KR102046878B1 - Composition comprising the extract of buckwheat for immune activity - Google Patents
Composition comprising the extract of buckwheat for immune activity Download PDFInfo
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- KR102046878B1 KR102046878B1 KR1020180011531A KR20180011531A KR102046878B1 KR 102046878 B1 KR102046878 B1 KR 102046878B1 KR 1020180011531 A KR1020180011531 A KR 1020180011531A KR 20180011531 A KR20180011531 A KR 20180011531A KR 102046878 B1 KR102046878 B1 KR 102046878B1
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- buckwheat
- extract
- cells
- present
- increase
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Abstract
본 발명은 메밀 추출물의 면역증진 용도를 규명한 것으로, 보다 구체적으로, 메밀 추출물을 유효성분으로 포함하는 면역기능 증강용 식품 조성물 및 면역증강용 약학 조성물에 관한 것이다. 본 발명에 따른 메밀 추출물은 산화질소의 생성을 증가시키고 사이토카인인 TNF-a 또는 IL-6의 생성을 증가시켜 대식세포의 활성을 증가시켜 면역증진 효과를 유도할 수 있으며, 자연살해세포 활성을 증가시키고, 비장 및 흉선의 중량을 증가시키며 백혈구수를 증진시킬 수 있는 효과가 있어 면역증진을 필요로 하는 질환의 약학적 조성물로 활용할 수 있다. 또한 본 발명의 메밀 추출물은 세포 독성을 유발시키지 않아 체내 안정하므로 면역증진을 위한 식품용 조성물로도 활용할 수 있다.The present invention has been made to investigate the use of buckwheat extract for immune enhancement, and more particularly, to a food composition for enhancing immune function and a pharmaceutical composition for immuno-enhancing comprising buckwheat extract as an active ingredient. Buckwheat extract according to the present invention can increase the production of nitric oxide and increase the production of the cytokine TNF-a or IL-6 to increase the activity of macrophages to induce immune-promoting effect, natural killer cell activity It can increase the weight of the spleen and thymus, and enhance the white blood cell count, so that it can be used as a pharmaceutical composition of a disease requiring immunological enhancement. In addition, the buckwheat extract of the present invention does not cause cytotoxicity and thus is stable in the body, and thus may be utilized as a composition for food for immune promotion.
Description
본 발명은 메밀 추출물의 면역증강 용도를 규명한 것으로, 메밀 추출물을 유효성분으로 포함하는 면역증강용 식품 조성물 및 약학적 조성물에 관한 것이다.The present invention has been made to identify the use of buckwheat extracts for immuno-enhancing, and relates to a food composition and pharmaceutical composition for immuno-enhancing comprising buckwheat extract as an active ingredient.
면역계는 자연저항, 비특이성 면역체계 및 특이성 면역체계로 구분할 수 있다. 자연저항(1차 방어선)이란 미생물을 위시한 모든 침입자들을 그들의 종류에 관계없이 막아내는 해부생리학적 요소들을 말하며, 비특이적 면역(2차 방어선)은 자연저항을 돌파하여 체내로 들어온 침입자들을 제거하는 식세포로 구성된 방어체계를, 그리고 특이성 면역계(3차 방어선)는 림프구들로 구성된 면역체계를 말하는데, 이중 특이성 면역계는 기억능 및 자기와 비자기를 구분할 수 있는 능력을 지닌 고도로 발달한 면역체계이다. The immune system can be divided into natural resistance, nonspecific immune system and specific immune system. Natural resistance (primary line of defense) refers to anatomical physiological elements that block all invaders, including microorganisms, regardless of their type. Nonspecific immunity (secondary line of defense) is a phagocytic cell that breaks through natural resistance and removes invaders. The constructed defense system, and the specific immune system (tertiary line of defense) refers to an immune system composed of lymphocytes, which are highly developed immune systems with memory and the ability to distinguish between self and nonmagnetic.
또한, 백혈구는 2차 또는 3차 방어선을 구성하여 1차 방어선을 돌파하여 체내에 들어온 이물을 담당하게 되며, 세균, 바이러스 감염 또는 염증 반응 시, 대식세포 및 림프구 활성의 조절은 의약품의 치료 효과 결정에 있어서 중추적인 역할을 한다. 대식세포(Macrophage)는 다양한 기능을 가진 세포로 산화적 스트레스 상황에서 여러 가지 사이토카인(cytokine)과 일산화산소(NO)를 생성하여 면역체계에서 중요한 역할을 한다. 특히 대식세포에서 리포 다당류(Lipopolysaccharide; LPS), 사이토카인, TNF-α와 같은 자극에 의해 발현되는 iNOS는 장시간 동안 다량의 NO를 생산하여, NFκB 활성을 촉진시키는 것으로 알려져 있다. 활성화된 대식세포에 의한 슈퍼옥사이드 음이온(superoxide anion, O2-), 과산화수소(hydrogen peroxide, H2O2)와 같은 활성산소종(reactive oxygenspecies; ROS) 및 일산화질소(nitric oxide, NO)의 생산은 비특이적 면역에 있어서 중요한 세포독성 및 세포활성억제기작이다. In addition, leukocytes constitute a secondary or tertiary line of defense to break through the primary line of defense and take charge of foreign bodies entering the body.In the event of bacterial, viral infection or inflammatory reaction, the regulation of macrophage and lymphocyte activity determines the therapeutic effect of the drug. Plays a pivotal role in Macrophage is a multifunctional cell that plays an important role in the immune system by producing various cytokines and oxygen monoxide (NO) in oxidative stress situations. In particular, iNOS expressed by stimulation such as lipopolysaccharide (LPS), cytokine, TNF-α in macrophages is known to produce a large amount of NO for a long time, promoting NFκB activity. The production of reactive oxygenspecies (ROS) and nitric oxide (NO), such as superoxide anion (O2-), hydrogen peroxide (H2O2), by activated macrophages is associated with nonspecific immunity. Is an important cytotoxic and cell activity inhibitory mechanism.
또한, 대식세포는 항원을 제시하거나(antigen-presenting), 종양을 없애거나(tumoricidal) 미생물세포를 죽이는(microbicidal) 세포로서, 세포매개(cell-mediated) 또는 체액성 면역(humoral immunity)에 중심적인 역할을 하는 조절세포로, 활성화된 대식세포에서 생산되는 NO는 비특이적 숙주방어기작인 대식작용을 하고, 세균 및 암세포의 증식억제 활성을 보인다. 뿐만 아니라 대식세포는 많은 라이소솜을 가지고 있고 이들은 산성가수분해효소와 과산화효소를 함유하고 있다. 또한 유리면 과 플라스틱 표면에 강하게 부착하는 성질이 있으며 미생물이나 종양세포 등을 활발하게 탐식한다. 상기 세포는 IFN-γ등의 사이토카인 수용체를 가지고 있다. 이들은 보체성분, 인터페론, 인터루킨-1 및 종양괴사인자 같은 사이토카인을 생산하며 T-세포로부터 생산되는 여러 가지 사이토카인에 의해 기능이 증강될 수 있다.In addition, macrophages are cells that present antigen-presenting, tumor-free, or microbicidal, and are central to cell-mediated or humoral immunity. As a regulatory cell that plays a role, NO produced by activated macrophages acts as a macrophage, a nonspecific host defense mechanism, and exhibits proliferation inhibitory activity of bacteria and cancer cells. In addition, macrophages contain many lysosomes, which contain acidic hydrolase and peroxidase. In addition, it strongly adheres to the glass surface and plastic surface, and actively detects microorganisms and tumor cells. The cell has a cytokine receptor such as IFN-γ. They produce cytokines such as complement, interferon, interleukin-1, and tumor necrosis factor and can be enhanced by several cytokines produced from T-cells.
따라서 현재 전 세계적으로 면역력 증강을 통해 건강을 증진시키고자 많은 연구가 진행되고 있고, 최근에는 합성화합물에 대한 부작용 문제점들이 대두되고 있어 천연물로부터 면역증강을 효과적으로 도출할 수 있는 소재의 개발에 연구가 집중되고 있다. Therefore, a lot of research is being conducted to improve the health through the enhancement of immunity all over the world, and recently, the problem of side effects on synthetic compounds has emerged, so the research is concentrated on the development of the material that can effectively derive the immunity from natural products. It is becoming.
한편, 메밀은 마디풀과에 속하는 일년초로서 원산지는 동아시아의 북부 아무르강에서 만주 바아칼호 지역에 걸쳐 재배되고 있는 곡물이다. 우리나라에서는 가뭄이 심해 벼농사가 불가할 때 재배하는 구황식물로서 이용되었으며, 생육기간이 짧고, 척박한 땅에서도 잘 자라며, 과거로부터 오방지영물이라 하여 식물이상의 의미를 부여받아왔다. On the other hand, buckwheat is a year-old plant belonging to the family Madipuru, and its origin is grain grown in the Manchuria Bakal Lake region in the northern Amur River in East Asia. In Korea, the drought was severe, and it was used as a cultivated plant grown when rice farming was impossible. Its growth period is short and it grows well in the poor land.
메밀은 전통적으로 홍역, 궤양성 위장병 등 내과적 치료용과 타박상, 악성종기 등 외과적 치료용으로 사용되어 왔으며, 독소를 해독시키며 노폐물을 제거하는 생리적 기능이 보고되어 있다. 또한 메밀은 혈압강화 효능을 가진 루틴을 많이 함유하여, 동맥경화증, 폐출혈, 동상, 치질, 감기치료 등에 그 효과가 인정되어 임상적으로 이용되고 있다.Buckwheat has traditionally been used for medical treatments such as measles and ulcerative gastrointestinal diseases, as well as for surgical treatments such as bruises and malignant boils, and physiological functions of detoxifying and removing wastes have been reported. In addition, buckwheat contains a lot of routines having an effect of strengthening blood pressure, and its effects have been recognized clinically for treating atherosclerosis, pulmonary bleeding, frostbite, hemorrhoids, and colds.
그러나 아직까지 메밀이 면역증진 작용이 있음을 보고한 예가 없다.However, no reports have yet been reported that buckwheat has an immunostimulating effect.
이에 본 발명자들은 메밀 추출물이 면역증강 효과가 있음을 규명함으로써 본 발명을 완성하였다.The present inventors have completed the present invention by elucidating that buckwheat extract has an immune enhancing effect.
그러므로 본 발명의 목적은 메밀 추출물을 유효성분으로 포함하는 면역기능 증강용 식품 조성물을 제공하는 것이다.Therefore, it is an object of the present invention to provide a food composition for enhancing immune function comprising buckwheat extract as an active ingredient.
또한 본 발명의 다른 목적은 메밀 추출물을 유효성분으로 포함하는 면역증강용 약학 조성물을 제공하는 것이다.It is another object of the present invention to provide a pharmaceutical composition for immuno-enhancement comprising buckwheat extract as an active ingredient.
또한 본 발명의 다른 목적은 메밀 추출물을 유효성분으로 포함하는 자연살해세포 활성 증진용 조성물을 제공하는 것이다.It is another object of the present invention to provide a composition for enhancing natural killer cell activity, including buckwheat extract as an active ingredient.
상기와 같은 본 발명의 목적을 달성하기 위해서, 본 발명은 메밀 추출물을 유효성분으로 포함하는 면역기능 증강용 식품 조성물을 제공한다.In order to achieve the object of the present invention as described above, the present invention provides a food composition for enhancing immune function comprising buckwheat extract as an active ingredient.
본 발명의 일실시예에 있어서, 상기 추출물은 메밀에 물을 첨가하여 수득한 열수 추출물; 또는 상기 열수 추출물에 주정을 첨가하고 침전시킨 후, 침전물을 동결건조시켜 수득한 고분자 분획물을 포함하는 추출물일 수 있다.In one embodiment of the present invention, the extract is a hydrothermal extract obtained by adding water to buckwheat; Alternatively, the extract may include a polymer fraction obtained by adding ethanol to the hot water extract and precipitating the precipitate, and lyophilizing the precipitate.
본 발명의 일실시예에 있어서, 상기 메밀 추출물은 대식세포의 활성을 증가시키고, 산화질소의 생성을 증가시키며, 사이토카인인 TNF-a 또는 IL-6의 생성을 증가시키는 것일 수 있다.In one embodiment of the present invention, the buckwheat extract may be to increase the activity of macrophages, increase the production of nitric oxide, increase the production of the cytokine TNF-a or IL-6.
본 발명의 일실시예에 있어서, 상기 메밀 추출물은 자연살해세포(NK cell)의 활성을 증가시키고, 비장 및 흉선의 중량을 증가시키며, 백혈구 수를 증진시키는 활성을 갖는 것일 수 있다.In one embodiment of the present invention, the buckwheat extract may have activity to increase the activity of natural killer cells (NK cells), increase the weight of the spleen and thymus, and enhance the number of white blood cells.
또한, 본 발명은 메밀 추출물을 유효성분으로 포함하는 면역증강용 약학 조성물을 제공한다.The present invention also provides a pharmaceutical composition for immuno-enhancing comprising buckwheat extract as an active ingredient.
본 발명의 일실시예에 있어서, 상기 추출물은 메밀에 물을 첨가하여 수득한 열수 추출물; 또는 상기 열수 추출물에 주정을 첨가하고 침전시킨 후, 침전물을 동결건조시켜 수득한 고분자 분획물을 포함하는 추출물일 수 있다.In one embodiment of the present invention, the extract is a hydrothermal extract obtained by adding water to buckwheat; Alternatively, the extract may include a polymer fraction obtained by adding ethanol to the hot water extract and precipitating the precipitate, and lyophilizing the precipitate.
본 발명의 일실시예에 있어서, 상기 메밀 추출물은 대식세포의 활성을 증가시키고, 산화질소의 생성을 증가시키며, 사이토카인인 TNF-a 또는 IL-6의 생성을 증가시키는 것일 수 있다.In one embodiment of the present invention, the buckwheat extract may be to increase the activity of macrophages, increase the production of nitric oxide, increase the production of the cytokine TNF-a or IL-6.
본 발명의 일실시예에 있어서, 상기 메밀 추출물은 자연살해세포(NK cell)의 활성을 증가시키고, 비장 및 흉선의 중량을 증가시키며, 백혈구 수를 증진시키는 활성을 갖는 것일 수 있다.In one embodiment of the present invention, the buckwheat extract may have activity to increase the activity of natural killer cells (NK cells), increase the weight of the spleen and thymus, and enhance the number of white blood cells.
또한, 본 발명은 메밀 추출물을 유효성분으로 포함하는 자연살해세포 활성 증진용 조성물을 제공한다.The present invention also provides a composition for enhancing natural killer cell activity, including buckwheat extract as an active ingredient.
본 발명의 일실시예에 있어서, 상기 추출물은 메밀에 물을 첨가하여 수득한 열수 추출물; 또는 상기 열수 추출물에 주정을 첨가하고 침전시킨 후, 침전물을 동결건조시켜 수득한 고분자 분획물을 포함하는 추출물일 수 있다.In one embodiment of the present invention, the extract is a hydrothermal extract obtained by adding water to buckwheat; Alternatively, the extract may include a polymer fraction obtained by adding ethanol to the hot water extract and precipitating the precipitate, and lyophilizing the precipitate.
본 발명에 따른 메밀 추출물은 산화질소의 생성을 증가시키고 사이토카인인 TNF-a 또는 IL-6의 생성을 증가시켜 대식세포의 활성을 촉진시킴으로써 면역증진 효과를 유도할 수 있으며, 자연살해세포 활성을 증가시키고, 비장 및 흉선의 중량을 증가시키며 백혈구수를 증진시킬 수 있는 효과가 있음을 확인하여, 궁극적으로 의약학적 조성물로 활용할 수 있다. 또한 본 발명의 메밀 추출물은 세포 독성을 유발시키지 않아 체내 안정하므로 면역증진을 위한 식품용 조성물로도 활용할 수 있다.Buckwheat extract according to the present invention can increase the production of nitric oxide and increase the production of the cytokine TNF-a or IL-6 to promote the activity of macrophages to induce immune-promoting effect, natural killer cell activity Increasing the weight of the spleen and thymus and improving the white blood cell count, it can be used ultimately as a pharmaceutical composition. In addition, the buckwheat extract of the present invention does not cause cytotoxicity and thus is stable in the body, and thus may be utilized as a composition for food for immune promotion.
도 1은 본 발명에 따른 메밀 추출물의 세포 독성 여부를 MTT 분석을 통해 수행한 결과를 나타낸 것으로, 1a는 메밀의 주정추출물 결과를 나타낸 것이고, 1b는 메밀의 열수추출물 결과를 나타낸 것이다.
도 2는 본 발명의 메밀 추출물에 대한 처리 농도별 산화질소 생성정도를 분석한 결과를 나타낸 것으로, 2a는 메밀의 주정추출물 결과를 나타낸 것이고, 2b는 메밀의 열수추출물 결과를 나타낸 것이다.
도 3은 메밀 추출물의 처리 농도별 TNF-a 사이토카인의 생성정도를 분석한 결과를 나타낸 것으로, 3a는 메밀의 주정추출물 결과를 나타낸 것이고, 3b는 메밀의 열수추출물 결과를 나타낸 것이다.
도 4는 메밀 추출물의 처리 농도별 IL-6 사이토카인의 생성정도를 분석한 결과를 나타낸 것으로, 4a는 메밀의 주정추출물 결과를 나타낸 것이고, 4b는 메밀의 열수추출물 결과를 나타낸 것이다.
도 5는 메밀 추출물을 투여한 마우스군, 면역억제제 투여 마우스 군 및 면역증강 보조제를 투여한 마우스 군의 비장과 흉선의 중량 변화를 측정한 결과이다.
도 6은 메밀 추출물을 투여한 마우스군, 면역억제제 투여 마우스 군 및 면역증강 보조제를 투여한 마우스 군의 총 백혈수 수치를 분석한 결과이다.
도 7은 메밀 추출물 처리에 따른 NK 세포활성도 및 그란자임(granzyme) 생산정도를 분석한 결과이다.1 shows the results of the cytotoxicity of the buckwheat extract according to the present invention through MTT analysis, 1a shows the results of alcohol extract of buckwheat, 1b shows the results of hot water extract of buckwheat.
Figure 2 shows the results of the analysis of the nitric oxide generation degree by the treatment concentration for the buckwheat extract of the present invention, 2a shows the results of alcohol extract of buckwheat, 2b shows the results of hot water extract of buckwheat.
Figure 3 shows the results of analyzing the degree of production of TNF-a cytokines for each treatment concentration of buckwheat extract, 3a shows the results of alcohol extract of buckwheat, 3b shows the results of hot water extract of buckwheat.
Figure 4 shows the results of analyzing the production of IL-6 cytokines according to the treatment concentration of buckwheat extract, 4a shows the results of alcohol extract of buckwheat, 4b shows the results of hot water extract of buckwheat.
5 is a result of measuring the weight change of the spleen and thymus of the mouse group, the immunosuppressive mouse group and the immuno-adjuvant adjuvant mouse group administered buckwheat extract.
Figure 6 shows the results of analyzing the total leukemia levels of the mouse group administered buckwheat extract, the mouse group administered with immunosuppressive agents and the mouse group administered with an adjuvant adjuvant.
Figure 7 is the result of analyzing the NK cell activity and granzyme production degree according to the buckwheat extract treatment.
본 발명은 메밀 추출물을 유효성분으로 포함하는 면역기능 증강용 식품 조성물 및 면역증강용 약학 조성물을 제공함에 특징이 있다.The present invention is characterized by providing a food composition for enhancing immune function and a pharmaceutical composition for immuno-enhancing comprising buckwheat extract as an active ingredient.
본 발명의 일실시예에서 메밀 추출물에 대한 면역활성과 관련된 효능을 분석하였는데, 이를 위해 대식세포 활성화 효과를 분석하였다. In one embodiment of the present invention was analyzed the efficacy associated with the immune activity against buckwheat extract, for this purpose the effect of macrophage activation.
대식세포의 활성화 효과는 NO의 생성능과 사이토카인의 생성능을 통해 확인하였는데, NO의 생성 촉진 효과는 대식세포 활성화의 지표로서, 대식세포에서 생성되는 NO는 침입한 병원체를 없애고 기본적인 숙주 방어체계를 작동시키는 역할을 한다. 특히 Th 세포의 분화단계 중 ThO 상태에서 Thl과 Th2로 나누어질 때, Th1로의 분화를 돕는 작용을 하는 것으로 알려져 있다. 또한 백혈구의 활성 중 암세포, 기생충 또는 박테리아를 살해하는 세포독성 활성은 NO를 유리시킴으로써 나타나는 것으로 보고되고 있다. 또한 기관지 염증에 NO가 T-cell과 내피세포간의 면역조절(immunomodulating)을 하며 강력한 혈소판 응집 억제작용이 있다는 것이 알려져 있다. 또한, TNF-a 및 IL-6와 같은 사이토카인의 분비를 증가시켜 면역증진 효과를 유도한다.The activation effect of macrophages was confirmed by NO and cytokine production ability. NO production promoting effect is an indicator of macrophage activation. NO produced in macrophages eliminates invading pathogens and operates basic host defense system. It plays a role. In particular, when Thhl is divided into Thl and Th2 in the ThO differentiation stage, it is known to have a function of helping to differentiate to Th1. In addition, cytotoxic activity of killing cancer cells, parasites or bacteria among the activities of leukocytes is reported to be released by the release of NO. It is also known that NO acts as an immune modulator between T-cells and endothelial cells in bronchial inflammation and has a potent platelet aggregation inhibitory effect. In addition, the secretion of cytokines such as TNF-a and IL-6 is increased to induce an immunostimulating effect.
이에 본 발명의 일실시예에서는 메밀의 추출물을 대식세포에 처리한 후, 산화질소 및 사이토카인의 생성 변화를 분석한 결과, 메밀 추출물의 처리 농도 의존적으로 대식세포에서 산화질소 및 사이토카인(TNF-a 및 IL-6)의 생성이 증가하는 것을 확인할 수 있었다.Therefore, in one embodiment of the present invention, after treating the extract of buckwheat to the macrophages, and analyzed the production changes of nitric oxide and cytokines, nitric oxide and cytokines (TNF-) in macrophages depending on the treatment concentration of buckwheat extract It was confirmed that the production of a and IL-6) is increased.
나아가 본 발명자들은 동물 실험을 통해 메밀의 추출물이 비장 및 흉선의 중량을 증진시킬 수 있다는 것을 확인하였고, 면역장기 지수도 증가시키는 효과가 있음을 확인함에 따라, 본 발명의 메밀 추출물이 면역계 주요 장기의 활성에 있어서 면역 증강제로서 작용함을 알 수 있었다.Furthermore, the inventors of the present invention have confirmed that the extract of buckwheat can enhance the weight of the spleen and thymus through animal experiments, and that the extract of the buckwheat extract of the present invention is effective in the major organs of the immune system. It was found to act as an immune enhancer in activity.
또한 본 발명의 메밀 추출물은 자연살해세포(NK) 세포를 활성화 시키는 작용이 있는데, 자연살해세포(NK; natural killer cell)는 선천면역세포 중의 하나로 암세포 또는 외부 침입 물질에 대해 선택적으로 세포 독성을 보이는 세포이다. 자연살해세포는 다른 면역세포와 달리 암세포 등 외부 침입 물질을 즉각적으로 감지하여 바로 제거할 수 있는데 이는 자연살해세포 표면에 존재하는 다양한 면역수용체를 통해 외부 물질과 정상세포를 구분할 수 있기 때문이다. 또한 자연살해세포는 IFN-γ나 TNF-α 같은 시토카인(cytokine) 생성을 통해 염증 및 면역반응을 조절 하는데 핵심적인 역할을 하며, 자연살해세포는 수지상 세포, 대식세포(macrophage), T세포와의 직접적 상호작용 및 시토카인을 통한 간접적 상호작용을 통해 면역반응을 조절하는데도 핵심적인 역할을 하는 것으로 알려져 있다. In addition, the buckwheat extract of the present invention has the action of activating natural killer cells (NK) cells, natural killer cells (NK) is one of the innate immune cells, showing selective cytotoxicity against cancer cells or external invasive substances. It is a cell. Unlike other immune cells, natural killer cells can immediately detect and remove foreign invading substances such as cancer cells because they can distinguish external cells from normal cells through various immunoreceptors on the surface of natural killer cells. In addition, natural killer cells play a key role in regulating inflammatory and immune responses through the production of cytokines such as IFN-γ and TNF-α. Natural killer cells play a role in dendritic cells, macrophages, and T cells. It is known to play a key role in regulating immune responses through direct interactions and indirect interactions with cytokines.
이러한 점에서 본원발명의 일실시예에 의하면, 본 발명의 메밀 추출물을 처리한 경우, 자연살해세포(NK) 세포를 활성화 시킬 수 있다는 것을 확인하였고, 또한, 면역억제제를 처리하여 면역이 억제된 상태에서 상기 추출물을 처리한 경우에도 NK 세포의 활성이 증진되는 것으로 나타났다.In this regard, according to one embodiment of the present invention, when the buckwheat extract of the present invention is treated, it was confirmed that the natural killer cells (NK) cells can be activated, and the immune suppressed state by treating the immunosuppressive agent In the case of treatment with the extract was shown to enhance the activity of NK cells.
따라서 본 발명에 따른 메밀 추출물은 자연살해세포를 활성화시킬 수 있으므로, 본 발명은 메밀 추출물을 유효성분으로 포함하는 자연살해세포의 활성 증진용 조성물을 제공할 수 있다.Therefore, the buckwheat extract according to the present invention can activate natural killer cells, the present invention can provide a composition for enhancing the activity of natural killer cells comprising buckwheat extract as an active ingredient.
앞서 기술한 바와 같이, 자연살해세포(NK세포; natural killer cell)는 암이나 바이러스 감염이 된 세포를 파괴함으로서 선천성 면역반응에서 중요한 역할을 수행하는 면역세포이다(J. Immunol., 136: 3910-3915, 1986; Melanoma Res., 13: 349-356, 2003; Lung Cancer, 35: 23-28, 2002). 또한, 자극(stimulation)을 통해 사이토카인(cytokine)을 분비하여 다른 면역 체계(immune system)를 동원(recruiting)함으로서 간접적인 면역반응을 수행하기도 한다. 이러한 NK 세포의 파괴능은 림포카인 활성세포(lymphokine activated killer cell, LAK) 및 종양침윤림프구(tumor infiltration lymphocytes, TIL)를 이용하여 고형암(solid tumor) 치료에 이용하거나, 공여자 임파구 주입(donor lymphocyte infusion)을 통한 면역치료법(Tilden. A. B. et al., J. Immunol., 136: 3910-3915, 1986; Bordignon C, et al., Hematologia 84: 1110-1149, 1999)을 수행함으로써, 골수이식이나 장기 이식시 발생하는 거부반응을 방지하기 위한 새로운 세포치료 요법으로 응용이 시도되고 있다. 또한, NK 세포의 활성 결함은 유방암(Konjevic G, et al., Breast Cancer Res. Treat., 66: 255-263, 2001), 흑색종암(RyukeY, et al., Melanoma Res., 13: 349-356, 2003), 폐암(Villegas FR, et al., Lung Cancer, 35: 23-28, 2002) 등 다양한 암 질환과 관련되어 있음이 보고되어 상기와 같은 질환들을 치료하기 위해 NK 세포 치료법이 사용되고 있다.As described above, natural killer cells (NK cells) are immune cells that play an important role in the innate immune response by destroying cancerous or virally infected cells (J. Immunol., 136: 3910-). 3915, 1986; Melanoma Res., 13: 349-356, 2003; Lung Cancer, 35: 23-28, 2002). In addition, by stimulation (cytokine) secretion (cytokine) by mobilizing other immune system (immune system) (recruiting) it also performs an indirect immune response. These NK cells can be used for the treatment of solid tumors using lymphokine activated killer cells (LAK) and tumor infiltration lymphocytes (TILs), or donor lymphocyte injections. bone marrow transplantation by performing immunotherapy (Tilden. AB et al., J. Immunol., 136: 3910-3915, 1986; Bordignon C, et al., Hematologia 84: 1110-1149, 1999). Applications have been attempted as novel cell therapy to prevent rejection in organ transplantation. In addition, the active defects of NK cells include breast cancer (Konjevic G, et al., Breast Cancer Res. Treat., 66: 255-263, 2001), melanoma cancer (RyukeY, et al., Melanoma Res., 13: 349- 356, 2003) and lung cancer (Villegas FR, et al., Lung Cancer, 35: 23-28, 2002), which have been reported to be associated with various cancer diseases, and NK cell therapy has been used to treat such diseases. .
또한, 자연살해세포(Natural killer cell, NK cell)는 정상인 혈액 내에 약 10~15% 가량 존재하며, 비자기(nonself)와 반응할 때 높은 살해능을 가진다. 각종 바이러스에 감염된 세포나 세균 침투, 혹은 비정상 세포의 생성에 있어, NK 세포는 비특이적으로 즉각적 반응하여 이물질을 제거한다. 그러나, 체내에 존재하는 NK 세포수는 그다지 많지 않기 때문에, 치료적 효과를 보이기 위해 필요한 유효 세포수를 증식시킬 수 있는 방법의 개발이 필요하고, 또한 NK 세포의 활성화를 촉진시키는 방법의 개발이 필요하다. In addition, natural killer cells (Natural killer cells, NK cells) are present in about 10 to 15% in the normal blood, and has a high killing capacity when reacting with nonself (nonself). In the production of cells infected with various viruses, bacterial penetration, or abnormal cells, NK cells react nonspecifically to remove foreign substances. However, since the number of NK cells present in the body is not so large, it is necessary to develop a method capable of proliferating the effective cell number necessary for showing a therapeutic effect, and also to develop a method for promoting the activation of NK cells. Do.
이러한 점에서 본원발명에 따른 메밀 추출물은 NK 세포를 효과적으로 활성화시킬 수 있고 나아가 본 발명의 메밀 추출물을 NK 세포에 처리하여 활성화된 NK 세포는 세포치료제로서도 사용 가능하다. In this regard, the buckwheat extract according to the present invention can effectively activate NK cells, and furthermore, activated NK cells by treating the buckwheat extract of the present invention to NK cells can be used as a cell therapy.
그러므로 본 발명의 메밀 추출물은 NK 세포 기능저하와 관련된 질환의 개선 및 치료를 위한 용도로 사용될 수 있다. NK 세포 기능저하와 관련된 질환으로는 이에 제한되지는 않으나, 면역질환 및 암을 포함할 수 있으며, 상기 암으로는 혈액암, 간암, 폐암, 신장암, 소아신경암, 피부암이 포함될 수 있다. Therefore, the buckwheat extract of the present invention can be used for the improvement and treatment of diseases related to NK cell dysfunction. Diseases related to NK cell dysfunction may include, but are not limited to, immune diseases and cancers, and the cancers may include blood cancer, liver cancer, lung cancer, kidney cancer, pediatric nerve cancer, and skin cancer.
또한 본 발명의 일실시예에서는 본 발명의 메밀 추출물이 백혈구의 수치를 증진시킬 수 있음을 확인하였다. In addition, in one embodiment of the present invention, it was confirmed that the buckwheat extract of the present invention can enhance the level of white blood cells.
백혈구는 앞서 종래기술에서도 언급된 바와 같이, 체내에 들어온 이물을 담당하게 되며, 세균, 바이러스 감염 또는 염증 반응 시 식세포 작용을 유도하여 면역방어 기능을 유도한다.Leukocytes, as mentioned in the prior art, are responsible for foreign substances that enter the body, and induces immune defense function by inducing phagocytic action during bacterial, viral infection or inflammatory reaction.
이러한 결과들을 통해 본 발명자들은 본 발명의 메밀 추출물이 면역증진 작용을 효과적으로 증진시킬 수 있음을 알 수 있었다.These results show that the present inventors can effectively enhance the immune enhancing effect of the buckwheat extract of the present invention.
따라서 본 발명은 메밀 추출물을 유효성분으로 포함하는 면역기능 증강용 식품 조성물을 제공할 수 있다.Therefore, the present invention can provide a food composition for enhancing immune function comprising buckwheat extract as an active ingredient.
본 발명에서 상기 메밀 추출물은 건조시킨 메밀을 통상적인 추출방법에 따라 제조할 수 있으며, 건조된 메밀에 물, 메탄올 등과 같은 C1 내지 C4의 저급 알콜 또는 이들의 혼합용매를 첨가하여 추출할 수 있으며, 냉침추출, 열수추출, 초음파 추출 및 환류냉각 추출 등의 추출방법으로 수행할 수 있다. 추출액은 여지로 감압 여과한 다음, 여과액을 농축하여 동결건조한 후 본 발명의 메밀 추출물로 수득할 수 있다.In the present invention, the buckwheat extract may be prepared according to a conventional extraction method of dried buckwheat, it can be extracted by adding C1 to C4 lower alcohol or a mixed solvent thereof, such as water, methanol, etc., Cold extraction, hot water extraction, ultrasonic extraction and reflux cooling extraction can be carried out by the extraction method. The extract may be filtered under reduced pressure, and then the filtrate may be concentrated and lyophilized to obtain the buckwheat extract of the present invention.
바람직하게 상기 본 발명에 따른 메밀 추출물은 메밀에 물을 첨가하여 수득한 열수 추출물일 수 있고, 또는 상기 수득한 열수 추출물에 다시 주정을 첨가하고 침전시킨 후, 침전물을 동결건조시켜 수득한 고분자 분획물, 즉 다당류 성분을 포함하는 분획물일 수 있다.Preferably, the buckwheat extract according to the present invention may be a hot water extract obtained by adding water to buckwheat, or the polymer fraction obtained by adding ethanol and precipitation to the obtained hot water extract, and then freezing and drying the precipitate, That is, it may be a fraction containing a polysaccharide component.
더 바람직하게 상기 메밀 추출물은 메밀 중량 대비 4~20배 부피의 물을 첨가하고 60~100℃의 온도에서 3~8시간 동안 환류 추출하여 수득한 메밀의 열수 추출물일 수 있고, 또는 상기 열수 추출물이 다시 80%의 주정을 첨가하여 12~24시간 침전시켜 침전된 메밀 유래 고분자 다당 성분 분획물일 수 있으며, 더욱 바람직하게 상기 메밀 추출물은 메밀 중량 대비 15~20배 부피의 물을 첨가하고 75~85℃의 온도에서 2~4시간 동안 환류 추출하여 수득한 메밀의 열수 추출물이거나 상기 열수 추출물에 다시 80%의 주정을 첨가하여 12시간 침전시켜 침전된 메밀 유래 다당 성분 분획물일 수 있다. 본 발명에서 상기 메밀 유래 다당 성분 분획물을 주정 추출물이라고 하였다.More preferably, the buckwheat extract may be a hot water extract of buckwheat obtained by adding 4-20 times the volume of water to the buckwheat weight and reflux extraction at a temperature of 60-100 ° C. for 3-8 hours, or the hot water extract is The buckwheat-derived polymer polysaccharide component fraction precipitated by adding 80% of alcohol and precipitated for 12 to 24 hours may be precipitated. More preferably, the buckwheat extract is added with a volume of 15-20 times the weight of buckwheat and is 75-85 ° C. It may be a hot water extract of buckwheat obtained by reflux extraction for 2 to 4 hours at a temperature of or buckwheat-derived polysaccharide component fraction precipitated by adding 80% alcohol to the hot water extract for 12 hours. In the present invention, the buckwheat-derived polysaccharide component fraction was called alcohol extract.
특히 본 발명자들은 메밀 열수추출물 및 상기 메밀의 열수추출물에 주정을 첨가하여 수득한 다당 성분을 함유한 주정 추출물에 대한 면역증진 효능을 분석하였는데, 그 결과 열수추출물 및 주정추출물 모두 대식세포의 활성을 증가시키고, 산화질소의 생성을 증가시키며, 사이토카인인 TNF-a 또는 IL-6의 생성을 증가시키는 것으로 나타났고, 또한, 자연살해세포(NK cell)의 활성도 월등하게 증진시키는 것으로 나타났다. In particular, the present inventors analyzed the immune-promoting efficacy of the buckwheat hot water extract and the ethanol extract containing the polysaccharide component obtained by adding ethanol to the hot water extract of buckwheat. As a result, both hot water extract and ethanol extract increased the activity of macrophages. It has been shown to increase the production of nitric oxide, to increase the production of the cytokine TNF-a or IL-6, and also to significantly enhance the activity of NK cells.
본 발명의 추출물을 첨가할 수 있는 식품으로는, 예를 들어, 각종 식품류, 음료, 껌, 차, 비타민 복합제, 건강 기능성 식품류 등이 있다. 또한, 면역력 증강 효과를 목적으로 식품 또는 음료에 첨가될 수 있다. 이 때, 식품 또는 음료 중의 상기 추출물의 양은 일반적으로 본 발명의 건강 기능 식품 조성물은 전체 식품 중량의 0.01 내지 50 중량%, 바람직하게는 0.01 내지 15 중량%로 가할 수 있으며, 건강 음료 조성물은 100 ㎖를 기준으로 0.02 내지 5 g, 바람직하게는 0.3 내지 1 g의 비율로 가할 수 있다. Examples of the food to which the extract of the present invention can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods. It may also be added to foods or beverages for the purpose of enhancing immunity. At this time, the amount of the extract in the food or beverage is generally added to the dietary supplement composition of the present invention to 0.01 to 50% by weight, preferably 0.01 to 15% by weight of the total food weight, the health beverage composition is 100 ml It can be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g based on the amount.
본 발명의 건강기능식품은 정제, 캡슐제, 환제, 액제 등의 형태를 포함한다. 본 발명의 건강 음료 조성물은 지시된 비율로 필수 성분으로서 상기 추출물을 함유하는 외에는 다른 성분에는 특별한 제한점이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등의 디사카라이드, 예를 들어 말토스, 슈크로스 등의 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 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 조성물 100 ㎖당 일반적으로 약 1 내지 20g, 바람직하게는 약 5 내지 12g이다. Health functional food of the present invention includes the form of tablets, capsules, pills, liquids and the like. The health beverage composition of the present invention is not particularly limited in the other components except the above-mentioned extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose, for example polysaccharides such as maltose and sucrose, and conventional sugars such as dextrin and cyclodextrin. 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 natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.
상기 외에 본 발명의 추출물은 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 추출물은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 조성물 100 중량부 당 0 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the extract 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 and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the extract of the present invention may contain fruit flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. 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 composition of the present invention.
또한 본 발명의 추출물을 함유하는 약학조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다. In addition, the pharmaceutical composition containing the extract of the present invention may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.
본 발명에 따른 추출물을 함유하는 약학조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있으며, 추출물을 포함하는 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜 (propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈 (tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.The pharmaceutical compositions containing extracts according to the present invention may be prepared in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Carriers, excipients and diluents which may be formulated and used in the composition comprising the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, Gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
본 발명의 추출물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나 바람직한 효과를 위해서, 본 발명의 추출물은 1일 0.0001 내지 100㎎/㎏으로, 바람직하게는 0.001 내지 10㎎/㎏으로 투여하는 것이 좋다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. Preferred dosages of the extracts of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the preferred effect, the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg per day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.
본 발명의 추출물은 쥐, 생쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하 주사에 의해 투여될 수 있다. The extract of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous injection.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.
<실시예 1><Example 1>
메밀 추출물의 제조Preparation of Buckwheat Extract
<1-1> 메밀 열수추출물 제조<1-1> Buckwheat hot water extract manufacturing
메밀은 한우리약초에서 깐메밀 (Fagopyrum esculentum Moench) 형태로 제공받아 사용하였으며, 50℃ 건조 오븐에서 건조하고 분쇄기를 이용하여 0.5 mm 이하가 되도록 분쇄하였다. 이후 메밀 100 g에 대해 20배 부피에 해당하는 물을 가하고 80℃의 온도에서 3시간 동안 환류 추출하여 메밀 열수 추출물을 수득하였다. 이후 추출물을 여과한 후, 회전 진공 증발기(rotary vacuum evaporator)를 이용하여 감압농축 한 다음, 72시간 동안 동결건조 하여 최종 메밀의 물 추출물을 수득하였고, 메밀 물 추출물을 하기에서 BWE (Buckwheat water extract)로 기재하였다.Buckwheat was provided in the form of Cango buckwheat (Fagopyrum esculentum Moench) from Hanwoo medicinal herbs, dried in a 50 ℃ drying oven and ground to less than 0.5 mm using a grinder. Then, water corresponding to a 20-fold volume of 100 g of buckwheat was added and refluxed for 3 hours at a temperature of 80 ° C. to obtain a buckwheat hydrothermal extract. Thereafter, the extract was filtered, concentrated under reduced pressure using a rotary vacuum evaporator, and then lyophilized for 72 hours to obtain a final buckwheat water extract, and the buckwheat water extract from BWE (Buckwheat water extract) It was described as.
<1-2> 다당 성분을 함유한 메밀의 주정추출물 제조<1-2> Alcohol extract of buckwheat containing polysaccharide component
메밀은 한우리약초에서 깐메밀 (Fagopyrum esculentum Moench) 형태로 제공받아 사용하였으며, 50℃ 건조 오븐에서 건조한 다음 분쇄기를 이용하여 1.0 mm 이하가 되도록 분쇄하였다. 이후 메밀 1000 g에 대해 20배 부피에 해당하는 물을 가하고 80℃의 온도에서 3시간 동안 환류 추출하여 메밀 열수 추출물을 수득하였다. 이후 추출물을 여과한 후, 회전 진공 증발기(rotary vacuum evaporator)를 이용하여 추출액의 1/10이 되도록 감압농축 한 다음, 감압농축된 메밀 열수 추출물에 다시 80%의 주정을 첨가하여 24시간 침전시킨 후 침전물을 동결건조하여 메밀의 주정 추출물을 수득하였다. 상기 방법으로 수득한 메밀 추출물은 다당을 주요성분으로 함유하고 있어 하기에서 BPF(Buckwheat polysaccharide fraction)로 기재하였다.Buckwheat was provided in the form of Cango buckwheat (Fagopyrum esculentum Moench) from Hanwoo medicinal herbs, dried in a 50 ℃ drying oven and then ground to less than 1.0 mm using a grinder. Then, water corresponding to a 20-fold volume was added to 1000 g of buckwheat and refluxed for 3 hours at a temperature of 80 ° C. to obtain a buckwheat hydrothermal extract. After filtering the extract, using a rotary vacuum evaporator (rotary vacuum evaporator) to concentrate to 1/10 of the extract under reduced pressure, and then precipitated for 24 hours by adding 80% alcohol to the concentrated buckwheat hot water extract The precipitate was lyophilized to obtain a ethanol extract of buckwheat. The buckwheat extract obtained by the above method contains polysaccharide as a main component, and is described below as Buckwheat polysaccharide fraction (BPF).
<실시예 2><Example 2>
메밀 추출물의 세포독성 분석Cytotoxicity Analysis of Buckwheat Extract
상기 실시예 1에서 수득한 본 발명의 메밀 추출물이 세포에 대한 독성이 있는지 확인하기 위하여 in vitro 내에서 세포독성 여부를 분석하였다. 이를 위해 murine macrophage 세포주인 Raw 264.7 세포를 KCLB에서 분양받아 사용하였는데, 세포배양용 플라스크(flask)에 세포를 분주한 후, DMEM 배지에 10% FBS(fetal bovine serum) 및 1% penicillin-streptomycin (10,000U/ml)을 첨가하고 37℃, 5% CO2 95% 습도조건이 유지되는 배양기에서 배양하여 사용하였다. The buckwheat extract of the present invention obtained in Example 1 was analyzed for cytotoxicity in vitro to determine whether the extract is toxic to cells. To this end, raw 264.7 cells, a murine macrophage cell line, were used in KCLB. After dispensing the cells in a cell culture flask, 10% FBS (fetal bovine serum) and 1% penicillin-streptomycin (10,000) were added to DMEM medium. U / ml) was added and used incubated in an incubator at 37 ℃, 5% CO 2 95% humidity conditions are maintained.
세포독성 평가는 MTT 분석법을 사용하였는데 상기 방법은 대사 과정이 온전한 세포의 미토콘드리아 내의 탈수소효소가 노란색 수용성 tetrazoliumsalt [3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide] (MTT)를 비수용성의 짙은 자주색 MTT formazan 결정으로 환원시키는 원리를 이용한 것으로 적절한 파장 (주로 500-600 nm)에서 흡광도를 측정하여 세포독성을 평가하는 방법이다. 면역반응의 지표로서 LPS (serotype: E.coli 055 : B5, Sigma Co., St. Louis, MO, USA)를 사용하였으며, 반응의 정도는 세포 증식의 증가 혹은 감소로 측정하였다. RAW 264.7 세포를 1×105 cells/well의 농도로 96well에 분주 후 24시간 동안 배양 후, 본 발명의 메밀 추출물을 각각 0.5, 1, 5, 10 μg/mL의 농도와 LPS 1 μg/mL의 농도로 처리하고 20시간 배양 후 0.5 mg/mL 농도의 MTT를 넣고 4시간 동안 37°C 배양기에서 반응시킨 후 DMSO(dimethyl sulfoxide)를 넣어 microplate reader를 이용하여 540 nm에서 흡광도를 측정하였고, 세포독성은 다음 식에 의해 계산하였다.Cytotoxicity assessment was performed using the MTT assay, in which the dehydrogenase in the mitochondria of cells with intact metabolism was soluble in yellow soluble tetrazoliumsalt [3- (4,5-dimethylthiazol-2-yl) -2-5-diphenyltetrazolium bromide] (MTT ) Is a method of evaluating cytotoxicity by measuring absorbance at an appropriate wavelength (mainly 500-600 nm) using the principle of reducing) to a water-insoluble dark purple MTT formazan crystal. LPS (serotype: E. coli 055: B5, Sigma Co., St. Louis, MO, USA) was used as an indicator of immune response, and the degree of response was measured by increasing or decreasing cell proliferation. After dispensing RAW 264.7 cells into 96well at a concentration of 1 × 10 5 cells / well, and incubating for 24 hours, the buckwheat extract of the present invention was 0.5, 1, 5, 10 μg / mL and
Cell viability (%) = sample 흡광도 / control 흡광도 × 100Cell viability (%) = sample absorbance / control absorbance × 100
분석 결과, 도 1에 나타낸 바와 같이 정상군을 100 %로 보았을 때 주정추출물(BPF)을 0.5, 1, 5, 10 μg/mL 농도별로 처리한 군에서 세포 생존율은 각각 112.5±6.0 %, 98.4±3.4 %, 109.5±5.6 %, 98.9±5.2 %로 나타나, 본 발명의 추출물은 세포 독성에 큰 영향을 주지 않는다는 것을 확인할 수 있었다(도 1a 참조).As a result, as shown in FIG. 1, the cell viability was 112.5 ± 6.0% and 98.4 ± in the group treated with 0.5, 1, 5, and 10 μg / mL of BPF when the normal group was seen as 100%. 3.4%, 109.5 ± 5.6%, 98.9 ± 5.2%, it was confirmed that the extract of the present invention does not significantly affect the cytotoxicity (see Figure 1a).
또한, 물 추출물(BWE)을 0.5, 1, 5, 10 μg/mL 농도별로 처리한 군에서 세포 생존율은 각각 103.1±2.5 %, 98.6±5.6 %, 94.3±6.3 %, 89.7±6.7 %로 모두 90 %이상의 세포 생존율을 나타내어, 본 발명의 추출물은 세포 독성에 큰 영향을 주지 않는다는 것을 확인할 수 있었다(도 1b 참조).In addition, in the group treated with 0.5, 1, 5, and 10 μg / mL concentrations of water extract (BWE), the cell survival rates were 103.1 ± 2.5%, 98.6 ± 5.6%, 94.3 ± 6.3%, and 89.7 ± 6.7%, respectively. Cell viability of more than%, it was confirmed that the extract of the present invention does not significantly affect the cytotoxicity (see Figure 1b).
<실시예 3><Example 3>
메밀 추출물의 산화질소(NO) 및 사이토카인 생성능 분석Analysis of Nitric Oxide (NO) and Cytokine Formation Capacity of Buckwheat Extract
<3-1> 산화질소 생성능 분석<3-1> Nitric Oxide Production Capacity Analysis
상기 실시예에서 사용한 세포실험군을 대상으로 세포 상등액을 수집한 후, 세포 상등액으로 면역세포 활성에 의해 분비되는 물질 중 NO(Nitric Oxide)의 양을 확인하였다. 염증성 매개 물질에 노출된 세포에 의해서 생성된 NO의 생성은 생체 방어와 자유기에 의해 유발된 조직 손상에 기여한다고 알려져 있으며 다양한 염증성 질환이 발병하게 되면 증가하는 것으로 알려져 있다. NO 활성도는 대사산물인 nitrate (NO3 -)와 nitrite (NO2 -)의 생성농도를 Griess 반응을 통해 확인하였는데, 구체적으로 RAW 264.7 세포를 1×105 cells/well의 농도로 96well에 분주 후 24시간 동안 배양한 다음, 본 발명의 추출물을 0.5, 1, 5, 10 μg/mL의 농도로 처리하고 LPS는 1 μg/mL의 농도로 처리한 다음 37°C, 5% CO2의 조건에서 20시간 배양하였다. 이후 세포 상등액과 세포 상등액을 동량에 해당하는 griess 시약을 첨가하여 15분 반응시키고 microplate reader를 이용하여 540 nm에서 흡광도를 측정하였다. Sodium nitrite (NaNO2)로 표준곡선을 작성하여 NO 함량을 정량하였다.After collecting the cell supernatant for the cell experiment group used in the above example, the amount of NO (Nitric Oxide) in the material secreted by the immune cell activity as the cell supernatant was confirmed. The production of NO produced by cells exposed to inflammatory mediators is known to contribute to tissue damage caused by biological defense and free radicals, and is known to increase when various inflammatory diseases develop. NO activity metabolite, nitrate and then dispensed into 96well a concentration of the were to produce a concentration of the check through the Griess reaction. Specifically, RAW 264.7 cells 1 × 10 5 cells / well ( NO 3 - -) , and nitrite (NO 2) After incubation for 24 hours, the extract of the present invention was treated at concentrations of 0.5, 1, 5, 10 μg / mL and LPS was treated at a concentration of 1 μg / mL and then at 37 ° C., 5% CO 2 . Incubated for 20 hours. Then, the cell supernatant and the cell supernatant were reacted for 15 minutes by adding the equivalent griess reagent, and the absorbance was measured at 540 nm using a microplate reader. A standard curve was prepared with sodium nitrite (NaNO 2 ) to quantify NO content.
분석 결과, 0.5, 1, 5, 10 μg/mL의 농도로 RAW 264.7 세포에 메밀 열수추출물 및 주정추출물을 각각 처리할 경우, 처리 농도 의존적으로 NO 생성이 증가되었으며, LPS로 활성화된 세포의 경우 NO 함량이 11.89±2.2 μM수준을 나타내었고, 메밀 주정추출물을 0.5, 1, 5, 10 μg/mL 처리 시에는 각각 12.92±1.1 μM, 14.46±0.8 μM, 16.42±2.6 μM, 16.23±1.5 μM으로 음성대조군에 비해 전체적으로 높은 함량을 나타내었다(도 2a 참조). 또한, 열수추출물의 경우 처리 농도 의존적으로 NO 생성이 증가되었으며, LPS로 활성화된 세포의 경우 NO 함량이 11.89±2.2 μM수준을 나타내었고, BWE 5 μg/mL의 농도에서 LPS를 처리한 것과 비슷한 수준으로 생성하였으며, 10 μg/mL의 농도에서 대조군에 비해 높은 NO 생성량을 확인할 수 있었다. BWE를 0.5, 1, 5, 10 μg/mL 처리하였을 때 각각 10.58±2.5 μM, 11.18±3.4 μM, 11.96±3.3 μM, 13.58±1.0 μM으로 증가하는 것으로 나타났다(도 2b 참조).As a result, treatment of buckwheat hot water extract and ethanol extract with RAW 264.7 cells at concentrations of 0.5, 1, 5, and 10 μg / mL increased NO production depending on treatment concentration, and NO for LPS-activated cells. The content was 11.89 ± 2.2 μM, and when 0.5, 1, 5, and 10 μg / mL of buckwheat liquor extracts were treated, 12.92 ± 1.1 μM, 14.46 ± 0.8 μM, 16.42 ± 2.6 μM, 16.23 ± 1.5 μM, respectively. The overall content was higher than that of the control group (see FIG. 2A). In addition, NO production increased depending on the treatment concentration in the case of hot water extract, and the NO content of the cells activated by LPS was 11.89 ± 2.2 μM, similar to that of LPS treatment at the concentration of
따라서 본 발명의 메밀 추출물은 면역계의 중요한 역할을 수행하는 대식세포의 활성을 증진시킴을 NO의 생성 증가를 통해 확인할 수 있었다.Therefore, the buckwheat extract of the present invention was confirmed by increasing the production of NO to enhance the activity of macrophages play an important role of the immune system.
<3-2> 사이토카인(TNF-α, IL-6) 생성능 분석<3-2> Analysis of cytokine (TNF-α, IL-6) production capacity
TNF(tumor necrosis factor)-α는 염증성 매개물질 중, 전구 염증성 사이토카인 중의 하나로 많은 감염성 질환 또는 자가면역성 질환에서 강력한 면역 매개 물질로 여겨지고 있는데, 이는 대식세포, 활성화 T세포, 비만세포에서 생산되며 중성구, 림프구, 단핵세포를 활성화시키는 작용을 한다. IL(interleukin)-6는 조혈작용과 면역반응을 조절하는데 관여하며 다른 사이토카인과 협동하여 상승작용을 나타내는 등 다양한 작용을 하며, 특정한 외부 물질 침입 시, 항체를 생성하는 B세포를 자극하여 면역글로불린의 합성을 촉진시킨다. 이에 본 발명의 메밀 추출물이 NO 생성 증진 이외에도 TNF-α, IL-6의 생성량을 증가시키는 작용이 있는지 확인하기 위해, 세포 상등액을 이용하여 Commercial competitive enzyme immunoassay kit (R&D system, Minneapolis, USA) 분석을 통해 TNF-α와 IL-6 등의 cytokine의 양을 측정하였다. Tumor necrosis factor (TNF) -α is one of the proinflammatory inflammatory cytokines and is considered to be a potent immune mediator in many infectious and autoimmune diseases. It acts to activate lymphocytes, monocytes. IL (interleukin) -6 is involved in regulating hematopoiesis and immune responses, and works synergistically with other cytokines to show synergistic effects.Immune globulin stimulates B cells that produce antibodies when invading certain foreign substances Promote the synthesis of In order to determine whether the buckwheat extract of the present invention has an effect of increasing the production of TNF-α and IL-6 in addition to enhancing NO production, a commercial competitive enzyme immunoassay kit (R & D system, Minneapolis, USA) analysis using a cell supernatant was performed. The amount of cytokine such as TNF-α and IL-6 was measured.
앞서 실험에 사용한 세포들을 대상으로 Anti-mouse TNF-α로 precoating된 96 well에 배양액 또는 표준시료와 TNF-α conjμgate를 각각 50 ul씩 첨가하여 실온에서 2시간 반응시켰다. 세척 버퍼로 4회 세척한 후 기질 용액 100 μl를 첨가하여 30분 간 빛을 차단한 조건 하에서 반응시킨 다음, 정지 용액 100 μl를 처리하고 microplate reader를 이용하여 450 nm에서 흡광도를 측정하였다. IL-6의 경우도 상기 방법과 동일하게 실험에 적용하였다.The cells used in the experiment were added 50 ul of the culture medium or the standard sample and the TNF-α conjμgate to 96 wells precoated with Anti-mouse TNF-α, and reacted at room temperature for 2 hours. After washing four times with the washing buffer, 100 μl of the substrate solution was added thereto, and reacted under light-blocking conditions for 30 minutes. Then, 100 μl of the stop solution was treated and the absorbance was measured at 450 nm using a microplate reader. IL-6 was also applied to the experiment in the same manner as the above method.
분석결과, 도 3에 나타낸 바와 같이, TNF-α의 생성은 LPS를 단독으로 처리 시 4864.1±91.6 pg/mL로 정상군에 비하여 유의적으로 높은 TNF-α를 생성하였으며, 메밀의 주정추출물을 0.5, 1, 5, 10 μg/mL 농도로 처리 시 각각 2049.41±156.0, 3160.1±531.8, 4676.7±167.5, 4881.8±64.0 pg/mL으로 나타났고, 추출물의 처리 농도 의존적으로 TNF-α 생성이 증가하였으며, 메밀 추출물 5, 10 μg/mL 농도 처리 군은 LPS를 단독으로 처리한 음성대조군과 비슷한 수준의 TNF-α가 생성된 것으로 나타나 메밀 추출물이 TNF-α 발현에 높은 영향을 주는 것으로 나타났다(도 3a 참조).As shown in FIG. 3, the production of TNF-α produced 4864.1 ± 91.6 pg / mL when treated with LPS alone, which produced significantly higher TNF-α compared to the normal group, and the ethanol extract of buckwheat was 0.5. , 1, 5, and 10 μg / mL treatment resulted in 2049.41 ± 156.0, 3160.1 ± 531.8, 4676.7 ± 167.5, and 4881.8 ± 64.0 pg / mL, respectively, and TNF-α production increased depending on the treatment concentration. The
또한, IL-6 생성을 측정한 결과 LPS를 단독으로 처리 시 7190.6±96.3 pg/mL로 정상군에 비하여 유의적으로 높은 TNF-α를 생성하였으며, 메밀 주정추출물을 0.5, 1, 5, 10 μg/mL 농도로 처리 시 각각 526.9±67.2, 2021.2±129.0, 7026.9±409.7, 8734.6±133.5 pg/mL으로 나타났고 0.5, 1 μg/mL의 농도에서 음성대조군에 비해 낮은 수준의 IL-6가 생성되었지만 5 μg/mL의 농도에서 비슷한 수준으로 나타났으며, 10 μg/mL의 농도에서 약 1544.0 μg/mL 높게 생성됨을 알 수 있었다. 따라서 본 발명의 메밀 추출물이 대식세포를 활성화시켜 IL-6의 생성을 촉진시킨다는 것을 확인할 수 있었다(도 4a 참조).In addition, IL-6 production resulted in significantly higher TNF-α compared to the normal group when treated with LPS alone at 7190.6 ± 96.3 pg / mL and 0.5, 1, 5, 10 μg of buckwheat liquor extracts. / mL concentrations of 526.9 ± 67.2, 2021.2 ± 129.0, 7026.9 ± 409.7, and 8734.6 ± 133.5 pg / mL, respectively, and produced lower levels of IL-6 than the negative control at concentrations of 0.5 and 1 μg / mL, respectively. At a concentration of 5 μg / mL, the results were similar, and about 1544.0 μg / mL was produced at a concentration of 10 μg / mL. Therefore, it was confirmed that the buckwheat extract of the present invention activates macrophages to promote the production of IL-6 (see FIG. 4A).
또한, 메밀의 열수추출물 역시 TNF-α의 생성은 LPS를 단독으로 처리하였을 때 4864.1±91.6 pg/mL로 정상군에 비하여 유의적으로 높은 TNF-α를 생성하였으며, BWE를 0.5, 1, 5, 10 μg/mL 농도로 처리하였을 때 각각 3133.6±44.9, 3480.0±374.6, 4366.4±296.2, 4696.3±226.4 pg/mL으로 LPS 처리군과 비슷한 수준으로 TNF-α의 생성이 증가하였으며, 이는 BWE가 Th-1 세포의 활성화 향상에 기여하여 면역증강에 도움을 줄 수 있음을 알 수 있었다(도 3b 참조). 또한, IL-6 생성을 측정한 결과 LPS를 단독으로 처리 시 7190.6±96.3 pg/mL로 정상군에 비하여 유의적으로 높은 IL-6를 생성하였으며, 특히 BWE 10 μg/mL의 농도에서 LPS를 처리한 군에 비하여 높은 IL-6를 생성하였다. BWE를 0.5, 1, 5, 10 μg/mL 농도로 처리하였을 때 각각 3064.6±124.7, 5771.3±126.7, 6948.3±84.5, 7480.2±183.4 pg/mL으로 나타나 BWE에 의해 활성화된 대식세포에서 면역매개인자인 IL-6의 생성을 촉진함으로써 면역활성을 증진시키는 효능이 있음을 알 수 잇었다(도 4b 참조).In addition, the production of buckwheat hot water extract also produced TNF-α significantly higher than that of the normal group at 4864.1 ± 91.6 pg / mL when treated with LPS alone, and BWE 0.5, 1, 5, When treated at a concentration of 10 μg / mL, TNF-α production was increased to 3133.6 ± 44.9, 3480.0 ± 374.6, 4366.4 ± 296.2, and 4696.3 ± 226.4 pg / mL, similar to those of the LPS treatment group, indicating that BWE increased Th- It can be seen that it can help to boost the immune system by contributing to the activation of the cells 1 (see Figure 3b). In addition, as a result of measuring IL-6 production, ILPS produced 7190.6 ± 96.3 pg / mL significantly higher IL-6 than normal group, especially LPS treatment at a concentration of 10 μg / mL BWE. It produced higher IL-6 compared to one group. When BWE was treated at concentrations of 0.5, 1, 5, and 10 μg / mL, 3064.6 ± 124.7, 5771.3 ± 126.7, 6948.3 ± 84.5, and 7480.2 ± 183.4 pg / mL, respectively, resulted in an immune mediator in BWE-activated macrophages. It was found that there is an effect of enhancing the immune activity by promoting the production of IL-6 (see Figure 4b).
따라서 이러한 결과를 통해 본 발명자들은, 숙주의 방어기구 일부로서 면역계에 매우 중요한 역할을 수행하며 외부물질 침입을 가장 먼저 인지하는 대식세포를 본 발명의 메밀 추출물이 효과적으로 활성화 시킬 수 있다는 것을 알 수 있었고, 이러한 대식세포의 활성화는 산화질소 생성증가 및 사이토카인(TNF-α 및 IP-6)의 생성을 증가시키는 결과를 통해 알 수 있었다.Therefore, through these results, the present inventors found that the buckwheat extract of the present invention can effectively activate macrophages that play a very important role in the immune system as part of the host's defense mechanism and recognize foreign material invasion first. Activation of these macrophages can be seen through the results of increased nitric oxide production and increased production of cytokines (TNF-α and IP-6).
따라서 본 발명의 메밀의 열수추출물 및 다당 성분을 함유한 주정추출물은 대식세포의 활성 촉진으로 인해 암세포와 각종 유해균의 성장을 억제시킬 수 있으며, 대식세포의 활성화를 통해 산화질소 및 사이토카인(TNF-α 및 IP-6)의 생성을 증가시켜 외부 항원으로부터 자극받지 않아도 체내에서 면역반응이 일어나는 초기에 생체 방어에 유리한 작용하여 면역반응을 증강시킬 수 있음을 알 수 있었다. Therefore, the buckwheat extract of buckwheat and the alcohol extract containing the polysaccharide component of the present invention can inhibit the growth of cancer cells and various harmful bacteria due to the promotion of macrophages, and nitric oxide and cytokines (TNF-) through the activation of macrophages. Increasing the production of α and IP-6) was found to be able to enhance the immune response by acting in favor of biological defense in the early stage of the immune response in the body without being stimulated by an external antigen.
<실시예 4><Example 4>
메밀 추출물 처리에 의한 면역억제 동물모델에서 비장 및 흉선 중량변화 측정 Measurement of Spleen and Thymus Weight Changes in Immunosuppressed Animal Models by Buckwheat Extract Treatment
메밀 추출물의 면역증강 효과를 동물모델을 대상으로 확인하였으며, 실험을 위해 사용한 동물들은 무균 환경에서 사육된 6주령의 암컷 Balb/c 마우스를 (주)새론바이오에서 구입하여 고형 사료와 물을 자유로이 공급하면서 실험 전 약 1주간 순화시킨 후 실험에 사용하였다. 마우스 사육조건은 온도 22±2 ℃, 습도는 40-60%로 유지하였고, 명암 주기 (Light and dark cycle)는 12시간 간격으로 조절하였다. 상기 조건으로 사육한 정상 마우스 군을 다음과 같이 5개 군으로 분류하였다. Immunostimulating effect of buckwheat extract was confirmed in animal models. Animals used for experiments were purchased from Salon Bio Co., Ltd., 6-week-old female Balb / c mice raised in a sterile environment, and were freely supplied with solid feed and water. While purified about 1 week before the experiment was used in the experiment. Mouse breeding conditions were maintained at a temperature of 22 ± 2 ℃, humidity 40-60%, and the light and dark cycle was adjusted every 12 hours. Normal mouse groups reared under the above conditions were classified into five groups as follows.
즉, 정상군 (normal group)과 면역억제 모델군 (CTX군; model control군)은 증류수를 처리하였고, 양성대조군은 CFX 50 mg/kg으로 처리하였으며, BPF 처리군은 본 발명의 메밀 추출물을 각각 50, 100 mg/kg으로 4주간 경구 투여하였다. 면역억제 동물모델 실험을 위하여 실험 종료 1주일 전 정상군은 매일 1회씩 7일간 생리식염수를 복강 투여하였고, 면역억제 모델군 (model control군)을 비롯하여 양성대조군 (CFX군) 및 BPF군은 CTX 100 mg/kg을 복강 주사하여 면역력을 억제한 후 경구 투여하였다. 마지막 투여가 끝난 24시간 후, 마우스의 혈액을 채취한 후 비장세포의 증식능과 사이토카인, 말초혈액 중 백혈구를 측정하기 위해서 각 동물의 비장가 흉선을 적출하였다. 이후 무균적으로 비장과 흉선을 적출하여 무게를 측정한 뒤, 실험동물 체중의 차이에 따른 변이를 없애고 이를 표준화하기 위하여 적출한 비장과 흉선의 무게와 마우스의 체중을 바탕으로 아래의 공식에 따라 비장 및 흉선 지수를 계산하였다.That is, the normal group and the immunosuppressive model group (CTX group; model control group) were treated with distilled water, the positive control group was treated with
비장 또는 흉선 지수 = 비장 또는 흉선 무게(mg) / 마우스 체중(g)Spleen or Thymus Index = Spleen or Thymus Weight (mg) / Mouse Weight (g)
분석 결과, 비장의 무게는 정상군에 비하여 CTX군이 18.5 % 감소하였으며, CTX군에 비하여 CFX군, 50-BPF, 100-BPF군은 각각 36.9, 32.5, 41.4 % 증가한 것으로 나타났다. 또한 흉선의 무게는 정상군에 비하여 CTX군이 51.3 % 감소하였으며, CTX군에 비하여 CFX군, 50-BPF, 100-BPF군은 각각 46.8, 47.4, 57.6 % 증가한 것으로 나타났다(도 5 참조).As a result, the weight of spleen was decreased by 18.5% in the CTX group compared to the normal group, and by 36.9, 32.5 and 41.4% in the CFX, 50-BPF, and 100-BPF groups, respectively. In addition, the weight of the thymus was reduced by 51.3% in the CTX group compared to the normal group, 46.8, 47.4, 57.6% in the CFX group, 50-BPF, 100-BPF group was increased compared to the CTX group (see Fig. 5).
비장은 혈액을 여과할 뿐 아니라 2차 림프조직으로 작용하며 혈액 속 항원을 제거하고 B 세포와 T 세포를 활성화 시키는 중요한 장기이며 따라서 비장세포수의 변화는 림프구의 변화를 나타낸다고 볼 수 있다. 또한, 비장과 흉선의 무게가 증가하는 현상은 비특이적인 면역반응 및 특이적 면역반응의 증가에 있어 중요한 지표로 인식되고 있음을 볼 때, 본 발명에 따른 메밀 추출물이 비장과 흉선의 중량을 증가시킬 수 있는 작용이 있음을 확인함으로써, 메밀 추출물이 면역계 주요 장기의 활성변화에 있어서 면역증강제로서의 역할을 수행할 수 있음을 알 수 있었다. The spleen not only filters blood, but also acts as a secondary lymphoid tissue and removes antigens from the blood and activates B and T cells. Therefore, the change in the number of splenocytes indicates a change in lymphocytes. In addition, the increase in the weight of the spleen and thymus is recognized as an important indicator in the increase of non-specific and specific immune response, the buckwheat extract according to the present invention will increase the weight of the spleen and thymus By confirming that there is a possible action, it was found that buckwheat extract can play a role as an adjuvant in changing the activity of major organs of the immune system.
<실시예 5>Example 5
메밀 추출물 처리에 의한 총 백혈구 수 변화분석Analysis of Total Leukocyte Count Changes by Buckwheat Extract Treatment
세균 또는 외상 신호로 인해 다양한 세포와 혈장내의 단백질 보체계가 활성되면 전염증성 사이토카인과 케모카인이 생성되고, 백혈구와 내피세포에서의 부착 물질들이 증가하게 된다. 이에 본 발명자들은 메밀 추출물 처리에 따른 동물 모델에서 백혈구 수에 미치는 변화를 분석하기 위해 상기 실시예에서 실험한 동물 마우스를 대상으로 에테르 마취 하에 개복하고 간문맥으로부터 채혈을 한 후, 면역지표로써 혈중 대표인자인 백혈구를 녹십자 의료재단에 의뢰하여 분석하였다.Bacterial or traumatic signals activate the protein complement system in various cells and plasma to produce proinflammatory cytokines and chemokines and increase adhesions in leukocytes and endothelial cells. Therefore, the inventors of the present invention, in order to analyze the effect on the leukocyte count in the animal model according to the buckwheat extract treatment, the animal mice tested in the above example was opened under ether anesthesia and blood collected from the portal vein, and then the representative factors in the blood as an immunomarker Phosphorus white blood cells were analyzed by the Green Cross Medical Foundation.
한편, 다량의 CTX 투여는 면역억제에 의한 백혈구 및 호중구 수 감소를 유발한다고 알려져 있다. 이에 본 실험에서는 CTX 100 mg/kg으로 면역억제 마우스 모델을 만들고 CFX 및 BPF를 경구 투여한 후 혈액중의 백혈구 수를 녹십자 의료재단에 분석 의뢰하여 측정한 결과, 도 6에 나타낸 바와 같이, CTX군의 백혈구 수는 정상군에 비해 37.6 % 감소되었고, CTX군에 비하여 CFX군은 23.6 % 증가한 것으로 나타났으며 50-BPF 및 100-BPF는 각각 27.3 % 및 42.1 %증가한 것으로 나타났다. 따라서 이러한 결과를 통해 본 발명의 추출물은 CTX에 의해 저하된 백혈구 수를 효과적으로 증가시킬 수 있어, 면역활성의 회복능이 우수하다는 것을 알 수 있었다. On the other hand, administration of a large amount of CTX is known to cause a decrease in the number of leukocytes and neutrophils by immunosuppression. In this experiment, an immunosuppressive mouse model was prepared with
<실시예 6><Example 6>
메밀 추출물 처리에 의한 NK 세포활성 분석Analysis of NK Cell Activity by Buckwheat Extract Treatment
NK(Natural killer) 세포의 세포독성은 NK세포에 의해 파괴된 종양세포 YAC-1 (NK-sensitive cell line)로부터 유리된 lactate dehydrogenase (LDH)를 측정하는 방법을 이용하여 분석하였다. NK세포 활성 변화를 측정하기 위해 100 mg/kg 용량으로 메밀 추출물을 2주간 식이한 마우스의 비장을 적출하여 이용하였다. 96 well plate에 각 well 당 1×103 cells/well씩 분주한 후 effector 세포로 이용하였다. Target 세포는 YAC-1 세포를 이용하였으며 effector 세포와 target 세포의 비율을 12.5:1, 25:1, 50:1로 조정하고 6시간 배양하였다. 배양 후 900 g로 10분간 원심분리 후 상등액과 lactate dehydrogenase (LDH) 용액(Pierce Biotechnology)과 혼합 후 microplate reader를 이용하여 680 nm 흡광도에서 측정하여 계산하고 YAC-1 세포의 사멸 정도를 NK cell activity (%)로 나타내었다. NK cell 활성과 동시에 NK 세포가 배출하는 물질인 granzyme B의 농도를 측정하였다.Cytotoxicity of natural killer (NK) cells was analyzed using the method of measuring lactate dehydrogenase (LDH) released from tumor cells YAC-1 (NK-sensitive cell line) destroyed by NK cells. In order to measure the change in NK cell activity, the spleens of mice fed with buckwheat extract for 2 weeks at 100 mg / kg dose were used. After dispensing 1 × 10 3 cells / well into each well in a 96 well plate, it was used as effector cells. The target cells were YAC-1 cells, and the ratio of effector cells to target cells was adjusted to 12.5: 1, 25: 1, 50: 1 and incubated for 6 hours. After incubation for 10 minutes at 900 g, the mixture was mixed with supernatant and lactate dehydrogenase (LDH) solution (Pierce Biotechnology), and then measured by absorbing at 680 nm using a microplate reader. %). Simultaneously with NK cell activity, the concentration of granzyme B, a substance that NK cells release, was measured.
NK cell cytotoxic activity (%) = [(effector cell spontaneous release target cell spontaneous release) / (target cell maximum release target spontaneous release)] × 100NK cell cytotoxic activity (%) = [(effector cell spontaneous release target cell spontaneous release) / (target cell maximum release target spontaneous release)] × 100
CTX의 투여에 의한 YAC-1 세포의 살해활성 및 granzyme 생산을 조사한 결과, 도 7에 나타낸 바와 같이, 본 발명의 메밀 추출물을 처리한 군은 NK세포의 활성을 정상군에 비해 유의적으로 증가시켰는데, 세포의 비율이 25:1일 경우 정상군에 비해 약 6배, 50:1인 경우 약 4배 높은 활성을 보이면서 통계적으로 유의한 차이를 보였다(p<0.01). 또한 NK 세포 활성과 동시에 NK 세포가 면역 작용 시 배출하는 물질인 granzyme B의 농도를 측정한 결과, 12.5:1, 25:1, 50:1의 세포농도에서 정상군에 비하여 각각 42.2, 36.4, 32.4 % 증가하는 것으로 나타났다. As a result of investigating the killing activity and granzyme production of YAC-1 cells by the administration of CTX, as shown in Figure 7, the group treated with buckwheat extract of the present invention significantly increased the activity of NK cells compared to the normal group When the ratio of the cells is 25: 1, the activity was about 6 times higher than that of the normal group, and when the ratio of 50: 1 was about 4 times higher, there was a statistically significant difference (p <0.01). In addition, the concentration of granzyme B, a substance released by NK cells during immunization at the same time as NK cell activity, was determined to be 42.2, 36.4, 32.4, respectively, at cell concentrations of 12.5: 1, 25: 1, 50: 1 Was found to increase by%.
상기 실험 결과를 통해 12.5:1의 세포농도에서 본 발명의 메밀 추출물의 NK 세포 활성이 가장 높았음을 알 수 있었고, 본 발명의 추출물이 NK 세포활성을 효과적으로 증진시키고 또한 면역을 활성화시켜 각종 질환으로부터 체내 방어기작을 활성화 시켜 건강을 유지 및 향상시킬 수 있음을 알 수 있었다.The experimental results showed that the NK cell activity of the buckwheat extract of the present invention was the highest at a cell concentration of 12.5: 1, and the extract of the present invention effectively promotes NK cell activity and activates immunity from various diseases. It was found that by activating the body's defense mechanisms can maintain and improve health.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.
Claims (10)
상기 조성물은 대식세포의 활성을 증가시키고, 산화질소의 생성을 증가시키며, 사이토카인인 TNF-a 또는 IL-6의 생성을 증가시키는 것을 특징으로 하는 면역기능 증강용 식품 조성물.The method of claim 1,
The composition is to increase the activity of macrophages, increase the production of nitric oxide, food composition for enhancing immune function, characterized in that to increase the production of the cytokine TNF-a or IL-6.
상기 조성물은 자연살해세포(NK cell)의 활성을 증가시키고, 비장 및 흉선의 중량을 증가시키며, 백혈구 수를 증진시키는 활성을 갖는 것을 특징으로 하는 면역기능 증강용 식품 조성물.The method of claim 1,
The composition is to increase the activity of natural killer cells (NK cells), increase the weight of the spleen and thymus, food composition for immune function enhancement characterized in that it has the activity to enhance the white blood cell count.
상기 조성물은 대식세포의 활성을 증가시키고, 산화질소의 생성을 증가시키며, 사이토카인인 TNF-a 또는 IL-6의 생성을 증가시키는 것을 특징으로 하는 면역증강용 약학 조성물.The method of claim 5,
The composition is to increase the activity of macrophages, increase the production of nitric oxide, increase the production of cytokine TNF-a or IL-6, characterized in that for enhancing the pharmaceutical composition.
상기 조성물은 자연살해세포(NK cell)의 활성을 증가시키고, 비장 및 흉선의 중량을 증가시키며, 백혈구 수를 증진시키는 활성을 갖는 것을 특징으로 하는 면역증강용 약학 조성물.The method of claim 5,
The composition increases the activity of natural killer cells (NK cells), increases the weight of the spleen and thymus, and has an activity to enhance the leukocytes, characterized in that the pharmaceutical composition for immuno-enhancing.
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| KR20050052839A (en) * | 2003-12-01 | 2005-06-07 | 주식회사 태평양 | Skin compositions for exteral application, containing plant extracts |
| KR100740716B1 (en) | 2005-10-18 | 2007-07-18 | 안동대학교 산학협력단 | Composition comprising the extract of Buckwheat for prevention and control of thrombosis |
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Non-Patent Citations (2)
| Title |
|---|
| J Korean Soc Food Sci Nutr. 2012, Vol.41, No.12, pp.1671-1676 1부.* |
| Korean J. Food & Nutr. 2008, Vol.21, No.3, pp.269-274 1부.* |
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