Composition for improving cognitive function using single or complex extract of acanthopanax koreanum, astragalus membranaceus, and / or momordica charantia

Abstract

The present invention discloses a composition for improving cognitive function using a single or complex extract of Acanthopanax koreanum, Astragalus membranaceus, and / or Momordica charantia. The composition increases, in an in-vitro efficacy test, the cell viability of SH-SY5Y cells, which are human-derived neuroblastomas treated with scopolamine that is an oxidative stress-inducing drug, increases the expression of an apoptosis inhibition-related biomarker (Bcl-2), an autophagy-enhancing biomarker (Beclin-1), and an acetylcholine biosynthesis-related enzyme (ChAT), which are neurotransmitters, also increases the expression of the antioxidant-related biomarker HO-1, and increases the inactivation of GSK-3β, which is a therapeutic target for Alzheimer's disease, and furthermore, even in an in-vivo efficacy test, increases the expression of Bcl-2, Beclin-1, ChAT, and HO-1 in the hippocampus and cerebrum of an animal model with reduced cognitive function and memory induced by administration of scopolamine, and inhibits the expression of iNOS, which is an enzyme involved in NO biosynthesis that induces tissue damage.

Description

Composition for improving cognitive function using single or combined extracts of Som-O-Gal-Pi, Hwang-Gi, and / or Yeo-Ju

[0001] The present invention relates to a composition for improving cognitive function using a single or combined extract of Acanthopanax koreanum, Astragalus membranaceus and / or Momordica charantia.

[0002] As the average life expectancy of people increases due to advancements in medicine and economic growth, problems associated with the aging of society are emerging as important issues. In particular, degenerative brain diseases and cognitive impairments due to aging are emerging as a social and economic burden on families and society.

[0003] The global prevalence of dementia is approximately 5-10% in people aged 65 and older, and the prevalence nearly doubles with each subsequent five-year increase (New Researcher Support Project Report, 2017). Alzheimer's disease (AD), which accounts for more than 50% of dementia in the elderly, is characterized by two distinct symptoms: the accumulation of extracellular amyloid-β plaques and intracellular neurofibrillary tangles (NFTs).

[0004] In neurodegenerative diseases such as Alzheimer's and Parkinson's, the brain exhibits oxidative damage because it is particularly sensitive to oxidative stress. Oxidative stress is also a major factor in the development of Alzheimer's, as the brain consumes approximately 20% of the body's oxygen consumption. Heme oxygenase-1 (HO-1), one of the enzymes that mitigates oxidative stress, maintains high levels of mRNA expression, particularly in the hippocampus and cerebellum. This is thought to enable rapid protein expression in response to oxidative stress when needed (Rev. Neurosci. 25:269-280, 2014; J. Neurosci. Res. 68:65-75, 2002).

[0005] Nitric oxide (NO), produced by cells, is involved in various signal transmissions and acts as a neurotransmitter in the nervous system. It is synthesized by nitric oxide synthase (NOS). Among them, an increase in inducible nitric oxide synthase (iNOS) and the resulting excessive production of nitric oxide exhibit cytotoxicity and cause tissue damage, inducing apoptosis and thus the death of nerve cells (Neuropharmacology 33:1425-1430, 1994; Neuron 13:487-494, 1994).

[0006] The Bcl-2 family proteins, which include more than 20 proteins, are divided into anti-apoptotic proteins and pro-apoptotic proteins, among which Bcl-2 is an anti-apoptotic protein and is known to promote nerve cell growth, neural network, and synaptic plasticity (Pharmachological Research 151:104547, 2020).

[0007] Beclin-1's autophagy-inducing function is important for maintaining neuronal homeostasis. Beclin-1 fragments that were degraded by caspases and rendered non-functional were detected in the brains of Alzheimer's disease patients, and when caspase activity was inhibited to prevent Beclin-1 degradation, apoptosis and neurodegeneration were observed to be alleviated (Neurobiol Dis 43:68-78, 2011; Molecular Neurodegeneration. 13:68, 2018).

[0008] Meanwhile, in Alzheimer's disease, the function of cholinesterase is increased by 40-90%, and the activity of acetylcholine is significantly reduced, which causes memory decline in patients. Acetylcholine, produced by choline acetyltransferase (ChAT), is a neurotransmitter involved in memory formation. Furthermore, acetylcholinesterase acts to convert beta-amyloid monomers into oligomers, which increases beta-amyloid accumulation in the brain and enhances its toxic effects (Biochemistry 42:10863-10873, 2003). Therefore, the majority of currently used Alzheimer's disease treatments are cholinesterase inhibitors (ChEIs) that inhibit the breakdown of acetylcholine, and some are antagonists of the N-methyl-D-aspartate (NMDA) receptor. However, these only alleviate symptoms and do not prevent the progression of Alzheimer's disease, leading to persistent cognitive decline and various side effects with long-term use (Intl J Mol Sci. 21:3833, 2020). Therefore, the search for natural products with fewer side effects and the potential to prevent the progression of cognitive decline is necessary.

[0009] Glycogen synthase kinase 3β (GSK-3β) is constitutively active in most tissues but is inactivated by phosphorylation at the Ser9 position. GSK-3β is a negative regulator of glucose homeostasis and is involved in energy metabolism, inflammation, endoplasmic reticulum stress, mitochondrial dysfunction, and apoptosis pathways, as well as regulating neural development and neuroplasticity. GSK-3β is known to be highly active in diseases such as Alzheimer's disease and to be involved in the abnormal phosphorylation of tau protein, making its inactivation an important therapeutic target for diseases such as Alzheimer's disease (Front Cell Neurosci . 2020 Mar 17; 14:19).

[0010] Native Korean plants have long been used for a variety of purposes in folk and Oriental medicine, and recent research has revealed their active ingredients. In particular, with the increasing number of reports of materials containing active ingredients capable of penetrating the blood-brain barrier, the potential for developing natural medicines and health functional foods to improve cognition and memory is growing.

[0011] Meanwhile, Acanthopanax koreanum is a deciduous shrub in the Araliaceae family. It is a Jeju specialty and a Korean endemic plant. It was registered as a geographical indication collective mark by the Korean Intellectual Property Office in 2016. According to Materia Medica and the Chinese Pharmacopoeia, the root bark is used as a medicinal ingredient. Its immune, anticancer, antistress, antihistamine, antidiabetic, detoxifying, antiobesity, anti-inflammatory, and analgesic effects have been reported, and a 2012 patent (application number: 1020120138538) confirmed its efficacy in preventing and treating neuropsychiatric disorders.

[0012] Astragalus membranaceus is a perennial herb in the Fabaceae family. According to several traditional Korean medical books, including Donguibogam, Gyeongakjeonseo, Hyangyakjipseongbang, and Bencao Gangmok, the roots of Astragalus membranaceus and Mongolian Astragalus membranaceus have been used as medicinal herbs. Its anti-obesity, anti-inflammation, neuroprotective, cell proliferation inhibitory, and anti-tumor effects have been reported, and its neuroprotective effects have been confirmed through several patents (application numbers: 1020040027034, 102013004336, 102015018792, 1020170114787, 1020170132831).

[0013] Momordica charantia (Cucurbitaceae) is an annual herb. According to the Chinese herbal medicine book, its fruit, called "gogwa," is used for medicinal purposes. Its efficacy has been reported to include hypoglycemic, antiviral, antitumor, immunomodulatory, antiaging, and antibacterial properties. In a 2012 Ministry of Education, Science and Technology project (Unique Number: 1345175157), its Alzheimer's prevention and antiaging activities were confirmed.

[0014] The present invention discloses cognitive function improvement activity, etc. of extracts of the Korean ginseng root or a complex extract of Korean ginseng root with astragalus membranaceus and / or yam.

[0015] The purpose of the present invention is to provide a composition for improving cognitive function using extracts of the Korean fig.

[0016] Other or specific purposes of the present invention will be presented below.

[0017] The present invention has been completed by confirming in the following examples and experimental examples that the extract of Acanthopanax japonica, the two- or three-complex extract of Acanthopanax japonica and / or the extract of Astragalus membranaceus and the two- or three-complex extract of the extract of Acanthopanax japonica and the two- or three-complex extract of the extract of Astragalus membranaceus and / or the two- or three-complex extract of the extract of Astragalus membranaceus and ... It is.

[0018] The present invention is provided based on these experimental results, and in one aspect, the present invention can be understood as a composition for improving cognitive function (or a composition for improving memory or a composition for protecting nerve cells) comprising as an active ingredient (i) an extract of Acanthopanax spp., (ii) an extract of Astragalus membranaceus, (iii) an extract of Yeoju, (iv) a complex extract of Acanthopanax spp. and Astragalus membranaceus or a complex of an extract of Acanthopanax spp. and Astragalus membranaceus, (v) a complex extract of Acanthopanax spp. and Astragalus membranaceus or a complex of an extract of Acanthopanax spp. and Astragalus membranaceus, or a complex extract ... It can be identified as a composition for improving diseases involving nerve cell death, which contains as active ingredients a complex of extracts of Atractylodes japonica, Astragalus membranaceus extract, and Yeoju extract.

[0019] In this specification, "extract" means an extract obtained by leaching the stem, leaf, fruit, flower, root, etc. of an extraction target plant using water, lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof, an extract obtained by using a supercritical extraction solvent such as carbon dioxide or pentane, or a fraction obtained by fractionating the extract, and any method such as cold immersion, reflux, heating, ultrasonic radiation, or supercritical extraction may be applied as the extraction method, taking into consideration the polarity, degree of extraction, and degree of preservation of the active substance. In the case of a fractionated extract, it means a fraction obtained by suspending the extract in a specific solvent and then mixing and allowing it to settle with a solvent of different polarity, and a fraction obtained by adsorbing the crude extract on a column filled with silica gel or the like and using a hydrophobic solvent, a hydrophilic solvent, or a mixed solvent thereof as a mobile phase. In addition, the meaning of the extract includes a concentrated liquid extract or solid extract from which the extraction solvent has been removed by a method such as freeze-drying, vacuum drying, hot air drying, or spray drying. Preferably, it means an extract obtained using water, ethanol, or a mixed solvent thereof as an extraction solvent, and more preferably, it means an extract obtained using a mixed solvent of water and ethanol as an extraction solvent.

[0020] In this specification, “improvement of cognitive function” means improvement of cognitive function or improvement of memory when neuronal death is inhibited as a result of inhibition of neuronal death, and thus improvement of cognitive function can be understood to mean the same thing as improvement of memory or protection of neuronal cells.

[0021] Also, in this specification, "disease involving neuronal cell death" includes degenerative brain diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, and ischemic brain diseases such as vascular dementia.

[0022] Also, in this specification, “improvement of a disease involving death of nerve cells” means alleviation of symptoms of a disease involving death of brain nerve cells, treatment, and prevention (inhibition or delay of onset) of such a disease.

[0023] Also, in this specification, “active ingredient” means an ingredient that exhibits the desired activity alone or can exhibit the activity together with a carrier that is inactive in itself.

[0024] The composition of the present invention may contain an effective ingredient in any amount (effective amount) depending on the intended use, formulation, mixing purpose, etc., as long as it can exhibit an effect of improving cognitive function, etc., and a typical effective amount will be determined within the range of 0.001 wt % to 20.0 wt % based on the total weight of the composition. Here, the "effective amount" refers to the amount of the effective ingredient included in the composition of the present invention that can exhibit the intended functional and pharmacological effects, such as improving cognitive function, when the composition of the present invention is administered to a mammal, preferably a human, which is the subject of application, for an administration period recommended by a medical professional, etc. Such an effective amount can be experimentally determined within the normal ability range of a person skilled in the art.

[0025] In addition, the composition of the present invention may additionally contain any compound or natural extract that has already been verified for safety and known to have the corresponding activity in the art, in order to enhance or reinforce the effect of improving cognitive function, or to improve the convenience of taking or ingesting through the addition of similar activities such as improving exercise capacity, in addition to the effective ingredient. Such compounds or extracts include compounds or extracts listed in the official regulations of each country's pharmacopoeia (in Korea, the "Korean Pharmacopoeia"), each country's health functional food code (in Korea, the "Health Functional Food Standards and Specifications" notified by the Ministry of Food and Drug Safety), compounds or extracts that have been approved for product use in accordance with the laws of each country regulating the manufacture and sale of pharmaceuticals (in Korea, the "Pharmaceutical Affairs Act"), and compounds or extracts that have been individually recognized for functionality in accordance with the laws of each country regulating the manufacture and sale of health functional foods (in Korea, the "Health Functional Food Act").

[0026] For example, green tea extract / theanine complex, EPA and DHA-containing fats, ginkgo leaf extract, fibroin enzyme hydrolysate, red ginseng (red ginseng concentrate) with recognized "memory improvement" functionality under the Korean Health Functional Food Act, Lactobacillus helveticus fermented product, Platycodon grandiflorum extract, Angelica gigas Nakai root extract or powder, phosphatidylserine with recognized "cognitive ability improvement" functionality, maca gelatinized powder, creatine, Hovenia dulcis fruit extract powder, and Cordyceps sinensis fermented extract with recognized "exercise ability improvement" functionality would be such compounds or extracts.

[0027] In a specific embodiment, the composition of the present invention can be considered as a food composition.

[0028] The food composition of the present invention can be manufactured in any form, for example, beverages such as tea, juice, carbonated beverages, and electrolyte beverages, processed milk such as milk and yogurt, foods such as gum, rice cakes, Korean traditional sweets, bread, confectionery, and noodles, and health functional food preparations such as tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, and bars.

[0029] In addition, the food composition of the present invention may be classified into any product category, as long as it complies with the applicable laws and regulations at the time of manufacturing and distribution, in terms of legal and functional classification. For example, it may be a health functional food according to the Korean "Health Functional Food Act," or a confectionery, legume, tea, beverage, special-purpose food, etc. according to each food type according to the food code of the Korean "Food Sanitation Act" (MFDS ​​Notice "Food Standards and Specifications").

[0030] The food composition of the present invention may contain food additives in addition to its effective ingredients. Food additives can generally be understood as substances added to, mixed with, or infiltrated into food during the manufacturing, processing, or preservation of food. Since they are consumed daily and over a long period of time with food, their safety must be guaranteed. The Food Additive Codex, which is based on the laws of each country that regulate the manufacturing and distribution of food (in Korea, the “Food Sanitation Act”), provides limited regulations on food additives with guaranteed safety in terms of ingredients or functions. The Korean Food Additive Codex (Ministry of Food and Drug Safety Notice “Food Additive Standards and Specifications”) classifies food additives into chemically synthesized products, natural additives, and mixed preparations in terms of ingredients. These food additives are classified into sweeteners, flavoring agents, preservatives, emulsifiers, acidulants, and thickeners in terms of functions.

[0031] Sweeteners are used to impart an appropriate sweetness to foods, and both natural and synthetic sweeteners can be used in the food composition of the present invention. Preferably, a natural sweetener is used. Examples of natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

[0032] Flavoring agents are used to enhance taste or aroma, and both natural and synthetic flavors can be used. Natural flavoring agents are preferred. When using natural flavoring agents, they can also serve the purpose of enhancing nutrition in addition to flavor. Natural flavoring agents can be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or from green tea leaves, Polygonum multiflorum, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. Also, flavoring agents obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo biloba, etc. can be used. Natural flavoring agents can be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents can be used, such as esters, alcohols, aldehydes, and terpenes.

[0033] Preservatives that can be used include calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc.; emulsifiers that can be used include acacia gum, carboxymethylcellulose, xanthan gum, pectin, etc.; and acidulants that can be used include citric acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, etc. In addition to the purpose of enhancing taste, acidulants can be added to ensure that the food composition has an appropriate acidity for the purpose of inhibiting the growth of microorganisms. Thickeners that can be used include suspending agents, sedimentation agents, gel-forming agents, and puffing agents.

[0034] In addition to the food additives described above, the food composition of the present invention may include physiologically active substances or minerals known in the art and guaranteed to be safe as food additives for the purpose of supplementing and reinforcing functionality and nutrition.

[0035] Examples of such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, and dibenzoylthiamine, and examples of minerals include calcium preparations such as calcium citrate, magnesium preparations such as magnesium stearate, iron preparations such as ferrous citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, and zinc.

[0036] The food composition of the present invention may include the aforementioned food additives in an appropriate amount that can achieve the purpose of addition depending on the product type.

[0037] With regard to other food additives that may be included in the food composition of the present invention, reference may be made to the food codes or food additive codes of each country.

[0038] The composition of the present invention may be considered as a pharmaceutical composition in other specific embodiments.

[0039] The pharmaceutical composition of the present invention may be prepared as an oral or parenteral formulation, depending on the route of administration, by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. The route of administration may be any suitable route, including topical, oral, intravenous, intramuscular, and direct absorption through mucosal tissues, and a combination of two or more routes may also be used.

[0040] Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or formulation, and specific examples can be found in the pharmacopoeias of each country, including the “Korean Pharmacopoeia.”

[0041] When the pharmaceutical composition of the present invention is prepared as an oral dosage form, it can be prepared in the form of powder, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, suspensions, wafers, etc., using a suitable carrier and a method known in the art. Examples of suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol; starches such as corn starch, potato starch, and wheat starch; cellulosics such as cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyols, vegetable oils, ethanol, and glycerol. When formulating, appropriate binders, lubricants, disintegrants, colorants, diluents, etc. may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch peristalsis, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax, etc., and lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts and calcium salts thereof, polyethylene glycol, etc., and disintegrants include starch, methyl cellulose, agar, bentonite, xanthan gum, starch, alginic acid, or sodium salts thereof. Other diluents include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine.

[0042] When the pharmaceutical composition of the present invention is prepared as a parenteral dosage form, it can be formulated in the form of injections, transdermal administration, nasal inhalants, and suppositories using a suitable carrier according to a method known in the art. When formulated as an injection, an aqueous isotonic solution or suspension can be used as a suitable carrier, and specifically, an isotonic solution such as PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, or 5% dextrose can be used. When formulated as a transdermal dosage form, it can be formulated in the form of an ointment, cream, lotion, gel, external solution, paste, liniment, aerosol, etc. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, or carbon dioxide, and in the case of formulating it as a suppository, the carrier can be witepsol, tween 61, polyethylene glycol, cacao butter, laurin butter, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene stearate, sorbitan fatty acid ester, or the like.

[0043] Specific formulations of pharmaceutical compositions are known in the art and can be found, for example, in Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.

[0044] The preferred dosage of the pharmaceutical composition of the present invention may range from 0.001 mg / kg to 10 g / kg per day, preferably from 0.001 mg / kg to 1 g / kg, depending on the patient's condition, weight, sex, age, severity of the condition, and route of administration. Administration may be administered once daily or divided into several doses. Such dosage should not be construed as limiting the scope of the present invention in any way.

[0045] As described above, according to the present invention, a composition for improving cognitive function using extracts of the Korean ginseng root, etc. can be provided.

[0046] The composition of the present invention can be commercialized as a food or pharmaceutical product.

[0047] Figure 1 compares the effects of single and combined extracts of the roots of Atractylodes japonica, Astragalus membranaceus root, and Jujube fruit on the survival of SH-SY5Y cells.

[0048] Figure 2 compares the effects of single and combined extracts of the roots of Astragalus membranaceus, Astragalus membranaceus root, and Yeoju fruit on the expression of proteins in the anti-apoptotic (Bcl-2), autophagy (Beclin-1), and synaptic transmission (ChAT) pathways.

[0049] Figure 3 shows the evaluation of the intracellular antioxidant efficacy and the effect on cell activity according to the treatment concentration of the complex extract.

[0050] Figure 4 shows the intracellular efficacy of the complex extract according to the treatment concentration, comparing the expression of proteins in the anti-apoptotic (Bcl-2), autophagy (Beclin-1), and synaptic transmission (ChAT) pathways.

[0051] Figure 5 shows the evaluation of the efficacy in the mouse hippocampus according to the dose of the complex extract.

[0052] Figure 6 shows the evaluation of the efficacy in the mouse brain according to the dosage of the complex extract.

[0053] The present invention will be described below with reference to examples. However, the scope of the present invention is not limited to these examples.

[0054]

[0055] <Example> Cognitive function improvement activity of single or combined extracts of Astragalus membranaceus, Astragalus membranaceus, and / or Yeoju

[0056] <Example 1> Preparation of single or combined extracts of Astragalus membranaceus, Astragalus membranaceus, and / or Yeoju

[0057] In this example, single extracts of Acanthopanax koreanum (hereinafter AKR), Astragalus membranaceus (hereinafter AMR), and Momordica charantia (hereinafter MCF) and two kinds of combined extracts ((AKR+AMR+MCF)50E) were prepared as follows. 50% ethanol in an amount 10 times the dry weight was added to each Acanthopanax koreanum stem or root segment, Astragalus membranaceus root segment, and Momordica charantia fruit segment or a mixture thereof (dry weight ratio?), and then extraction was performed at 50°C for 6 hours using a reflux condenser. The extract was filtered using a filter paper, concentrated using a rotary vacuum evaporator, and dried using a freeze dryer to obtain a solid extract.

[0058] The obtained extracts are as shown in Table 1 below, consisting of two single extracts of Atractylodes japonica, two mixed extracts of three types, and four mixed extracts of two types, for a total of eight types. The mixed extracts were obtained using Atractylodes japonica root, Astragalus membranaceus root, and Yeoju fruit.

[0059] The mixing ratio of each raw material for single or combined extracts is as shown in Table 1 below.

[0060] The mixing ratio of raw materials ( Dry weight ratio? ) AKR (Astragalus membranaceus) AMR (Mixed Root) MCF (MCF) Manufacturing Example 11 (Root) -- Manufacturing Example 21 (Stem) -- Manufacturing Example 3-1 -- Manufacturing Example 4 -- Manufacturing Example 5211 Manufacturing Example 6122 Manufacturing Example 7101- Manufacturing Example 812- Manufacturing Example 93-1 Manufacturing Example 101-2

[0061] <Example 2> In vitro efficacy evaluation 1. Evaluation of neuronal cell protection efficacy

[0062] To determine whether single or combined extracts, such as the extract of Acanthopanax japonica, exhibit neuroprotective effects, cell viability was analyzed using the MTT reagent (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide). Human neuroblastoma SH-SY5Y cells were cultured in 96-well plates, treated with the extracts of Example 1 at various concentrations, and incubated for 1 hour. Scopolamine, a stress-inducing drug, was treated (at what concentration?) and incubated for an additional 24 hours. After treating with the MTT reagent and reacting for 4 hours, the formazan produced with DMSO was sufficiently dissolved and measured at 540 nm to compare cell viability. Scopolamine is a muscarinic cholinergic receptor antagonist that induces cholinergic dysfunction and oxidative stress in the brain, leading to memory impairment. It is a drug that is mainly used to induce dementia in cell and animal models, and was used to stress the cells.

[0063] The results are shown in Figures 1 to 4. Figure 1 shows the results of evaluating the neuroprotective efficacy of extracts of the stem or root of Acanthopanax rhizome, Figure 2 shows the results of evaluating the neuroprotective efficacy of extracts of Acanthopanax rhizome, Astragalus membranaceus root, and Yeoju fruit alone and a combination of these three, Figure 3 shows the results of evaluating the neuroprotective efficacy of extracts of Acanthopanax rhizome and Astragalus membranaceus root alone and a combination of two extracts, and Figure 4 shows the results of evaluating the neuroprotective efficacy of extracts of Acanthopanax rhizome and Yeoju fruit alone and a combination of two extracts.

[0064] Referring to FIGS. 1 to 4, it can be seen that not only the extracts of the roots of the Korean angelica tree, the roots of the astragalus membranaceus, and the fruit of the Yeoju tree alone, but also the combined extracts of two or three types of the roots of the Korean angelica tree, the roots of the astragalus membranaceus, and / or the fruit of the Yeoju tree all exhibited neuroprotective effects in a generally concentration-dependent manner.

[0065] Based on these experimental results, subsequent experiments were conducted on samples with relatively excellent activity. Specifically, the single extract used was the extract of the root of Acanthopanax japonica, and the composite extracts used were the composite extract of Acanthopanax japonica root, Astragalus membranaceus root, and Yeoju fruit in a 2:1:1 mixing ratio of Preparation Example 5 for the three-composite extract of Acanthopanax japonica root, Astragalus membranaceus root, and Yeoju fruit, the composite extract of the 10:1 mixing ratio of Preparation Example 7 for the two-composite extract of Acanthopanax japonica root and Astragalus membranaceus root, and the composite extract of the 3:1 mixing ratio of Preparation Example 9 for the two-composite extract of Acanthopanax japonica root and Yeoju fruit.

[0066] 2. Measurement of expression of biomarkers related to cell death inhibition (Bcl-2), autophagy promotion biomarkers (Beclin-1), and enzymes related to acetylcholine biosynthesis (ChAT), a neurotransmitter.

[0067] The effects of single or combined extracts of Acanthopanax japonica on the expression of biomarkers related to inhibition of apoptosis (Bcl-2), promotion of autophagy (Beclin-1), and enzymes related to acetylcholine biosynthesis (ChAT), a neurotransmitter, were evaluated. SH-SY5Y cells were cultured in 6-well plates, treated with the extracts of Example 1 at various concentrations, and cultured for 1 hour. After scopolamine treatment, the cells were further cultured for 24 hours. After the reaction was completed, the cells were harvested and lysed in RIPA buffer, and the same amount of protein quantified by the Bradford method was subjected to SDS-PAGE (Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis) and Western blotting. The expression patterns were confirmed using antibodies against Bcl-2, Beclin-1, and ChAT.

[0068] The results are shown in Figures 5 to 9. Figure 5 shows the results for the extract of the root of Acanthopanax rhizome, Figure 6 shows the results for the extracts of Acanthopanax rhizome, Astragalus membranaceus root, and Yeoju fruit alone or in combination, and Figure 7 shows the results for the extracts of Acanthopanax rhizome, Astragalus membranaceus root, and Yeoju fruit in combination. Figure 8 shows the results for the extracts of the root of Acanthopanax rhizome and Astragalus membranaceus root in combination, and Figure 9 shows the results for the extracts of the root of Acanthopanax rhizome and Yeoju fruit in combination.

[0069] Referring to Figures 5 to 9, it can be confirmed that not only the extract of the root of Acanthopanax japonica alone, but also the combined extracts of Acanthopanax japonica root, Astragali root, and / or two or three kinds of Yeoju fruit, increase the expression of biomarkers related to cell death inhibition, etc., which are reduced by scopolamine treatment, in a generally concentration-dependent manner (NC: scopolamine treatment group). SAK3 (4-5 μM), used as a positive control (PC), is a T-type calcium channel enhancer, a compound that has been reported to have enhanced efficacy in scopolamine-treated SH-SY5Y cells and in an animal model of dementia.

[0070] 3. Measurement of the antioxidant-related biomarker HO-1

[0071] HO-1, an antioxidant biomarker, was measured by enzyme-linked immunosorbent assay (ELISA) using an HO-1 ELISA kit. In this experiment, two or three compound extracts of the relatively active extracts of Acanthopanax japonica root, Astragali root, and / or Yeoju fruit were used.

[0072] SH-SY5Y cells were cultured in 12-well plates, treated with the extracts of Example 1 at various concentrations (10–200 μg / mL), and incubated for 1 hour. As a positive control, SAK3 was treated at 400 nM. Stress was induced with scopolamine, and the cells were further incubated for 24 hours. After the reaction was completed, the cells were harvested, lysed in RIPA buffer, and the expression level of HO-1 protein was analyzed by comparing it with a standard substance compared to the protein quantified by the Bradford method.

[0073] The results are shown in Figures 10 to 12. Figure 10 shows the results for a three-composite extract of the root of Atractylodes japonica, the root of Astragalus membranaceus, and the fruit of Yeoju. Figure 11 shows the results for a two-composite extract of the root of Atractylodes japonica and the root of Astragalus membranaceus, and Figure 12 shows the results for a two-composite extract of the root of Atractylodes japonica and the fruit of Yeoju.

[0074] Referring to Figures 10 to 12, it can be seen that the two and three types of composite extracts generally increase the expression of the antioxidant-related biomarker HO-1, which was reduced by scopolamine treatment, in a concentration-dependent manner (NC: scopolamine treatment group).

[0075] 4. Measurement of GSK-3β inactivation, a therapeutic target for Alzheimer's disease and other diseases

[0076] To evaluate GSK-3β inactivation, a therapeutic target for Alzheimer's disease and other diseases, a GSK-3β ELISA kit was used. In this experiment, two or three compound extracts of the relatively active extracts of the root of Acanthopanax japonica, the root of Astragali, and / or the fruit of Yeoju were used.

[0077] SH-SY5Y cells were cultured in 96-well plates, treated with the extracts of Example 1 at various concentrations (10–200 μg / mL), and incubated for 1 hour. SAK3 (10–20 nM) was used as a positive control. The cells were further incubated for 24 hours after scopolamine treatment. After the reaction was completed, the cells were fixed, and the absorbance value of the inactive GSK-3β (p-GSK-3β(S9)) protein was compared to the absorbance value of the GSK-3β protein.

[0078] The results are presented together in Figures 10 to 12. Figures 10 to 12 show that the two and three types of combined extracts generally increased the degree of GSK-3β inactivation reduced by scopolamine treatment in a concentration-dependent manner.

[0079] <Example 3> In vivo efficacy evaluation

[0080] In vivo efficacy evaluation was performed on a three-composite extract (mixing ratio 2:1:1) of the roots of Atractylodes japonica, Astragalus membranaceus root, and Jujube fruit, which showed the highest relative activity.

[0081] After acclimatization for one week, 10-week-old male ICR mice were divided into five groups (N; Normal group, NC; Negative Control group, PC; Positive Control group (Donepezil 3 mg / kg), Ex-L; Extracts-Low dose group (complex extract 300 mg / kg), Ex-H; Extracts-High dose group (complex extract 500 mg / kg)) based on their average body weight. Donepezil, the projection drug of the positive control group, is a cholinesterase inhibitor that helps maintain the normal level of the neurotransmitter acetylcholine and is used to treat mild and moderate Alzheimer's disease symptoms.

[0082] Drinking water (N, NC), control (PC), and complex extracts (Ex-L, Ex-H) were administered orally for 4 weeks. Starting 2 weeks before euthanasia, scopolamine (NC, PC, Ex-L, Ex-H) was administered intraperitoneally 30 minutes after oral administration to induce cognitive and memory impairment. After the experiment, blood, liver, and brain were extracted, and only the cerebrum and hippocampus were isolated and secured. Organ index and blood biochemical evaluation results confirmed that there was no toxicity due to the complex extract intake.

[0083] Meanwhile, Western Blot was performed to evaluate the efficacy in the hippocampus and cerebral regions. Each brain tissue was homogenized and lysed with RIPA buffer, and then quantified using the Bradford method. Equal amounts of protein were subjected to SDS-PAGE electrophoresis, and the expression patterns of key biomarkers in the anti-apoptotic (Bcl-2), autophagy (Beclin-1), synaptic transmission (ChAT), antioxidant (HO-1), and inflammatory (iNOS) pathways were confirmed using the same Western Blot method as in Example 2.

[0084] The expression patterns in the hippocampus and the cerebrum are shown in Fig. 13 and Fig. 14, respectively. In both the hippocampus and the cerebrum, administration of the three types of complex extracts increased the expression of apoptosis inhibition-related biomarker (Bcl-2), autophagy promotion biomarker (Beclin-1), acetylcholine biosynthesis-related enzyme (ChAT), and antioxidant-related biomarker (HO-1), which were reduced by scopolamine administration, in a dose-dependent manner, and decreased the expression of iNOS, an NO biosynthesis enzyme that induces tissue damage, which was increased by scopolamine administration.

[0085] The present invention can be commercialized as a functional food or drug with cognitive ability improvement functionality and used in industry.

Claims

1. A composition for improving cognitive function or memory, comprising as an active ingredient (i) an extract of Acanthopanax spp., (ii) an extract of Astragalus membranaceus, (iii) an extract of Yeoju, (iv) a complex extract of Acanthopanax spp. and Astragalus membranaceus or a complex of an extract of Acanthopanax spp. and Astragalus membranaceus, (v) a complex extract of Acanthopanax spp. and Yeoju, or a complex extract of an extract of Acanthopanax spp. and Yeoju, or (vi) a complex extract of Acanthopanax spp., Astragalus membranaceus and Yeoju, or a complex of an extract of Acanthopanax spp., Astragalus membranaceus and Yeoju, as well as an extract of Yeoju.

2. In paragraph 1, The above extract is a composition that is an extract of water, ethanol or a mixed solvent thereof.

3. In paragraphs 1 and 2, A composition characterized in that the above cognitive function improvement is due to inhibition of neuronal cell death.

4. In any one of paragraphs 1 to 3, The above composition is a food composition.

5. In any one of paragraphs 1 to 3, The above composition is a pharmaceutical composition.

6. A pharmaceutical composition for improving a disease involving the death of nerve cells, comprising as an active ingredient (i) an extract of Acanthopanax spp., (ii) an extract of Astragalus membranaceus, (iii) an extract of Yeoju, (iv) a complex extract of Acanthopanax spp. and Astragalus membranaceus or a complex of an extract of Acanthopanax spp. and Astragalus membranaceus, (v) a complex extract of Acanthopanax spp. and Yeoju, or a complex extract of an extract of Acanthopanax spp. and Yeoju, or (vi) a complex extract of Acanthopanax spp., Astragalus membranaceus and Yeoju, or a complex of an extract of Acanthopanax spp., Astragalus membranaceus and Yeoju, A composition according to claim 1, wherein the disease involving the death of the above-mentioned nerve cells is Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis (MS), or amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease.

7. In paragraph 6, The above extract is a composition that is an extract of water, ethanol or a mixed solvent thereof.

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