained by drying the extract, and modified or purified products thereof.

[0050] The above composition may additionally include a strawberry tree extract.

[0051] The aforementioned Rubus sorbifolius var. myriadenus is a deciduous broad-leaved shrub reaching a height of 1 to 2 meters. It possesses dark red glandular hairs and hook-shaped thorns, with glandular hairs densely covering the entire young branches. The leaves are alternate and pinnately compound, with 3 to 5 leaflets, though the lower ones number 5 to 7. The leaflets are broadly lanceolate, gradually tapering to a point at the tip and rounded at the base; they feature double serrations along the margins and are characterized by being hairless except along the veins. White flowers bloom in April, borne in compound corymbs at the tips of branches, and are covered with glandular and soft hairs. The fruit is an aggregate, oval-shaped capsule that ripens to white and is edible.

[0052] As described above, even when treated with only the extract of *Young-aja* alone, the neuroprotective effect is excellent; however, when the extract of *Dogwood* is included as an active ingredient, the neuroprotective effect can be further enhanced by inhibiting the death of neurons induced by oxidative stress due to the synergistic effect resulting from the mixing of active ingredients contained in each natural extract, thereby demonstrating an excellent neuroprotective effect.

[0053] The composition of the present invention may contain 1 to 5 parts by weight of a dogwood extract per 100 parts by weight of a koji extract. Within the above range, compared to the case where the koji extract is included alone, when the dogwood extract is used as a mixed extract, the neuroprotective effect by inhibiting the death of nerve cells induced by oxidative stress may be further enhanced due to the synergistic effect of the koji extract and the dogwood extract.

[0054] The above composition may exhibit a neuroprotective effect by inhibiting the death of neurons induced by oxidative stress. Due to this effect, it may exhibit a preventive or therapeutic effect against degenerative brain diseases.

[0055] The above degenerative brain disease may be one or more selected from the group consisting of stroke, paralysis, memory loss, memory impairment, dementia, amnesia, Parkinson's disease, Alzheimer's disease, Pick's disease, and Creutzfeld-Jakob disease, but is not limited to the above examples, and all degenerative brain diseases may exhibit preventive and therapeutic effects without limitation.

[0056] A functional food composition for the prevention of degenerative brain diseases according to another embodiment of the present invention may include a neuroprotective composition.

[0057] The food composition according to the present invention may be provided in the form of powder, granules, tablets, capsules, syrup, or beverage, and may be used in combination with other foods or food additives, and may be used appropriately according to conventional methods. The amount of the mixture of active ingredients may be appropriately determined according to the purpose of use, for example, for prevention, health, or therapeutic treatment.

[0058] As a specific example, processed foods capable of enhancing the effects of improving eyesight and preventing or improving eye diseases can be manufactured using the above food composition. Such processed foods include, for example, snacks, beverages, alcoholic beverages, fermented foods, canned goods, processed dairy products, processed meat products, noodles, etc. Snacks include biscuits, pies, cakes, bread, candy, jelly, gum, cereal, etc. Beverages include drinking water, carbonated beverages, functional ion drinks, juices (e.g., apple, pear, grape, aloe, tangerine, peach, carrot, tomato juice, etc.), sikhye, etc. Alcoholic beverages include sake, whiskey, soju, beer, spirits, fruit wine, etc. Fermented foods include soy sauce, doenjang, gochujang, etc. Canned goods include canned seafood (e.g., canned tuna, mackerel, saury, conch, etc.), canned livestock (canned beef, pork, chicken, turkey, etc.), and canned agricultural products (canned corn, peaches, pineapple, etc.). Processed dairy products include cheese, butter, yogurt, etc. Processed meat products include pork cutlets, beef cutlets, chicken cutlets, sausages, sweet and sour pork, chicken nuggets, marinated beef slices, etc. Noodles include sealed packaged fresh noodles, etc. In addition, the above composition may be used in retort foods, soups, etc.

[0059] A pharmaceutical composition for the prevention and treatment of degenerative brain diseases according to another embodiment of the present invention may include a neuroprotective composition.

[0060] The above pharmaceutical composition may further comprise a component selected from the group consisting of pharmaceutically acceptable carriers, excipients, and diluents. Specifically, the above pharmaceutical composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, and sterile injectable solutions, each according to conventional methods.

[0061] In the present invention, carriers, excipients, and diluents that may be included in the pharmaceutical composition may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, the composition is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the extracts and fractions thereof. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, syrups, etc., and may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. As non-aqueous solvents and suspending agents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, Witepsol, Macrogol, Tween 61, cocoa oil, laurin oil, glycerogelatin, etc. may be used.

[0062] The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" in this invention refers to an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dose level may be determined based on factors including the severity of the disease, drug activity, the patient's age, weight, health, gender, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and elimination rate, the duration of treatment, drugs combined or used concurrently with the composition of the present invention, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered alone or in combination with known immunotherapies. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking all of the above factors into consideration.

[0063] The above pharmaceutical composition may be a formulation selected from the group consisting of solutions, suspensions, syrups, emulsions, liposomes, powders, granules, tablets, sustained-release formulations, eye drops, capsules, contact lens cleaners, and contact lens lubricants, but is not limited to the above examples, and any formulation to enhance the preventive or therapeutic effect of dry eye syndrome may be used without limitation.

[0064] Preparation Example

[0065] Preparation of Youngja extract

[0066] Dried Adenophora seeds were ground, the ground Adenophora seeds were placed in 1 L of 70% ethanol solution, and refluxed by sonication for 24 hours. After filtration through a glass filter funnel, the extract was concentrated using a rotary vacuum evaporator (Buchi Labortechnik, Flavil, Switzerland) and then freeze-dried using a freeze dryer (SP Industries, Inc., Warminster, Pennsylvania, USA). The final yield of the Adenophora extract was 33.95%, and it was stored at -80°C until use in the experiment and dissolved in distilled water immediately before use.

[0067] Preparation of strawberry tree extract

[0068] A strawberry tree extract was prepared using the leaves of the strawberry tree in the same manner as the aforementioned cinnamon extract.

[0069] The above extracts of *Youngja* and *Dog's-tree* were mixed in the proportions shown in Table 1 below to be used as a mixed extract. The above mixed extract was prepared by mixing freeze-dried powdered extracts in the proportions shown in Table 1 below and dissolving them in distilled water.

[0070] RE1 RE2 RE3 RE4 RE5 RE6 Young children 100 100 100 100 100 100 Pulsatilla koreana - 0.5 1 3 5 7

[0071] (Unit weight parts)

[0072] Experimental Example

[0073] Evaluation test of neuroprotective ability

[0074] To evaluate neuroprotective activity, the toxicity of the neuroprotective composition was confirmed in PC-12 cells. Specifically, PC-12 cells were seeded into a 96-well plate at a density of 5.0 × 10⁴ cells / well and cultured for 3 hours. After the culture was finished, the medium was removed from the wells. Subsequently, RE1 to RE6 extracts were added to the culture medium at a concentration of 100 μg / mL. 100 μL of the prepared culture medium was dispensed into each well and cultured for 24 hours.

[0075] To induce oxidative stress after culture, 400 μM H2O2 was added and reacted for 1 hour, after which MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution was added to each well and reacted for 3 hours. 50 μL of DMSO was dispensed into each well to dissolve the purple formazan, and absorbance was measured at 570 nm using a microplate reader (Infinite M200; Tecan Austria GmbH, Grodig, Austria). Cytotoxicity was analyzed based on the absorbance measurements.

[0076] The test results are as shown in Figure 1. The control group was the group without extract treatment, and after culture, H2O2 was not treated. As the comparison group, 400 Mm of H2O2 was treated to the group without extract treatment after culture.

[0077] In the group treated solely with the Young-aja extract, slight cytotoxicity was observed at a concentration of 100 μg / mL. However, while cell viability was confirmed to be 100% at concentrations of 1 μg / mL, 5 μg / mL, and 10 μg / mL, slight cytotoxicity was observed after 50 μg / mL.

[0078] Subsequently, regarding the complex extract, RE2 and RE6 showed higher cell viability compared to the control group, but it can be confirmed that excellent cell viability is exhibited when used within the mixing range of the present invention.

[0079] Based on the above test results, the cytoprotective effect was confirmed. The neuroprotective effect was determined by the cell survival rate of the extract-treated group versus the cell survival rate of the control group; it was confirmed that RE3 to RE5 exhibited superior neuroprotective effects compared to RE1.

[0080] Although preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention as defined in the following claims also fall within the scope of the present invention.

Claims

Claim 1 A neuroprotective composition comprising 1 to 5 parts by weight of a Chinese dogwood extract per 100 parts by weight of a Chinese dogwood extract. Claim 2 A neuroprotective composition according to claim 1, wherein the extract is extracted with a solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, and mixed solvents thereof. Claim 3 In claim 1, the composition is a neuroprotective composition that exhibits a neuroprotective effect by inhibiting the death of neurons induced by oxidative stress. Claim 4 In claim 1, the composition is a neuroprotective composition exhibiting a preventive or therapeutic effect against degenerative brain diseases. Claim 5 A neuroprotective composition according to claim 4, wherein the degenerative brain disease is one or more selected from the group consisting of stroke, paralysis, memory loss, memory impairment, dementia, amnesia, Parkinson's disease, Alzheimer's disease, Pick's disease, and Creutzfeld-Jakob disease. Claim 6 A functional food composition for the prevention of degenerative brain diseases comprising a neuroprotective composition according to any one of claims 1 to 5. Claim 7 A pharmaceutical composition for the prevention and treatment of degenerative brain diseases comprising a neuroprotective composition according to any one of claims 1 to 5.

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