KR101049493B1 - Compositions for improving memory power and learning ability comprising extract from lepidium apefalum as active ingredient - Google Patents
Compositions for improving memory power and learning ability comprising extract from lepidium apefalum as active ingredient Download PDFInfo
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- KR101049493B1 KR101049493B1 KR1020100092045A KR20100092045A KR101049493B1 KR 101049493 B1 KR101049493 B1 KR 101049493B1 KR 1020100092045 A KR1020100092045 A KR 1020100092045A KR 20100092045 A KR20100092045 A KR 20100092045A KR 101049493 B1 KR101049493 B1 KR 101049493B1
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- dementia
- apefalum
- lepidium
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- A—HUMAN NECESSITIES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
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- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2200/00—Function of food ingredients
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- A61K2236/30—Extraction of the material
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- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
Abstract
Description
본 발명은 정력자 추출물을 유효성분으로 포함하는 기억력 및 학습 능력 증진용 조성물에 관한 것이다.
The present invention relates to a composition for improving memory and learning ability comprising a sperm extract as an active ingredient.
인간의 뇌에는 모두 1000억 개의 신경세포가 존재하며 이 들은 각 각 주위의 다른 신경세포들과 평균 1만개 정도의 시냅스(synapse)라고 하는 부위로 연결되어 서로 네트워크를 형성하고 있다. 이들 시냅스를 통하여 신경세포들 사이에 긴밀한 정보전달이 이루어지게 되며, 이 때 매개 역할을 하는 것이 바로 신경전달물질(neurotransmitter)이다. 신경세포들은 신경전달물질을 방출하여 수용체(receptor)를 자극시키고 이를 통하여 서로 정보를 교환하는 것이다. 인간의 두뇌가 기억을 저장하는 방식은 바로 시냅스의 연결강도를 지속적인 화학적, 전기적 자극을 통하여 강화하는 과정으로 이를 장기강화(long - term potentiation, LPT)라고 하며 이러한 지속적 자극을 통한 시냅스간의 신경전달 네트워크가 강화되는 시냅스의 특성을 시냅스가소성(synaptic plasticity)이라고 한다.There are 100 billion neurons in the human brain, each of which is connected to other neurons around each other by an average of 10,000 synapses, forming a network. Through these synapses, close communication between the neurons is achieved, and at this time, a neurotransmitter is a mediator. Neurons emit neurotransmitters to stimulate receptors and thereby exchange information with each other. The way the human brain stores memory is the process of strengthening the synaptic connection strength through continuous chemical and electrical stimulation. This is called long-term potentiation (LPT). Synaptic plasticity is called synaptic plasticity.
신경전달물질에 의하여 시냅스(synapse)에서 정보 전달이 화학적으로 이루어진다는 사실은 20세기 초에 실험적으로 증명되었다. 현재 알려져 있는 신경전달물질로는 아세틸콜린(acetylcholine, ACh), 놀에피네프린(norepinephrine), 도파민(dopamine, DA), 가바(GABA), 글라이신 및 글루타민산 등이 있다(표 1).The fact that neurotransmitters chemically communicate information at synapses was experimentally demonstrated in the early 20th century. Currently known neurotransmitters include acetylcholine (ACh), norepinephrine, norepinephrine, dopamine (DA), GABA, glycine and glutamic acid (Table 1).
(ACh)(ACh)
중뇌신경계Midbrain nervous system
(간, 소화액분비), 평활근이나 골격근의 수축 및 이완, 기억(Liver, digestive secretion), contraction and relaxation of smooth and skeletal muscle, memory
(pirenzepine), 항치매제(pirenzepine), anti-dementia
(tacrine)(tacrine)
(5-HT)(5-HT)
(imipramine)(imipramine)
(DA)(DA)
(선조체, 측좌핵)(Frame, nucleus)
(haloperidol) 항파킨슨약(haloperidol) anti-Parkinson's medicine
(L-dopa)(L-dopa)
(NA)(NA)
(교감신경)(Sympathetic)
중뇌신경계Midbrain nervous system
(desipramine)(desipramine)
(Gly)(Gly)
(strychine)(strychine)
(GABA)(GABA)
골격근 긴장도Skeletal muscle tone
트란키라이저Tranquilizer
(benzodiazepine)(benzodiazepine)
근이완약Muscle relaxant
(baclofen)(baclofen)
(Glu)(Glu)
(Enk)(Enk)
(morphine)(morphine)
(NO)(NO)
(nitroglycerin)(nitroglycerin)
노인성 치매의 일종인 알츠하이머병으로부터 유발되는 기억력 및 학습능력의 상실 등 인지 기능 장애는 대뇌 기저부의 아세틸콜린성 신경세포의 손상에서 비롯된다는 연구결과에 의해 무스카린성 아세틸콜린 수용체(muscarine acetylcholine receptor)에 대한 효능제(agonist), 아세틸콜린 생성촉진제, 아세틸콜린에스터라제 저해제 등 여러 가지 작용기전에 따라 아세틸콜린성 신경세포의 기능을 강화시켜줄 수 있는 약물들이 개발되어 왔다. 현재 아세틸콜린에스터라제 저해제로 타크린(tacrine)이 개발되어 있으나 위장장애와 같은 일부 부작용이 나타나고 있어 천연 식물소재로부터 저해활성소재를 탐색하여 활용할 필요가 있다.Cognitive dysfunction, such as the loss of memory and learning ability caused by Alzheimer's disease, a type of senile dementia, results from damage to acetylcholine neurons in the base of the cerebral base. Agonists, acetylcholine production promoters, and acetylcholinesterase inhibitors have been developed to enhance the function of acetylcholine neurons according to various mechanisms of action. Currently, tacrine has been developed as an acetylcholinesterase inhibitor, but some side effects such as gastrointestinal disorder have appeared, so it is necessary to search for and utilize inhibitory active materials from natural plant materials.
다닥냉이의 씨앗인 정력자 (Lepidium apefalum wild.)는 한국, 중앙아시아, 히말라야, 중국 등지에 분포한다. 북아메리카가 원산지이고 들 또는 인가 주변의 빈터에서 자란다. 종자는 갈색의 작은 원반 모양이고 가장자리에 흰색의 막질 날개가 있다. 정력자에는 지방유, 시니그린(Sinigrin) 및 당류등이 들어있다. 한방에서 말린 종자인 정력자를 기침과 천식, 폐결핵, 삼출성흉막염, 몸이 붓고 소변을 잘 보지 못하는 증세 및 심장 쇠약 등에 사용한다. Lepidium apefalum wild., The seed of the horseradish, is found in Korea, Central Asia, the Himalayas, and China. It is native to North America and grows in the clearings around fields or licensees. Seeds are small brown discoid, with white membranous wings at the edge. Energizers include fatty oils, Sinigrins and sugars. Energizer, a dried seed, is used for coughing, asthma, pulmonary tuberculosis, exudative pleurisy, swelling and poor urination, and heart failure.
인간의 두뇌에 긍정적인 효과를 주는 플라보노이드로는 콩에서 발견되는 이소플라본(isoflavones), 차와 코코아, 붉은 포도주에서 발견되는 플라바놀 (flavanols), 그리고 안소시아닌 (anthocyanins) 등이 있으며 그 작용 기전은 이소플라본의 경우 뇌에서 에스트로젠의 작용을 모방하며 플라바놀과 안소시아닌은 미토젠 활성 단백질 키나아제(mitogen-activated protein kinase, MAPK) 경로와 PI3키나아제 (phosphoinositide 3-kinase, PI3 kinase)/Akt 신호전달 단계 반응과 같은 뉴런의 신호전달 경로와 상호 작용한다. MAPK와 PI3 키나아제 경로 둘 모두 뇌의 해마와 피질에서 기억 저장 작용을 하는 것으로 알려져 있다. 즉 이 들 경로의 키나아제들을 활성화시킴으로써 기억과 학습을 증진시키는 잠재력을 갖는다. 또한 기억과 연관된 유전자의 발현에 관련되는 cAMP response element binding (CREB)과 같은 단백질의 활성화를 촉진하는 것으로 알려져 있다. 영국의 킹스 컬리지 런던(kings college london0 연구팀에 의하면 브로콜리, 감자, 오렌지, 사과 및 무에 함유되어 있는 성분들이 알츠하이머병 치료제와 동일한 방식으로 작용한다는 사실을 밝혀내었으며, 이 들 성분들이 신경전달물질인 아세틸콜린을 파괴시키는 효소인 아세틸콜린에스터라제를 저해한다는 사실을 밝혀내었다. Flavonoids that have a positive effect on the human brain include isoflavones found in soybeans, flavonols found in tea and cocoa, red wine, and anthocyanins. Silver isoflavones mimic the action of estrogens in the brain, while flavanols and ansocyanins are mitogen-activated protein kinase (MAPK) pathways and PI3 kinase (PI3 kinase) / Akt signaling. It interacts with signaling pathways in neurons, such as the delivery stage response. Both MAPK and PI3 kinase pathways are known to have memory storage functions in the hippocampus and cortex of the brain. It has the potential to enhance memory and learning by activating these pathway kinases. It is also known to promote the activation of proteins such as cAMP response element binding (CREB), which is involved in the expression of genes associated with memory. Researchers from Kings College London in the UK have found that the ingredients in broccoli, potatoes, oranges, apples and radishes work the same way as Alzheimer's. It has been shown to inhibit acetylcholinesterase, an enzyme that destroys acetylcholine.
따라서, 생약재 및 식물소재 등으로부터 아세틸콜린에스터라제 저해제 및 수용체 활성을 촉진시킬 수 있는 작용제(agonist) 및 길항제(antagonist)와 기억력 및 인지기능을 향상에 관련된 단백질을 활성화시키는 기능들을 탐색하고, 이로부터 선정된 천연식물 소재들을 이용하여 기억력 및 인지기능장애를 치료 및 예방할 수 있는 물질의 필요성이 대두되고 있다.
Accordingly, the present inventors searched for agonists and antagonists capable of promoting acetylcholinesterase inhibitors and receptor activity from herbal medicines and plant materials, and functions for activating proteins related to improving memory and cognitive function. There is a need for a material that can treat and prevent memory and cognitive dysfunction using selected natural plant materials.
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.
Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.
본 발명자들은 기억력 및 학습능력을 효과적으로 증진시킬 수 있으며 인체에 안전한 물질, 특히 식물-유래 물질을 개발하고자 예의 연구 노력하였고, 그 결과 종래부터 한약재로 사용되고 있는 정력자가 기억력 및 학습능력을 증진시키는데 매우 유효하다는 것을 규명함으로써, 본 발명을 완성하게 되었다.The present inventors have made diligent research efforts to develop a memory-safe substance, especially a plant-derived substance, which can effectively improve memory and learning ability, and as a result, it is very effective to enhance the memory and learning ability of the energetic energy that has been used as a traditional herbal medicine. By clarifying that the present invention has been completed.
따라서 본 발명의 목적은 정력자 추출물을 유효성분으로 포함하는 기억력 및 학습 능력 증진용 조성물을 제공하는 데 있다.Therefore, it is an object of the present invention to provide a composition for enhancing memory and learning ability comprising a sperm extract as an active ingredient.
본 발명의 다른 목적은 정력자 추출물을 유효성분으로 포함하는 인지기능장애 예방 또는 치료용 조성물을 제공하는 데 있다.Another object of the present invention to provide a composition for the prevention or treatment of cognitive dysfunction comprising a sperm extract as an active ingredient.
본 발명의 또 다른 목적은 정력자 추출물을 유효성분으로 포함하는 기억력 및 학습 능력 증진용 식품 조성물을 제공하는 데 있다.Still another object of the present invention is to provide a food composition for improving memory and learning ability including the sperm extract as an active ingredient.
본 발명의 또 다른 목적은 정력자 추출물을 유효성분으로 포함하는 인지기능장애 예방 또는 치료용 약제학적 조성물을 제공하는 데 있다.Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating cognitive dysfunction, which includes an extract as an active ingredient.
본 발명의 또 다른 목적은 정력자 추출물을 유효성분으로 포함하는 인지기능장애 예방 또는 개선용 식품 조성물을 제공하는 데 있다.
Still another object of the present invention is to provide a food composition for preventing or improving cognitive dysfunction, which includes sperm extract as an active ingredient.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.
Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.
본 발명의 일 양태에 따르면, 본 발명은 정력자 (Lepidium apefalum)추출물을 유효성분으로 포함하는 기억력 및 학습 능력 증진용 조성물을 제공한다.
According to an aspect of the present invention, the present invention provides a composition for enhancing memory and learning ability, including Lepidium apefalum extract as an active ingredient.
본 발명의 다른 양태에 따르면, 본 발명은 정력자 추출물을 유효성분으로 포함하는 인지기능장애 예방 또는 치료용 조성물을 제공한다.
According to another aspect of the present invention, the present invention provides a composition for the prevention or treatment of cognitive dysfunction, comprising a sperm extract as an active ingredient.
본 발명자들은 기억력 및 학습능력을 효과적으로 증진시킬 수 있으며 인체에 안전한 물질, 특히 식물-유래 물질을 개발하고자 예의 연구 노력하였고, 그 결과 종래부터 한약재로 사용되고 있는 정력자가 기억력 및 학습능력을 증진시키는데 매우 유효하다는 것을 규명하였다.The present inventors have made diligent research efforts to develop a memory-safe substance, especially a plant-derived substance, which can effectively improve memory and learning ability, and as a result, it is very effective to enhance the memory and learning ability of the energetic energy that has been used as a traditional herbal medicine. It was identified.
본 발명의 조성물에서 이용되는 정력자 추출물은 정력자에 추출용매를 처리하여 수득하는 경우에는, 다양한 추출용매가 이용될 수 있다. 바람직하게는, 극성 용매 또는 비극성 용매를 이용할 수 있다. 극성 용매로서 적합한 것은, (i) 물, (ii) 알코올(바람직하게는, 메탄올, 에탄올, 프로판올, 부탄올, 노말-프로판올, 이소-프로판올, 노말-부탄올, 1-펜탄올, 2-부톡시에탄올 또는 에틸렌글리콜), (iii) 아세트산, (iv) DMFO(dimethyl-formamide) 및 (v) DMSO(dimethyl sulfoxide)를 포함한다. 비극성 용매로서 적합한 것은, 아세톤, 아세토나이트릴, 에틸 아세테이트, 메틸 아세테이트, 플루오로알칸, 펜탄, 헥산, 2,2,4-트리메틸펜탄, 데칸, 사이클로헥산, 사이클로펜탄, 디이소부틸렌, 1-펜텐, 1-클로로부탄, 1-클로로펜탄, o-자일렌, 디이소프로필 에테르, 2-클로로프로판, 톨루엔, 1-클로로프로판, 클로로벤젠, 벤젠, 디에틸 에테르, 디에틸 설파이드, 클로로포름, 디클로로메탄, 1,2-디클로로에탄, 어닐린, 디에틸아민, 에테르, 사염화탄소 및 THF를 포함한다.When the sperm extract used in the composition of the present invention is obtained by treating the sperm with an extraction solvent, various extraction solvents may be used. Preferably, a polar solvent or a nonpolar solvent can be used. Suitable polar solvents include (i) water, (ii) alcohols (preferably methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, 1-pentanol, 2-butoxyethanol Or ethylene glycol), (iii) acetic acid, (iv) dimethyl-formamide (DMFO) and (v) dimethyl sulfoxide (DMSO). Suitable as nonpolar solvents are acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1- Pentene, 1-chlorobutane, 1-chloropentane, o -xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloro Methane, 1,2-dichloroethane, anneal, diethylamine, ether, carbon tetrachloride and THF.
보다 바람직하게는, 본 발명에서 이용되는 추출용매는 (a) 물, (b) 탄소수 1-4의 무수 또는 함수 저급 알코올 (메탄올, 에탄올, 프로판올, 부탄올 등), (c) 상기 저급 알코올과 물과의 혼합용매, (d) 아세톤, (e) 에틸 아세테이트, (f) 클로로포름, (g) 부틸아세테이트, (h) 1,3-부틸렌글리콜, (i) 헥산 및 (j) 디에틸에테르를 포함한다. 가장 바람직하게는, 본 발명의 추출물은 물, 메탄올, 에탄올 또는 이의 조합을 정력자에 처리하여 수득한 것이다.More preferably, the extraction solvent used in the present invention is (a) water, (b) anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) the lower alcohol and water Mixed solvent with (d) acetone, (e) ethyl acetate, (f) chloroform, (g) butyl acetate, (h) 1,3-butylene glycol, (i) hexane and (j) diethyl ether Include. Most preferably, the extract of the present invention is obtained by treating the energizer with water, methanol, ethanol or a combination thereof.
본 명세서에서 사용되는 용어 ‘추출물’은 상술한 바와 같이 당업계에서 조추출물(crude extract)로 통용되는 의미를 갖지만, 광의적으로는 추출물을 추가적으로 분획(fractionation)한 분획물도 포함한다. 즉, 정력자 추출물은 상술한 추출용매를 이용하여 얻은 것뿐만 아니라, 여기에 정제과정을 추가적으로 적용하여 얻은 것도 포함한다. 예컨대, 상기 추출물을 일정한 분자량 컷-오프 값을 갖는 한외 여과막을 통과시켜 얻은 분획, 다양한 크로마토그래피 (크기, 전하, 소수성 또는 친화성에 따른 분리를 위해 제작된 것)에 의한 분리 등, 추가적으로 실시된 다양한 정제 방법을 통해 얻어진 분획도 본 발명의 정력자 추출물에 포함되는 것이다.As used herein, the term "extract" has the meaning commonly used as a crude extract in the art as described above, but also broadly includes a fraction additionally fractionating the extract. That is, the sperm extract includes not only those obtained by using the aforementioned extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, fractions obtained by passing the extract through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), etc. The fraction obtained through the purification method is also included in the sperm extract of the present invention.
본 발명에서 이용되는 정력자 추출물은 감압 증류 및 동결 건조 또는 분무 건조 등과 같은 추가적인 과정에 의해 분말 상태로 제조될 수 있다.The sperm extract used in the present invention may be prepared in powder form by an additional process such as distillation under reduced pressure and freeze drying or spray drying.
본 발명의 조성물은 기억력 및 학습능력 증진 뿐 만 아니라 인지능력장애 예방 및 치료에 매우 효과적으로 작용한다.The composition of the present invention works very effectively in preventing and treating cognitive impairment as well as enhancing memory and learning ability.
상기 본원발명의 효과들은 신경세포, 바람직하게는 대뇌 기저부의 아세틸콜린성 신경세포의 보호 및 손상을 억제시킴으로써 발휘된다.The effects of the present invention are exerted by inhibiting the protection and damage of neurons, preferably acetylcholinergic neurons in the base of the cerebral base.
아세틸콜린(Acetylcholine)은 신경말단에서 분비되너 신경의 자극을 근육에 전달하는 신경전달물질로 자극이 전달이 끝나면 아세틸콜린스테라아제(Acetylcholinesterase)에 의하여 콜린과 아세테이트로 분해된다.Acetylcholine is a neurotransmitter that is secreted at the nerve endings and delivers nerve stimuli to muscles. After stimulation is completed, acetylcholine is broken down into choline and acetate by acetylcholinesterase.
본 발명의 바람직한 구현예에 따르면, 본 발명은 아세틸콜린스테라아제(Acetylcholinesterase)의 활성을 억제시킴으로써 아세틸콜린스테라아제에 의한 아세틸콜린의 분해를 억제하여 신경세포의 보호 및 손상을 억제시킬 수 있다.According to a preferred embodiment of the present invention, the present invention can inhibit the degradation of acetylcholine by acetylcholinesterase by inhibiting the activity of acetylcholinesterase to inhibit the protection and damage of nerve cells.
또한, 본 발명은 항산화 활성을 통하여 신경세포의 손상으로 인한 기억력 감퇴 및 학습능력 저하를 억제할 뿐 만 아니라 신경세포의 손상을 예방하여 기억력 및 학습능력을 향상시킬 수 있는 효과를 발휘한다.In addition, the present invention not only suppresses memory loss and learning ability degradation due to nerve cell damage through antioxidant activity, but also prevents nerve cell damage, thereby exerting the effect of improving memory and learning ability.
본 발명의 명세서에서 사용하는 용어 “활성산소종”은 유기호흡을 하는 생물에게 있어 필수적인 산소가, 세포 내의 효소 그리고 대부분의 전자 운반 과정 혹은 에너지대사 과정 중에 불완전하게 환원되거나 펩티드 성장인자, 사이토카인들 및 다양한 작용의 자극에 의해 발생되는 산소부산물들을 의미한다. 즉, 활성산소종은 프리 래디칼(free radical: 어떠한 원자 또는 분자가 외곽궤도에 짝 없는 전자를 가지고 있는 형태)을 가져 안정되지 못한 상태를 의미하며, 그로 인해 강한 활성을 가진다. As used herein, the term “reactive oxygen species” means that oxygen, which is essential for organisms that induce organic respiration, is incompletely reduced, or peptide growth factors, cytokines, or enzymes in cells and during most electron transport or energy metabolism processes. And oxygen by-products generated by stimulation of various actions. In other words, reactive oxygen species have a free radical (a form in which an atom or molecule has electrons unpaired in its outer orbit), which means that it is unstable, and thus has strong activity.
활성산소종은 생산이 과잉되면 생체에 대해 독성 즉, 산화적 손상(oxidative stress)을 가져온다 하여 유해산소라고 명명되기도 한다. 세포내의 거대한 분자(단백질, 지질 등)를 산화시킴으로써 세포의 항상성을 파괴하고, 세포를 사멸시키는 등의 작용으로 세포조직 내에 치명적인 손상을 유발하며, 암, 근이형증, 알츠하이머병, 파킨슨씨병, 허혈성질환, 동맥경화증과 같은 다양한 퇴행성 질병의 원인과 일반적 노화와 관계가 있기 때문이다. Reactive oxygen species are sometimes referred to as harmful oxygen because excessive production leads to toxicity to the living organism, that is, oxidative stress. By oxidizing huge molecules (proteins, lipids, etc.) in the cell, it destroys the homeostasis of cells and kills the cells, causing fatal damage in cell tissues, cancer, muscular dysplasia, Alzheimer's disease, Parkinson's disease, This is because it is related to the causes of various degenerative diseases such as atherosclerosis and general aging.
예컨대, 활성산소종에는 수퍼옥사이드 아니온 래디칼(superoxide anion radical: O2-), 하이드로겐 퍼옥사이드(hydrogen peroxide: H2O2), 하이드록시 래디칼(hydroxyl radical: OH) 외에도 리피드 퍼옥사이드(lipid peroxide), 리트릭 옥사이드(Nitric oxide: NO), 퍼옥시니트리트(peroxinitrite: NO3 2-), 티올퍼옥시 래디칼(thiol peroxy radical: R-SO2-)등이 있으며, 이 중 H2O2가 TGF, -1, PDGF 및 EGF등의 자극에 의해 생성되는 주종으로, 2차 신호전달 물질로서 가장 주목받는다. 그 이유는 전이금속과 반응하지 않는 한 상대적으로 안정하고, 독성이 적으며, 쉽게 확산되어 세포막을 통과할 수 있기 때문에 외부자극에 반응하여 빠른 시간 내에 생성 소멸되어야 하는 신호전달 물질로 적합한 특성을 가지기 때문이다.For example, reactive oxygen species include lipid peroxide (lipid) in addition to superoxide anion radical (O 2- ), hydrogen peroxide (H 2 O 2 ), hydroxy radical (OH), and the like. peroxide, nitric oxide (NO), peroxynitrite (NO 3 2- ), thiol peroxy radical (R-SO 2- ), among others H 2 O It is the main species produced by stimulation such as bivalent TGF, -1, PDGF, and EGF, and attracts the most attention as a secondary signaling substance. The reason is that it is relatively stable, less toxic, and easily diffuses and can pass through cell membranes unless it reacts with transition metals. Because.
본 발명의 바람직한 구현예에 따르면, 본 발명은 항산화 활성을 가지며, 보다 바람직하게는 활성산소종(Reactive oxygen species)의 발생을 억제 또는 제거하여, 궁극적으로 신경세포의 손상을 억제하여 기억력 및 학습능력 증진 뿐 만 아니라 인지기능장애를 예방 및 치료가 가능한 조성물이다.According to a preferred embodiment of the present invention, the present invention has antioxidant activity, more preferably inhibits or eliminates the generation of reactive oxygen species, and ultimately inhibits the damage of neurons to memory and learning ability It is a composition capable of preventing and treating cognitive dysfunction as well as enhancement.
본 발명은 NMDA (N-methyl-D-aspartate) 리셉터와 결함하여 신경세포의 사멸을 억제함으로써 신경세포의 보호 및 손상을 억제시킨다. 그러므로, 본 발명은 NMDA 리셉터와 결합하여 신경세포 손상으로 인한 기억력 및 학습능력의 저하를 억제시킬 수 있을 뿐 만 아니라 인지능력장애를 예방 및 치료가 가능하다.The present invention inhibits neuronal cell death by inhibiting neuronal cell death by inhibiting NMDA (N-methyl-D-aspartate) receptor, thereby inhibiting neuronal cell protection and damage. Therefore, the present invention can be combined with the NMDA receptor not only to suppress the decrease in memory and learning ability due to neuronal damage, but also to prevent and treat cognitive impairment.
본 발명의 바람직한 구현예에 따르면, 본 발명은 NMDA (N-methyl-D-aspartate) 리셉터의 길항제(antagonist)로 작용하여 신경세포의 손상을 억제시킴으로써 기억력 및 학습능력을 향상시키고 인지능력장애를 예방 및 치료할 수 있다.According to a preferred embodiment of the present invention, the present invention acts as an antagonist of the N-methyl-D-aspartate (NMDA) receptor to inhibit neuronal damage, thereby improving memory and learning ability and preventing cognitive impairment. And treatment.
본 발명의 명세서에서 ‘인지기능장애’는 기억처리, 지각 및 문제해결과 같은 인지기능을 발휘하지 못하는 질환을 의미한다.'Cognitive dysfunction' in the specification of the present invention refers to a disease that does not exhibit cognitive functions such as memory processing, perception and problem solving.
본 발명은 인지기능장애를 예방 및/또는 치료할 수 있는 조성물이다. 보다 구체적으로, 본 발명은 아세틸콜린스테라아제(Acetylcholinesterase)의 활성을 억제, 항산화 활성(예컨대, 활성산소종) 및 NMDA (N-methyl-D-aspartate) 리셉터와의 결합을 통하여 인지기능장애를 예방 및/또는 치료할 수 있는 조성물이다.The present invention is a composition capable of preventing and / or treating cognitive dysfunction. More specifically, the present invention inhibits the activity of acetylcholinesterase, prevents cognitive dysfunction through the combination of antioxidant activity (eg, reactive oxygen species) and NMDA (N-methyl-D-aspartate) receptor And / or compositions that can be treated.
본 발명의 바람직한 구현예에 따르면, 본 발명에서의 인지기능장애는 치매(dementia), 학습장애(learning disorder), 실인증(agnosia), 건망증(amnesia), 실어증(aphasia), 실행증(apraxia) 또는 섬망(delirium)을 의미하며, 보다 바람직하게는 AIDS 유발 치매(AIDS dementia complex), 빈스완거 병(Binswanger's disease), 루이소체 치매(dementia with Lewy Bodies), 전두측두엽성 치매(frontotemporal dementia), 경도 인지 장애(mild cognitive impairment), 다발성 경색성 치매(multi-infarct dementia), 픽병(Pick's disease), 의미치매(semantic dementia), 알츠하이머성 치매(Alzheimer's dementia) 또는 혈관성 치매(vascular dementia)을 의미한다.
According to a preferred embodiment of the present invention, the cognitive impairment in the present invention is dementia, learning disorder, agnosia, amnesia, aphasia, apraxia or Delirium, more preferably AIDS dementia complex, Binswanger's disease, dementia with Lewy Bodies, frontotemporal dementia, mild perception Mild cognitive impairment, multi-infarct dementia, Pick's disease, semantic dementia, Alzheimer's dementia or vascular dementia.
본 발명의 조성물은 약제학적 조성물로 제조될 수 있다.The composition of the present invention may be prepared as a pharmaceutical composition.
본 발명의 바람직한 구현예에 따르면, 본 발명의 조성물은 (a) 상술한 본 발명의 정력자 추출물의 약제학적 유효량; 및 (b) 약제학적으로 허용되는 담체를 포함하는 약제학적 조성물이다. 본 명세서에서 용어 “약제학적 유효량”은 상술한 정력자 추출물의 효능 또는 활성을 달성하는 데 충분한 양을 의미한다.According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a pharmaceutically effective amount of the sperm extract of the present invention as described above; And (b) a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to achieve the efficacy or activity of the aforementioned sperm extract.
본 발명의 조성물이 약제학적 조성물로 제조되는 경우, 본 발명의 약제학적 조성물은 약제학적으로 허용되는 담체를 포함한다. 본 발명의 약제학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences (19th ed., 1995)에 상세히 기재되어 있다.When the composition of the present invention is made into a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
본 발명의 약제학적 조성물은 경구 또는 비경구 투여할 수 있으며, 바람직하게는 경구 투여 방식으로 적용된다.The pharmaceutical composition of the present invention may be administered orally or parenterally, and preferably applied by oral administration.
본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. 본 발명의 약제학적 조성물의 일반적인 투여량은 성인 기준으로 0.001-100 ㎎/kg 범위 내이다.Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. Typical dosages of the pharmaceutical compositions of the invention are in the range of 0.001-100 mg / kg on an adult basis.
본 발명의 약제학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액, 시럽제 또는 유화액 형태이거나 엑스제, 산제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.
The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.
본 발명의 조성물은 식품 조성물로 제공될 수 있다.The composition of the present invention may be provided as a food composition.
본 발명의 조성물이 식품 조성물로 제조되는 경우, 유효성분으로서 정력자 추출물뿐 만 아니라, 식품 제조 시에 통상적으로 첨가되는 성분을 포함하며, 예를 들어, 단백질, 탄수화물, 지방, 영양소, 조미제 및 향미제를 포함한다. 상술한 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스, 올리고당 등; 및 폴리사카라이드, 예를 들어 덱스트린, 사이클로덱스트린 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 향미제로서 천연 향미제 [타우마틴, 스테비아 추출물 (예를 들어 레바우디오시드 A, 글리시르히진 등]) 및 합성 향미제(사카린, 아스파르탐 등)를 사용할 수 있다. 예컨대, 본 발명의 식품 조성물이 드링크제로 제조되는 경우에는 본 발명의 정력자 추출물 이외에 구연산, 액상과당, 설탕, 포도당, 초산, 사과산, 과즙, 두충 추출액, 대추 추출액, 감초 추출액 등을 추가로 포함시킬 수 있다.
When the composition of the present invention is prepared as a food composition, as an active ingredient, not only sperm extract, but also components commonly added in food production, for example, proteins, carbohydrates, fats, nutrients, seasonings and Contains flavors. Examples of the above carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and xylitol, sorbitol, erythritol. As the flavoring agent, natural flavoring agents (tauumatin, stevia extract (for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is prepared with a drink, in addition to the energetic extract of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, tofu extract, jujube extract, licorice extract, etc. Can be.
본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:
(ⅰ) 본 발명은 정력자 추출물을 유효성분으로 포함하는 기억력 및 학습 능력 증진용 조성물 및 인지기능장애 예방 및 치료용 조성물을 제공한다.(Iii) The present invention provides a composition for improving memory and learning ability, and a composition for preventing and treating cognitive dysfunction, which includes a sperm extract as an active ingredient.
(ⅱ) 본 발명은 아세틸콜린스테라아제의 활성을 억제, 항산화 활성(예컨대, 활성산소종) 및 NMDA 리셉터와의 친화력을 통하여 신경세포, 특히 대뇌 기저부의 신경세포의 손상을 억제시킬 수 있는 효과를 가진다.(Ii) The present invention has the effect of inhibiting the activity of acetylcholinesterase, antioxidant activity (e.g., reactive oxygen species) and affinity with NMDA receptors to inhibit the damage of nerve cells, especially nerve cells at the base of the cerebrum. Have
(ⅲ) 본 발명은 신경세포를 보호 및 손상의 예방 효과를 통하여 기억력 및 학습 능력을 증진시킬 수 있을 뿐 만 아니라 인지기능장애로 질환을 예방 및 치료 할 수 있는 효능을 발휘한다.(Iii) The present invention not only enhances memory and learning ability through protecting and protecting nerve cells, but also exerts effects of preventing and treating diseases with cognitive impairment.
(ⅳ) 또한, 본 발명은 기억력 및 학습 능력의 증진 효능, 또는 인지기능장애를 예방 및 치료 효능을 가지는 정력자 추출물의 의약 및 식품으로서의 기초적인 자료를 제공한다.
(Iii) The present invention also provides basic data as medicines and foods of sperm extracts having enhanced efficacy of memory and learning ability, or prevention and treatment of cognitive dysfunction.
도 1은 본 발명에서 정력자 에탄올 추출물의 AChE를 저해시키는 효과를 나타낸 결과이다.
도 2는 본 발명에서 정력자 에탄올 추출물의 항산화 활성 효과를 나타낸 결과이다.
도 3은 본 발명에서 정력자 에탄올 추출물의 NMDA 리셉터 결합 효과를 나타낸 결과이다.Figure 1 is a result showing the effect of inhibiting the AChE of sperm ethanol extract in the present invention.
Figure 2 is the result showing the antioxidant activity of the sperm ethanol extract in the present invention.
Figure 3 is a result showing the binding effect of the NMDA receptor ethanol extract ethanol in the present invention.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .
실시예Example
본 명세서 전체에 걸쳐, 특정 물질의 농도를 나타내기 위하여 사용되는 “%“는 별도의 언급이 없는 경우, 고체/고체는 (중량/중량) %, 고체/액체는 (중량/부피) %, 그리고 액체/액체는 (부피/부피) %이다.
Throughout this specification, "%" used to denote the concentration of a particular substance is intended to include solids / solids (wt / wt), solid / liquid (wt / The liquid / liquid is (vol / vol)%.
실험재료 및 방법Materials and Methods
본 실험에서는 동인한방제약에서 정력자를 구입하여 사용하였다. 건조된 정력자에 80% 에탄올을 가해 COSMOS-660 초고속 진공 저온 추출기로 95℃에서 6시간 동안 추출 및 농축하였다. 농축된 시료는 -80℃에 보관하여 결빙시킨 후 동결건조기로 건조하여 분말형태의 시료가 실험에 사용되었다. 시료는 실험직전에 3차 증류수 또는 DMSO (dimethyl sulfoxide)로 녹여 시료 검액으로 하였다.
In this experiment, energetics were purchased from Dongin Oriental Medicine. 80% ethanol was added to the dried energizer and extracted and concentrated at 95 ° C. for 6 hours using a COSMOS-660 ultrafast vacuum cold extractor. The concentrated sample was stored at -80 ° C, frozen and dried with a lyophilizer, and a powdered sample was used for the experiment. The sample was dissolved in tertiary distilled water or DMSO (dimethyl sulfoxide) immediately before the experiment to prepare a sample sample.
AChE (Acetylcholinesterase) 저해활성의 측정Measurement of AChE (Acetylcholinesterase) Inhibitory Activity
정력자추출물의 AChE (Acetylcholinesterase) 저해활성의 측정은 Ellman 등의 방법을 변형하여 사용하였다(Ellman, G. L., et al, Biochem. Pharmacol., 7:88-95, (1961)). AChE 저해활성은 AChE가 아세틸티오콜린(acetylthiocholine)을 가수분해할 때 생성되는 반응산물인 티오콜린(thiocholine)이 DTNB(5,5'-dithio bis(-2-nitrobenzoic acid))와 반응하여 노란색의 NTB(2-nitrobenzoic acid)를 생성하므로, 이때 생성되는 NTB를 412 ㎚에서 흡광도를 측정하여 관찰하였다.Determination of AChE (Acetylcholinesterase) inhibitory activity of sperm extract was used by modifying the method of Ellman et al . (Ellman, GL, et al, Biochem. Pharmacol. , 7: 88-95, (1961)). AChE inhibitory activity is yellow when the reaction product, thiocholine, produced when AChE hydrolyzes acetylthiocholine, reacts with DTNB (5,5'-dithio bis (-2-nitrobenzoic acid)). Since NTB (2-nitrobenzoic acid) is produced, the resulting NTB was observed by measuring absorbance at 412 nm.
즉, 500 ㎕ 큐벳(cuvette)을 반응조로 하여 0.1 M SPB(sodium phosphate buffer; pH 8.0) 445 ㎕와 시료검액 25 ㎕(최종농도 100 ㎍/㎖)를 첨가하고, 커플링제인 0.001 M DTNB 18 ㎕(Sigma Aldrich, St.Louis, MO, USA)와 기질인 0.0075 M 아세틸티오콜린 요오드 6 ㎕(Sigma Aldrich, St.Louis, MO, USA)를 넣은 후 25℃의 항온조에서 4분간 반응시켰다. 반응 즉시 효소인 아세티콜린스테아제 6 ㎕(0.072 unit(Sigma Aldrich, St.Louis, MO, USA)를 첨가하여 25℃의 항온조에서 12분 동안 반응시킨 후 412 ㎚에서 흡광도를 측정하였다.That is, 500 μl cuvette was used as a reaction tank, and 445 μl of 0.1 M sodium phosphate buffer (pH 8.0) and 25 μl of sample sample solution (final concentration 100 μg / ml) were added, and the coupling agent was 0.001 M DTNB 18 μl. (Sigma Aldrich, St.Louis, MO, USA) and 6 μl (Sigma Aldrich, St.Louis, MO, USA) of 0.0075 M acetylthiocholine iodine (Sigma Aldrich, St.Louis, MO, USA) were added and reacted for 4 minutes in a 25 ° C incubator. Immediately after the reaction, 6 µl (0.072 unit (Sigma Aldrich, St. Louis, MO, USA), an enzyme, aceticocholinesterase was added thereto, and reacted for 12 minutes in a 25 ° C incubator, and the absorbance was measured at 412 nm.
모든 실험군은 3회 중복으로 실시하여 흡광도의 평균값을 구했으며, 정력자추출물의 AChE 저해활성 효과는 정력자추출물 대신 동량의 0.1 M SPB(pH 8.0)를 넣은 대조군의 흡광도 값과 정력자추출물을 넣은 실험군의 흡광도 값을 이용하여 아래와 같이 계산하여 산출하였다. 한편, 양성 대조구는 단계별 농도의 타크린(Sigma Aldrich, St.Louis, MO, USA)을 사용하였다.All the experimental groups were repeated three times to obtain the average absorbance. The AChE inhibitory activity of the sperm extract was obtained by adding the absorbance value and the sperm extract of the control group with the same amount of 0.1 M SPB (pH 8.0) instead of the sperm extract. Using the absorbance value of the experimental group was calculated by calculating as follows. On the other hand, the positive control was used tacrine (Sigma Aldrich, St. Louis, MO, USA) of the concentration level.
항산화 활성의 측정Determination of Antioxidant Activity
정력자추출물의 항산화 활성은 Cayman사의 항산화 어세이 키트를 이용하여 ABTS(2,2‘-azino-di-[3-ethylbenzthiazoline silphonate])가 메트마이오글로빈(metmyoglobin)에 의해서 ABTS+로 산화되는 것을 억제하는 정도를 405 ㎚에서 흡광도를 측정하여 관찰하였다.Antioxidant activity of sperm extracts inhibits the oxidation of ABTS (2,2'-azino-di- [3-ethylbenzthiazoline silphonate]) to metsoglobin by ABTS + using Cayman's antioxidant assay kit The degree was observed by measuring the absorbance at 405 nm.
즉, 메트미오글로빈과 과산화수소를 반응시키면 페릴 마이오글로빈(ferryl myoglobin) 라디칼이 생성되는데, 이 때 생성된 페릴마이오글로빈 라디칼은 ABTS를 라디칼 양이온을 가진 ABTS+로 산화시키고 이것은 405㎚에서 녹색을 나타낸다. 정력자추출물 속에 존재하는 항산화제는 전자공여 라디칼 소거에 의해 이 반응을 억제함으로써 녹색을 띄는 ABTS 라디칼 생성을 억제하고 이를 흡광도 값으로 측정하여 나타낼 수 있다. 이 때, ABTS의 산화를 억제하는 정력자추출물 속 항산화제의 양은 토코페롤 유사체인 트롤록스(Trolox)와 비교하여 표기하였다.In other words, the reaction of metomioglobin with hydrogen peroxide produces a perryl myoglobin radical, which produces perylmyoglobin radicals, which oxidize ABTS to ABTS + with a radical cation, which is green at 405 nm. Antioxidants present in the sperm extract can be suppressed by the electron donor radical scavenging to inhibit the production of green ABTS radicals and measured by absorbance values. At this time, the amount of antioxidant in the sperm extract inhibiting the oxidation of ABTS was expressed in comparison with Trolox, a tocopherol analogue.
96 웰에 정력자추출물 10 ㎕와 메트마이오글로빈 10 ㎕(Cayman Chemical Company, MI, USA), 크로모겐 150 ㎕(Cayman Chemical Company, MI, USA)를 넣고 40 ㎕의 과산화수소(Cayman Chemical Company, MI, USA)를 첨가하여 반응시켰다. 뚜껑을 덮고 상온에서 5분간 반응시킨 뒤 405 ㎚에서 흡광도를 측정하였다. 모든 실험군은 3회 중복으로 실시하여 흡광도의 평균값을 구했으며, 정력자 추출물의 항산화 활성은 시료 대신 농도별로 희석된 동량의 트롤록스 표준 용액(Trolox standard solution, Cayman Chemical Company, MI, USA)을 넣어 측정한 후, 시료를 넣은 실험군의 흡광도 값과 비교하여 나타내었다.
Into a 96 well, add 10 μl of sperm extract, 10 μl of metioglobin (Cayman Chemical Company, MI, USA), 150 μl of chromogen (Cayman Chemical Company, MI, USA), and 40 μl of hydrogen peroxide (Cayman Chemical Company, MI, USA). ) Was added to react. After the lid was reacted at room temperature for 5 minutes, the absorbance was measured at 405 nm. All experiment groups were repeated three times to obtain the average absorbance.The antioxidant activity of the sperm extract was added to the same amount of Trolox standard solution (Trolox standard solution, Cayman Chemical Company, MI, USA) instead of the sample. After the measurement, it was shown by comparing with the absorbance value of the experimental group put the sample.
NMDA 리셉터 친화성 테스트NMDA Receptor Affinity Test
NMDA 수용체의 친화력은 마우스의 대뇌에서 분리한 NMDA 수용체와 [3H]-글리신과의 결합력을 식물추출물이 저해하는 정도를 측정함으로써 알아보았다.The affinity of the NMDA receptor was determined by measuring the extent to which plant extracts inhibited the binding of NMDA receptor and [ 3 H] -glycine isolated from the mouse brain.
96 웰 플레이트에 마우스의 대뇌에서 분리한 멤브레인 180 ㎕와 농도별로 희석된 정력자 추출물 10 ㎕, 300 nM [3H]-글리신(Sigma Aldrich, St.Louis, MO, USA) 10 ㎕를 넣고, 얼음 위에서 40분간 반응시켰다. 반응이 끝난 후, 글래스 피버 필터(glass fiber filter; 1450-421 Filtermat A)를 이용하여 셀 하베스터(MicroBeta FilterMate-96 Harvester, USA)에서 필터링 한 후 오븐에서 완전히 건조시켰다. 건조 후, 칵테일 용액을 넣고 바이크로베타 카운터(MicroBeta counter; Wallac 1450 MicroBeta counter, Perkin-Elmer, USA)를 이용하여 방사능을 측정하였으며, 이를 이용하여 특이적인 결합을 계산하였다. 모든 실험군은 3회 중복으로 실시하여 평균값을 구했으며, 비-특이적인 결합의 계산을 위해서 1 mM의 D-세린을 사용하였다. 식물 추출물의 특이적인 결합과 NMDA 수용체의 친화력은 다음과 같이 계산하여 산출하였다.In a 96 well plate, 180 μl of membranes isolated from the cerebrum of the mouse and 10 μl of sperm extract diluted by concentration, and 10 μl of 300 nM [ 3 H] -glycine (Sigma Aldrich, St. Louis, MO, USA) were placed on ice. The reaction was carried out for 40 minutes. After the reaction, the glass fiber filter (glass fiber filter; 1450-421 Filtermat A) was filtered in a cell harvester (MicroBeta FilterMate-96 Harvester, USA) and dried in an oven completely. After drying, the cocktail solution was added and radioactivity was measured using a MicroBeta counter (Wallac 1450 MicroBeta counter, Perkin-Elmer, USA), and specific binding was calculated using this. All experimental groups were averaged by performing three replicates, and 1 mM of D-serine was used for the calculation of non-specific binding. Specific binding of plant extracts and affinity of NMDA receptors were calculated as follows.
특이적인 결합 = 총 결합 - 비-특이적인 결합Specific binding = total binding-non-specific binding
(* CPM : Counts per minute)
(* CPM: Counts per minute)
통계처리Statistical processing
본 연구의 실험결과는 평균과 표준편차로 표시하고 유의성 검정은 시그마 플롯(Sigma Plot)을 이용하여 Student's t-테스트를 실시하였다.
The experimental results of this study are expressed as mean and standard deviation, and the significance test was performed by Student's t- test using Sigma Plot.
결과 및 고찰Results and Discussion
AChE (Acetylcholinesterase) 저해활성의 측정Measurement of AChE (Acetylcholinesterase) Inhibitory Activity
아세틸콜린은 신경말단에서 분비되어 신경의 자극을 근육에 전달하는 신경전달물질로 자극의 전달이 끝나면 AChE에 의해서 콜린과 아세테이트로 분해된다.Acetylcholine is a neurotransmitter that is secreted at the nerve endings and delivers nerve impulses to muscles. After stimulus delivery, acetylcholine is broken down into choline and acetate by AChE.
노인성 치매로 인해 유발되는 기억력 상실 및 학습력 저하와 같은 인지기능 장애는 대뇌 기저부의 아세틸콜린성 신경세포 손상으로부터 기인된다는 가설이 가장 확실시 되고 있으며, 현재 전 세계적으로 가장 활발하게 연구, 개발되고 있으며 FDA의 허가를 받은 치매 개선제는 모두 AChE 저해제이다.The hypothesis that cognitive impairment, such as memory loss and learning deterioration caused by senile dementia, is most likely to result from acetylcholine neuronal damage in the base of the cerebral base, is currently the most active research and development worldwide and is approved by the FDA. All dementia improvers received were AChE inhibitors.
따라서 본 실험에서는 효소활성을 평가함으로써 AChE 저해활성이 있는 식물자원을 탐색하였다.Therefore, in this experiment, we investigated plant resources with AChE inhibitory activity by evaluating enzymatic activity.
정력자의 에탄올 추출물을 양성대조구로 사용한 타크린과 비교하여 AChE 저해활성을 [도 1]에 나타내었다. 정력자 추출물을 최종농도 각각 0.01 ㎎/㎖, 0.1 ㎎/㎖, 1 ㎎/㎖ 및 10 ㎎/㎖의 4개의 농도에서 아세틸콜린에스테라제 저해효과를 실험하였다. 정력자의 저해효과는 각각 20.2%, 34.4%, 72.4% 및 120%의 저해능을 나타내었다. 추출물의 농도에 의존적으로 저해활성이 증가되었다. 특히, 1 ㎎/㎖의 시료 농도에서 약 72.4%의 저해효과를 나타내어 정력자 추출물이 AChE 활성 억제 작용에 비교적 우수한 천연물로 확인되었다.
AChE inhibitory activity is shown in [FIG. 1] compared to tacrine using ethanol extract of sperm as a positive control. Energizer extracts were tested for acetylcholinesterase inhibitory effects at four concentrations of 0.01 mg / ml, 0.1 mg / ml, 1 mg / ml and 10 mg / ml, respectively. The inhibitory effect of sperm showed 20.2%, 34.4%, 72.4%, and 120%, respectively. Inhibitory activity was increased depending on the concentration of the extract. In particular, it showed an inhibitory effect of about 72.4% at a sample concentration of 1 mg / ㎖, the sperm extract was found to be a relatively good natural product for inhibiting AChE activity.
항산화 활성의 측정Determination of Antioxidant Activity
활성산소종(Reactive oxygen species)은 세포의 지질, 단백질 및 DNA를 공격하여 과산화지질과 산화단백질 등을 생성하고 노화를 촉진하며, 뇌 조직에 산화적 손상과 뇌세포의 파괴를 일으켜 치매를 유발하는 것으로 보고되고 있다. 따라서 활성산소를 제거하는 것이 치매를 예방하고 치료하는데 매우 중요한 것으로 여겨진다.Reactive oxygen species attack lipids, proteins, and DNA of cells to produce lipid peroxides and oxidative proteins, promote aging, and cause dementia by causing oxidative damage and destruction of brain cells in brain tissue. It is reported. Therefore, the removal of free radicals is considered to be very important in preventing and treating dementia.
일반적으로 활성산소종은 자동산화 반응을 통해 연쇄적으로 일어나기 때문에 체내에서는 항산화제가 자동산화반응 중에 생성되는 라디칼에 수소원자를 공여함으로써 이 반응을 종결시킨다.In general, reactive oxygen species occur in series through an autooxidation reaction, so the antioxidants in the body terminate the reaction by donating hydrogen atoms to radicals generated during the autooxidation reaction.
이렇게 항산화제는 전자 전달계에서 라디칼 소거에 의해서 산화를 억제하게 되는데, 본 실험에서는 지실 에탄올추출물의 항산화 활성을 측정함으로써 기억력 증진에 도움을 주는 천연물질을 탐색하고자 하였다.Antioxidants inhibit oxidation by radical scavenging in the electron transport system. In this experiment, we tried to search for natural substances that help improve memory by measuring the antioxidant activity of fruit ethanol extract.
식물추출물에 대한 항산화 활성은 총 항산화력을 측정하는 Cayman사의 항산화제 어세이 키트를 이용하여 측정하였으며 실험결과는 도 2에 나타내었다.Antioxidant activity against plant extracts was measured using Cayman's Antioxidant Assay Kit, which measures the total antioxidant power, and the experimental results are shown in FIG. 2.
식물추출물 10 ㎍/㎖ 농도에서 항산화 활성의 측정하였다. 항산화 활성이 높은 물질일수록 ABTS 라디칼 (녹색)의 생성을 억제하여 O.D. 값이 낮게 측정된다. 정력자는 표준물질인 트롤록스와 비교했을 때 트롤록스 0.225 mM보다 O.D. 값이 낮은 것으로 미루어 상당한 항산화 활성을 나타내는 것이 확인되었다.
Antioxidant activity was measured at the concentration of 10 ㎍ / ㎖ plant extracts. Higher antioxidant activity inhibits the production of ABTS radicals (green), resulting in lower OD values. Energists were found to exhibit significant antioxidant activity in light of lower OD values than 0.225 mM of trolox when compared to trolox, a standard.
NMDA 리셉터 친화성 테스트NMDA Receptor Affinity Test
최근 글루타메이트 및 NMDA (N-methyl-D-aspartate) 등 각종 흥분성 아미노산에 의한 뇌세포의 손상을 방지함으로써 치매를 제어하고자 하는 연구가 주목 받고 있다.Recently, research to control dementia by preventing damage to brain cells by various excitatory amino acids such as glutamate and NMDA (N-methyl-D-aspartate) has attracted attention.
NMDA 수용체는 신경세포에서 가장 중요한 Ca2+ 채널로 작용하여 NMDA 등 각종 NMDA 작용제(agonist)에 의해 신경세포 내로 Ca2+의 유입을 촉진시킴으로써 신경세포의 사멸(apoptosis)을 유도한다.NMDA receptors act as the most important Ca 2+ channel in neurons and induce apoptosis of neurons by promoting the influx of Ca 2+ into neurons by various NMDA agonists such as NMDA.
따라서 식물추출물과 NMDA 수용체와의 친화력을 측정함으로써 우수한 효능을 가진 NMDA 수용체의 길항제(antagonist)를 식물 소재로부터 탐색하고자 하였다. Therefore, the antagonists of NMDA receptors with excellent efficacy by measuring the affinity between plant extracts and NMDA receptors were investigated.
정력자 에탄올추출물을 대상 시료로 하여 이러한 시료가 마우스의 대뇌에서 분리한 NMDA 수용체와 이 수용체의 글리신 결합 사이트에 선택적인 리간드로 알려져 있는 [3H]-글리신과의 결합을 저해하는 효과를 지표로 하여 NMDA 수용체에 대한 친화력을 탐색해 보았다.Enzymatic ethanol extract was used as a target sample to inhibit the binding of NMDA receptors isolated from mouse cerebrum and [ 3 H] -glycine, which is known as a selective ligand to glycine binding site. The affinity for the NMDA receptor was explored.
각 식물추출물을 각각 0.01, 0.1, 1 및 10 ㎎/㎖ 농도로 희석하여 수용체에 대한 친화력을 검색한 결과는 도 3에 나타내었으며, 정력자 추출물은 NMDA 수용체에 대하여 농도 의존적으로 결합하였다. 정력자의 수용체 친화력은 각각 0.01, 0.1, 1 및 10 ㎎/㎖ 농도에서 각각 36.1, 40.5, 44.1 및 73.3%%를 나타냈다. 특히, 10 ㎎/㎖의 고농도에서 44.1%로 수용체 결합을 저해하였다. 천연식물 정력자의 NMDA 수용체 글리신 바인딩 사이트에 대한 친화력을 검색하는 연구는 향후 치매 치료제의 연구개발에 있어서 유용하게 활용할 수 있는 중요한 지표가 될 수 있을 것이라 사료된다.
Each plant extract was diluted to 0.01, 0.1, 1 and 10 mg / ㎖ concentrations to search for affinity for the receptor is shown in Figure 3, the sperm extract bound to the NMDA receptor concentration dependent. Receptor affinity of the sperm was 36.1, 40.5, 44.1 and 73.3 %% at 0.01, 0.1, 1 and 10 mg / ml concentrations, respectively. In particular, receptor binding was inhibited by 44.1% at a high concentration of 10 mg / ml. The search for affinity of NMDA receptor glycine binding site of natural plant sperm may be an important indicator that can be usefully used in the research and development of dementia treatment in the future.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.
Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
Claims (11)
Enhancement ( lepidium apefalum ) composition for enhancing memory and learning ability comprising the extract as an active ingredient.
A pharmaceutical composition for preventing or treating cognitive disorders comprising Lepidium apefalum extract as an active ingredient.
The composition of claim 1 or 2, wherein the composition inhibits the activity of acetylcholinesterase.
The composition of claim 1 or 2, wherein the composition has antioxidant activity.
The composition of claim 4, wherein the composition inhibits or eliminates the generation of reactive oxygen species.
The composition of claim 1 or 2, wherein said composition acts as an antagonist of an N-methyl-D-aspartate (NMDA) receptor.
The composition of claim 2, wherein the cognitive impairment is dementia, learning disability, blindness, forgetfulness, aphasia, executive or delirium.
The method of claim 2, wherein the cognitive dysfunction is AIDS-induced dementia, Vincewanger disease, Lewy body dementia, frontal temporal dementia, mild cognitive impairment, multiple infarct dementia, PEEK disease, semantic dementia, Alzheimer's dementia or vascular dementia Characterized in that the composition.
A food composition for improving memory and learning ability, comprising sperm extract ( Lepidium apefalum ) as an active ingredient.
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CN201180044377.1A CN103153323B (en) | 2010-09-17 | 2011-09-16 | Memory and learning capacity enhancing compositions |
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Cited By (4)
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CN102895344A (en) * | 2011-07-29 | 2013-01-30 | 苏州知微堂生物科技有限公司 | Preparation technology and production method for integrated new formulation of descurainia and jujube decoction |
KR20200099429A (en) | 2019-02-14 | 2020-08-24 | 동의대학교 산학협력단 | A method for manufacturing a black sesame oil and a composition for improving cognitive function and memory using the same |
KR102345335B1 (en) * | 2020-10-06 | 2021-12-29 | 동의대학교 산학협력단 | Anti-stress compositon containing lepidium apetalum extracts |
KR20220057884A (en) | 2020-10-30 | 2022-05-09 | 국립낙동강생물자원관 | Composition for improving learning ability, cognitive function and memory, including evening primrose extract |
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JP2010001282A (en) * | 2008-05-23 | 2010-01-07 | Kinjirushi Kk | Isothiocyanate-containing composition, food, foodstuff, medicine, cosmetic, daily necessary and miscellaneous good |
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JP2010001282A (en) * | 2008-05-23 | 2010-01-07 | Kinjirushi Kk | Isothiocyanate-containing composition, food, foodstuff, medicine, cosmetic, daily necessary and miscellaneous good |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102895344A (en) * | 2011-07-29 | 2013-01-30 | 苏州知微堂生物科技有限公司 | Preparation technology and production method for integrated new formulation of descurainia and jujube decoction |
KR20200099429A (en) | 2019-02-14 | 2020-08-24 | 동의대학교 산학협력단 | A method for manufacturing a black sesame oil and a composition for improving cognitive function and memory using the same |
KR102345335B1 (en) * | 2020-10-06 | 2021-12-29 | 동의대학교 산학협력단 | Anti-stress compositon containing lepidium apetalum extracts |
KR20220057884A (en) | 2020-10-30 | 2022-05-09 | 국립낙동강생물자원관 | Composition for improving learning ability, cognitive function and memory, including evening primrose extract |
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