KR102728914B1 - Composition for Protection of Brain Neuronal Cells Using an Extract of Clausena excavata - Google Patents

Abstract

The present invention discloses a composition for protecting brain neurons using an extract of Clausena excavata, which has a concentration-dependent inhibitory effect on HT22 cells, a mouse hippocampal cell line, without being cytotoxic.

Description

{Composition for Protection of Brain Neuronal Cells Using an Extract of Clausena excavata}

The present invention relates to a composition for protecting brain nerve cells using an extract of Clausena excavata.

As many countries around the world gradually enter an aging society, the number of people suffering from various degenerative brain and nervous system diseases such as stroke, Parkinson's disease (PD), and Alzheimer's disease (dementia) is rapidly increasing, and the cost is also astronomical. According to the 2019 Statistics Korea data, the mortality rate due to brain diseases is ranked 3rd, following malignant neoplasms (cancer), heart disease, and cerebrovascular diseases. The brain, which controls the information essential for maintaining life, causes various neurological disorders that have a fatal impact on life when there is an abnormality in the nerve signals. In the current situation where there is no effective method to treat brain diseases, it places a considerable mental burden on the surrounding families due to the decline in quality of life and the enormous expenditure of medical expenses. Recognizing the seriousness of this problem, countries around the world are seeking concentrated solutions with nationwide attention.

Neurons constantly die during the process of development and synapse restructuring, and cell death caused by stress and cytotoxic drugs is one of the main factors of degenerative brain diseases. Degenerative brain diseases are brain diseases that occur with age and are known to be caused by the accumulation of environmental and genetic factors. Specific nerve cell groups in the brain and spinal cord gradually lose their functions, and they are caused by the death of nerve cells that are most important for information transmission in the brain nervous system, problems in the formation or function of synapses that transmit information between nerve cells, and abnormal increases or decreases in the electrical activity of nerves. Neurons in the brain and spinal cord perform very diverse functions depending on their location, and depending on which part of the nerve cells are damaged and lose their functions first, and the form in which this dysfunction progresses, they show very diverse clinical aspects. Degenerative brain diseases can be classified by considering the main symptoms and the brain area affected, and include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease. These degenerative brain diseases are caused by the loss of function of the cranial nerves, resulting in symptoms of memory and cognitive impairment (Clin. Interv. Aging. 2007, 2:347).

Among various factors, oxidative stress is known to be closely related to the causes of degenerative neurological diseases. Although the brain accounts for only 2% of body weight, it consumes about 20% of total oxygen consumption, so it has a high oxygen utilization rate and is the part of the body where the most active oxygen is generated. According to recent studies, chronic stress and oxidative stress have been reported to induce oxidative stress in the hypothalamus-pituitary-adrenal cortex, hippocampus, striatum, substantia nigra, and forebrain, increasing cell death and decreasing neurons and growth factors, resulting in degenerative brain diseases (Floyd RA. Proc Soc Exp Biol Med. 1999 Dec;222(3):236-45.;Wang JY et al., Curr Pharm Des. 2006;12(27):3521-33).

In particular, oxidative stress in neurons causes apoptosis by releasing cytochrome C from mitochondria and activating caspase-3. In addition, reactive oxygen species activate NMDA receptors, which induces an increase in Ca2+ ions through the metabotrophical cascade, and the increase in intracellular Ca2+ activates caspase-2, which damages DNA (Bonini P et al., J Neurosci Res. 2004 Jan 1;75(1):83-95.; Polster BM and Fiskum G. J Neurochem. 2004 Sep;90(6):1281-9.; Gorman AM et al., Neuroreport. 1998 Jul 13;9(10):R49-55). Cell death occurs due to excitatory neurotoxicity caused by disruption of Ca2+ ion homeostasis, dysfunction of the endoplasmic reticulum and mitochondria, and DNA damage caused by oxidative stress (Wei et al., 1999, Toxicology 134:117-26.).

Glutamate is a representative excitatory neurotransmitter in the central nervous system and plays a very important role in neurotransmission at the synapses of nerve cells, formation and growth of neurons, behavior, learning, and memory. However, excessive glutamate is known to induce oxidative stress within cells and damage nerve cells by reducing the activity of intracellular antioxidant enzymes, glutathione peroxidase and glutathione reductase, thereby inhibiting glutathione production, which can cause acute or chronic degenerative brain diseases (Neurochem. Int. 2004, 45:583-595; Nat. Rev. Neurosci. 2007, 8:803).

HT22 cells, a mouse hippocampal cell line, have been widely used as an in vitro model to elucidate the mechanisms of neurotoxicity induced by oxidative stress (Life Sci. 84: 267-271). HT22 cells lack ionotropic glutamate receptors, and thus glutamate treatment can reveal cell damage caused by oxidative stress rather than receptor hyperexcitation (Neurosci. Lett. 2004, 362:253).

In the present invention, the neuroprotective effect of Clausena excavata extract was confirmed.

The purpose of the present invention is to provide a composition for protecting brain nerve cells using an extract of Clausena excavata.

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

As confirmed in the examples below, the present inventors examined whether Clausena excavata extract had cytotoxicity against HT22 cells and whether it had an effect of inhibiting glutamate-induced cell death using HT22 cells, a mouse hippocampal cell line. They confirmed that it had a concentration-dependent effect of inhibiting HT22 cell death without cytotoxicity.

In consideration of these experimental results, in one aspect, the present invention can be understood as a composition for protecting brain nerve cells comprising a Clausena excavata extract as an effective ingredient, in another aspect, it can be understood as a composition for improving a disease involving death of brain nerve cells comprising a Clausena excavata extract as an effective ingredient, and in yet another aspect, it can be understood as a composition for improving cognitive ability comprising a Clausena excavata extract as an effective ingredient.

In this specification, the "Clausena excavata extract" means an extract obtained by leaching the stem, leaf, fruit, flower, root, or mixture thereof of Clausena excavata, which is an extraction target, with water, a lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butylacetate, 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 as the extraction method, any method such as cold steeping, reflux, heating, ultrasonic radiation, or supercritical extraction can be applied considering the polarity, extraction degree, and preservation degree 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 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 freeze-drying, vacuum drying, hot air drying, spray drying or the like. Preferably, it means an extract obtained by using water, ethanol or a mixed solvent thereof as an extraction solvent, and more preferably, it means an extract obtained by using a mixed solvent of water and ethanol as an extraction solvent.

In addition, in this specification, the term "active ingredient" means an ingredient that exhibits the desired activity alone or can exhibit activity together with a carrier that is inactive on its own.

In addition, in this specification, "protection of brain nerve cells" includes the meaning of inhibiting death of brain nerve cells due to aging, in addition to the meaning of inhibiting death of brain nerve cells in diseases that entail death of brain nerve cells as defined below. Since inhibition of death of brain nerve cells directly leads to improvement in cognitive ability, the protection of brain nerve cells can also be understood to mean improvement in cognitive ability.

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

Also, in this specification, "disease involving death of brain nerve cells" 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.

In the composition of the present invention, the effective ingredient may be included in any amount (effective amount) depending on the intended use, formulation, mixing purpose, etc., as long as it can exhibit a brain nerve cell protection effect, 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, which can exhibit the intended functional and pharmacological effects, such as a brain nerve cell protection effect, 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 suggested 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.

In addition to the effective ingredient, the composition of the present invention may additionally contain any compound or natural extract that has already been verified as safe and known to have the corresponding activity in the art in order to enhance or reinforce the brain nerve cell protection effect or to improve the convenience of taking or ingesting through the addition of similar activities such as anti-stress activity, fatigue improvement activity, etc. Such compounds or extracts include compounds or extracts listed in the pharmacopoeia of each country (in Korea, the “Korean Pharmacopoeia”), the codes of health functional foods of each country (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 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 whose functionality is recognized in accordance with the laws of each country regulating the manufacture and sale of health functional foods (in Korea, the “Health Functional Food Act”). For example, Lactobacillus Helveticus fermented product, Platycodon grandiflorum extract (DRJ-AD), Angelica root extract, Angelica extract powder, and phosphatidylserine, which have been recognized for their functionality as “improving cognitive ability” under the Korean “Health Functional Food Act,” and fermented amino acid complex, extract of the fruit of the Chinese quince tree, and extract of the Rhodiola rosea, which have been recognized for their functionality as “improving fatigue,” and L-theanine, ashwagandha extract, hydrolysed milk protein, and Glycyrrhiza uralensis leaf extract, which have been recognized for their functionality as “anti-stress,” are examples of such compounds or extracts.

One or more of these compounds or natural extracts may be included in the composition of the present invention together with the active ingredient.

The composition of the present invention, in a specific embodiment, can be understood as a food composition.

The food composition of the present invention can be manufactured in any form, and for example, can be manufactured into beverages such as tea, juice, carbonated beverages, and electrolyte beverages, processed dairy products 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, liquid, powder, flakes, paste, syrup, gel, jelly, and bars.

In addition, the food composition of the present invention may have any product classification as long as it complies with the laws and regulations in effect 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」 (KFDA Notice 「Food Standards and Specifications」).

The food composition of the present invention may contain food additives in addition to its effective ingredients. Food additives can be generally understood as substances that are added to, mixed in with, or infiltrated into food during the manufacturing, processing, or preservation of food, and since they are consumed with food every day and for a long period of time, their safety must be guaranteed. In the food additive code according to the laws of each country that regulate the manufacturing and distribution of food (in Korea, the “Food Sanitation Act”), food additives whose safety is guaranteed are limitedly stipulated in terms of ingredients or functions. In the Korean Food Additive Code (the “Food Additive Standards and Specifications” notified by the Ministry of Food and Drug Safety), food additives are stipulated by classifying them into chemical synthetic products, natural additives, and mixed preparations in terms of ingredients, and these food additives are classified into sweeteners, flavoring agents, preservatives, emulsifiers, acidulants, and thickeners in terms of functions.

Sweeteners are used to provide appropriate sweetness to foods, and can be natural or synthetic. Preferably, a natural sweetener is used, and examples of natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents can be used to improve taste or aroma, and both natural and synthetic ones can be used. It is preferable to use natural ones. When using natural ones, in addition to flavor, the purpose of enhancing nutrition can also be used. Natural flavoring agents can be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or from green tea leaves, dandelions, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. In addition, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo nuts, etc. can be used. Natural flavoring agents can be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents can be used, and synthetic flavoring agents can be esters, alcohols, aldehydes, terpenes, etc.

Preservatives that can be used include calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. Also, emulsifiers that can be used include acacia gum, carboxymethyl cellulose, xanthan gum, pectin, etc. Also, 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 the food composition to have an appropriate acidity for the purpose of inhibiting the growth of microorganisms.

As thickening agents, suspending agents, sedimentation agents, gel forming agents, and bulking agents can be used.

In addition to the food additives described above, the food composition of the present invention may contain 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.

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 minerals include calcium preparations such as calcium citrate, magnesium preparations such as magnesium stearate, iron preparations such as ferrous citrate, chromium chloride, potassium iodine, selenium, germanium, vanadium, and zinc.

The food composition of the present invention may contain the food additives described above in an appropriate amount that can achieve the purpose of addition depending on the product type.

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.

The composition of the present invention may be understood as a pharmaceutical composition in other specific embodiments.

The pharmaceutical composition of the present invention may be prepared as an oral formulation or a parenteral formulation according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to an effective ingredient. Here, the route of administration may be any appropriate route, including a topical route, an oral route, an intravenous route, an intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be combined. An example of a combination of two or more routes is a case where two or more formulations of drugs according to the route of administration are combined, for example, a case where one drug is first administered via the intravenous route and another drug is secondarily administered via the topical route.

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.”

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; celluloses 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, glycerol, etc. In the case of formulation, suitable binders, lubricants, disintegrants, colorants, diluents, etc. may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, waxes, etc. Lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talc, stearic acid, magnesium salts and calcium salts thereof, polyethylene glycol, etc. Disintegrants include starch, methyl cellulose, agar, bentonite, xanthan gum, starch, alginic acid or sodium salts thereof. In addition, diluents include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, etc.

When the pharmaceutical composition of the present invention is prepared as a parenteral formulation, it can be formulated in the form of injections, transdermal administration, nasal inhalers, 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 administration, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, pastes, liniments, aerosols, etc. In the case of nasal inhalers, they 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 suppositories, the carriers that can be used include witepsol, Tween 61, polyethylene glycols, cacao butter, laurin butter, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, and sorbitan fatty acid esters.

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.

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

As described above, according to the present invention, a composition for protecting brain nerve cells using a Clausena excavata extract can be provided, and this composition for protecting brain nerve cells can be commercialized as a food or a pharmaceutical product.

Figure 1 shows the results showing that Clausena excavata extract does not exhibit cytotoxicity to brain neurons and has a protective effect against glutamate-induced neurotoxicity.

Hereinafter, the present invention will be described with reference to examples and experimental examples. However, the scope of the present invention is not limited to these examples and experimental examples.

<Example> Preparation of Clausena excavata extract

Clausena excavata extract was prepared by adding 70% ethanol in an amount 10 times the weight of the dried powder of Clausena excavata leaves, stirring for 1 day, filtering, and concentrating the filtrate under reduced pressure and freeze-drying.

<Experimental example> Experiment on the protection and activity of brain nerve cells

1. Experimental method

1.1 Cell culture

HT22 cells were used in DMEM medium supplemented with 10% FBS (Fetal Bovine Serum) and 1% P/S (penicillin/streptomycin), and cultured in an incubator maintained at 37°C and 5% _CO2 . When the cell density reached 70-80% saturation, cells were harvested using 0.05% Trypsin-EDTA solution and used in the experiment.

1.2 Toxicity assessment for nerve cells

The toxicity of the example sample to HT22 cells was evaluated using the MTT assay. HT22 cells were dispensed into a 96-well plate at a concentration of 5 × 10 ⁴ cells/mL (100 μL) and cultured for 24 hours. The example sample was dissolved in the medium immediately before use to prepare the highest concentration group, which was then diluted stepwise to prepare each concentration. The prepared test substance was treated to the cultured cells at 100 μL for each concentration three times and cultured for 24 hours. After incubation, 20 μL of MTT solution (5 mg/mL in PBS) was treated to each well and then cultured at 37°C for 3 hours. After incubation, the medium and MTT solution were carefully removed, 100 μL of DMSO was added to dissolve the formed formazan cyrstal, and then the absorbance was measured at 540 nm.

1.3 Evaluation of inhibitory activity against glutamate-induced neurotoxicity

The protective effect of the test sample against glutamate-induced neurotoxicity was evaluated using the HT22 MTT assay. HT22 cells were dispensed into a 96-well plate at a concentration of 5 × 10 ⁴ cells/mL (100 μL) and cultured for 24 hours. The sample was dissolved in a medium containing 8 mM glutamate immediately before use to prepare the highest concentration group, which was then diluted stepwise to prepare each concentration. The prepared test substance was treated to the cultured cells at 100 μL for each concentration three times and cultured for 24 hours. After incubation, 20 μL of MTT solution (5 mg/mL in PBS) was treated to each well and then cultured at 37°C for 3 hours. After incubation, the medium and MTT solution were carefully removed, 100 μL of DMSO was added to dissolve the formed formazan cyrstal, and the absorbance was measured at 540 nm.

1.4 Cell viability (%) and protective effect (%)

Cell viability (%) and protective effect (%) were calculated using the optical density (OD) values of each group according to the following formula ( , OD of untreated control; , OD of the group treated with glutamate only; , OD of the glutamate+sample treatment group).

2. Results

The cytotoxicity results of the example samples and the inhibitory effect on glutamate-induced neurotoxicity are shown in Figure 1.

The sample of the example was not cytotoxic to HT22 cells at 25 to 200 μg/mL. When HT22 cells were treated with 4 mM glutamate alone, cell viability decreased to 24.9%. When 4 mM glutamate and 1 to 20 μg/mL of the extract were treated together, cell viability recovered to 96.1%.

Claims (8)

A food composition for improving cognitive ability containing Clausena excavata extract as an active ingredient.
In the first paragraph,
A composition characterized in that the above extract is an extract obtained by extracting Clausena excavata leaves with water, ethanol or a mixed solvent thereof.
delete delete A pharmaceutical composition for improving a disease involving death of brain nerve cells, comprising an extract of Clausena excavata as an active ingredient,
The disease that entails the death of the above brain nerve cells is a degenerative brain disease.
The composition wherein the above degenerative brain disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis or amyotrophic lateral sclerosis.
In paragraph 5,
A composition characterized in that the above extract is an extract obtained by extracting Clausena excavata leaves with water, ethanol or a mixed solvent thereof.

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