KR20100000693A - Composition comprising the extract of platycodon grandiflorum for the improvement of age-related decrease in neurogenesis - Google Patents
Composition comprising the extract of platycodon grandiflorum for the improvement of age-related decrease in neurogenesis Download PDFInfo
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- KR20100000693A KR20100000693A KR1020080060286A KR20080060286A KR20100000693A KR 20100000693 A KR20100000693 A KR 20100000693A KR 1020080060286 A KR1020080060286 A KR 1020080060286A KR 20080060286 A KR20080060286 A KR 20080060286A KR 20100000693 A KR20100000693 A KR 20100000693A
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Images
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- 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
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- 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
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/302—Foods, ingredients or supplements having a functional effect on health having a modulating effect on age
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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
- A23V2250/00—Food ingredients
- A23V2250/20—Natural extracts
- A23V2250/21—Plant extracts
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Abstract
Description
본 발명은 도라지 추출물을 유효성분으로 함유하는 신경줄기세포 분화 촉진용 기능성 식품에 관한 것이다.The present invention relates to a functional food for promoting neural stem cell differentiation containing bellflower extract as an active ingredient.
신경조직은 다른 조직과 달리 발생과정 중에서 분열, 분화 및 시넵스 형성 등의 일련 과정이 전부 이루어지는 조직으로 알려져 있으며, 출생 후에는 거의 분화가 이루어지지 않는 것으로 알려져 왔다. Unlike other tissues, the neural tissue is known to be a tissue in which a series of processes such as division, differentiation, and synapse formation are all performed during development, and it has been known that little differentiation occurs after birth.
한편 최근의 연구보고에 의하면 신경세포의 경우 매우 제한된 부위에서 신경세포의 재생이 나타나며, 그 부위는 외측뇌실의 아랫부분인 뇌실밑구역 (subventricular zone)과 해마(hippocampus)의 치아이랑(dentate gyrus)에서 과립세포(granule cell)의 밑부분인 과립밑구역 (subgranular zone)으로 알려져 있다 (Kempermann et al., 2003; Larsson et al., 2004; Paizanis et al., 2007).Recent studies have shown that neurons regenerate at very limited sites, which are located in the subventricular zone, the lower part of the lateral ventricles, and in the dental gyrus of the hippocampus. Is known as the subgranular zone at the base of granule cells (Kempermann et al., 2003; Larsson et al., 2004; Paizanis et al., 2007).
뇌실밑구역 (subventricular zone)에서 생성된 세포는 rostral migratory stream을 통해 뇌의 앞쪽 부분으로 이동을 하며, 결국 후각망울 (olfactory bulb)에 있는 과립세포(granule cell)로 분화 하거나 이 부위에 있는 신경아교세포(neuroglia)로 분화한다 (Pencea et al., 2001; Menezes et al., 2002; Bedard and Parent, 2004; Martoncikova et al., 2006).Cells produced in the subventricular zone migrate through the rostral migratory stream to the anterior part of the brain, resulting in differentiation into granule cells in the olfactory bulb or neuroglial cells in this region. (neuroglia) (Pencea et al., 2001; Menezes et al., 2002; Bedard and Parent, 2004; Martoncikova et al., 2006).
과립밑구역 (subgranular zone)에서 생성되는 세포는 과립밑구역에서 위쪽으로 이동을 해서 치아이랑(dentate gyrus)의 과립세포가 되는 것으로 알려져 있으며 (Kuhn et al., 1996; Reznikov, 1991; Eckenhoff and Rakic, 1988), 이렇게 생성된 과립세포는 해마의 CA3부분과 연결되어 해마의 고유기능과 연관이 된다(Li and Pleasure, 2005).Cells produced in the subgranular zone are known to migrate upward in the subgranular zone to become granular cells of the dentate gyrus (Kuhn et al., 1996; Reznikov, 1991; Eckenhoff and Rakic). , 1988), the granule cells thus produced are linked to the CA3 part of the hippocampus, which is associated with the intrinsic function of the hippocampus (Li and Pleasure, 2005).
한편, 신경세포는 신경계 질환에서 뿐 아니라 정상 성인의 뇌에서도 매일 많은 숫자가 죽어가며 노화에 따라서 죽는 신경세포수는 기하급수적으로 증가한다 (Yuan and Yankner, Nature. 407, 802-809, 2000). 특히, 해마는 외현적 기억에 필수불가결한 부위로 알려져 있으며, 노화에 따라서 손상을 가장 쉽게 받는 부위로 알려져 있다 (Scoviller and Milner, J Neurol Neurosurg Psychiatry 20, 11-21, 1957). 따라서 이들 부위에서 신경줄기세포의 발생과 분화를 증진하면 신경재생을 촉진할 수 있을 뿐 아니라 노화로 인해 나타나는 신경세포사를 어느 정도 보상할 수 있다. On the other hand, neurons die every day, not only in neurological diseases, but also in the brains of normal adults, and the number of neurons dying with age increases exponentially (Yuan and Yankner, Nature. 407, 802-809, 2000). In particular, the hippocampus is known as an indispensable site for external memory, and is known as the site most easily damaged by aging (Scoviller and Milner, J Neurol Neurosurg Psychiatry 20, 11-21, 1957). Therefore, by promoting the development and differentiation of neural stem cells in these areas can not only promote nerve regeneration but also compensate to some extent neuronal death caused by aging.
도라지는 초롱꽃과에 속하는 다년생 식물로써 오래전부터 우리나라에서 부식 뿐만아니라 약용식물로 이용되어 왔다. 천연에 존재하는 도라지는 진정, 해열, 진 통작용이 있으며, 항염증 작용이 있는 것으로 알려져 있으며(Lee, Kor J Phamarongr. 5, 49-56, 1974) 인체의 결장암 세포와 직장암 세포를 대상으로 한 실험에서는 암세포의 증식억제 효과가 매우 높다는 보고도 있다(Hwang, Korean J Food Nut. 5, 13-19, 1992; Lee et al., Korean University Medical Journal 20, 39-45, 1983; Lee et al., Korean University Medical Journal 25, 365-370, 1988). 이러한 도라지는 트리터피노이드(triterpenoid)계 사포닌과 당질, 섬유질을 상당히 높게 함유하고 있어서 한방 약제로 이용되며(Lee, Chiese pharmaceutics, p129, 1981), 약리 성분 분석을 통하여 사포게닌(sapogenin)의 구조가 밝혀져 있는 것으로 알려져 있다(Kubota et al, Chemical Coommun. 22, 1314-1320, 1969; AAkiyama et al, Chem Phrm Bull. 20, 1945-1951, 1972).Bellflower is a perennial plant belonging to the campanula, and has been used not only for corrosion but also for medicinal plants in Korea. Natural bellflower is known to have calming, antipyretic, analgesic and anti-inflammatory effects (Lee, Kor J Phamarongr. 5, 49-56, 1974). In experiments, it has been reported that cancer cell proliferation inhibitory effect is very high (Hwang, Korean J Food Nut. 5, 13-19, 1992; Lee et al., Korean University Medical Journal 20, 39-45, 1983; Lee et al. , Korean University Medical Journal 25, 365-370, 1988). These bellflowers contain triterpenoid-based saponins, sugars, and fibers, which are quite high, and are used as herbal medicines (Lee, Chiese pharmaceutics, p129, 1981). (Kubota et al, Chemical Coommun. 22, 1314-1320, 1969; AAkiyama et al, Chem Phrm Bull. 20, 1945-1951, 1972).
본 발명자들은 도라지 추출물이 노화 과정에 있는 마우스에서 해마의 치아이랑 부위에서 신생 줄기세포의 수를 현저하게 증가시키는 것을 확인하였고, 이로써 노화에 의해 신경세포의 재생 및 분화 등이 감소되는 현상을 개선할 수 있음을 확인함으로써 본 발명을 완성하게 되었다.The present inventors confirmed that the bellflower extract significantly increases the number of neoplastic stem cells in the dentate gyrus of the hippocampus in mice undergoing aging, thereby improving the phenomenon in which neuronal regeneration and differentiation are reduced by aging. By confirming that the present invention can be completed.
상기한 과제를 해결하기 위한 일례로서, 본 발명은 도라지 추출물을 포함하 고 신경줄기세포의 분화와 증식 촉진능을 갖는 기능성 식품을 제공한다.As an example for solving the above problems, the present invention provides a functional food containing bellflower extract and having the ability to promote differentiation and proliferation of neural stem cells.
이하 본 발명의 기능성 식품을 구체적으로 설명하면 다음과 같다.Hereinafter, the functional food of the present invention will be described in detail.
상기 신경줄기세포는 해마(hippocampus) 치아이랑(dentate gyrus) 과립밑구역(subgranular zone) 유래 신경줄기세포를 의미하며, 줄기세포의 분화는 줄기세포의 생성, 분열, 분화 및 이동을 포함한다.The neural stem cells refer to neural stem cells derived from the hippocampus dental gyrus subgranular zone, and the differentiation of stem cells includes generation, division, differentiation and migration of stem cells.
본원에서 정의되는 추출물은 물, 메탄올, 부탄올 또는 이들의 혼합용매로부터 선택되어진 극성용매, 바람직하게는 에탄올에 가용된 추출물을 포함한다.Extracts as defined herein include extracts soluble in polar solvents, preferably ethanol, selected from water, methanol, butanol or mixed solvents thereof.
또한 본원에서 정의되는 도라지는 초롱꽃과(Campanulaceae)인 도라지(Platycodon grandiflorum) 뿌리 및 동종 식물의 뿌리로부터 얻은 삼출액, 즉 수지상 유액을 건조한 것을 포함한다.Bellflower as defined herein also includes dried exudates obtained from the roots of the Platycodon grandiflorum (Campanulaceae) and the roots of the same plants, ie, dendritic emulsions.
이하, 본 발명의 추출물을 수득하는 방법을 상세히 설명한다.Hereinafter, the method for obtaining the extract of the present invention will be described in detail.
본 발명의 도라지 추출물은, 건조된 도라지의 뿌리를 세절하여 건조 중량의 약 1 내지 20배, 바람직하게는 약 2 내지 6배의 물, C1 내지 C4의 저급 알콜 또는 이들의 혼합용매, 바람직하게는 물 또는 에탄올로, 20 내지 150℃, 바람직하게는 70 내지 120℃ 추출온도에서 약 1시간 내지 10일, 바람직하게는 약 2시간 내지 5시간 동안 냉침추출, 열수추출, 초음파 추출, 환류냉각 추출 등의 추출방법, 바람직하게는 냉침 추출방법을 이용하여 수득한 추출액을 여과, 감압농축 또는 건조하여 수득할 수 있다.The bellflower extract of the present invention is chopped from the roots of dried bellflower, which is about 1 to 20 times the dry weight of water, preferably about 2 to 6 times the water, C1 to C4 lower alcohol, or a mixed solvent thereof, preferably Water or ethanol, cold sewage extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, etc. for about 1 hour to 10 days, preferably about 2 hours to 5 hours at an extraction temperature of 20 to 150 ℃, preferably 70 to 120 ℃ Extraction method obtained by the extraction method, preferably cold extraction method can be obtained by filtration, concentrated under reduced pressure or dried.
본 발명의 기능성 식품은 상기한 도라지 추출물을 포함하며, 이외에 식품학적으로 허용 가능한 식품보조 첨가제를 포함할 수 있다.Functional food of the present invention includes the above-mentioned bellflower extract, in addition to food acceptable food supplements.
본 발명의 기능성 식품은 줄기세포 재싱 및 분화 촉진을 위한 목적으로 다양한 식품 및 음료 등에 다양하게 이용될 수 있다. 본 발명의 도라지 추출물을 첨가할 수 있는 식품으로는, 예를 들어, 각종 식품류, 음료, 껌, 차, 비타민 복합제, 건강보조 식품류 등이 있고, 분말, 과립, 정제, 캡슐 또는 음료인 형태로 사용할 수 있다.The functional food of the present invention can be used in various foods and beverages for the purpose of promoting stem cell ashing and differentiation. Foods to which the bellflower extract of the present invention may be added include, for example, various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, and may be used in the form of powders, granules, tablets, capsules, or beverages. Can be.
본 발명의 도라지 추출물 자체는 독성 및 부작용은 거의 없으므로 예방 목적으로 장기간 복용 시에도 안심하고 사용할 수 있는 성분이다. Bellflower extract itself of the present invention is a component that can be used with confidence even when taken for a long time for the purpose of prevention because there is little toxicity and side effects.
본 발명의 기능성 식품은, 줄기세포의 재생 및 분화 촉진을 목적으로 상기 도라지 추출물을 다양한 제형의 식품 또는 음료에 첨가하여 제조될 수 있다. 이 때, 식품 또는 음료 중의 상기 도라지 추출물의 양은 일반적으로 전체 식품 중량의 0.01 내지 100 중량%로 가할 수 있으며, 건강 음료로 제조할 경우 전체 조성물 100 ㎖를 기준으로 0.02 내지 10 g, 바람직하게는 0.3 내지 1 g의 비율로 가할 수 있다. The functional food of the present invention may be prepared by adding the bellflower extract to foods or beverages of various formulations for the purpose of promoting regeneration and differentiation of stem cells. At this time, the amount of the bellflower extract in the food or beverage can generally be added in 0.01 to 100% by weight of the total food weight, when prepared as a health beverage 0.02 to 10 g, preferably 0.3 based on 100 ml of the total composition To 1 g.
본 발명의 기능성 식품을 음료로 제조할 경우 지시된 비율로 필수 성분으로서 상기 추출물을 함유하는 것 외에 액체성분에는 특별한 제한점은 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. When the functional food of the present invention is prepared as a beverage, the extract contains the extract as an essential ingredient in the indicated proportions, and there is no particular limitation in the liquid component, and it contains various flavors or natural carbohydrates as additional ingredients, as in general beverages. can do.
상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등의 디사카라이드, 예를 들어 말토스, 슈크로스 등의 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트 리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 기능성 식품 100 ㎖당 일반적으로 약 1 내지 20g, 바람직하게는 약 5 내지 12g이다.Examples of the above-mentioned natural carbohydrates are conventional monosaccharides such as disaccharides such as glucose and fructose, such as maltose, sucrose and the like, and polysaccharides such as dextrin, cyclodextrin and the like. Sugars and sugar alcohols such as xylitol, sorbitol and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the functional food of the present invention.
상기 외에 본 발명의 기능성 식품은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 기능성 식품은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. In addition to the above, the functional food of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the functional food of the present invention may contain fruit flesh for the production of natural fruit juice and fruit juice beverage and vegetable beverage.
이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 기능성 식품 100 중량부 당 0 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다.These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the functional food of the present invention.
상술한 바와 같이, 본 발명의 도라지 추출물은 노화시에 나타나는 신경줄기세포의 분화 및 증식의 감소를 현저하게 억제함으로써 노화에 의한 신경세포 분화 및 증식을 촉진시키기 위한 기능성 식품으로 유용하게 이용될 수 있다. As described above, the bellflower extract of the present invention can be usefully used as a functional food for promoting neuronal differentiation and proliferation by aging by remarkably inhibiting the reduction of the differentiation and proliferation of neural stem cells during aging.
이하, 본 발명을 실시예 및 실험예에 의해 구체적으로 설명하겠는바, 다음 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 한정되는 것은 아니다. Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples, but the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.
실시예Example 1. 도라지 추출물의 제조 1. Preparation of Bellflower Extract
1-1. 도라지의 추출물의 제조1-1. Preparation of Extracts of Bellflower
2007년 2월 서울특별시 소재 한약재 시장에서 건조된 도라지를 구입한후, 분말화 하여 100% 에탄올 2 L를 가하여 일주일간 냉침시켰다. 상기 냉침이 끝난 에탄올 추출물은, 감압플라스크에 감압장치(aspirator)를 연결해 플라스크 내부를 감압시킨 다음, 감압플라스크에 연결된 여과기에 넣고 여과하였다. In February 2007, dried bellflower was purchased at the Herbal Medicine Market in Seoul, and then powdered and added to 2 L of 100% ethanol for 1 week. After the cold ethanol extract was connected to a pressure reducing flask (aspirator) to depressurize the inside of the flask, and then filtered into a filter connected to the pressure reducing flask.
상기 여과되어진 여액을 감압농축기(BUCHI 011, SWITZERLAND)로 감압농축하여 도라지 추출물 67 g을 얻어 실험예의 시료로 사용하였다.The filtered filtrate was concentrated under reduced pressure with a vacuum concentrator (BUCHI 011, SWITZERLAND) to obtain 67 g of bellflower extract, which was used as a sample in the experimental example.
실험예Experimental Example 1. 도라지 추출물의 줄기세포분화 측정 1. Measurement of Stem Cell Differentiation of Bellflower Extract
도라지 추출물의 줄기세포분화 촉진 효과를 확인하기 위하여 C57BL/6J 마우스를 대상으로 줄기세포의 분화를 5-bromo-2-deoxyguanosine (BrdU), doublecortin (DCX), Ki-67(proliferation marker)로 표지하는 방법을 이용하여 하기와 같이 실험하였다.To identify the stem cell differentiation promoting effect of the bellflower extract, the differentiation of stem cells in C57BL / 6J mice was labeled with 5-bromo-2-deoxyguanosine (BrdU), doublecortin (DCX), and Ki-67 (proliferation marker). The experiment was conducted as follows.
도라지 추출물의 줄기세포분화 촉진 효능을 측정하기 위하여 동물 실험을 하 기와 같은 방법으로 수행하였다. In order to measure the stem cell differentiation promoting effect of the bellflower extract was carried out in the same manner as the animal experiment.
실험동물은 체중 25 내지 30 g의 수컷 12개월령 C57BL/6J 마우스 20마리를 사용하였고, 오전 7시부터 오후 7시까지 빛을 가하는 일정한 명암주기 하에서 온도 23± 2℃, 상대습도 55 ± 10%에서 사육하였으며, 음식과 물은 자유로이 섭취하게 하였다. Experimental animals used 20 male 12-month-old C57BL / 6J mice, weighing 25 to 30 g, at a temperature of 23 ± 2 ° C. and a relative humidity of 55 ± 10% under constant light and dark cycles from 7 am to 7 pm. It was raised, and food and water were taken freely.
상기 실시예 1에서 얻은 도라지 추출물 25 mg을 증류수 2 ml에 용해한 것을 식도용 바늘을 이용하여 0.2 ml을 4주 간 경구투여(용량은 체중 ㎏ 당 100 mg이 되도록 투여)하였으며, 대조군은 용매인 증류수를 동량 투여하였다. 20일 동안 투여 후에 줄기세포를 표지하기 위해 BrdU (Sigma, USA) 12.5 mg을 생리식염수 1 ml에 용해한 다음, 8시간 간격으로 0.1 ml을 3회 복강투여하였다.25 mg of bellflower extract obtained in Example 1 was dissolved in 2 ml of distilled water, orally administered 0.2 ml for 4 weeks using an esophageal needle (dose was administered to 100 mg / kg body weight), and the control group was distilled water as a solvent. Was administered in equal amounts. After 20 days of administration, 12.5 mg of BrdU (Sigma, USA) was dissolved in 1 ml of saline solution to label stem cells, and then 0.1 ml of three doses were administered at 8 hour intervals.
대조군 및 실험군을 마지막 BrdU 투여 4시간 후에 티오펜탈 소듐(thiopental sodium, 유한양행, 한국)을 체중 ㎏ 당 각각 30 ㎎의 용량으로 복강 내 주사하여 마취시킨 다음, 1,000㎖ 당 헤파린 1,000 IU를 함유한 4 ℃의 생리식염수를 좌심실로 주입하여 관류 세척하였다. 관류 세척이 끝난 동물은 바로 4℃의 4% 파라포름알데하이드(in 0.1 M phosphate buffer; PB, pH 7.4)로 관류 고정을 하였다. The control and experimental groups were anesthetized by intraperitoneal injection of thiopental sodium at 30 mg / kg body weight 4 hours after the last BrdU administration, and then containing 1,000 IU of heparin per 1,000 ml. Physiological saline at 4 ° C. was injected into the left ventricle and washed perfusion. After the perfusion wash, the animals were directly perfused with 4% paraformaldehyde (in 0.1 M phosphate buffer; PB, pH 7.4) at 4 ° C.
관류 고정이 끝난 동물을 뼈절단기를 이용하여 머리뼈공간을 열어 뇌를 적출한 다음, 동일 고정액에서 6 시간동안 후고정하였다. 후고정이 끝난 뇌는 30% 슈크로스 용액(in 0.1 M phosphate buffer)에 넣어 바닥에 가라앉을 때까지 침강 시킨 후, 슬라이드 마이크로톰(sliding microtome, Reichert-Jung, Germany)으로 조직을 30 ㎛ 두께로 잘라 보존액(storing solution)이 들어있는 6웰 플레이트에 넣어 사 용시까지 4℃에서 보관하였다. After perfusion fixation, the animals were opened by using a bone cutter to remove the brain, and then fixed in the same fixative for 6 hours. After fixation, the brain was settled in a 30% sucrose solution (in 0.1 M phosphate buffer) until it settled to the bottom, and the tissue was cut into a 30 μm thickness using a sliding microtome (Reichert-Jung, Germany). 6-well plate containing a storage solution (storing solution) was stored at 4 ℃ until use.
각 조직절편 중에서 해마부위(hippocampus)가 잘 나와 있는 조직을 선택하였고, 조직에 묻어있는 보존액을 세척하기 위해 0.01M PBS로 10분씩 3회 세척한 다음, 조직절편에 존재하는 내인성 페록시다아제(peroxidase)를 제거하기 위하여 0.3% H2O2(in PBS)로 30분 동안 반응시켰다. 그 다음, 65℃ 50% 포름아마이드(foramide) (in 2X standard citate solution) 용액에 2시간 반응시킨 다음에 37℃ 2N HCl에 30분간 반응시켜 DNA를 변성(denaturation)시켰다. 비특이적인 면역반응을 방지하기 위하여, 상기 반응된 조직절편을 3%의 normal goat serum으로 30분간 반응시켰다. 상기 3%의 normal goat serum과 반응을 한 조직절편을 줄기세포를 표지하기 위해 1:1000으로 희석한 1차 항체인 rabbit anti-BrdU(Chmicon Internationl, USA), goat anti-DCX(SantaCruz, USA) 또는 rabbit anti-Ki-67(Abcam, UK)와 4℃에서 48시간 반응시켰다. 반응이 끝난 조직절편은 각각 1:200으로 희석시킨 2차 항체인 anti-rabbit IgG 또는 anti-goat IgG(Vector)로 2시간 반응시킨 후, 1:200으로 희석시킨 3차 항체인 ABC용액(Vector)으로 2시간 반응시켰으며, 3,3'-diaminobezidine (DAB)를 기질로 발색하였다. Among the tissue sections, tissues with well-formed hippocampus were selected, and washed three times with 0.01 M PBS for 10 minutes to wash the stock solution on the tissue, and then endogenous peroxidase ( In order to remove peroxidase) it was reacted with 0.3% H 2 O 2 (in PBS) for 30 minutes. Then, the mixture was reacted with 65 ° C. 50% formamide (in 2X standard citate solution) solution for 2 hours and then reacted with 37 ° C. 2N HCl for 30 minutes to denature DNA. In order to prevent nonspecific immune responses, the reacted tissue sections were reacted with 3% normal goat serum for 30 minutes. Rabbit anti-BrdU (Chmicon Internationl, USA), goat anti-DCX (SantaCruz, USA), which is a primary antibody diluted 1: 1000 in order to label stem cells of tissue sections reacted with the 3% normal goat serum Or rabbit anti-Ki-67 (Abcam, UK) was reacted for 48 hours at 4 ℃. After completion of the reaction, the tissue sections were reacted with anti-rabbit IgG or anti-goat IgG (Vector), a secondary antibody diluted 1: 200, respectively, and then reacted with ABC solution (Vector), a third antibody diluted 1: 200. ), And 3,3'-diaminobezidine (DAB) was developed as a substrate.
각 단계 사이에는 0.01M PBS를 사용하여 10분씩 3회 세척하였다.Each step was washed three times for 10 minutes using 0.01 M PBS.
상기 3차 반응이 끝난 조직절편은 젤라틴 코팅된 슬라이드 글라스(slide glass)에 도말한 후 실온에서 12시간 동안 건조하였으며, 상기 건조한 조직절편은 통상적인 탈수·투명화 과정을 거쳐 봉입하였다.After tertiary reaction, the tissue sections were plated on a gelatin-coated slide glass and dried at room temperature for 12 hours, and the dried tissue sections were enclosed through a conventional dehydration and transparency process.
각 조직들은 디지털 카메라(Axiocam, Cal Zeiss, Germany)가 부착되어 있는 악시오엠1 현미경(AxioM1 microscope, Carl Zeiss, Germany)으로 치아이랑 전체를 200배로하여 사진 촬영하여 도 1에 나타내었다. (도 1 참조) Each tissue was photographed using axioM1 microscope (Carl Zeiss, Germany) attached to a digital camera (Axiocam, Cal Zeiss, Germany) with a total of 200 times the entire tooth gyrus and shown in FIG. (See Figure 1)
도 1에서 나타나는 바와 같이 A, C 및 E는 용매투여군의 치아이랑 전체 영역의 사진이고 B, D 및 F는 도라지추출물 투여군의 치아이랑 전체 영역으로, 진하게 염색된 세포를 이미지 분석기(Optimas 6.5, CyberMetrics, USA) 프로그램을 사용하여 신경세포의 수를 계수하여 도 2에 나타냈다. (도 2 참조) As shown in FIG. 1, A, C and E are photographs of the entire area of the teeth of the solvent-administered group and B, D and F are the entire areas of the tooth and the entire area of the bellflower extract-administered group, and image stained cells (Optimas 6.5, CyberMetrics , USA) program was used to count the number of neurons is shown in FIG. (See Figure 2)
각 군에 대한 유의성의 검증을 위하여 student t-test를 수행하였으며, 데이터는 평균과 표준평균오차로 나타내었다. 치아이랑 부위를 악시오엠1 현미경(AxioM1 microscope, Carl Zeiss, Germany)을 이용하여 사진 촬영하였고, 그 결과를 도 1에 나타내었다. Student t-test was performed to verify the significance of each group, and the data were expressed as mean and standard mean error. The dentate gyrus was photographed using an AxioM1 microscope (Carl Zeiss, Germany), and the results are shown in FIG. 1.
실험결과, 도 1에서는 용매를 투여한 대조군(A)의 경우 매우 적은 수의 세포에서 BrdU에 염색성을 확인할 수 있었던 반면에, 도라지 추출물 100 ㎎/㎏을 투여한 실험군에서(B)는 치아이랑에서 다수의 BrdU에 염색된 세포를 확인할 수 있었다. 내인성 신경줄기세포의 신경세포로의 분화에 대한 표지자인 DCX에 대한 염색성은 용매를 투여한 대조군(C)의 경우에도 소수의 DCX 염색성을 보이는 세포가 관찰된 반면, 도라지추출물을 100mg/kg을 투여한 실험군에서(D)는 DCX에 염색된 세포가 다수 관찰되었다. 또한, 신경세포의 증식 표지자로 알려진 Ki-67의 경우에도 용매를 투여한 대조군(E)의 경우, 극소수의 세포에서만 면역반응성을 확인할 수 있었지만, 도라지 추출물을 투여한 군에서는 (F) 대조군에 비교하였을 때 보다 많은 수의 Ki-67 반응성을 보이는 세포들이 확인되었다.As a result, in FIG. 1, the control group (A) administered with the solvent was able to confirm staining of BrdU in a very small number of cells, whereas in the experimental group administered with 100 mg / kg of bellflower extract (B) A number of cells stained with BrdU could be confirmed. Dyeing of DCX, a marker for differentiation of endogenous neural stem cells into neurons, was observed in the control group (C) with solvent, while a few cells showing DCX staining were observed, whereas 100 mg / kg of bellflower extract was administered. In the experimental group (D), a large number of cells stained with DCX were observed. In addition, in the case of Ki-67, which is known as a proliferation marker of neurons, the control group (E) was able to confirm immunoreactivity only in a small number of cells, but compared to the (F) control group in the bellflower group. As a result, more Ki-67 responsive cells were identified.
또한, 도 2에서 도라지 추출물의 투여에 따른 BrdU에 염색된 세포를 계수로 확인한 결과, 100 mg/kg의 도라지 추출물을 투여한 경우 대조군에 대해 치아이랑 절편 당 약 7.5개의 세포가 증가(180%)하여 있는 것을 확인할 수 있었으며, DCX에 염색된 세포는 도라지 추출물을 투여한 경우 치아이랑 절편당 약 3.7개 가량 증가(155%)하였으며, Ki-67에 염색된 세포는 도라지 추출물을 투여한 경우 절편당 약 5.6개가 증가(800%)하는 것을 확인할 수 있었다. 이로 인하여 도라지추출물의 투여가 신경줄기세포의 분화 및 증식에 탁월한 효과가 있는 것을 확인할 수 있었다.In addition, as a result of confirming the cells stained with BrdU according to the administration of the bellflower extract in Figure 2, the administration of 100 mg / kg bellflower extract increased about 7.5 cells per tooth gyrus section for the control group (180%) The cells stained with DCX increased about 3.7 per tooth gyrus section (155%) when the bellflower extract was administered, and the cells stained with Ki-67 were sectioned when the bellflower extract was administered. It was confirmed that about 5.6 increased (800%). Therefore, it was confirmed that the administration of bellflower extract has an excellent effect on the differentiation and proliferation of neural stem cells.
하기에 본 발명의 추출물을 포함하는 조성물의 제제예를 설명하나, 본 발명은 이를 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다.Hereinafter, the preparation examples of the composition including the extract of the present invention, but the present invention is not intended to limit it, but is intended to explain in detail only.
제제예Formulation example 1. One. 산제의Powder 제조 Produce
도라지 추출물 100 mg Bellflower Extract 100 mg
유당 100 mg Lactose 100 mg
탈크 10 mg Talc 10 mg
상기의 성분들을 혼합하고 기밀포에 충진하여 산제를 제조한다. The above ingredients are mixed and filled in an airtight cloth to prepare a powder.
제제예Formulation example 2. 정제의 제조 2. Preparation of Tablets
도라지 추출물 100 mg Bellflower Extract 100 mg
옥수수전분 100 mg Corn starch 100 mg
유당 100 mg Lactose 100 mg
스테아린산 마그네슘 2 mg 2 mg magnesium stearate
상기의 성분들을 혼합한 후 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조한다. After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.
제제예Formulation example 3. 캡슐제의 제조 3. Preparation of Capsule
도라지 추출물 100 mg Bellflower Extract 100 mg
결정성 셀룰로오스 3 mg 3 mg of crystalline cellulose
락토오스 14.8 mg Lactose 14.8 mg
마그네슘 스테아레이트 0.2 mg Magnesium Stearate 0.2 mg
통상의 캡슐제 제조방법에 따라 상기의 성분을 혼합하고 젤라틴 캡슐에 충전하여 캡슐제를 제조한다. According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.
제제예Formulation example 4. 4. 액제의Liquid 제조 Produce
도라지 추출물 100 mg Bellflower Extract 100 mg
이성화당 10 g 10 g of isomerized sugar
만니톨 5 g 5 g of mannitol
정제수 적량 Purified water
통상의 액제의 제조방법에 따라 정제수에 각각의 성분을 가하여 용해시키고 레몬향을 적량 가한 다음 상기의 성분을 혼합한 다음 정제수를 가하여 전체를 100 ㎖로 조절한 후 갈색병에 충진하여 멸균시켜 액제를 제조한다. According to the conventional method of preparing a liquid solution, each component is added and dissolved in purified water, lemon flavor is added, the above ingredients are mixed, and then, purified water is added to adjust the total amount to 100 ml, and then sterilized by filling into a brown bottle. Manufacture.
제제예Formulation example 5. 건강 보조 식품의 제조 5. Preparation of dietary supplements
도라지 추출물 2500 ㎎ Bellflower Extract 2500 mg
비타민 혼합물 적량 Vitamin mixture proper amount
비타민 A 아세테이트 70 ㎍ 70 μg of Vitamin A Acetate
비타민 E 1.0 ㎎ Vitamin E 1.0 mg
비타민 B 10.13 ㎎ Vitamin B 10.13 mg
비타민 B 20.15 ㎎ Vitamin B 20.15 mg
비타민 B 60.5 ㎎ Vitamin B 60.5 mg
비타민 B 120.2 ㎍ 120.2 μg of vitamin B
비타민 C 10 ㎎ Vitamin C 10 mg
비오틴 10 ㎍ 10 μg biotin
니코틴산아미드 1.7 ㎎ Nicotinic Acid 1.7 mg
엽산 50 ㎍ 50 μg folic acid
판토텐산 칼슘 0.5 ㎎ Calcium Pantothenate 0.5mg
무기질 혼합물 적량 Mineral mixture
황산제1철 1.75 ㎎ Ferrous Sulfate 1.75 mg
산화아연 0.82 ㎎ Zinc Oxide 0.82 mg
탄산마그네슘 25.3 ㎎ Magnesium carbonate 25.3 mg
제1인산칼륨 15 ㎎ Potassium monophosphate 15 mg
제2인산칼슘 55 ㎎
구연산칼륨 90 ㎎ Potassium Citrate 90 mg
탄산칼슘 100 ㎎ Calcium Carbonate 100 mg
염화마그네슘 24.8 ㎎ Magnesium chloride 24.8 mg
상기의 비타민 및 미네랄 혼합물의 조성비는 비교적 건강식품에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 그 배합비를 임의로 변형 실시하여도 무방하며, 통상의 건강식품 제조방법에 따라 상기의 성분을 혼합한 다음, 과립을 제조하고, 통상의 방법에 따라 건강식품 조성물 제조에 사용할 수 있다. Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.
도 1은 도라지 추출물의 신경세포재생촉진 효과를 확인하기 위해 용매(물) (A, C 및 E) 또는 도라지 추출물 100 mg/kg (B, D 및 F)을 4주간 경구 투여 한 다음, 희생 직전 BrdU를 8시간 간격으로 3회 투여하고, 8시간 경과 후에 치아이랑 조직을 채취하여 BrdU 항체(A 및 B), DCX 항체 (C 및 D) 및 Ki-67 항체 (E 및 F)로 염색하여 관찰한 이미지 사진이며, 1 is oral administration of a solvent (water) (A, C and E) or 100 mg / kg (B, D and F) for 4 weeks to confirm the neuronal cell regeneration promoting effect of the bellflower extract, immediately before sacrifice BrdU was administered three times at 8-hour intervals, and after 8 hours, gingival tissue was collected and stained with BrdU antibodies (A and B), DCX antibodies (C and D), and Ki-67 antibodies (E and F). One image photo,
도 2는 도라지 추출물을 용매 또는 도라지 추출물을 100 mg/kg으로 경구 투여 한 다음, BrdU를 8시간 간격으로 3회 투여하고, 8시간 경과 후에 BrdU, DCX 및 Ki-67에 염색된 세포수를 나타낸 그래프이다.Figure 2 is oral administration of bellflower extract solvent or bellflower extract at 100 mg / kg, then three times the BrdU every 8 hours, showing the number of cells stained BrdU, DCX and Ki-67 after 8 hours It is a graph.
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