KR101940158B1 - Peanut sprouts mixtures having immune enhancing activity - Google Patents
Peanut sprouts mixtures having immune enhancing activity Download PDFInfo
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- KR101940158B1 KR101940158B1 KR1020170094658A KR20170094658A KR101940158B1 KR 101940158 B1 KR101940158 B1 KR 101940158B1 KR 1020170094658 A KR1020170094658 A KR 1020170094658A KR 20170094658 A KR20170094658 A KR 20170094658A KR 101940158 B1 KR101940158 B1 KR 101940158B1
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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
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G2/00—Vegetative propagation
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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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
- A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
- A23L17/40—Shell-fish
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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
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/40—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by drying or kilning; Subsequent reconstitution
- A23L3/44—Freeze-drying
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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
- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
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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
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Polymers & Plastics (AREA)
- Nutrition Science (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Food Science & Technology (AREA)
- Botany (AREA)
- Biodiversity & Conservation Biology (AREA)
- Mycology (AREA)
- Ecology (AREA)
- Forests & Forestry (AREA)
- Environmental Sciences (AREA)
- Marine Sciences & Fisheries (AREA)
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- Developmental Biology & Embryology (AREA)
- Microbiology (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
Description
본 발명은 면역 증강 효과를 갖는 콩나물 혼합물에 관한 것으로, 보다 상세하게는 콩을 발아시킨 후 고농도의 산소수로 재배한 고농도 산소수 콩나물에 표고버섯과 건굴을 혼합하여 동결건조시킴으로써 세포독성을 가지지 않으면서 면역 관련 사이토카인들과 유전자 및 단백질들을 유도하고, 면역세포들을 증식시킴으로써 면역 증진 효능을 갖는 면역 증강 효과를 갖는 콩나물 혼합물에 관한 것이다.More particularly, the present invention relates to a soybean sprout mixture having an immunopotentiating effect, and more particularly, to a soybean sprout mixture having an immunopotentiating effect, and more particularly to a soybean sprout mixture having a high concentration of oxygenated soybean sprouts, Related immunosuppressive effect by inducing secretory-related cytokines, genes and proteins, and proliferating immune cells.
일상생활에서 쉽게 접할 수 있는 음식인 콩나물은 우리나라 고유의 전통식품으로 생육기간이 짧고 재배가 쉬우며 저렴한 가격으로 대중적인 음식으로써 오랜 기간 이용되어 왔다. 또한 단백질, 비타민, 무기질 등을 공급해주는 콩나물은 약재로도 이용되어 왔으며 특히 숙취해소로 알려진 아스파라긴과 항암 작용이 있는 이소플라본 등을 함유하고 있다.Bean sprouts, which are easily accessible in everyday life, are Korean traditional foods that have been used for a long time as a popular food with low growth period, easy cultivation and low price. Soybean sprouts that provide protein, vitamins, minerals, etc. have also been used as medicines. Especially, they contain asparagine, which is known to relieve hangover, and isoflavone, which has anticancer action.
콩나물은 같은 품종이더라도 생산지, 수확 후의 건조 및 저장조건 등에 따라 발아율, 신장율 및 생산수율 등에 현저한 차이가 존재하기 때문에 대한민국 등록특허 제10-0159834호(유기게르마늄콩나물재배방법), 대한민국 등록특허 제10-0011630호(식물성장조절제를살포하여콩나물을재배하는방법) 등과 같이 다양한 방법을 이용하여 이용하여 콩나물의 생산성이나 품질을 향상시킨 기술이 개시되어 있다. Since the bean sprouts have a remarkable difference in germination rate, elongation rate and production yield depending on the production site, drying and storage conditions after harvest even in the same kind of varieties, Korean Patent No. 10-0159834 (Organic Germanium bean sprout cultivation method), Korean Patent No. 10- 0011630 (a method of cultivating a soybean sprout by spraying a plant growth regulator), etc., to improve productivity and quality of bean sprouts.
콩나물에는 면역력 증강 등 다양한 기능과 효능을 갖는 물질이 함유되어 있는 것으로 알려져 있으며, 이에 따라 대한민국 공개특허 제10-1997-0025399호(콩나물또는숙주나물재배용키틴유도체혼합조성물의제조방법과그사용법), 대한민국 공개특허 제10-2007-0012580호(인삼, 홍삼엑기스를 이용재배한 기능성 콩나물 혼합조성물), 대한민국 공개특허 제10-2011-0013763호(녹차와 영지버섯의 혼합환을 사용한 콩나물의 재배방법) 등에는 콩나물이 갖는 효능을 강화시키거나 추가적인 다른 효능을 발휘하도록 콩나물에 다른 재료를 혼합한 혼합물을 얻는 다양한 시도가 있다.The bean sprouts are known to contain substances having various functions and effects such as immunity enhancement. Accordingly, Korean Patent Laid-Open Publication No. 10-1997-0025399 (a method for producing a chitin derivative mixed composition for cultivating bean sprouts or host herb and its use) Korean Patent Laid-Open No. 10-2007-0012580 (mixed ginseng and functional bean sprouts grown using red ginseng extract), Korean Patent Laid-Open No. 10-2011-0013763 (cultivation method of bean sprouts using mixed rings of green tea and gingko mushrooms) There are various attempts to obtain a mixture of bean sprouts mixed with other ingredients to enhance the efficacy of bean sprouts or to exhibit additional other effects.
한편, 표고버섯은 다당류에 의한 항암효과 및 항바이러스, 혈장 콜레스테롤 함량저하, 동맥경화, 고혈압, 등과 같은 성인병 치료 및 예방의 효과가 입증되었으며, 표고버섯 추출물은 항 산화성, 아질산염 소거작용이 우수한 것으로 보고되어 세계 적으로 식용으로 광범위하게 이용되고 있다. On the other hand, shiitake mushroom has proved to be effective in treating and preventing adult diseases such as anticancer effects and antiviral, plasma cholesterol content reduction, arteriosclerosis and hypertension, and the mushroom extract has excellent antioxidant and nitrite scavenging activity And is widely used for edible purposes worldwide.
또한 굴에는 면역력을 증강시키는 미네랄과 아연이 포함되어 있어 체내에서 백혈구를 생성하고 활성화시켜 면역체계에 도움을 준다.In addition, the oysters contain minerals and zinc that enhance the immune system, which helps the immune system to produce and activate white blood cells in the body.
본 발명은 상기와 같은 점을 인식하여 안출된 것으로 본 발명의 목적은 콩을 발아시킨 후 고농도의 산소수로 재배한 고농도 산소수 콩나물에 표고버섯과 건굴을 혼합하여 동결건조시킴으로써 세포독성을 가지지 않으면서 면역 관련 사이토카인들과 유전자 및 단백질들을 유도하고, 면역세포들을 증식시킴으로써 면역 증진 효능을 갖는 면역 증강 효과를 갖는 콩나물 혼합물을 제공하기 위한 것이다. The present invention has been made in recognition of the above points, and it is an object of the present invention to provide a soybean sprouts which is germinated by soybean and then cultured in a high concentration of oxygenated water and is free of cytotoxicity Inducing immunosuppressive effect by inducing secretory-related cytokines, genes and proteins, and proliferating immune cells.
상기와 같은 목적을 달성하기 위하여 본 발명에 따른 면역 증강 효과를 갖는 콩나물 혼합물은, 산소수를 발아된 콩에 살포하여 재배된 콩나물에 표고버섯과 건굴을 혼합하여 동결건조시킨 것을 특징으로 한다.In order to achieve the above-mentioned object, the bean sprouts mixture having immunity enhancing effect according to the present invention is characterized in that oxygenated water is sprayed on the germinated soybeans and lyophilized is obtained by mixing the shiitake mushroom and the gringo mushroom.
또한, 본 발명에 따른 면역 증강 효과를 갖는 콩나물 혼합물은, 콩나물 : 표고버섯 : 건굴은 10 : 1 : 1의 중량 비율로 혼합되는 것을 특징으로 한다.Further, the bean sprouts mixture having an immunostimulating effect according to the present invention is characterized by being mixed at a weight ratio of bean sprouts: shiitake: ginseng at a weight ratio of 10: 1: 1.
또한, 본 발명에 따른 면역 증강 효과를 갖는 콩나물 혼합물은, 상기 산소수는 용존산소 농도가 10 ppm ~ 20 ppm인 것을 특징으로 한다.Further, in the bean sprouts mixture having an immunostimulating effect according to the present invention, the oxygen water has a dissolved oxygen concentration of 10 ppm to 20 ppm.
또한, 본 발명에 따른 면역 증강 효과를 갖는 콩나물 혼합물은, 상기 콩나물은 20~30 ℃ 암실에서 2~6시간 간격으로 5~15분씩 상기 산소수를 발아콩에 5~7일간 살수하여 재배된 것을 특징으로 한다.The bean sprouts mixture having immunity enhancing effect according to the present invention is characterized in that the soybean sprouts are cultivated by sprinkling the germinated soybeans on soybean sprouts for 5 to 7 days in a dark room at 20 to 30 DEG C for 5 to 15 minutes at intervals of 2 to 6 hours .
상기와 같은 구성에 의하여 본 발명에 따른 면역 증강 효과를 갖는 콩나물 혼합물은 콩을 발아시킨 후 고농도의 산소수로 재배한 고농도 산소수 콩나물에 표고버섯과 건굴을 혼합하여 동결건조시킴으로써 세포독성을 가지지 않으면서 면역 관련 사이토카인들과 유전자 및 단백질들을 유도하고, 면역세포들을 증식시킴으로써 면역 증진 효능을 갖는 콩나물 혼합물을 얻을 수 있는 장점을 갖는다.According to the present invention, the bean sprouts mixture having immunity enhancing effect according to the present invention is prepared by mixing shiitake mushroom and gingko mushroom with high-concentration oxygenated bean sprouts cultivated with high concentration of oxygen water after germination of soybean, Inducing secretin-related cytokines, genes and proteins, and proliferating immune cells, thereby obtaining a bean sprouts mixture having immunity-promoting effect.
도 1은 본 발명의 일실시예에 따른 면역 증강 효과를 갖는 콩나물 혼합물의 마우스 대식세포에 대한 세포 독성 효과를 나타낸 그래프
도 2는 본 발명의 일실시예에 따른 면역 증강 효과를 갖는 콩나물 혼합물의 인간 세포주인 B raji 세포에서 증식능을 나타낸 그래프
도 3은 본 발명의 일실시예에 따른 면역 증강 효과를 갖는 콩나물 혼합물의 마우스 대식세포에 대한 전염증성 사이토카인을 나타낸 그래프
도 4는 본 발명의 일실시예에 따른 면역 증강 효과를 갖는 콩나물 혼합물의 마우스 대식세포에서 콩나물 혼합물의 면역 증강 효과를 iNOS 유전자 및 단백질 발현 그리고 산화질소의 발현을 측정하여 나타낸 그래프1 is a graph showing cytotoxic effects of a mixture of bean sprouts having an immunostimulating effect on mouse macrophages according to an embodiment of the present invention
FIG. 2 is a graph showing the proliferative activity in B raji cells, which is a human cell line of a mixture of bean sprouts having an immunostimulating effect according to an embodiment of the present invention
FIG. 3 is a graph showing proinflammatory cytokines to mouse macrophages of a mixture of bean sprouts having an immunostimulating effect according to an embodiment of the present invention
FIG. 4 is a graph showing the immunoenhancing effect of a mixture of bean sprouts in mouse macrophages of a bean sprouts mixture having an immunostimulating effect according to an embodiment of the present invention, by measuring iNOS gene and protein expression and expression of nitric oxide
이하에서는 실시예를 참조하여 본 발명에 따른 면역 증강 효과를 갖는 콩나물 혼합물을 보다 상세하게 설명하기로 한다.Hereinafter, the bean sprouts mixture having the immunostimulating effect according to the present invention will be described in more detail with reference to examples.
도 1은 본 발명의 일실시예에 따른 면역 증강 효과를 갖는 콩나물 혼합물의 마우스 대식세포에 대한 세포 독성 효과를 나타낸 그래프이고, 도 2는 본 발명의 일실시예에 따른 면역 증강 효과를 갖는 콩나물 혼합물의 인간 세포주인 B raji 세포에서 증식능을 나타낸 그래프이며, 도 3은 본 발명의 일실시예에 따른 면역 증강 효과를 갖는 콩나물 혼합물의 마우스 대식세포에 대한 전염증성 사이토카인을 나타낸 그래프이고, 도 4는 본 발명의 일실시예에 따른 면역 증강 효과를 갖는 콩나물 혼합물의 마우스 대식세포에서 콩나물 혼합물의 면역 증강 효과를 iNOS 유전자 및 단백질 발현 그리고 산화질소의 발현을 측정하여 나타낸 그래프이다.FIG. 1 is a graph showing the cytotoxic effect of a bean sprouts mixture having an immunostimulating effect on mouse macrophages according to an embodiment of the present invention. FIG. 2 is a graph showing a cytotoxic effect of a bean sprout mixture having an immunostimulating effect according to an embodiment of the present invention FIG. 3 is a graph showing proinflammatory cytokines to mouse macrophages of a mixture of bean sprouts having an immunostimulating effect according to an embodiment of the present invention. FIG. FIG. 2 is a graph showing the immunological enhancement effect of a mixture of bean sprouts in a mouse macrophage of a bean sprouts mixture having an immunostimulating effect according to an embodiment of the present invention, by measuring iNOS gene and protein expression and expression of nitric oxide. FIG.
<실시예1 : 콩나물 혼합물의 제조>≪ Example 1: Preparation of bean sprouts mixture >
산소수를 발아된 콩에 살포하여 재배된 콩나물에 표고버섯과 건굴을 혼합하여 동결건조시켜 콩나물 혼합물을 제조한다. 콩나물 혼합물은 콩나물 : 표고버섯 : 건굴은 10 : 1 : 1의 중량 비율로 혼합된다.Oxygen water is sprayed on the germinated soybean, and the bean sprouts cultivated are mixed with shiitake mushrooms and gingko leaves and lyophilized to prepare bean sprouts mixture. The bean sprouts mixture is mixed at a weight ratio of bean sprouts: shiitake: gingko at a weight ratio of 10: 1: 1.
산소수를 이용한 콩나물은 용존산소 농도가 10 ppm ~ 20 ppm인 산소수를 20~30 ℃ 암실에서 2~6시간 간격으로 5~15분씩 발아콩에 살수하여 5~7일간 살수하여 재배된다.Oxygenated bean sprouts are grown by sprinkling 5 ~ 15 minutes of germinated soybean spikes at 20 ~ 30 ℃ for 2 ~ 6 hours in 20 ~ 30 ℃ for 5 ~ 7 days.
<실시예2 : 콩나물 혼합물을 이용한 면역 증강 효과에 대한 실험>≪ Example 2: Experiment on immunity enhancement effect using bean sprouts mixture >
2. 재료 및 방법 2. Materials and Methods
- 세포 배양- Cell culture
Balb/c male 8주령에서 12주령 사이의 마우스 femur와 Tibia에서 골수를 분리하였다. 10% FBS와 1% PS가 첨가 된 MEM alpha 배지를 사용하였으며 대식세포로 분화시키기 위해 30% L929 conditioned medium을 사용하였다. 6일간 37℃ 및 5% CO2 조건에서 배양하였다.Balb / c male Bone marrow was isolated from mouse femur and Tibia between 8 and 12 weeks of age. MEM alpha medium supplemented with 10% FBS and 1% PS was used and 30% L929 conditioned medium was used to differentiate into macrophages. And cultured for 6 days at 37 ° C and 5% CO 2 .
- 콩나물 혼합물 처리 조건- Treatment conditions of bean sprouts mixture
고농도 산소수로 재배한 콩나물과 표고버섯 및 건굴을 10 : 1 : 1로 혼합한 뒤 동결건조 시킨 혼합물을 0.1 mg/ml, 0.5 mg/ml, 1 mg/ml의 농도로 BMDMs에 처리하였다.The mixture of bean sprouts cultivated with high concentration oxygenated water, shiitake mushroom and dried ginger was mixed at 10: 1: 1 and the lyophilized mixture was treated with BMDMs at concentrations of 0.1 mg / ml, 0.5 mg / ml and 1 mg / ml.
- 세포 독성검사 (Cell viability)- Cell viability test
세포에 대한 콩나물 혼합물의 독성평가를 위해 MTT 방법을 시행하였다. BMDMs을 48 웰플레이트(well plate)에 1x106 cells/ml로 첨가하고 콩나물 혼합물을 농도별로 처리한 후 24시간 후에 MTT 용액을 200 μl/well 넣고 37℃ 및 5% CO2 인큐베이터(incubator)에서 빛을 차단해주었다. 4시간 후에 상층액은 버리고 DMSO 200 μl/well 넣은 후 보라색 침전물이 녹으면 570 nm에서 흡광도로 측정 후, 대조군(control)의 상대적 정량값으로 그래프화 하였다.To evaluate the toxicity of the bean sprouts mixture to the cells, MTT method was used. BMDMs were added to a 48-well plate at a concentration of 1 × 10 6 cells / ml, and the bean sprouts mixture was treated at different concentrations. After 24 hours, 200 μl / well of MTT solution was added and incubated at 37 ° C. in a 5% CO 2 incubator . After 4 hours, the supernatant was discarded and 200 μl / well of DMSO was added. After the purple precipitate was dissolved, the absorbance was measured at 570 nm, and the resultant was quantified as a relative quantitative value of the control.
- T cell, B cell 증식 측정- T cell, B cell proliferation measurement
인간 세포주인 B Raji 세포에서 콩나물 혼합물에 대한 증식능을 알아보기 위해 Water Soluble Tetrazolium Salts (WST-1) 방법을 시행하였다. B 세포를 48 웰플레이트(well plate)에 1x104 cells/ml로 첨가하고 2시간 뒤 콩나물 혼합물을 농도별로 처리한 후 72시간 후에 WST-1 용액을 10 μl/well 넣고 37℃ 및 5% CO2 인큐베이터(incubator)에서 1시간 동안 빛을 차단해주었다. 450nm로 측정 후, 대조군(control)의 상대적 정량값으로 그래프화 하였다. Water Soluble Tetrazolium Salts (WST-1) method was used to investigate the proliferative activity of the bean sprouts mixture in the human cell line B Raji cells. Adding B cells to 1x10 4 cells / ml in 48-well plate (well plate), and 2 hours after treatment the sprouts mixture by concentration of WST-1 solution after 72 hours into 10 μl / well 37 ℃ and 5% CO 2 Light was blocked for 1 hour in an incubator. After measurement at 450 nm, the relative quantitation values of the control (control) were plotted.
- ELISA- ELISA
콩나물 혼합물의 전염증성 사이토카인을 측정하기 위해 ELISA를 시행하였다. BMDMs을 48 웰플레이트(well plate)에 1x106 cells/ml로 첨가하고 콩나물 혼합물을 농도별로 처리한 후 24시간 후에 상층액 내의 사이토카인을 ELISA kit를 이용하여 측정하였다. ELISA microplate에 capture antibody로 anti-mouse IL-6 및 TNF-α를 분주하여 4℃에서 하룻밤 동안 코팅(coating)시키고, 이를 0.05% Tween 20이 포함된 PBST로 세척한 후 1% BSA 용액으로 1시간 동안 블록킹(blocking)하였다. PBST로 세척 한 뒤, 각 배양 상층액을 1% BSA에 3배 희석하여 분주하고 실온에서 2시간 반응시켰다. 다시 PBST로 세척한 뒤 희석한 각각의 biotinylated anti-mouse detection antibody를 첨가하고 2시간 반응 후, streptavidin-horseradish peroxidase conjugate를 첨가하여 20분 반응 시켰다. 그 후, 이를 다시 PBST로 세척 한 다음, substrate reagent 용액을 첨가하여 실온에서 20분 동안 암반응 시킨 뒤 2N H2SO4로 반응을 종료시킨 후 450 nm에서 흡광도를 측정하였다.ELISA was performed to measure the proinflammatory cytokine of the bean sprouts mixture. BMDMs were added to a 48 well plate at a concentration of 1 × 10 6 cells / ml, and the mixture of bean sprouts was treated by concentration. After 24 hours, the cytokine in the supernatant was measured using an ELISA kit. ELISA microplate was coated with anti-mouse IL-6 and TNF-α by a capture antibody overnight at 4 ° C, washed with PBST containing 0.05%
- Real-time PCR- Real-time PCR
콩나물 혼합물을 마우스 대식세포에 처리하여 iNOS에 대한 유전자 발현을 Real-time PCR을 통해 확인하였다. BMDMs을 6 웰플레이트(well plate)에 1x106 cells/ml로 24시간 배양한 후 콩나물 혼합물 1 mg/ml을 2시간, 4시간, 8시간 동안 처리해주었다. 이후 TRIzol 용액을 이용하여 세포내 RNA를 분리하고 RNA 정량값을 토대로 RT premix를 이용하여 cDNA를 합성하였다. 합성된 cDNA는 primer를 이용하여 Real time PCR을 통해 증폭시켰다. 사용된 primer는 Mouse iNOS이고 대조군(Control) 유전자인 GAPDH와 비교하여 상대적 양을 비교하였다. The gene expression for iNOS was confirmed by real-time PCR by treating the soybean sprout mixture with mouse macrophages. BMDMs were cultured in a 6-well plate at 1 × 10 6 cells / ml for 24 hours, and 1 mg / ml of the bean sprouts mixture was treated for 2 hours, 4 hours, and 8 hours. Subsequently, the RNA was isolated using TRIzol solution and cDNA was synthesized using RT premix based on RNA quantification value. The synthesized cDNA was amplified by real time PCR using primer. The primers used were mouse iNOS and compared with the control (GAPDH) gene.
- Western Blotting- Western Blotting
마우스 대식세포에 콩나물 혼합물을 처리했을 때 iNOS 단백질 발현량을 확인하고자 하였다. 마우스 대식세포를 6 웰플레이트(well plate)에 1x106 cells/ml로 24시간 배양한 후 콩나물 혼합물을 농도별로 24시간 동안 처리해주었다. 배지를 제거하고 Protein lysis buffer를 이용하여 단백질을 추출한 후 정량하여 30 ug의 단백질을 SDS-PAGE gel에서 분리하였다. 단백질을 멤브레인(membrane)으로 옮긴 후 1차, 2차 항체를 순차적으로 처리하여 BIO-RAD, ECL solution detection kit로 발현되면 이미지 분석 기계(Chemidoc 기계)로 측정하였다.To examine the expression level of iNOS protein in mouse macrophages treated with a mixture of bean sprouts. Mouse macrophages were cultured in a 6- well plate at 1x10 6 cells / ml for 24 hours, and then the bean sprouts mixture was treated for 24 hours at various concentrations. After removing the medium and extracting proteins using Protein lysis buffer, 30 ug of proteins were separated on SDS-PAGE gel. Proteins were transferred to membranes, and the primary and secondary antibodies were sequentially treated and analyzed by BIO-RAD and ECL solution detection kit using an image analyzer (Chemidoc machine).
- 산화질소(nitric oxide) 생성량 측정- Measurement of nitric oxide production
마우스 대식세포를 96 웰플레이트(well plate)에 1×106 cells/ml로 분주한 다음 24시간 배양하였다. 배양 후 IFN-γ (100 ng/ml), LPS (100 ng/ml) 및 콩나물 혼합물을 농도별로 처리하고 24시간 후에 상층액에서 산화질소의 농도를 Griess시약 (1% sulfanilamide, 0.1% N-1-Naphytylethylenediamine dihydrochloride, 5% phosphoric acid)을 이용하여 흡광도 577 nm에서 측정하였다.Mouse macrophages were plated at a density of 1 × 10 6 cells / ml in a 96-well plate and cultured for 24 hours. After incubation, the concentrations of IFN-γ (100 ng / ml), LPS (100 ng / ml) and bean sprouts were treated at different concentrations and after 24 hours the concentration of nitric oxide in the supernatant was measured using Griess reagent (1% sulfanilamide, 0.1% - Naphytylethylenediamine dihydrochloride, 5% phosphoric acid) at an absorbance of 577 nm.
2. 실험 결과2. Experimental results
- 콩나물 혼합물의 세포 독성 평가- Evaluation of cytotoxicity of bean sprouts mixture
MTT assay를 통해 대식세포에 콩나물 혼합물에 대한 세포 독성 평가를 실시한 결과 모든 농도에서 독성이 없는 것으로 확인 되었다 (도 1 참조).Cytotoxicity assays for macrophage mixed bean sprouts with MTT assay showed no toxicity at all concentrations (see Figure 1).
- 콩나물 혼합물에 대한 면역 세포의 증식능 평가- Evaluation of the proliferative capacity of immune cells against bean sprouts mixture
면역세포의 증식에 미치는 콩나물 혼합물의 증식효과를 탐색하기 위해 B Raji 세포에 콩나물 혼합물을 농도별로 처리한 후 WST-1 방법으로 면역세포의 증식능을 확인하였다. B 세포에서 콩나물 혼합물을 0.1 mg/ml로 처리하였을 때 최적의 증식능을 보였다. 따라서 면역 세포는 콩나물 혼합물에 대한 증식 효과를 보임을 확인하였다 (도 2 참조).In order to investigate the proliferative effect of the bean sprouts mixture on the proliferation of immune cells, B raji cells were treated with a mixture of bean sprouts at various concentrations and then the proliferative capacity of the immune cells was confirmed by the WST-1 method. The optimal proliferative capacity was shown when B cells were treated with 0.1 mg / ml bean sprouts mixture. Thus, it was confirmed that the immune cells showed a proliferative effect on the bean sprouts mixture (see FIG. 2).
- 콩나물 혼합물의 전염증성 사이토카인 분비능 평가- Assessment of proinflammatory cytokine secretion ability of bean sprouts mixture
대식세포(Macrophage)는 내재면역뿐만 아니라 적응면역 등 다양한 숙주 반응에 관여하여 숙주방어와 항상성 유지에 관여하는 것으로 알려져 있으며, 면역 반응 시에는 interleukin-6(IL-6) 및 tumor necrosis factor-α(TNF-α)와 같은 사이토카인을 생산하여 감염 초기에 생체 방어에 중요한 역할을 하는 세포로 알려져 있다. 콩나물 혼합물에 의한 대식세포의 면역 증강 효과를 확인하기 위해 활성이 증가된 대식세포로부터 분비되는 면역 활성 사이토카인인 IL-6 및 TNF-α의 분비량을 ELISA로 분석하였다. Macrophage is known to be involved in various host responses such as immune response as well as endogenous immunity and is involved in host defense and homeostasis. Interleukin-6 (IL-6) and tumor necrosis factor-α TNF-α) and is known to play an important role in biological defense in the early stages of infection. The secretion of IL-6 and TNF-α, the immunologically active cytokines secreted from macrophages with increased activity, was analyzed by ELISA to confirm the immunopotentiation effect of macrophages by the bean sprouts mixture.
대식세포에 콩나물 혼합물을 0.1 mg/ml, 0.5 mg/ml, 1 mg/ml로 처리하고 24시간 후에 상층액에서 사이토카인을 ELISA로 확인해본 결과 IL-6와 TNF-α 모두 농도 의존적으로 발현이 증가했음을 볼 수 있었다. 콩나물 혼합물은 면역증강 사이토카인들의 생성을 증가시킴에 따라 생체 면역 기능을 전반적으로 향상시킴을 확인할 수 있었다. (도 3 참조)The expression of cytokines in the supernatant was assayed by ELISA after 24 hours of treatment with 0.1 mg / ml, 0.5 mg / ml, and 1 mg / ml of soybean sprout mixture in macrophages. The expression of both IL-6 and TNF- Increase. The bean sprouts mixture increased the production of immunopotentiating cytokines, and it was confirmed that the bioimmune function was improved overall. (See Fig. 3)
- 콩나물 혼합물의 iNOS 유전자 발현 및 NO 생성능 평가- Evaluation of iNOS gene expression and NO production ability of bean sprouts mixture
일산화질소 (NO)는 선천면역에서 항균 및 항바이러스 효과를 가지며, IFN-r로 유도되는 iNOS는 염증 관련 유전자로 아르기닌으로부터 NO 합성에 관여한다. 생체 내에서 고농도의 산화질소 생성은 혈관확장과 더불어 생체 면역 반응을 촉진하게 된다. 이와 같은 이유로 산화질소는 면역 반응의 증가를 의미하는 물질이다. Nitric oxide (NO) has antimicrobial and antiviral effects on innate immunity, and iNOS induced by IFN-r is involved in NO synthesis from arginine as an inflammation-related gene. The production of nitric oxide at high concentration in vivo accelerates the vital immune response as well as vasodilation. For this reason, nitric oxide is a substance that means an increase in the immune response.
대식세포에 콩나물 혼합물을 1 mg/ml로 시간별로 처리한 후 real-time PCR로 iNOS의 mRNA 발현을 확인한 결과 시간 의존적으로 증가하였고, 8시간째에 최적의 발현량을 보였다. (도 4의 A부분 참조)The expression of iNOS mRNA was measured by real-time PCR after a 1-mg / ml mixture of macrophage and bean sprouts was time-dependent, and the expression level was optimal at 8 hours. (See part A of FIG. 4)
또한 콩나물 혼합물을 농도별로 처리하여 iNOS의 단백질 발현량을 western blotting으로 확인하였더니 농도 의존적으로 발현이 증가했음을 확인하였다. (도 4의 B부분 참조)In addition, the expression of iNOS protein was confirmed by western blotting by treating the mixture of bean sprouts with concentration, and the expression was increased in a concentration dependent manner. (See part B of Fig. 4)
더불어 대식세포에서 콩나물 혼합물에 의한 산화질소 생성 정도를 관찰하기 위하여 배양 상층액에서 Griess 반응으로 NO 생성을 측정하였다. 대식세포를 활성화시키는 LPS (100 ng/ml)는 양성 대조군으로서 비교하였다. 콩나물 혼합물을 1 mg/ml로 처리한 경우에는 콩나물 혼합물만으로도 NO를 생성했고, IFN-γ의 존재 하에서는 콩나물 혼합물의 농도 의존적으로 NO를 생성하였다. (도 4의 C부분 참조)In addition, NO production was measured by Griess reaction in culture supernatants to observe the degree of nitric oxide production by macrophage mixture. Macrophage activating LPS (100 ng / ml) was compared as a positive control. When the bean sprouts mixture was treated with 1 mg / ml, only the bean sprouts mixture produced NO, and in the presence of IFN-γ, NO was produced depending on the concentration of the bean sprouts mixture. (See part C of Fig. 4)
상술한 바와 같이 본 발명에 따른 면역 증강 효과를 갖는 콩나물 혼합물은 세포독성을 가지지 않으면서 면역 관련 사이토카인들과 유전자 및 단백질들을 유도하고, 면역세포들을 증식시킴으로써 면역 증진 효능을 가진다.As described above, the bean sprouts mixture having an immunostimulating effect according to the present invention has immune enhancement effect by inducing immune related cytokines, genes and proteins without cell cytotoxicity and proliferating immune cells.
이상에서 살펴본 바와 같이 본 발명은 바람직한 실시 예를 들어 도시하고 설명하였으나, 상기한 실시 예에 한정되지 아니하며 본 발명의 정신을 벗어나지 않는 범위 내에서 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자에 의해 다양한 변경과 수정이 가능하다 할 것이다.While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be taken by way of limitation, Various changes and modifications may be made by those skilled in the art.
Claims (4)
콩나물 : 표고버섯 : 건굴은 10 : 1 : 1의 중량 비율로 혼합되고,
상기 콩나물은 20~30 ℃ 암실에서 2~6시간 간격으로 5~15분씩 용존산소 농도가 10 ppm ~ 20 ppm인 산소수를 발아콩에 5~7일간 살수하여 재배된 것을 특징으로 하는 면역 증강 효과를 갖는 콩나물 혼합물.Oxygen water was sprayed on the sprouted soybeans, and the bean sprouts were mixed with shiitake mushroom and gingko mushroom and lyophilized,
Bean sprouts: Shiitake mushrooms: Gingko are mixed at a weight ratio of 10: 1: 1,
The bean sprouts are grown by sprinkling oxygenated water having a dissolved oxygen concentration of 10 ppm to 20 ppm in germinated soybeans for 5 to 7 days in a dark room at 20 to 30 캜 for 5 to 15 minutes at intervals of 2 to 6 hours. ≪ / RTI >
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