KR100596318B1 - Healthy foods containing butterbur extract for memory ability improvement - Google Patents
Healthy foods containing butterbur extract for memory ability improvement Download PDFInfo
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- KR100596318B1 KR100596318B1 KR1020040047683A KR20040047683A KR100596318B1 KR 100596318 B1 KR100596318 B1 KR 100596318B1 KR 1020040047683 A KR1020040047683 A KR 1020040047683A KR 20040047683 A KR20040047683 A KR 20040047683A KR 100596318 B1 KR100596318 B1 KR 100596318B1
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- butterbur
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- A—HUMAN NECESSITIES
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Abstract
본 발명은 기억력 증강기능을 갖는 새로운 건강기능식품의 소재인 머위 추출물을 주·부원료로 이용하여 제조되거나 각종 기호성 식품에 첨가하여 제조된 건강기능식품에 관한 것이다.The present invention relates to a health functional food prepared by using the butterbur extract which is a material of a new health functional food having a memory enhancing function as a main ingredient, or added to various taste foods.
본 발명의 머위 추출물을 함유하는 건강기능식품은 청소년, 성인 및 노인층의 광범위한 계층까지 기억력 증강을 기대 할 수 있다.Health functional foods containing butterbur extract of the present invention can be expected to enhance memory up to a wide range of adolescents, adults and the elderly.
머위 추출물, 기억력 증강기능Butterbur Extract, Memory Enhancement
Description
도 1은 머위 조추출물을 음수로 투여한 실험동물과 대조군의 LTP(Long Term Potentiation) 유발 정도를 비교한 그래프,1 is a graph comparing the degree of induction of LTP (Long Term Potentiation) of a control animal and a control group administered with crude coltsfoot extract negatively,
도 2는 머위 조추출물을 사료로 투여한 결과에 대한 LTP 유발 정도 비교 그래프,2 is a graph comparing the degree of LTP induction for the result of administering crude butterbur extract as a feed,
도 3은 머위 조추출물 투여가 뇌 피질의 BDNF(Brain Derived Neurotropic Factor) 발현에 미치는 영향을 나타낸 사진,Figure 3 is a photograph showing the effect of the administration of butterbur crude extract on the expression of Brain Derived Neurotropic Factor (BDNF) in the brain cortex,
도 4는 머위 조추출물 투여가 뇌 해마의 CREB(cAMP Responsive Element Binding Protein) 발현에 미치는 영향을 나타낸 사진,Figure 4 is a photograph showing the effect of crude butterbur extract administration on the expression of cREB (cAMP Responsive Element Binding Protein) in the brain hippocampus,
도 5는 머위 조추출물을 음수로 투여한 정상 랫드에 대한 모리스 수미로 학습 결과를 나타낸 그래프,5 is a graph showing the results of learning with Morris Sumi for normal rats treated with colostrum crude extract negatively,
도 6은 머위 조추출물을 음수로 투여한 정상 노령 랫드에 대한 모리스 수미로 학습 결과를 나타낸 그래프, 6 is a graph showing the learning results of Morris Sumi for normal old rats treated with crude coltsfoot extract;
도 7은 정상 노령 랫드의 작업 기억에 대한 머위 추출물의 효과를 나타낸 그래프,7 is a graph showing the effect of coltsfoot extract on working memory in normal aged rats,
도 8은 머위 조추출물을 투여한 노령 랫드의 유영 궤적 사진, 8 is a swimming trajectory photograph of the old rats administered the crude coltsfoot extract,
도 9는 머위 조추출물을 용량별 음수로 투여한 정상 랫드에 대한 모리스 수미로 학습 결과를 나타낸 그래프, 9 is a graph showing the learning results of Morris Sumi for normal rats administered crude butterbur extract by dose,
도 10은 머위 분획추출물을 용량별 음수로 투여한 정상 랫드에 대한 모리스 수미로 학습 결과를 나타낸 그래프,10 is a graph showing the learning results of Morris Sumi for normal rats administered with butterbur extract in a negative dose,
도 11은 머위 분획추출물을 용량별 음수로 투여한 모래쥐에 대한 수동회피반응을 나타낸 그래프이다.FIG. 11 is a graph illustrating passive avoidance of sand rats treated with butterbur extract as a negative dose.
본 발명은 기억력 증강 효과를 갖는 새로운 건강기능식품의 소재인 머위 추출물을 함유하는 기억력 증강을 위한 건강기능식품에 관한 것이다.The present invention relates to a health functional food for memory enhancement containing butterbur extract which is a material of a new health functional food having a memory enhancing effect.
인지기능은 일상적인 생활의 활동에 결정적인 영향을 미치는 정신적 과정(central process)의 하나로, 집중력, 단기기억(short term memory, working memory) 장기기억(long term memory), 논리성(reasoning), 운동조절능력 및 작업의 계획성 등 정신적인 과정들이 포함되어 있으며, 이러한 과정들이 명확하게 운영되도록 하는 효율성은 매일 우리의 지적인 활동능력과 직접적인 관계가 있어 궁극적으로는 QOL(Quality of Life)에 중요한 영향을 준다.Cognitive function is one of the central processes that have a decisive influence on the activities of daily living. It is concentration, short term memory, working memory, long term memory, reasoning, and motor control. And mental processes, such as the planning of work, and the effectiveness of these processes to operate clearly has a direct impact on our intellectual ability each day and ultimately has a significant impact on the quality of life.
현대사회는 무한 경쟁시대로서 현대인은 하루도 쉬지 아니하고 자기 자신 또는 타인과의 경쟁을 끊임없이 하고 있어, 스트레스를 받는 것이 살아가면서 하루 세 끼 밥을 먹는 것과 마찬가지로 피할 수 없는 일이 되었다. 이런 스트레스는 기억에 관계하는 뇌 '해마'의 노화를 촉진하여 기억의 감퇴를 야기한다. Modern society is an age of endless competition, and modern people are constantly competing with themselves or others without taking a day off, and stressfulness is inevitable as if they are eating three meals a day. This stress promotes aging of the brain's hippocampus, which is related to memory, leading to memory loss.
그러므로 현대인들에 있어 삶의 질과 밀접한 관련이 있는 기억력의 감퇴 현상을 방지하기 위해 미리 예방하고 효과적으로 대처하는 것이 중요하다. 대처 방안으로 꾸준히 운동을 하는 방법도 있으나 극소수 사람들에 불과하며, 약물로 해결하고자 하는 방법은 약물에 대한 거부감 및 약물 남용의 문제점이 있다. 그러므로 기억력 증강 효과를 갖는 기능성 소재를 찾아 수요자가 손쉽게 접근할 수 있는 건강기능식품을 개발하는 것이 요구되고 있다. Therefore, it is important for modern people to prevent and effectively cope with the loss of memory, which is closely related to the quality of life. As a coping method, there is a method of exercising steadily, but only a few people, and there are problems of drug rejection and drug abuse. Therefore, it is required to find a functional material having a memory enhancing effect and to develop a health functional food that can be easily accessed by consumers.
지금까지 기억력 증진과 관련된 기능성 소재로서 기억학습중추신경계의 대사량을 증가시킨다고 판단되어지는 약물에 대한 연구들이 있어 왔다. 이들은 대개 감마아미노부트릭산(α-amino butyric acid)의 유도체들로서, 이와 같은 약물들은 콜린계에 작용하여 아세틸콜린이라는 신경전달물질의 합성과 배출을 촉진시킬 수 있다고 알려져 있고, 현재까지 개발된 약물들에는 아세틸콜린 합성전구체(acetylcholine precursor), 수용체 활성제(Receptor agonist), 니코틴 등이 있으나, 그 효과가 일시적이고 제한적이어 더 많은 연구가 필요하다.Until now, there have been studies on drugs that are thought to increase the metabolic capacity of the memory learning central nervous system as a functional material related to memory enhancement. These are usually derivatives of α-amino butyric acid, and these drugs are known to act on the choline system and promote the synthesis and release of neurotransmitters called acetylcholine. These include acetylcholine precursors, receptor agonists, nicotine, etc., but their effects are temporary and limited, and more research is needed.
그리고 비타민 C나 E, 베타카로틴, 세닐린(seniline), 은행잎 추출물(Egb 761)등은 산화억제 기능을 통해 뇌 기능 장애에 효과를 나타낸다고 추측되어지나, 아직까지 체계적인 연구는 부족하여 그 효과에 대해서는 좀 더 많은 조사가 필요한 상태이다.And vitamin C or E, beta-carotene, seniline (seniline), ginkgo biloba extract (Egb 761), etc. are thought to be effective in the brain dysfunction through the antioxidant function, but the systematic research is lacking so far about the effect More research is needed.
중추신경계의 대사량 촉진제 또는 항산화제를 이용한 기능성 소재에 대한 연구가 이루어지고 있으며, 천연소재로부터 부작용이 적은 효과적인 소재를 찾기 위한 연구가 활발히 이루어지고 있으나, 아직 명확한 효과를 가진 소재는 개발되어 있지 않은 상태이다.Research on functional materials using metabolic accelerators or antioxidants of the central nervous system has been conducted, and active research is being conducted to find effective materials with fewer side effects from natural materials. to be.
또한 이에 대한 천연소재의 개발에는 일차 검증된 기능성 소재가 반드시 신경계질환들의 혈뇌장벽(BBB ; Blood Brain Barrier)을 통과해야만 효과를 발휘 할 수 있으나, BBB에 대한 연구가 현재 매우 낮은 수준에 있다는 점도 개발의 어려운 점의 하나이다.In addition, the development of natural materials for this can only be effective if the first validated functional material must cross the blood brain barrier (BBB) of neurological diseases, but research on BBB is also at a very low level. Is one of the difficult points.
현재 BBB 문제를 우회하는 유일한 대안은 in vivo 실험을 통한 경험적 효과검증이다. 이러한 점에서 수 천년 동안 경험적으로 검증되어 온 뇌 신경계 관련 천연물들은 기억력 증강과 관련된 건강기능식품으로의 개발이 기대되어진다.Currently, the only alternative to bypassing the BBB problem is empirical validation through in vivo experiments. In this regard, natural products related to the brain's nervous system, which have been empirically tested for thousands of years, are expected to be developed as health functional foods related to memory enhancement.
한편 머위는 국화과(숙근초)에 속하며 습한 들이나 논, 밭 및 산기슭 등지에 자생하는 봉두근, 사두초, 야남곡으로 불리기도 하는 식물로써 어린잎과 어린 꽃줄기를 식용으로 이용하며, 기침, 담, 기관지천식, 건위, 해열, 종기와 뱀·벌레 물린 상처 치료 및 화상에 좋으며, 중독(복어탕)과 주독을 푸는 효과가 있는 것으로 알려져 있고, 머위의 꽃봉오리는 건위, 진해, 해열, 고혈압에 효과가 있으며, 이 외에도 식욕을 증진하는 데 사용되기도 하며, 차로 만들어 이용하기도 하고 술에 담그어 약술을 만들기도 하고 어린 꽃봉오리를 베어서 음건하여 튀김을 만들어 먹기도 하지만, 머위의 생리활성에 관한 연구 결과들은 매우 빈약한 실정이다.Butterbur is a plant belonging to the chrysanthemum family (Sukgeuncho) and is known as Bongdu-geun, Sadocho, and Yamangok, which grows in moist fields, paddy fields, fields, and foothills, and uses young leaves and young flowers for food. It is good for bronchial asthma, dry stomach, fever, boil and snake and insect bite wounds, and it is known to be effective in relieving poisoning (fuguintang) and poison, and the buds of butterbur are effective for stomach, Jinhae, fever and high blood pressure. In addition, it can be used to enhance appetite, make tea, use alcohol to make medicine, and cut young flower buds to dry it to make fried foods. It is a poor situation.
이에 본 발명자들은 부작용이 거의 없는 천연물에서 in vivo 실험을 통한 효과검증으로 혈뇌장벽의 문제를 해결하고 기억력을 증강시킬 수 있는 기능성 소재를 찾기 위해 연구하던 중 머위 추출물을 동물에 투여하여 기억력의 증강 효과를 확인하고 기억력의 전기생리학적 파라메타인 장기간 강화(LTP ; Long Term Potentiation)가 유의성 있게 증가되며, 기억학습의 신호전달 기전과 밀접한 관련이 있는 BDNF(Brain Derived Neurotropic Factor) 및 CREB(cAMP Responsive Element Binding Protein)의 발현이 증가되어 기억력 개선 효과가 나타나는 것을 확인하여 본 발명을 완성하였다.In this regard, the present inventors have been researching to find a functional material that can solve the problem of blood brain barrier and enhance memory by in vivo experiments on natural products with little side effects. Long Term Potentiation (LTP), an electrophysiological parameter of memory, is significantly increased, and Brain Derived Neurotropic Factor (BDNF) and CAMP Responsive Element Binding are closely related to the signaling mechanisms of memory learning. Protein) was confirmed to increase the expression of memory improvement effect was completed the present invention.
본 발명은 천연물에서 기억력을 증강시킬 수 있는 소재로서 머위 추출물을 주·부원료로 하여 제조되거나 기호성 식품에 첨가하여 제조된 기억력 증강을 위한 건강기능식품을 제공하는 것을 목적으로 한다.
It is an object of the present invention to provide a health functional food for memory enhancement prepared by preparing butterbur extract as a main ingredient or as a main ingredient or adding to a favorite food as a material capable of enhancing memory in natural products.
상기 목적을 달성하기 위하여 본 발명은 기억력의 증강을 위한 머위 추출물 함유 건강기능식품을 제공한다. In order to achieve the above object, the present invention provides a dietary supplement containing butterbur extract for the enhancement of memory.
이와 같은 본 발명을 더욱 상세히 설명하면 다음과 같다.Referring to the present invention in more detail as follows.
본 발명의 머위 추출물은 기억력의 증강 효과를 갖는 것으로서, 이와 같은 본 발명에 사용되는 머위는 한국산 머위(Petasites japonicus) 및 서양 머위(Petasites officinalis)를 모두 포함한다.Butterbur extract of the present invention has an effect of enhancing memory, the butterbur used in the present invention includes both Korean butterbur (Petasites japonicus) and Western butterbur (Petasites officinalis).
본 발명에서 기억력의 증강 효과를 갖는 머위 추출물은 머위에 물 또는 알코올을 첨가하여 추출한 머위의 조추출물 뿐만 아니라 상기 추출물에 에틸아세테이트를 첨가하여 에틸아세테이트 분획을 제거하고 수(Water)층에서 분리된 머위 분획추출물을 함유하는 것을 의미한다.In the present invention, the coltsfoot extract has an effect of enhancing memory, as well as the crude extract of coltsfoot extract by adding water or alcohol to the coltsfoot, to remove the ethyl acetate fraction by adding ethyl acetate to the extract and the coltsfoot separated from the water layer. It means containing a fraction extract.
상기 머위 조추출물은 머위에 물을 첨가하여 열수추출하거나 알코올을 첨가하여 냉침 또는 저온 가열하여 추출함으로서 제조되는 것으로서 감압 건조하여 분말 형태로 제조될 수 있다. 바람직하게는 상기 머위 조추출물을 추출하는데 첨가되는 알코올은 5 내지 100% 에탄올 또는 메탄올 중에서 선택하여 사용할 수 있으며, 더욱 바람직하게는 70% 에탄올을 사용하는 것이 좋다.The crude butterbur extract is prepared by extracting hot water by adding water to the butterbur or extracting it by cold or low temperature heating by adding alcohol, and dried under reduced pressure to prepare a powder form. Preferably, the alcohol added to extract the crude butterbur extract may be selected from 5 to 100% ethanol or methanol, and more preferably 70% ethanol.
또한 본 발명은 상기 머위에 물 또는 알코올 수용액을 첨가하여 추출한 다음 상기 추출물에 에틸아세테이트를 첨가하여 에틸아세테이트 분획을 제거하고 수(Water)층을 분리하여 수층에서 수득되는 추출물에서 머위 분획추출물을 제조한다.In addition, the present invention is extracted by adding water or alcohol aqueous solution to the butterbur, and then added ethyl acetate to the extract to remove the ethyl acetate fraction and to separate the water (Water) layer to prepare a butterbur extract from the extract obtained in the aqueous layer .
상기 머위 분획추출물은 컬럼박층크로마토그래프 확인 시험에서 이동상을 부탄올:메탄올:수산화암모늄:물=3:2:1:2의 비율로 하여 전개한 다음 254nm의 UV를 쬐 이면 Rf 값 0.5~0.8에서 암갈색으로 흡수가 일어나며, 10% 황산에 적신 후 100℃로 가열하면, Rf 값 0.6에서 흑갈색 반점을 나타내고, 요오드 발색 시 Rf 값 0.5~0.8에서 갈색 반점을 나타내며, 닌히드린발색시약과 드라겐드로프 발색시약의 정색 반응에서 음성을 나타낸다.The butterbur fraction extract was developed in a column thin layer chromatograph identification test at a ratio of butanol: methanol: ammonium hydroxide: water = 3: 2: 1: 2, and then exposed to UV at 254 nm, at an R f value of 0.5 to 0.8. Absorption occurs in dark brown, and when soaked in 10% sulfuric acid and heated to 100 ° C, it shows dark brown spots at R f value of 0.6, brown spots at R f value of 0.5 to 0.8 when iodine is developed, and ninhydrin coloring reagent and dragend Negative reaction in color reaction of rope color reagent.
또한 본 발명에 따른 머위 분획추출물은 수용액에서 pH가 5.0~8.0이며, 헥산, 에틸아세테이트, 에탄올, 아세톤, 에테르, 벤젠에 불용이며 메탄올에 약간(0.1%) 녹고 증류수에는 용해되는 성질을 가진다.In addition, the butterbur fraction extract according to the present invention has a pH of 5.0 to 8.0 in an aqueous solution, insoluble in hexane, ethyl acetate, ethanol, acetone, ether, benzene, slightly dissolved in methanol (0.1%), and dissolved in distilled water.
본 발명의 기억력 증강을 위한 건강기능식품은 상기 머위 조추출물 및 머위의 분획추출물이 식품의 주·부원료로 하여 제조되거나 다른 식품에 첨가됨으로서 제조될 수 있다. 이 때 식품학적으로 허용되는 식품보조첨가제를 사용할 수 있다.뇌 기능과 관련된 기능성 소재의 작용은 장기간 강화(LTP ; Long Term Potentiation) 변화와 해마 및 내측피질(inferior temporal cortex)과 같은 국소적 영역에서의 BDNF(Brain Derived Neurotropic Factor)발현증가 및 TrkB수용체 활성화와 관련되어 있으며, BDNF의 박탈은 학습 및 기억의 손상을 가져오므로 기억학습에는 BDNF가 필수적인 요소이다. 또한 CREB(cAMP Responsive Element Binding Protein)은 뇌 손상 보호 외에도 공간(space) 및 사회적(social) 학습 등 여러 가지 다양한 기억 형성의 조절인자로 알려져 있다.The health functional food for enhancing memory of the present invention may be prepared by preparing the crude extract of coltsfoot and the fraction extract of coltsfoot as the main and secondary ingredients of the food or by adding to other foods. At this time, a food-acceptable food supplement may be used. The action of functional materials related to brain function is characterized by changes in long term terminology (LTP) and in local areas such as the hippocampus and the inferior temporal cortex. It is associated with increased Brain Derived Neurotropic Factor (BDNF) expression and TrkB receptor activation, and BDNF is essential for memory learning because deprivation of BDNF causes learning and memory impairment. In addition, CAMP Responsive Element Binding Protein (CREB) is known to be a regulator of various memory formation, such as space and social learning, in addition to protecting brain damage.
상기 머위 조추출물을 랫드에 투여하여 모리스 수미로 학습 후 뇌 기능에 미치는 결과를 살펴 본 결과 기억력의 전기생리학적 파라메타인 LPT(Long Term Potentiation)를 유의성 있게 증가시키고(도 1 및 도 2), 기억학습의 신호전달 기전과 밀접한 관련이 있는 BDNF(Brain Derived Neurotropic Factor) 및 CREB(cAMP Responsive Element Binding Protein)의 발현을 증가시키므로(도 3 및 도 4) 상기 머위 추출물이 기억력 증강기능을 가짐을 확인하였다. As a result of examining the effect on the brain function after learning with Morris Sumy by administering the crude coltsfoot extract to rats, LPT (Long Term Potentiation), an electrophysiological parameter of memory, was significantly increased (FIGS. 1 and 2) and memory Since the expression of Brain Derived Neurotropic Factor (BDNF) and CAMP Responsive Element Binding Protein (CREB), which are closely related to the signaling mechanism of learning, is increased (FIGS. 3 and 4), it was confirmed that the coltsfoot extract has a memory enhancing function. .
본 발명의 머위 조추출물 및 머위 분획추출물을 랫드에 투여하여 뇌 기능에 미치는 영향을 살펴 본 결과 기억력 증강 효과를 나타내었다(도 5). 특히 머위 조추출물에 비해 머위 분획추출물의 기억력 증강 효과가 뛰어나므로 머위 분획추출물에 기억력 증강활성성분이 농축되어 있음을 알 수 있다(도 9 내지 도 11). 또한 머위 조추출물을 노령 랫드에 투여하여 모리스 수미로 학습을 시킨 결과 기억력 개선효과를 나타냈다(도 6 내지 도 8).As a result of examining the effects on the brain function by administering the crude butterbur extract and the butterbur fraction extract of the present invention to rats (Fig. 5). In particular, since the memory effect of the butterbur fraction extract is superior to the crude butterbur extract, it can be seen that the memory-enhancing active ingredient is concentrated in the butterbur fraction extract (FIGS. 9 to 11). In addition, administration of the crude butterbur extract to the elderly rats were learned to Morris Sumi showed a memory improvement effect (Figs. 6 to 8).
그러므로 본 발명의 머위 추출물은 기억력 증강을 위한 새로운 건강기능식품의 소재로서 청소년, 성인 및 노인층의 광범위한 계층까지 기억력 증강을 위해 유용하게 사용할 수 있으며, 향후 노인성 뇌질환(건망증, 치매, 알츠하이머병 등)의 예방 및 개선을 위한 건강기능식품으로 개발될 수 있다.Therefore, the butterbur extract of the present invention can be usefully used for memory enhancement to a wide range of adolescents, adults, and the elderly as a material of new dietary supplement for memory enhancement, and future senile brain diseases (forgetfulness, dementia, Alzheimer's disease, etc.) It can be developed as a dietary supplement for the prevention and improvement of health.
본 발명의 머위 추출물은 급성독성 및 아급성 독성시험 결과 식품에 첨가하여 사용하기에 적합한 것이다.Butterbur extract of the present invention is suitable for use in addition to food results of acute toxicity and subacute toxicity test.
본 발명에서 기억력 증강기능이 기대되는 머위 추출물의 유효량은 성인(60kg 체중)에 대해 1일 0.8g 내지 3.2g 이고, 바람직하기로는 1일 1.6g 내지 3.2g이다In the present invention, the effective amount of the extract of butterbur which is expected to enhance memory is 0.8g to 3.2g per day for adults (60kg body weight), preferably 1.6g to 3.2g per day.
이하 본 발명을 실시예에 의하여 상세히 설명한다. 하기 실시예는 본 발명을 예시하기 위한 것이며, 본 발명의 내용이 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention.
<실시예 1> 머위 조추출물의 제조Example 1 Preparation of Butterbur Extract
건조되지 않은 머위 1.8t을 깨끗이 세척하고 길이 10cm 내외로 잘라 건조시킨 머위 98kg을 얻었으며, 이것에 70%의 에탄올 370L를 가하여 55℃~60℃ 사이의 온도에서 8시간동안 섞으면서 추출한 후 다시 같은 양의 에탄올을 가하여 재추출하였다. 이 추출액을 감압농축기로 농축하여 수분함량 약 31%(67~68˚Brix)의 머위 조추출물 42kg(수율 42%)를 얻었다.1.8t of undried butterbur was washed cleanly and cut into 10cm length to obtain 98kg of butterbur dried. 70% 370L of ethanol was added thereto, and the mixture was extracted by mixing for 8 hours at a temperature between 55 ℃ ~ 60 ℃ and then again the same. Amount of ethanol was added and reextracted. The extract was concentrated in a vacuum concentrator to obtain 42 kg of crude butterbur extract (yield 42%) having a water content of about 31% (67-68 ° Brix).
<실시예 2> 머위의 분획추출물 제조Example 2 Preparation of Fraction Extract of Butterbur
실시예 1에서 제조된 머위의 조추출물 4kg에 증류수 20L를 현탁하여 에틸아세테이트 18L로 2회 진탕추출 한 후 에틸아세테이트 가용성분획(36L)을 제거하고 분리된 수층을 50℃에서 감압 농축하여 머위 분획추출물(pH : 6.7) 2.8kg(수율 70%)을 얻었다.Suspend 20L of distilled water in 4 kg of crude extract prepared in Example 1, shake twice with 18L of ethyl acetate, remove ethyl acetate soluble fraction (36L), and concentrate the separated aqueous layer under reduced pressure at 50 ° C. (pH: 6.7) 2.8 kg (yield 70%) were obtained.
상기 머위 분획추출물을 실리카겔 TLC (Merck)에 점적한 다음 이동상을 부탄올:메탄올:수산화암모늄:물=3:2:1:2의 비율로 하여 전개시켰다.The butterbur fraction extract was added to silica gel TLC (Merck) and the mobile phase was developed at a ratio of butanol: methanol: ammonium hydroxide: water = 3: 2: 1: 2.
컬럼박층크로마토그래프 확인 시험에서 상기 분획추출물은 254nm의 UV를 쬐여 Rf 값 0.6에서 암갈색으로 흡수가 일어나는 것을 확인하였으며, 10% 황산에 적신 후 100℃로 가열하였을 때 Rf 값 0.6에서 흑갈색 반점을 나타내었다. 또한 상기 머 위 분획추출물은 Rf 값 0.6에서 요오드 발색으로 갈색 반점을 나타내었으며, 닌히드린발색시약과 드라겐드로프 발색시약의 정색 반응에서 음성을 나타내었다.In the column thin layer chromatography confirmation test, the fraction extract was exposed to dark brown at R f value of 0.6 by UV exposure at 254 nm, and dark brown spots at R f value of 0.6 when heated to 100 ° C. after soaking in 10% sulfuric acid. Indicated. In addition, the coltsfoot extract showed brown spots with iodine coloration at Rf value 0.6, and was negative in color reaction of ninhydrin colorant and dragendrop colorant.
<실시예 3> 머위 추출물 투여에 의한 LTP(Long Term Potentiation)의 변화실험Example 3 Change Experiment of Long Term Potentiation by Administration of Butterbur Extract
(1) 머위 추출물 투여에 의한 LTP 변화(1) Changes in LTP by Administration of Butterbur Extract
가. 실험재료 및 실험동물 end. Experimental Materials and Animals
실험재료는 상기 실시예 1에서 제조된 머위 조추출물을 사용하였고, 실험동물은 정상성장기 랫드(Sparague Dawley rat, 웅성)를 사용하였다. Experimental material was used the crude colts extract prepared in Example 1, the experimental animals were used as a normal growth rat (Sparague Dawley rat, male).
나. 실험방법I. Experiment method
머위 추출물의 기억력 개선에 미치는 영향과 관련된 LTP를 관찰하기 위해 실험동물에 상기 머위 조추출물을 3주 동안 음수[25%(2.5g/l)농도]를 통하여 투여하였고, 4주째부터 모리스 수미로 학습(Morris Water Maze)을 실시하였으며, 5주째에 뇌의 해마에서 LTP 측정실험을 실시하였다.In order to observe the LTP related to the effect of improving the memory of the coltsfoot extract, the crude coltsfoot extract was administered to the experimental animals through negative water [25% (2.5g / l) concentration for 3 weeks] (Morris Water Maze) was performed, and LTP measurement experiments were performed in the hippocampus at 5 weeks.
상기 모리스 수미로 학습(Morris Water Maze)의 방법은 실험동물을 동서남북 중 임의의 두 위치에서 출발시켜 목표물인 플랫폼을 찾아가도록 매일 일정시간에 하루 2회씩 5일 동안 수영학습을 시키는 것으로 이루어 졌다. The method of learning Morris Water Maze was made by swimming experiments twice a day for 5 days at a certain time every day to find a target platform starting from two locations in North, South, East and West.
상기 모리스 수미로 학습을 한 실험동물을 20% 우레탄(7 ml/kg, Sigma, USA)으로 복강내 주사하여 마취시킨 후 뇌 정위장치(stereotaxic, Stoelting, USA)에 고정시켜 두개골 절개술(craniotomy)을 실시하였다.The experimental animals trained with Morris Sumi were anesthetized by intraperitoneal injection with 20% urethane (7 ml / kg, Sigma, USA), and then fixed in a brain stereotactic device (stereotaxic, Stoelting, USA) for craniotomy. Was carried out.
LTP의 측정을 위하여 기록용 tungsten-미세전극은 해마 CA1 세포에 위치시키고 자극용 전극은 치상회(dentate gyrus)의 과립형 세포에 위치 시켰다. 자극 전극은 샤퍼측지섬유 경로(Schaffer collateral 경로) 위에 놓이게 하며, 기록 전극은 CA1 신경 세포의 Dendrite 부위에 놓이게 한다. LTP를 유도하기 위해 자극 전극을 통해 경련성 자극을 가하고 LTP가 얻어지면 2시간 동안 관찰하여 기록하였다.For the measurement of LTP, the recording tungsten-microelectrode was placed on hippocampal CA1 cells and the stimulation electrode was placed on granular cells of dentate gyrus. The stimulation electrode is placed on the Schaffer collateral pathway and the recording electrode is placed on the Dendrite site of CA1 neurons. Convulsive stimulation was applied through the stimulation electrode to induce LTP, and when LTP was obtained, it was observed and recorded for 2 hours.
다. 실험결과All. Experiment result
도 1에서 알 수 있는 바와 같이 대조군인 정상음료 투여군(n=8, n은 실험동물의 수, 이하 같다)은 평균 9.42±1.79%의 potential의 증가를 나타냈으며, 머위 조추출물 함유 음료 투여 군(n=8)은 25.10±4.4%의 LTP 증가를 나타냈다. 실험기간 동안 두 군 모두 유의한 차이(*p<0.05, **p<0.01 vs control)를 유지하였다.As shown in FIG. 1, the control group of normal beverage (n = 8, n is the number of experimental animals, which is equal to or less than the mean) showed an increase of potential of 9.42 ± 1.79% on average, and the beverage administration group containing colostrum crude extract ( n = 8) showed an LTP increase of 25.10 ± 4.4%. Both groups maintained significant differences (* p <0.05, ** p <0.01 vs control) during the experimental period.
도 1에서 화살표(↑) 표기는 경련성 자극을 준 시점을 나타낸 것이며, 데이터는 평균±표준편차로 나타낸 것이고, student's t-test로 유의성을 검정하였다. 하나의 * 표기는 p<0.05인 부분을, 두 ** 표기는 p<0.01인 부분을 나타낸 것이고, M은 머위 조추출물을 나타낸 것이다.The arrow (↑) notation in FIG. 1 represents the time point of convulsive stimulation, the data is expressed as mean ± standard deviation, and the significance was tested by student's t-test. One * notation denotes a portion where p <0.05, two ** notations denote a portion where p <0.01, and M denotes butterbur crude extract.
(2) 머위 추출물 함유 사료 투여에 의한 LTP 변화(2) Changes in LTP by Administration of Feed containing Butterbur Extract
가. 실험재료 및 실험동물end. Experimental Materials and Animals
실험재료는 하기 표1과 같은 조성을 갖는 머위 조추출물 함유 사료를 사용하였다.Experimental material was used feed containing crude butter extract having the composition shown in Table 1.
실험동물은 상기 실험 (1)과 같은 랫드를 사용하였다.Experimental animals used the same rats as in Experiment (1).
나. 실험방법I. Experiment method
사료로 공급되는 실시예 1에서 제조된 머위 조추출물이 LTP에 미치는 영향을 평가하기 위하여, 5주째에 실험동물을 마취하여 뇌 해마에서의 LTP의 변화를 기록하였다.In order to evaluate the effect of the crude butterbur extract prepared in Example 1 to feed on LTP, experimental animals were anesthetized at 5 weeks and the change in LTP in the brain hippocampus was recorded.
실험은 상기 (1)의 방법과 같은 방법으로 이루어졌다.The experiment was conducted in the same manner as in the method of (1) above.
다. 실험결과All. Experiment result
도 2에 나타난 바와 같이, 사료를 4주간 공급 투여한 후, 실험동물의 뇌 해마에서의 LTP의 변화(5주째)를 기록한 결과, 대조군인 일반 정상사료 투여군(n=8)은 10.55±5.57%, 실시예 1에서 제조된 머위 조추출물 제외사료(대두유, 소맥배아유, 비타민mix, α-tocopherol 등 포함) 투여군(n=6)은 22.53± 3.91%의 증가를, 표 1의 머위 조추출물 함유 사료 투여군(n=6)은 37.89± 7.04%의 증가를 나타내었 으며, 자극(TBS)후 발생된 LTP는 정상사료 투여군과 머위 추출물 함유 사료 투여군 모두에서 2시간 이상 유지되었다. 이와 같은 결과는 정상음료 및 머위 조추출물 함유 음료를 투여한 실험동물군의 비교에서도 동일한 경향을 보였다.As shown in FIG. 2, after feeding the feed for 4 weeks, the change in LTP in the brain hippocampus of the experimental animal (5 weeks) was recorded. As a control, the normal normal feed group (n = 8) was 10.55 ± 5.57%. , Crude butter extract extract prepared in Example 1 (including soybean oil, wheat germ oil, vitamin mix, α-tocopherol, etc.) administration group (n = 6) increased by 22.53 ± 3.91%, containing the butterbur crude extract of Table 1 The feed group (n = 6) showed an increase of 37.89 ± 7.04%, and LTP generated after stimulation (TBS) was maintained for more than 2 hours in both the normal feed group and the feed group containing butterbur extract. These results showed the same trend in the comparison of the experimental animals group administered the normal beverage and the beverage extract containing coltsfoot extract.
도 2에서 화살표(↑)는 10분 동안 base-line 기록 후 TBS 자극이 주어진 시점을 나타낸 것이다. 모든 결과는 평균±표준편차로 나타내었으며, student's t-test로 유의성(*, p<0.05; **, p<0.01)을 검정하였다. M은 머위 조추출물을 나타낸 것이다.Arrow (↑) in Figure 2 shows the time point given the TBS stimulation after baseline recording for 10 minutes. All results were expressed as mean ± standard deviation, and the significance (*, p <0.05; **, p <0.01) was tested by student's t- test. M represents coltsfoot crude extract.
따라서 본 발명의 머위 추출물은 상기 실시예 3의 LTP 변화검정 동물실험에서 기억력 증강에 효과가 있음을 확인할 수 있으며, 본 발명의 머위 추출물은 기억력의 증강이 기대되는 새로운 건강기능식품의 소재임을 알 수 있다.Therefore, the butterbur extract of the present invention can be confirmed that the LTP change test in the animal experiment of Example 3 has an effect on memory enhancement, the butterbur extract of the present invention can be seen that the material of the new health functional food is expected to enhance memory have.
<실시예 4> 머위 추출물 투여가 동물의 뇌 BDNF 및 CREB의 발현에 미치는 영향Example 4 Effect of Butterbur Extract Administration on the Expression of Brain BDNF and CREB in Animals
기억학습에 대한 CREB 및 BDNF 방출의 활성 의존적 조절의 분자기전에 머위 추출물의 투여가 영향을 미치는지 확인하였다.The effect of administration of coltsfoot extract on the molecular mechanism of activity dependent regulation of CREB and BDNF release on memory learning was determined.
가. 실험재료 및 실험동물end. Experimental Materials and Animals
실험재료는 실시예 1에서 제조된 머위 조추출물을 사용하였고, 실험동물은 정상성장기 랫드(Sparague Dawley rat, 웅성)를 사용하였다.Experimental material was used crude butter extract prepared in Example 1, the experimental animal was used as a normal growth rat (Sparague Dawley rat, male).
나. 실험방법I. Experiment method
3주동안 실험동물에 음수를 통하여 상기 머위 조추출물을 투여(in 0.5% CMC) 하였고, 4주째에 실험동물의 LTP data를 기록 분석하였다. LTP기록 후 가능한 빨리 전체 뇌로부터 대뇌 피질과 해마를 분리하고, 그 분리된 샘플에서 total RNA를 분리한 후, 스펙트로포토메타(spectro photometer)를 이용하여 그 RNA의 양을 측정하였다. 또 샘플로부터 일정량을 취하여 MMVL reverse transcriptase를 사용하여, cDNA를 합성하였으며 이와 같이 만들어진 샘플을 사용하여 BDNF 및 CREB의 발현 정도를 각각 측정하였다.The colostrum crude extract was administered (in 0.5% CMC) through negative water for 3 weeks, and the LTP data of the experimental animals were recorded and analyzed at 4 weeks. After the LTP recording, the cerebral cortex and hippocampus were separated from the whole brain as soon as possible, and total RNA was separated from the separated sample, and then the amount of the RNA was measured using a spectrophotometer. In addition, cDNA was synthesized using a MMVL reverse transcriptase from a sample, and the expression levels of BDNF and CREB were measured using the samples thus prepared.
다. 실험결과All. Experiment result
도 3 및 도 4의 사진에서 확인할 수 있는 바와 같이, 대조군(n=6)에 비해 머위 조추출물 투여 시험군(n=7)에서 현저히 BDNF(피질, 도 3) 및 CREB(해마, 도 4)의 발현이 증가하였음을 알 수 있다. 따라서 본 발명의 머위 추출물이 BDNF 및 CREB mRNA의 발현에 관여했음을 나타낸 것으로서, 본 발명의 머위 추출물은 기억력의 증강 효과를 갖는 새로운 건강기능식품의 소재임을 알 수 있다.As can be seen in the photos of FIGS. 3 and 4, BDNF (cortex, FIG. 3) and CREB (hippocampus, FIG. 4) were significantly higher in the coltsfoot extract administration test group (n = 7) compared to the control (n = 6). It can be seen that the expression of increased. Therefore, as the butterbur extract of the present invention was shown to be involved in the expression of BDNF and CREB mRNA, the butterbur extract of the present invention can be seen that the material of a new dietary supplement with a memory enhancing effect.
<실시예 5> 머위 추출물의 학습능력 및 기억력 개선 효과Example 5 Improvement of Learning and Memory of Butterbur Extract
가. 실험재료 및 실험동물end. Experimental Materials and Animals
실험재료는 상기 실시예 1의 머위 조추출물을 사용하였고, 실험동물은 정상성장기 랫드(Sparague Dawley rat, Wister rat, 웅성, 180~200g 체중)를 준비하였다.Experimental material was used as the crude colts extract of Example 1, the experimental animals were prepared in normal growth rats (Sparague Dawley rat, Wister rat, male, 180 ~ 200g body weight).
나. 실험방법I. Experiment method
실험동물에 실시예 1에서 제조된 머위 조추출물을 3주 동안 음수[25%(2.5g/l)농도]를 통하여 투여하였고, 4주째부터 실험동물에 대해 상기 실시예 3과 같은 방법으로 모리스 수미로 학습을 실시(4 trials/day)하였다.Crude crude extract prepared in Example 1 was administered to the experimental animals through a negative water [25% (2.5g / l) concentration for 3 weeks, and from the fourth week to the experimental animal Morris Sumi in the same manner as in Example 3 Learning was conducted (4 trials / day).
학습을 실시한 실험동물들에 대하여 학습이 완료되어 실험동물이 플랫폼에 도착하기까지의 시간(Escape time)이 10초 내외로 되었을 때부터 24시간 경과 후 프로브 테스트(probe trial test; 목표물이 있었던 1/4분면 영역 또는 목표물 주변반경 25cm 이내에 랫드가 들어와 머무른 시간 및 거리를 측정하여 비교하는 실험)를 1회 실시하였다. 이 때의 작업 기억(working memory) 또는 1차 학습을 완료 후 플랫폼을 다른 위치로 옮겼을 때 나타나는 전후의 시간 차이(short term working memory) 등을 비교 측정하여 도 5에 나타내었다.Probe trial test was performed 24 hours after the completion of the study and the time until the animals arrived at the platform (Escape time) was about 10 seconds. One experiment was performed to measure and compare the time and distance the rats entered and stayed within the quadrant area or 25 cm around the target. The working memory or the time difference (short term working memory) before and after appearing when the platform is moved to another location after completion of the first learning is shown in FIG. 5.
다. 실험결과All. Experiment result
도 5에서 보는 바와 같이 정상 랫드에서 머위 조추출물은 학습과 작업기억(working memory) 능력 모두의 개선에 효과적이었다. 이 결과에서 본 발명의 머위 추출물은 기억능력의 증강 효과를 갖는 새로운 건강기능식품의 소재임을 알 수 있다.As shown in FIG. 5, coltsfoot extract in normal rats was effective in improving both learning and working memory ability. In this result it can be seen that the butterbur extract of the present invention is the material of a new health functional food having an enhancement effect of memory capacity.
도 5에서 M은 머위 조추출물이고 C는 대조군을 나타내는 것이다.In Figure 5, M is the coltsfoot crude extract and C represents a control.
<실시예 6> 노령 랫드에 대한 학습능력 및 기억력 개선 효과Example 6 Effects of Learning and Memory Improvement on Aged Rats
본 실시예는 머위 추출물이 청소년뿐만 아니라 성인 및 노인층의 광범위한 계층까지 기억력 증강기능이 기대되는지를 알아보기 위하여 노령 정상 랫드에 대한 학습 및 기억력 증강을 측정한 것이다.This example is to measure the learning and memory enhancement for elderly normal rats to determine whether the butterbur extract is expected to enhance the memory function to a wide range of adults and elderly people as well as adolescents.
가. 실험재료 및 실험동물end. Experimental Materials and Animals
실험재료는 상기 실시예 1의 머위 조추출물을 사용하였고, 실험동물로 22개월령 랫드(88주령, Sparague Dawley rat ,n=16)를 준비하였다.Experimental material was used the crude colts extract of Example 1, and 22 months old rats (88 weeks old, Sparague Dawley rat, n = 16) was prepared as experimental animals.
나. 실험방법I. Experiment method
실험동물에 상기 머위 조추출물을 12주동안 음수[25%(2.5g/l)농도]를 통하여 투여하고 상기 실시예 5와 같은 방법으로 모리스 수미로 학습에서 1차 학습에 대한 프로브 시험을 실시하여 작업기억을 측정하였으며 자발 운동량은 프로브 시험 24시간 후에 4시간동안 대조군과 비교 측정하였다. The colostrum crude extract was administered to experimental animals through a negative water [25% (2.5 g / l) concentration] for 12 weeks, and the probe test for the first learning in the Morris Sumiro learning in the same manner as in Example 5 Working memory was measured and spontaneous momentum was measured for 4 hours after the probe test compared to the control group.
다. 실험결과All. Experiment result
도 6에 도시된 바와 같이, 모리스 수미로 학습에서 유의적(n=7∼8, * p<0.05 vs control)인 기억력의 차이를 보였다.As shown in FIG. 6, Morris Sumi showed significant memory differences in learning (n = 7-8, * p <0.05 vs control).
도 7에 도시된 바와 같이 프로브 시험에 의한 작업 기억(working memory)도 대조군에 비해 약 1.4배 증가하였다.As shown in FIG. 7, the working memory by the probe test also increased about 1.4 times compared to the control.
또한 도 8에 도시된 바와 같이 실험동물이 플랫폼 일사분면 영역(quadrant) 내에서 단위시간당 유영(遊泳)한 거리가 대조군에 비해 유의적으로 증가했음을 보여준다. 투여기간 동안 두 군간에 체중 및 음수량의 유의적인 차이는 관찰되지 않았다.In addition, as shown in Figure 8 shows that the experimental animal swims per unit time in the platform quadrant area (quadrant) significantly increased compared to the control. No significant difference in body weight and drinking volume was observed between the two groups during the administration period.
도 6, 도 7 및 도 8에서 MH는 머위 조추출물을 나타내는 것이다.6, 7 and 8, M H represents the butterbur crude extract.
이와 같은 실험을 통하여 본 발명의 머위 추출물은 노인층까지 기억력 증강 기능을 나타냄으로 광범위한 계층을 대상으로 기억력 감퇴증상을 보이는 질병을 예방 빛 개선하는 건강기능식품으로 사용될 수 있다.Through such experiments, the butterbur extract of the present invention can be used as a health functional food to improve the prevention of diseases showing memory loss symptoms for a wide range of classes by showing the memory enhancement function to the elderly.
<실시예 7> 머위 조추출물의 학습능력 및 단기기억력 개선효과Example 7 Improvement of Learning and Short-term Memory of Butterbur Extract
가. 실험재료 및 실험동물end. Experimental Materials and Animals
실험재료는 상기 실시예 1에서 제조된 머위 조추출물을 사용하였고, 실험동물은 정상성장기 랫드(Wister rat, 수컷)를 준비하였다.Experimental material was prepared by extracting the coltsfoot extract prepared in Example 1, the experimental animals were prepared in normal growth rats (Wister rat, male).
나. 실험방법I. Experiment method
실시예 1에서 제조된 머위 조추출물을 실험동물에 3주 동안 용량별(200mg/kg, 400mg/kg)로 음수를 통하여 투여하였으며, 4주째부터 실험동물에 대해 모리스 수미로 학습을 실시하였다.The crude butterbur extract prepared in Example 1 was administered to the experimental animals by means of negative doses (200mg / kg, 400mg / kg) for 3 weeks, and from the fourth week, the study was conducted with Morris Sumi.
상기 모리스 수미로 학습은 실험동물을 10초 이내에 목표물을 찾아가도록 1주간 1일 4회씩 반복해서 수영학습 시킨 뒤 24시간이 경과하여 목표물인 플랫폼을 반대 위치로 옮긴 후 모리스 수미로 학습을 실시했을 때 첫 번째와 두 번째 목표물을 찾아가는 시간(Escape time)의 차이(Learning Index)를 비교 측정하였다.The Morris Sumi learning is to learn the swimming animals repeatedly swimming four times a day for 1 week to find the target within 10 seconds and after 24 hours to move the platform to the opposite position when learning with Morris Sumi We compared and measured the learning index of the escape time for the first and second targets.
다. 실험결과All. Experiment result
상기 시험에서는 Learning index가 증가할수록 단기기억력이 향상된 것으로 도 9에서 알 수 있는 바와 같이 대조군의 Learning Index 시간은 대조군에 비해 머위 분획 추출물 200mg/kg 투여량에서는 1.38배, 400mg/kg 투여량에서 1.87배의 기억력 증강 효과를 나타내었으므로 머위 조추출물이 투여한 실험동물에서 용량 의존 적인(n=8, * p<0.01 vs control, ** p<0.05 vs control) 단기기억력의 증강 효과가 있음을 알 수 있다. In this test, as the learning index increases, the short-term memory is improved. As can be seen in FIG. 9, the learning index time of the control group is 1.38 times at the 200 mg / kg dose of Butterbur extract, and 1.87 times at the 400 mg / kg dose as compared to the control group. As a result of the memory enhancing effect, the effect of dose-dependent (n = 8, * p <0.01 vs control, ** p <0.05 vs control) short-term memory was found to be increased in the experimental animals administered with crude butterbur extract. have.
도 9에서 M은 머위 조추출물이고 C는 대조군을 나타내는 것이다.In Figure 9, M is the coltsfoot crude extract and C represents a control.
<실시예 8> 머위 분획추출물의 학습능력 및 단기기억력 개선효과Example 8 Improvement of Learning and Short-term Memory of Butterbur Extract
가. 실험재료 및 실험동물end. Experimental Materials and Animals
실험재료는 상기 실시예 2에서 제조된 머위 분획추출물을 사용하였고, 실험동물은 정상성장기 랫드(Sparague Dawley rat, 수컷)를 준비하였다.Experimental material was used in the coltsfoot extract prepared in Example 2, the experimental animals were prepared from normal growth rats (Sparague Dawley rat, male).
나. 실험방법I. Experiment method
실시예 2에서 제조된 머위 분획추출물을 실험동물에 4주 동안 용량별(120mg/kg, 240mg/kg, 480mg/kg)로 음수를 통하여 투여하였으며, 5주째부터 실험동물에 대해 모리스 수미로 학습을 실시하였다.Butterbur fraction extract prepared in Example 2 was administered to the experimental animals for 4 weeks by dose (120mg / kg, 240mg / kg, 480mg / kg) through the drinking water, and from the fifth week to study with Morris Sumi Was carried out.
상기 모리스 수미로 학습은 실험동물을 10초 이내에 목표물을 찾아가도록 1주간 1일 4회씩 반복해서 수영학습 시킨 뒤 24시간이 경과하여 목표물인 플랫폼을 반대 위치로 옮긴 후 모리스 수미로 학습을 실시했을 때 첫 번째와 두 번째 목표물을 찾아가는 시간(Escape time)의 차이(Memory Index)를 비교 측정하였다.The Morris Sumi learning is to learn the swimming animals repeatedly swimming four times a day for 1 week to find the target within 10 seconds and after 24 hours to move the platform to the opposite position when learning with Morris Sumi We compared and measured the difference between the escape times of the first and second targets (memory index).
다. 실험결과All. Experiment result
도 10에서 알 수 있는 바와 같이 대조군의 Memory Index 시간은 16.58± 5.56(sec)이나, 머위 분획추출물을 투여한 실험동물은 120mg/kg 투여용량에서 32.46±15.15(sec), 240mg/kg 투여용량에서 45.28±5.90*(sec), 480mg/kg 투여용량에서 33.48±17.06(sec)으로 240mg/kg까지 용량 의존적인(n=5, * p<0.05 vs control) 단기기억력의 증강 효과를 나타내었다. As can be seen in Figure 10, the Memory Index time of the control group is 16.58 ± 5.56 (sec), but the experimental animal administered the butterbur fraction extract was 32.46 ± 15.15 (sec), 240 mg / kg dose at 120 mg / kg dose At 45.28 ± 5.90 * (sec), 480mg / kg dose, 33.48 ± 17.06 (sec) up to 240mg / kg showed dose-dependent (n = 5, * p <0.05 vs control) short-term memory.
도 10에서 MW는 머위의 분획추출물을 나타낸 것이다.MW in Figure 10 shows the fraction extract of butterbur.
상기 실시예 7과 8을 비교한 결과 머위 조추출물의 경우 200mg/kg ~ 400mg/kg 투여량에서 각각 1.38배 및 1.87배의 증강 효과를 보인 반면, 머위 분획추출물의 경우 120mg/kg, 240mg/kg의 투여량에서 각각 1.96배 및 2.73배의 증강 효과를 보여, 가장 현저한 증강 효과를 보인 추출물의 투여용량(240mg/kg)이 약 2배나 적게 소요된 반면에 기억력 증강 효과에 있어서는 240mg/kg 일 때 대조군의 2.73배로 머위 조추출물의 경우보다 1.5배 정도나 더 높은 기억력 증강 효과를 나타낸 것을 알 수 있다.As a result of comparing Examples 7 and 8, the crude extract of the butterbur extract showed an enhancement effect of 1.38 times and 1.87 times at 200 mg / kg to 400 mg / kg dose, respectively, while the extract of the butterbur extract was 120 mg / kg and 240 mg / kg. At doses of 1.96 and 2.73 times, respectively, the most remarkable enhancement was the dose of extract (240mg / kg), which was about twice as low, while the memory enhancement effect was 240mg / kg. It can be seen that the memory enhancement effect was about 1.5 times higher than that of the crude butterbur extract at 2.73 times of the control group.
<실시예 9> 머위 분획추출물의 수동회피반응에 의한 기억력 개선효과Example 9 Memory Improvement Effect of Butterbur Fraction Extract by Passive Avoidance
가. 실험재료 및 실험동물end. Experimental Materials and Animals
실험재료는 상기 실시예 2에서 제조된 머위의 분획추출물을 사용하였고, 실험동물은 정상의 성장기 모래쥐(Mongolian gerbil, 수컷)를 준비하였다.Experimental material was used for extracting the fraction of butterbur prepared in Example 2, the experimental animals prepared a normal growth sand rat (Mongolian gerbil, male).
나. 실험방법I. Experiment method
실험동물에 머위의 분획추출물을 5주 동안 용량별(125mg/kg, 250mg/kg)로 음 수를 통하여 투여하였으며, 6주째부터 실험동물에 대해 Step down latency test(수동회피반응: passive avoidance test)를 실시하였다.Fractional extracts of butterbur were administered to the experimental animals through a negative dose (125mg / kg, 250mg / kg) for 5 weeks, and from the 6th week, the step down latency test (passive avoidance test) was performed on the experimental animals. Was carried out.
상기 수동회피반응시험은 실험동물이 그리드(Grid)로 내려올 때 0.4mA의 전기쇼크를 가하여 기억시키고 24시간 경과 후 플랫폼 위에서 그리드로 내려오지 않고 머무르는 시간(Latency time)을 비교 측정하였다.The passive avoidance test was performed by applying an electric shock of 0.4 mA when the test animal descended to the grid, and comparing and measuring the latency time of staying on the platform after 24 hours without descending to the grid.
다. 실험결과All. Experiment result
상기 시험에서는 Latency time이 증가할수록 기억력이 향상된 것으로 도 11에서 알 수 있는 바와 같이 대조군의 수동회피반응 시간은 54.8±17.6(sec)이나, 머위 분획추출물을 투여한 실험동물은 125mg/kg 투여용량에서 101.6±21.5*(sec), 250mg/kg 투여용량에서 133.6±39.2*(sec)로 머위 분획추출물을 투여한 실험동물에서 용량 의존적으로(n=8~10, * p<0.05 vs control) 유의적인 기억력의 증강 효과를 나타내었다.In this test, as the latency time increased, the memory was improved. As can be seen in FIG. 11, the passive avoidance reaction time of the control group was 54.8 ± 17.6 (sec), but the experimental animals administered the butterbur fraction extract at 125 mg / kg dose. Dose-dependent (n = 8 ~ 10, * p <0.05 vs control) in experimental animals administered with Butterbur fraction extract at 101.6 ± 21.5 * (sec), 250 mg / kg dose at 133.6 ± 39.2 * (sec) It has been shown to enhance memory.
도 11에서 MW는 머위 분획추출물을 나타낸 것이다.In Figure 11, MW shows the butterbur fraction extract.
<실시예 10> 안전성(급성독성 및 아급성 독성) 실험Example 10 Safety (Acute Toxicity and Subacute Toxicity) Experiments
본 발명의 머위 추출물이 인체에 독성이 있는지를 확인하기 위하여 다음과 같은 동물 실험을 하였다.In order to confirm whether the extract of butterbur of the present invention is toxic to the human body, the following animal experiment was performed.
(1) 급성독성 시험(1) Acute Toxicity Test
가. 실험재료 및 실험방법end. Experimental Materials and Methods
실험재료는 상기 실시예 1에서 제조된 머위 조추출물을 사용하였고, 실험동물은 ICR 마우스 14마리를 준비하였다.Experimental material was prepared from crude coltsfoot extract prepared in Example 1, the experimental animals were prepared 14 ICR mice.
나. 실험방법I. Experiment method
실험동물에 머위 조추출물을 용량별로(수분함량 23%; 2g/kg, 4g/kg) 음수를 통하여 투여하고, 투여 전후 실험동물의 체중변화 및 생존여부를 72시간동안 관찰하였다. Crude crude extract was administered to the experimental animals by dose (water content 23%; 2g / kg, 4g / kg) through the negative, and the weight change and survival of the experimental animals before and after administration were observed for 72 hours.
다. 실험결과All. Experiment result
72시간 내에 실험동물 모두가 생존하였으며 평균 체중 증가량도 2g/kg 및 4g/kg의 머위 조추출물 투여군에서 각각 3.3g, 2.5g으로 정상증가량의 오차 범위에 들어 있었으며, 6g/kg b.w. 투여량에서도 LD50를 관찰 할 수 없었다. 참고로 한국 식약청(KFDA)의 안전성 기준은 2g/kg bw이하이므로, 머위 추출물은 안전할 것으로 판단된다.All animals survived within 72 hours, and the average weight gain was 3.3 g and 2.5 g, respectively, in the control group of 2 g / kg and 4 g / kg crude butterbur extract, respectively, and LD was increased even at 6 g / kg bw dose. 50 could not be observed. For reference, the Korea Food and Drug Administration (KFDA) safety standards are less than 2g / kg bw, so the coltsfoot extract is considered safe.
(2) 아급성 독성시험(2) Subacute Toxicity Test
가. 실험재료 및 실험동물end. Experimental Materials and Animals
상기 (1)의 시험과 동일한 실험재료를 사용하였고, 실험동물은 ICR 마우스 13마리를 준비하였다.The same experimental material as in the test of (1) was used, and experimental animals were prepared with 13 ICR mice.
나. 실험방법I. Experiment method
실험동물에 2주동안 2g/kg bw/day의 투여량으로 경구 투여하여 평균체중 변화를 관찰하였다. The average body weight was observed by oral administration to the experimental animals at a dose of 2 g / kg bw / day for 2 weeks.
다. 실험결과All. Experiment result
실험동물의 평균체중 증가량의 범위에서 변화가 관찰되지 않았으며, 모두 생존하였다.No change was observed in the range of mean weight gain of the test animals, and all survived.
이러한 안전성 실험을 통하여 본 발명의 머위 추출물은 인체에 악영향을 미치지 않을 것으로 판단된다.Through such safety experiments it is determined that the butterbur extract of the present invention does not adversely affect the human body.
이하 본 발명에 따른 머위 조추출물 또는 머위 분획추출물을 츄잉 껌, 라면 등의 면류, 사탕, 과자류, 시리얼, 빵류 등 각종 고상 식품에 첨가하여 기억력 증강 효과를 갖는 건강기능식품을 제조할 수 있고, 또한 차 및 음료로도 제조할 수 있다.By adding the crude butterbur extract or butterbur fraction extract according to the present invention to various solid foods, such as chewing gum, ramen noodles, candy, confectionery, cereals, bread, etc., it is possible to prepare a health functional food having a memory enhancing effect, It can also be produced as tea and beverages.
또한 본 발명에 따른 기억력 증강기능을 갖는 머위 추출물 또는 머위 분획추출물을 사료에 첨가하여 동물의 사료로도 활용할 수 있다.In addition, the coltsfoot extract or coltsfoot fraction extract having a memory enhancing function according to the present invention can be added to the feed can also be used as animal feed.
이하 본 발명은 하기의 구체적인 실시예에 의하여 제한되는 것은 아니다.Hereinafter, the present invention is not limited by the following specific examples.
<제조실시예 1> 츄잉 껌의 제조Preparation Example 1 Preparation of Chewing Gum
껌베이스 20 중량%, 설탕 72 중량%, 향료 1 중량% 및 물 2 중량%와 상기 실시예 1에서 제조된 머위 조추출물 5 중량%를 배합하여 통상의 방법으로 츄잉껌을 제조하였다. Chewing gum was prepared in a conventional manner by combining 20% by weight of gum base, 72% by weight of sugar, 1% by weight of perfume, and 2% by weight of water, and 5% by weight of butterbur crude extract prepared in Example 1.
<제조실시예 2> 캔디의 제조Preparation Example 2 Preparation of Candy
설탕 50 중량%, 물엿 39.8 량% 및 향료 0.2 중량%와 상기 실시예 1에서 제조된 머위 조추출물 10 중량%를 배합하여 통상의 방법으로 캔디를 제조하였다.Candy was prepared by the conventional method by combining 50% by weight of sugar, 39.8% by weight of starch syrup, 0.2% by weight of fragrance, and 10% by weight of the crude butterbur extract prepared in Example 1.
<제조실시예 3> 캔디의 제조Preparation Example 3 Preparation of Candy
당알콜 45 중량%, 환원물엿 49.8 중량% 및 향료 0.2 중량%와 상기 실시예 2에서 제조된 머위 분획추출물 5 중량%를 배합하여 통상의 방법으로 캔디를 제조하였다. Candy alcohol was prepared by combining 45% by weight of sugar alcohol, 49.8% by weight of reduced starch syrup, 0.2% by weight of fragrance, and 5% by weight of butterbur fraction extract prepared in Example 2.
<제조실시예 4> 비스켓의 제조Preparation Example 4 Preparation of Biscuits
박력 1급 21.59 중량%, 중력 1급 22.22 중량%, 정백당 4.80 중량%, 식염 0.73 중량%, 포도당 0.78 중량%, 팜쇼트닝 11.15 중량%, 암모 1.54 중량%, 중조 0.17 중량%, 중아황산나트륨 0.16 중량%, 쌀가루 1.45 중량%, 비타민 B1 0.0001 중량%, 비타민 B2 0.0001 중량%, 밀크향 0.04 중량%, 물 21.3298 중량%, 전지분유 1.16 중량%, 대용분유 0.29 중량%, 제일인산칼슘 0.03 중량%, 살포염 0.29 중량% 및 분무유 7.27 중량%와 상기 실시예 2에서 제조된 머위 분획추출물 5 중량%를 배합하여 통상의 방법으로 비스켓을 제조하였다.Force 1st class 21.59 wt%, gravity first class 22.22 wt%, white sugar 4.80 wt%, salt 0.73 wt%, glucose 0.78 wt%, palm shortening 11.15 wt%, ammo 1.54 wt%, sodium bicarbonate 0.17 wt%, sodium bisulfite 0.16 wt% , Rice flour 1.45%, Vitamin B1 0.0001%, Vitamin B2 0.0001%, Milk flavor 0.04%, Water 21.3298%, Whole milk powder 1.16%, Substitute powder 0.29%, calcium phosphate 0.03%, Spray salt Biscuits were prepared in a conventional manner by combining 0.29 wt% and 7.27 wt% of spray oil with 5 wt% of the butterbur fraction extract prepared in Example 2.
<제조실시예 5> 음료의 제조Preparation Example 5 Preparation of Beverage
꿀 0.26 중량%, 치옥토산아미드 0.0002 중량%, 니코틴산아미드 0.0004 중량%, 염산리보플라빈나트륨 0.0001 중량%, 염산피리독신 0.0001 중량%, 이노시톨 0.001 중량%, 오르트산 0.002 중량% 및 물 89.7362 중량%와 상기 실시예 1에서 제조된 머위 조추출물 10 중량%을 배합하여 통상의 방법으로 음료를 제조하였다.0.26% by weight of honey, 0.0002% by weight of thioctoamide, 0.0004% by weight of nicotinic acid, 0.0001% by weight of riboflavin hydrochloride, 0.0001% by weight of pyridoxine hydrochloride, 0.001% by weight of inositol, 0.002% by weight of orthoic acid and 89.7362% by weight of
<제조실시예 6> 음료의 제조Preparation Example 6 Preparation of Beverage
설탕 7.78 중량%, 구연산 0.11 중량%, 향료 0.1 중량% 및 정제수 87.01 중량%와 상기 실시예 2에서 제조된 머위 분획추출물 5 중량%를 배합하여 통상의 방법으로 음료를 제조하였다. 7.78 wt% of sugar, 0.11 wt% of citric acid, 0.1 wt% of fragrance, and 87.01 wt% of purified water were mixed with 5 wt% of the butterbur fraction extract prepared in Example 2 to prepare a beverage.
이상에서 설명한 바와 같이 본 발명의 머위 추출물은 부작용이 거의 없는 천연물에서 in vivo 실험을 통한 효과검증으로 기억력 증강기능을 가지는 새로운 건강기능식품의 소재이다.As described above, the butterbur extract of the present invention is a material of a new health functional food having a memory enhancing function as an effect verification through in vivo experiments in natural products having almost no side effects.
그러므로 본 발명에 따른 머위 추출물을 함유하는 기억력 증강을 위한 건강기능식품은 LTP 유발 정도 평가, BDNF(Brain Derived Neurotropic Factor) 및 CREB(cAMP Responsive Element Binding Protein) 발현 영향 평가와 관련된 전기생리학적 연구에서 확인된 바와 같이 기억력의 증강 기능을 가지며, 청소년, 성인 및 노인층의 광범위한 계층까지 기억력 증강을 위해 유용하게 사용할 수 있고, 향후 노인성 뇌질환(건망증, 치매, 알츠하이머병 등)의 예방 및 개선을 위한 건강기능식 품으로도 유용하게 개발될 수 있다.Therefore, the health functional food for memory enhancement containing butterbur extract according to the present invention has been confirmed in the electrophysiological studies related to the evaluation of LTP induction, BDNF (Brain Derived Neurotropic Factor) and CREB (cAMP Responsive Element Binding Protein) expression effects As shown, it has a function of enhancing memory, and it can be usefully used to enhance memory of a wide range of adolescents, adults, and the elderly, and a health function for preventing and improving future senile brain diseases (forgetfulness, dementia, Alzheimer's disease, etc.) It can also be usefully developed as food.
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| JPH09140359A (en) * | 1995-11-27 | 1997-06-03 | Shinichiro Saito | Wild vegetable juice |
| KR20010105904A (en) * | 2000-05-19 | 2001-11-29 | 이진수 | a flavoring process for butterbur a stem |
| KR20030011464A (en) * | 2001-08-03 | 2003-02-11 | 성덕모 | Composition for healthy food carpable of removing toxic oxygen using wild plants and method for extracting wild plants |
| KR20030039726A (en) * | 2001-11-14 | 2003-05-22 | 한불화장품주식회사 | A cosmetic composition containing an extract of petasites japonicus |
| KR20040081602A (en) * | 2003-03-14 | 2004-09-22 | 충남대학교산학협력단 | Extract of Petasites japonicus having neuroprotective effect and antioxidant activity |
-
2004
- 2004-06-24 KR KR1020040047683A patent/KR100596318B1/en active IP Right Grant
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09140359A (en) * | 1995-11-27 | 1997-06-03 | Shinichiro Saito | Wild vegetable juice |
| KR20010105904A (en) * | 2000-05-19 | 2001-11-29 | 이진수 | a flavoring process for butterbur a stem |
| KR20030011464A (en) * | 2001-08-03 | 2003-02-11 | 성덕모 | Composition for healthy food carpable of removing toxic oxygen using wild plants and method for extracting wild plants |
| KR20030039726A (en) * | 2001-11-14 | 2003-05-22 | 한불화장품주식회사 | A cosmetic composition containing an extract of petasites japonicus |
| KR20040081602A (en) * | 2003-03-14 | 2004-09-22 | 충남대학교산학협력단 | Extract of Petasites japonicus having neuroprotective effect and antioxidant activity |
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| Publication number | Publication date |
|---|---|
| KR20050000354A (en) | 2005-01-03 |
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