KR100542731B1 - FUNCTIONAL FOODS CONTAINING Wasabia japonica - Google Patents
FUNCTIONAL FOODS CONTAINING Wasabia japonica Download PDFInfo
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- KR100542731B1 KR100542731B1 KR1020030044612A KR20030044612A KR100542731B1 KR 100542731 B1 KR100542731 B1 KR 100542731B1 KR 1020030044612 A KR1020030044612 A KR 1020030044612A KR 20030044612 A KR20030044612 A KR 20030044612A KR 100542731 B1 KR100542731 B1 KR 100542731B1
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- wasabi
- effect
- cholesterol
- present
- functional food
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Classifications
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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
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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
- A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
- A23L13/60—Comminuted or emulsified meat products, e.g. sausages; Reformed meat from comminuted meat product
- A23L13/65—Sausages
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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
- A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
- A23L13/60—Comminuted or emulsified meat products, e.g. sausages; Reformed meat from comminuted meat product
- A23L13/67—Reformed meat products other than sausages
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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
- A23V2200/302—Foods, ingredients or supplements having a functional effect on health having a modulating effect on age
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/3262—Foods, ingredients or supplements having a functional effect on health having an effect on blood cholesterol
Abstract
본 발명은 지방 대사 개선 효과가 있는 고추냉이를 함유하는 기능성 식품에 관한 것으로서, 구체적으로 본 발명은 지방 대사를 개선하기 위하여 항산화 효과 및 항고지혈증 효과가 있는 고추냉이를 함유하는 기능성 식품에 관한 것이다. The present invention relates to a functional food containing wasabi having an effect of improving fat metabolism, specifically, the present invention relates to a functional food containing wasabi having an antioxidant effect and antihyperlipidemic effect in order to improve fat metabolism.
본 발명에서 사용되는 고추냉이는 DPPH에 대한 수소 공여능이 뛰어나고, 직접적으로 산화 질소 생성을 억제하며, iNOS mRNA 발현 및 iNOS 발현을 억제하여 간접적으로 산화 질소 생성을 억제하고, 잔틴 산화제의 활성을 저해하여 슈퍼옥사이드 라디칼의 생성을 억제하는 항산화 효과를 가지고 있다. 또한, 본 발명에서 사용되는 고추냉이는 고밀도 지단배질-콜레스테롤의 함량은 증가시키고, 총 콜레스테롤 및 저밀도 지단백질-콜레스테롤의 함량은 감소시키는 항고지혈증 효과를 가지고 있다. 본 발명에서는 이러한 효과를 가지고 있는 고추냉이를 주성분으로 또는 부성분으로 함유하는 기능성 식품을 제공함으로써 지방 대사와 관련된 질병을 예방 또는 치료하는데 사용할 수 있도록 하였다.Wasabi used in the present invention has excellent hydrogen donating ability to DPPH, directly inhibits nitric oxide production, indirectly inhibits nitric oxide production by inhibiting iNOS mRNA expression and iNOS expression, and inhibits the activity of xanthine oxidant. It has an antioxidant effect that inhibits the production of superoxide radicals. In addition, wasabi used in the present invention has an antihyperlipidemic effect of increasing the content of high-density lipoprotein-cholesterol and reducing the content of total cholesterol and low-density lipoprotein-cholesterol. In the present invention, by providing a functional food containing horseradish as a main ingredient or as a minor ingredient having such an effect, it can be used to prevent or treat diseases related to fat metabolism.
고추냉이, 항산화 효과, 항고지혈증 효과, 지방 대사, 기능성 식품Wasabi, Antioxidant Effect, Antihyperlipidemic Effect, Fat Metabolism, Functional Food
Description
도 1은 본 발명에 따른 고추냉이 잎의 물 추출물이 산화 질소 생성을 억제 하는 효과가 있음을 보여주는 것이고, 1 shows that the water extract of horseradish leaves according to the present invention has the effect of inhibiting the production of nitric oxide,
도 2는 본 발명에 따른 고추냉이 잎의 물 추출물이 iNOS mRNA 발현을 억제하는 효과가 있음을 보여주는 것이고, Figure 2 shows that the water extract of horseradish leaves according to the present invention has an effect of inhibiting iNOS mRNA expression,
도 3은 본 발명에 따른 고추냉이 잎의 물 추출물이 iNOS 발현을 억제하는 효과가 있음을 보여주는 것이고, Figure 3 shows that the water extract of horseradish leaves according to the present invention has an effect of inhibiting iNOS expression,
도 4는 본 발명에 따른 고추냉이 잎 또는 뿌리가 총 콜레스테롤의 함량을 감소시키는 효과가 있음을 보여주는 것이고, Figure 4 shows that horseradish leaves or roots according to the present invention has an effect of reducing the content of total cholesterol,
도 5는 본 발명에 따른 고추냉이 잎 또는 뿌리가 고밀도 지단백질-콜레스테롤의 함량을 증가시키는 효과가 있음을 보여주는 것이고, Figure 5 shows that horseradish leaves or roots according to the present invention has the effect of increasing the content of high-density lipoprotein-cholesterol,
도 6은 본 발명에 따른 고추냉이 잎 또는 뿌리가 저밀도 지단백질-콜레스테롤의 함량을 감소시키는 효과가 있음을 보여주는 것이다. Figure 6 shows that horseradish leaves or roots according to the present invention has an effect of reducing the content of low-density lipoprotein-cholesterol.
본 발명은 지방 대사 개선 효과가 있는 고추냉이를 함유하는 기능성 식품에 관한 것으로서, 구체적으로 본 발명은 지방 대사를 개선하기 위하여 항산화 효과 및 항고지혈증 효과가 있는 고추냉이를 함유하는 기능성 식품에 관한 것이다. The present invention relates to a functional food containing wasabi having an effect of improving fat metabolism, specifically, the present invention relates to a functional food containing wasabi having an antioxidant effect and antihyperlipidemic effect in order to improve fat metabolism.
최근 식생활의 변화로 인한 질병의 발생률이 매년 증가하고 있어, 식생활과 질병과의 관계에 관한 많은 연구들이 수행되고 있다. 특히 생활 패턴의 서구화, 영양 섭취량의 증가, 동물성 지방 섭취량의 증가, 평균체중의 증가, 운동량 감소 또는 스트레스 증가로 인하여 지방 대사와 관련된 질병의 발생율이 증가하고 있다.Recently, the incidence of diseases due to dietary changes is increasing every year, and many studies on the relationship between diet and disease have been conducted. In particular, the incidence of diseases related to fat metabolism is increasing due to the westernization of life patterns, the increase in nutritional intake, the increase in animal fat intake, the increase in the average body weight, the decrease in the amount of exercise or the increase in stress.
지방 대사와 관련된 질병인 순환 장애, 동맥 경화, 노화, 고지혈증 또는 내분비 질환은 생체내에서 생성되는 유리 라디칼 및 지방 대사물질 특히 콜레스테롤로부터 유발된다. Diseases associated with fat metabolism, such as circulatory disorders, atherosclerosis, aging, hyperlipidemia or endocrine diseases, are derived from free radicals and fatty metabolites, especially cholesterol, produced in vivo.
생체내에서 생성되는 유리 라디칼은 생체막을 공격하여 지질 라디칼의 연쇄 반응을 통하여 과산화 지질을 축적시킴으로써 각종 질병을 일으킨다. 이러한 유리 라디칼이 체내에서 소량으로 생성될 경우에는 항산화제에 의해 물로 변환되어 소실된다. 그러나, 유리 라디칼이 과량으로 생성될 경우에는 별도로 항산화 작용을 하는 식품을 섭취해주어야 한다.Free radicals generated in vivo attack various biological membranes and cause various diseases by accumulating lipid peroxide through a chain reaction of lipid radicals. When these free radicals are produced in small amounts in the body, they are converted to water by antioxidants and lost. However, if free radicals are produced in excess, foods that have antioxidant activity should be consumed separately.
콜레스테롤은 뇌, 신경, 근육, 피부, 간, 장 및 심장 등 신체내 모든 부위에 있는 세포의 세포막의 주요 성분이며 부신피질과 고환 및 난소에서 생성되는 스테 로이드 호르몬과 비타민 D, 담즙산의 전구체로서 소장에서 흡수되거나 간에서 합성되는 지방 대사물질이다. 그러나, 콜레스테롤이 체내에서 과잉으로 존재할 경우, 혈중 지질이 높아 동맥 경화성 질환을 일으킬 수 있으며, 이외에도 협심증, 심근 경색증과 같은 관상 동맥 질환, 뇌경색과 같은 질환을 유발할 수 있다. Cholesterol is a major component of the cell membranes of cells in all parts of the body, including the brain, nerves, muscles, skin, liver, intestines, and heart, and is a precursor of steroid hormones, vitamin D, and bile acids, which are produced in the adrenal cortex, testes, and ovaries. Fat metabolites that are absorbed by or synthesized in the liver. However, when cholesterol is excessive in the body, high blood lipids may cause atherosclerotic diseases, and in addition, it may cause diseases such as coronary artery diseases such as angina pectoris and myocardial infarction and cerebral infarction.
고추냉이(Wasabia japonica)는 다년생 식물로 한국, 일본, 사할린 등 온·난대지방에 분포한다. 고추냉이는 예로부터 식용 식물로 향신료, 육류 요리, 생선 요리 등에 사용되어오고 있으며, 약용 식물로 혈액 순환 촉진, 식욕증진, 관절염의 치료, 방부 및 살균 등의 용도에 사용되어 왔다. 우리나라에서는 봄에 전초를 산유채, 봄에 잔뿌리를 제거하여 말린 뿌리를 산규근이라 하여 건위, 진통, 살균 등의 목적으로 사용되어오고 있다. Wasabia ( wasabia japonica ) is a perennial plant and is distributed in temperate and temperate regions such as Korea, Japan, and Sakhalin. Wasabi has been used as an edible plant for spices, meat dishes, fish dishes, etc., and has been used for medicinal plants for promoting blood circulation, increasing appetite, treating arthritis, preserving and sterilizing. In Korea, it is used for the purpose of dry stomach, analgesic, sterilization, etc., which is called "San Kyu-geun" by removing the rapeseed in spring and the grass roots in spring.
본 발명자들은 고추냉이에 항산화 효과 및 항고지혈증 효과가 있음을 밝혀내고, 이러한 효과를 가지고 있는 고추냉이를 함유하는 기능성 식품을 제공함으로써 본 발명을 완성하였다. The present inventors found that there was an antioxidant effect and an antihyperlipidemic effect on wasabi, and completed the present invention by providing a functional food containing wasabi having this effect.
본 발명의 목적은 고추냉이가 항산화 효과 및 항고지혈증 효과가 있음을 밝히고, 이러한 고추냉이를 이용하여 지방 대사 개선 효과가 있는 고추냉이를 함유한 기능성 식품을 제공하는 것이다.
It is an object of the present invention to find that horseradish has an antioxidant effect and antihyperlipidemic effect, and to provide a functional food containing horseradish having an effect of improving fat metabolism by using wasabi.
본 발명은 지방 대사 개선 효과가 있는 고추냉이를 함유하는 기능성 식품을 제공한다. 구체적으로 본 발명은 지방 대사를 개선하기 위하여 항산화 효과 및 항고지혈증 효과가 있는 고추냉이를 함유하는 기능성 식품을 제공한다.The present invention provides a functional food containing wasabi having an effect of improving fat metabolism. Specifically, the present invention provides a functional food containing horseradish having an antioxidant effect and an antihyperlipidemic effect in order to improve fat metabolism.
본 발명에서 사용되는 고추냉이는 항산화 효과가 있어 지방 대사를 개선할 수 있다. Wasabi used in the present invention has an antioxidant effect can improve fat metabolism.
구체적으로, 고추냉이로부터 제조된 물 추출물 또는 에탄올 추출물은 수소 공여능(hydrogen donating ability)이 뛰어나 DPPH(1,1-diphenyl-2-picrylhydrazyl)와의 산화 환원 반응에서 활성 산소로 인한 자유 라디칼을 소거(scavenging)할 수 있다. 활성 산소는 세포 파괴능이 매우 강하며 이들에 의해 야기되는 자유 라디칼 반응은 지질을 포함한 세포의 거대 분자를 파괴할 수 있다. 또한, 상기 자유 라디칼 반응으로부터 생성된 산소 자유 라디칼이 체내에서 과량으로 존재할 경우, 동맥 질환, 발암 및 노화 진행을 촉진할 수 있다. 따라서, 본 발명에 따른 고추냉이를 첨가한 식품은 활성 산소로 인한 자유 라디칼 반응을 억제시킴으로써 항산화 효과를 가지고 있다. Specifically, water extracts or ethanol extracts prepared from horseradish have excellent hydrogen donating ability and thus scavenging free radicals due to active oxygen in redox reaction with DPPH (1,1-diphenyl-2-picrylhydrazyl). )can do. Free radicals are very strong in breaking down cells, and the free radical reactions caused by them can destroy the macromolecules of cells, including lipids. In addition, when oxygen free radicals generated from the free radical reaction are present in the body in excess, it may promote arterial disease, carcinogenesis and aging progression. Therefore, the food containing wasabi according to the present invention has an antioxidant effect by inhibiting free radical reaction due to free radicals.
또한, 고추냉이부터 제조된 물 추출물 또는 에탄올 추출물은 산화 질소(nitrogen oxide, NO) 소거 효과가 있어 항산화 효과를 가지고 있다. 산화 질소는 반응성 산소종(reactive oxygen species)의 일종으로 산화 질소 합성제(nitric oxide synthethase)에 의해 합성되며, 그 중에서도 iNOS에 의해 다량 합성되는 것으로 알려져 있다. iNOS는 리포폴리사카라이드(lipopolysaccharide), 인터페론-γ(interferon-γ), 인터루킨-1(interleukine-1) 및 TNF-α(tumor necrosis factor-α) 등의 자극에 의해 대식세포, 혈관 평활근 세포, 내피세포, 간세포 또는 심근 세포에서 다량의 산화 질소를 생성하는 것으로 알려져 있다. 생성된 산화 질소는 전염증성 또는 항염증성 작용과 같은 다양한 생리적 효과를 가지고 있으나, 생체내에서 고농도로 존재할 경우 숙주세포의 파괴, 쇼크에 의한 혈관 확장, 염증 반응 유발에 의한 조직의 상해를 초래할 수 있다. 본 발명에 따른 고추냉이를 함유하는 기능성 식품은 LPS와 같은 자극에 의한 산화 질소의 생성량을 억제시킴으로써 항산화 효과를 가지고 있다. In addition, water extract or ethanol extract prepared from wasabi has an antioxidant effect because it has a nitric oxide (nitrogen oxide, NO) scavenging effect. Nitric oxide is a kind of reactive oxygen species, which is synthesized by nitric oxide synthethase, and is known to be synthesized in large quantity by iNOS. iNOS is induced by macrophages, vascular smooth muscle cells, by stimulation of lipopolysaccharide, interferon-γ, interleukine-1 and TNF-α (tumor necrosis factor-α). It is known to produce large amounts of nitric oxide in endothelial cells, hepatocytes or cardiomyocytes. The produced nitric oxide has various physiological effects such as proinflammatory or anti-inflammatory action, but when present in high concentrations in vivo, it may cause destruction of host cells, expansion of blood vessels by shock, and injury of tissues by inducing inflammatory reactions. . Functional food containing wasabi according to the present invention has an antioxidant effect by suppressing the amount of nitrogen oxide produced by stimulation such as LPS.
또한, 고추냉이로부터 제조된 물 추출물 또는 에탄올 추출물은 상기 산화 질소를 생성하는 iNOS mRNA 유전자 발현 및 iNOS 발현을 억제하여 산화 질소의 생성량을 줄일 수 있다. 구체적으로, 세포주에 LPS만 단독으로 처리할 경우에는 iNOS mRNA 유전자 발현 및 iNOS 발현이 강하게 유도되었다. 그러나, 세포주에 고추냉이로부터 제조된 물 추출물 또는 에탄올 추출물과 LPS를 동시에 처리할 경우에는 iNOS mRNA의 발현 및 iNOS 발현이 억제되었다. 따라서, 본 발명에 따른 고추냉이를 함유하는 기능성 식품은 iNOS mRNA 발현 및 iNOS 발현을 억제하여 산화 질소 생성량을 줄임으로써 항산화 효과를 가지고 있다.In addition, the water extract or ethanol extract prepared from wasabi may reduce the production of nitric oxide by inhibiting iNOS mRNA gene expression and iNOS expression to produce the nitric oxide. Specifically, iNOS mRNA gene expression and iNOS expression were strongly induced when only LPS was treated in the cell line. However, iNOS mRNA expression and iNOS expression were inhibited when LPS and LPS and water extract prepared from horseradish at the same time. Therefore, the functional food containing wasabi according to the present invention has an antioxidant effect by inhibiting iNOS mRNA expression and iNOS expression to reduce the amount of nitric oxide produced.
또한, 고추냉이로부터 제조된 물 추출물 또는 에탄올 추출물은 잔틴 산화제(xanthine oxidase)의 활성을 억제하여 이로부터 생성되는 슈퍼옥사이드 라디칼(superoxide radical) 함량을 줄일 수 있다. 잔틴 산화제는 자유 라디칼을 생성하는 효소로, 잔틴을 기질로 하여 요산을 생성하면서 자유 라디칼인 슈퍼옥사이드 라디칼을 생성하는 것으로 알려져 있다. 수퍼옥사이드 라디칼이 체내에서 여러 요인에 의해 증가되면 동맥질환, 발암 및 노화 진행을 촉진할 수 있음이 여러 연구 보고에서 제시되고 있다. 따라서, 본 발명에 따른 고추냉이를 함유하는 기능성 식품은 잔틴 산화제의 활성을 억제하여 슈퍼옥사이드 라디칼의 생성을 줄임으로써 항산화 효과를 가지고 있다.In addition, the water extract or ethanol extract prepared from wasabi may inhibit the activity of xanthine oxidase to reduce the superoxide radical content produced therefrom. Xanthine oxidants are enzymes that produce free radicals, and are known to produce superoxide radicals, which are free radicals, while producing uric acid based on xanthine as a substrate. Increasing the number of superoxide radicals in the body by various factors has been suggested in several studies to promote arterial disease, carcinogenesis and aging progression. Therefore, the functional food containing wasabi according to the present invention has an antioxidant effect by reducing the production of superoxide radicals by inhibiting the activity of xanthine oxidant.
또한, 본 발명에서 사용되는 고추냉이는 항고지혈증 효과가 있어 지방 대사를 개선할 수 있다.In addition, horseradish used in the present invention has an antihyperlipidemic effect and can improve fat metabolism.
구체적으로, 고추냉이로부터 제조된 물 추출물 또는 에탄올 추출물은 고밀도 지단백질-콜레스테롤(high density lipoprotein-cholesterol)의 함량은 증가시키면서 저밀도 지단백질-콜레스테롤(low density lipoprotein-cholesterol)의 함량을 억제시킴으로써 항고지혈증 효과를 가지고 있다. 고밀도 지단백질-콜레스테롤은 간과 소장에서 합성되어 세포막, 조직 등에 침착된 유리형의 콜레스테롤을 흡착 제거하는 역할을 가지고 있어 생체에 유익하다. 그러나 저밀도 지단백질-콜레스테롤은 혈액 중에 과량으로 존재할 경우 콜레스테롤의 축적 및 혈액 점도의 상승을 통합 복합적인 작용으로 동맥 경화 및 혈전 형성을 촉진하며 심혈관 질환의 발생을 야기시키는 것으로 알려져 있다. 고추냉이로부터 제조된 물 추출물 또는 에탄올 추출물을 고지혈증에 걸린 랫트에 각각 투여한 결과, 총 콜레스테롤의 함량은 대조군에 비하여 감소하였다. 또한, 고추냉이로부터 제조된 물 추출물 또는 에탄올 추출물을 투여한 랫트에서는 대조군에 비하여 고밀도 지단백질-콜레스테롤의 함량은 증가하였으나, 저밀도 지단백질-콜레스테롤의 함량은 감소하였다. 따라서, 본 발명에 따른 고추냉이를 함유한 기능성 식품은 항고지혈증 효과가 있어 지방 대사 개선 효과를 가지고 있다.Specifically, water extract or ethanol extract prepared from horseradish has an antihyperlipidemic effect by increasing the content of high density lipoprotein-cholesterol while suppressing the content of low density lipoprotein-cholesterol. Have. High-density lipoprotein-cholesterol, which is synthesized in the liver and small intestine, has a role of adsorption and removal of free cholesterol deposited on cell membranes and tissues, which is beneficial to the living body. However, low-density lipoprotein-cholesterol is known to promote arteriosclerosis and thrombus formation by integrating a combination of cholesterol accumulation and elevated blood viscosity when present in excess in blood and cause cardiovascular disease. As a result of administering water extract or ethanol extract prepared from wasabi to rats suffering from hyperlipidemia, the total cholesterol content was decreased compared to the control group. In addition, the rats administered water extract or ethanol extract prepared from horseradish increased the content of high-density lipoprotein-cholesterol, but the low-density lipoprotein-cholesterol content decreased compared to the control group. Therefore, the functional food containing wasabi according to the present invention has an antihyperlipidemic effect and has an effect of improving fat metabolism.
본 발명에서 사용되는 고추냉이는 세절하여 그대로 사용할 수도 있고, 분말 또는 액상('즙'을 포함) 형태로 첨가할 수 있다. Wasabi used in the present invention may be used as it is chopped, and may be added in the form of powder or liquid (including 'juice').
상기 고추냉이는 통상적인 식품에 주성분 또는 부성분으로 첨가할 수 있다. 바람직하게는 항산화 효과 또는 항고지혈증 효과와 관련하여 햄버거 패트, 소세지, 어묵 및 치킨을 포함한 즉석 식품 또는 된장, 고추장, 간장, 시금장, 냉면, 김치, 국수 및 떡을 포함한 전통 식품에 첨가할 수 있다. The wasabi may be added as a main ingredient or a subcomponent to a conventional food. Preferably, it can be added to instant foods including hamburger pat, sausage, fish paste and chicken or traditional foods including miso, red pepper paste, soy sauce, spinach, cold noodles, kimchi, noodles and rice cakes in relation to antioxidant or antihyperlipidemic effects. .
첨가되는 고추냉이의 함량은 미감 및 향취에 따라 조절할 수 있다. 바람직하게는 0.1∼1 중량%로 첨가한다.The content of wasabi may be adjusted according to taste and flavor. Preferably it is added at 0.1 to 1% by weight.
이하, 본 발명을 실시예에 의하여 상세히 설명한다. 그러나, 본 발명이 하기 실시예에 의하여 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of examples. However, the present invention is not limited by the following examples.
<실시예 1> <Example 1>
고추냉이를 함유한 햄버거 패트의 제조Preparation of Hamburger Pats with Wasabi
단계 1: 액상 형태의 고추냉이 추출물 제조Step 1: Preparation of Wasabi Extract in Liquid Form
세절한 고추냉이 잎 부분 400g에 물을 첨가하고, 3회 반복 추출한 후 감압농축하여 물을 제거하였다. 얻어진 잔여물을 동결건조하여 고추냉이 잎의 물 추출물 8.34g을 제조하였다. 상기 물 대신에 에탄올을 사용하여 고추냉이 잎의 에탄올 추출물 7.71g을 제조하였다.Water was added to 400 g of fine wasabi leaf portions, and extracted three times, and then concentrated under reduced pressure to remove water. The residue obtained was lyophilized to prepare 8.34 g of water extract of horseradish leaves. Ethanol was used in place of the water to prepare 7.71 g of ethanol extract of horseradish leaves.
상기 고추냉이 잎 대신에 고추냉이 뿌리를 사용하고, 동일한 방법을 실시하여 고추냉이 뿌리의 물 추출물 6.56g 또는 고추냉이 뿌리의 에탄올 추출물 7.40g을 제조하였다.Wasabi roots were used instead of the wasabi leaves, and the same method was carried out to prepare 6.56 g of wasabi root extract or 7.40 g of ethanol extract of wasabi root.
단계 2: 고추냉이를 함유한 햄버거 패트의 제조Step 2: Preparation of Hamburger Pats with Wasabi
돼지 고기를 1/4 인치로 갈은 후, 하기 표 1에 기재되어 있는 양으로 소금(NaCl), 간장(Soy sauce), 후추(Pepper), 아질산 나트륨(Sodium nitrite), 설탕(Sugar), 분리한 콩 단백질(ISP, isolated soybean protein), L-아스코르브산(L-ascorbic acid), 밀렛 젤리(Millet jelly), 친수성 미네랄(water soluble mineral), 참기름(Sesame oil) 및 상기 단계 1에서 제조한 고추냉이 추출물을 첨가하였다. 상기 혼합물을 잘 혼합한 후, 70g 크기로 햄버거 패트 형태를 만들고 냉동시켰다. After grinding the pork to 1/4 inch, salt (NaCl), soy sauce, pepper (Sodium nitrite), sugar (Sugar), separation in the amount described in Table 1 below Isolated soybean protein (ISP), L-ascorbic acid, millet jelly, water soluble mineral, sesame oil and pepper prepared in step 1 Wasabi extract was added. After the mixture was mixed well, a burger pat was formed into a 70 g size and frozen.
<실시예 2> <Example 2>
고추냉이를 함유한 소세지의 제조Preparation of Sausage Containing Wasabi
단계 1: 액상 형태의 고추냉이 추출물 제조Step 1: Preparation of Wasabi Extract in Liquid Form
상기 실시에 1의 단계 1과 동일한 방법으로 액상 형태의 고추냉이 추출물을 제조하였다.Wasabi extract was prepared in the same manner as in step 1 of Example 1 in the liquid form.
단계 2: 고추냉이를 함유한 소세지의 제조Step 2: Preparation of Sausage Containing Wasabi
돼지고기를 3∼4cm로 모나게 썰어 소금과 질산염 및 아질산염(발색제)을 첨가하고, 24시간 정도 큐어링 한 다음, 이것을 갈아 조미료, 부재료와 상기 단계 1에서 제조한 고추냉이 추출물을 0.1∼1.0중량% 첨가하고 혼합한 후, 케이싱에 넣어 삶거나 훈연, 건조시켜 소세지를 제조하였다.Slice the pork into 3 to 4 cm, add salt, nitrate and nitrite (coloring agent), cure for about 24 hours, grind it and season it with 0.1 ~ 1.0 weight of horseradish extract prepared in step 1 After adding and mixing the%, boiled in a casing, smoked, dried to prepare a sausage.
본 발명에서 사용되는 고추냉이의 항산화 효과 및 항고지혈증 효과를 알아보 기 위하여 상기 실시예 1의 단계 1과 동일한 방법으로 고추냉이 잎의 물 추출물(WJL-W), 고추냉이 잎의 에탄올 추출물(WJL-E), 고추냉이 뿌리의 물 추출물(WJR-W) 또는 고추냉이 뿌리의 에탄올 추출물(WJR-E)을 제조하고 실험에 사용하였다.Water extract of wasabi leaves (WJL-W), ethanol extract of wasabi leaves (WJL) in the same manner as in step 1 of Example 1 to determine the antioxidant and antihyperlipidemic effect of wasabi used in the present invention -E), wasabi root water extract (WJR-W) or wasabi root ethanol extract (WJR-E) was prepared and used in the experiment.
<실험예 1>Experimental Example 1
고추냉이의 DPPH에 대한 수소 공여능 측정Determination of Hydrogen Donating Ability for DPPH in Wasabi
상기 실시예 1의 단계 1에서 제조한 고추냉이 추출물 WJL-W, WJL-E, WJR-W, WJR-E 각각에 메탄올을 첨가하여 100, 200, 400 및 800 ㎍/㎖ 농도의 희석액을 제조하였다. 상기 희석액에 5 ×10-4M DPPH 용액 500 ㎕을 동량으로 첨가하고, 10 초동안 진탕한 후 517nm에서 반응액의 흡광도를 측정하였다(시료군의 흡광도). 또한, 동량으로 5 ×10-4M DPPH 용액 500 ㎕를 대조군으로 사용하여 517 nm에서 반응액의 흡광도를 측정하였다(대조군의 흡광도). 항산화 효과는 대조군에 대하여 50%로 흡광도를 감소시키는 농도(IC50)로 측정하였다. 또한, 상기 시료군의 흡광도 및 대조군의 흡광도를 이용하여 하기 수학식 1에 의해 농도에 따른 DPPH의 소거능을 측정하였다. Methanol was added to each of the wasabi extracts WJL-W, WJL-E, WJR-W, and WJR-E prepared in Step 1 of Example 1 to prepare dilutions of 100, 200, 400, and 800 μg / ml. . 500 μl of a 5 × 10 −4 M DPPH solution was added to the diluted solution in the same amount, shaken for 10 seconds, and the absorbance of the reaction solution was measured at 517 nm (absorbance of the sample group). In addition, the absorbance of the reaction solution was measured at 517 nm using the same amount of 500 × 5 × 10 -4 M DPPH solution as a control (absorbance of the control group). Antioxidant effect was measured at a concentration (IC 50 ) that reduced the absorbance by 50% relative to the control. In addition, the scavenging ability of DPPH according to the concentration was measured by the following equation 1 using the absorbance of the sample group and the absorbance of the control group.
그 결과는 표 2에 나타내었다.The results are shown in Table 2.
상기 표 2에서 보여지는 바와 같이, 고추냉이 잎 또는 뿌리로부터 추출한 물 추출물과 에탄올 추출물의 농도에 따라 DPPH의 소거능이 증가함을 알 수 있다. 농도에 따른 DPPH의 소거능은 대조군에 대한 시료군의 DPPH의 소거능을 나타낸다. 따라서, 본 발명에서 사용되는 고추냉이는 대조군에 비하여 DPPH에 대한 수소 공여능이 뛰어남을 알 수 있다. 특히, 고추냉이 잎의 물 추출물과 고추냉이 잎의 에탄올 추출물의 IC50 은 각각 558.1 ㎍/㎖ 및 644.9 ㎍/㎖이었다. 따라서, 본 발명에 따른 고추냉이를 함유하는 김치는 DPPH에 대한 수소 공여능이 뛰어나 항산화 효과를 가지고 있다. As shown in Table 2, it can be seen that the scavenging ability of DPPH increases with the concentration of water extract and ethanol extract extracted from horseradish leaves or roots. The scavenging ability of DPPH according to the concentration indicates the scavenging ability of DPPH in the sample group relative to the control group. Thus, wasabi used in the present invention can be seen that the hydrogen donating ability for DPPH is superior to the control. In particular, the IC 50 of the water extract of wasabi leaves and the ethanol extract of wasabi leaves were 558.1 μg / ml and 644.9 μg / ml, respectively. Therefore, kimchi containing wasabi according to the present invention has an excellent hydrogen donating ability against DPPH and has an antioxidant effect.
<실험예 2>Experimental Example 2
고추냉이의 산화 질소 소거 효과 측정Measurement of Nitric Oxide Scavenging Effect of Wasabi
상기 실시예 1의 단계 1에서 제조한 고추냉이 잎의 물 추출물(WJL)을 이용하고, 통상적으로 알려진 산화 질소 소거능 측정 방법(Marcocci, Maguire, Droy-Kefaiz, Packer)을 이용하여 고추냉이의 산화 질소 소거능을 측정하였다. 구체적으로는 상기 추출물 4㎖에 25mM 소디움 니트로프루시드액(sodium nitroprusside) 0.5㎖를 첨가하고 25℃에서 2시간 30분 동안 반응시켰다. 반응 혼합물에 그리스 시약 100㎕(5% phosphoric acid 에 1% sulfanilamide이 있는 용액 + 1% α-naphthylamide 수용액)를 첨가하고 실온에서 10분 동안 방치한 후, 540nm에서 ELISA 판독기(ELISA reader)로 흡광도를 측정하였다(시료군의 흡광도). 또한, 반응 혼합물을 넣지 않은 것을 제외하고는 상기와 동일한 방법으로 대조군의 흡광도를 측정하였다(대조군의 흡광도). 이를 이용하여 하기 수학식 2에 의해 농도에 따른 산화질소의 소거능(% inhibition)을 측정하였다. Nitric oxide of horseradish using wasabi extract (WJL) prepared in step 1 of Example 1, using a commonly known method of measuring nitric oxide scavenging ability (Marcocci, Maguire, Droy-Kefaiz, Packer) Scavenging ability was measured. Specifically, 0.5 ml of 25 mM sodium nitroprusside was added to 4 ml of the extract and reacted at 25 ° C. for 2
그 결과를 표 3에 나타내었다.The results are shown in Table 3.
상기 표 3에서 보여지는 바와 같이, 고추냉이 잎 또는 뿌리로부터 추출한 물 추출물과 에탄올 추출물의 농도에 따라 산화 질소의 소거능이 증가함을 알 수 있다. 농도에 따른 산화 질소의 소거능은 대조군에 대한 시료군의 산화 질소의 소거능을 나타낸다. 따라서, 본 발명에서 사용되는 고추냉이는 대조군에 비하여 산화 질소에 대한 수소 공여능이 뛰어남을 알 수 있다. As shown in Table 3, it can be seen that the scavenging ability of nitric oxide increases with the concentration of water extract and ethanol extract extracted from horseradish leaves or roots. The scavenging ability of nitric oxide according to the concentration indicates the scavenging ability of nitric oxide in the sample group to the control group. Thus, wasabi used in the present invention can be seen that the hydrogen donating ability to the nitrogen oxide is superior to the control.
<실험예 3>Experimental Example 3
고추냉이의 산화 질소 생성 억제 효과Inhibitory Effect of Wasabi on Nitric Oxide Production
고추냉이가 대식 세포에서 LPS 자극에 의한 산화 질소의 생성에 미치는 영향을 알아보기 위하여 하기와 같은 실험을 수행하였다.To investigate the effect of horseradish on the production of nitric oxide by LPS stimulation in macrophages, the following experiment was performed.
LPS 1 ㎍/㎖와 고추냉이 잎의 물 추출물(WJL) 100, 200, 400 및 800 ㎍/㎖의 혼합물을 각각 처리한 RAW 264.7 세포주를 37℃에서 배양하고, 통상적인 방법을 수행하여 세포 배양 상등액을 취하였다. 세포 배양 상등액 100㎕에 그리스 시약 100㎕(5% phosphoric acid 에 1% sulfanilamide이 있는 용액 + 1% α-naphthylamide 수용액)를 첨가하고, 96 웰 플레이트에서 10분 동안 반응시킨 후 540 nm에서 ELISA 판독기(ELISA reader)로 흡광도를 측정하였다. 대조군으로서 LPS 1 ㎍/㎖를 처리한 RAW 264.7 세포주에 대해서도 상기와 동일한 방법을 실시하고, 흡광도를 측정하였다. 그 결과를 도 1에 나타내었다.RAW 264.7 cell lines treated with a mixture of 1 μg / ml LPS and 100, 200, 400, and 800 μg / ml water extract of horseradish leaf (WJL), respectively, were incubated at 37 ° C., and cultured supernatant by conventional methods. Was taken. To 100 µl of the cell culture supernatant, 100 µl of the grease reagent (a solution containing 1% sulfanilamide in 5% phosphoric acid + an aqueous 1% α-naphthylamide solution) was added and reacted in a 96 well plate for 10 minutes, followed by an ELISA reader at 540 nm. The absorbance was measured with an ELISA reader. As a control, a RAW 264.7 cell line treated with 1 μg / ml of LPS was also subjected to the same method as above, and the absorbance was measured. The results are shown in FIG.
도 1에서 보여지는 바와 같이, LPS 1 ㎍/㎖를 단독으로 처리한 RAW 264.7 세포주로부터 생성된 산화 질소는 9.08 ±0.09 μM이었으나, LPS 1 ㎍/㎖와 고추냉이 잎의 물 추출물을 처리한 RAW 264.7 세포주로부터 생성된 산화 질소는 6.67 ±0.03 μM, 6.07 ±0.11 μM, 5.69 ±0.03 μM 및 5.32 ±0.08 μM로 27∼41%의 산화 질소 생성 억제 효과를 보여주었다. 따라서, 본 발명에 따른 고추냉이를 함유하는 기능성 식품은 산화 질소 생성 억제하여 항산화 효과를 가지고 있음을 알 수 있다.As shown in FIG. 1, the nitric oxide produced from the RAW 264.7 cell line treated with 1 μg / ml of LPS alone was 9.08 ± 0.09 μM, but RAW 264.7 treated with 1 μg / ml of LPS and water extract of horseradish leaves. Nitric oxide produced from the cell line showed 6.67 ± 0.03 μM, 6.07 ± 0.11 μM, 5.69 ± 0.03 μM, and 5.32 ± 0.08 μM, showing 27-41% inhibition of nitric oxide production. Therefore, it can be seen that the functional food containing wasabi according to the present invention has an antioxidant effect by inhibiting the production of nitric oxide.
<실험예 4>Experimental Example 4
고추냉이의 iNOS mRNA 발현 및 iNOS 발현 억제 효과Inhibitory Effects of Horseradish on iNOS mRNA and iNOS Expression
고추냉이가 대식 세포에서 LPS 자극에 의한 iNOS mRNA 발현 및 iNOS 발현에 미치는 영향을 알아보기 위하여 하기와 같은 실험을 수행하였다.To investigate the effect of horseradish on iNOS mRNA expression and iNOS expression by LPS stimulation in macrophages, the following experiment was performed.
LPS 1㎍/㎖와 고추냉이 잎의 물 추출물(WJL-W) 200, 400 및 800 ㎍/㎖의 혼합물을 각각 처리한 RAW 264.7 세포주를 37℃에서 24시간 동안 배양하였다. 하기에 제시되어 있는 방법을 이용하여 배양한 세포로부터 전체 RNA를 분리하고, RT-PCR 방법에 의해 iNOS mRNA를 분석하였다. RAW 264.7 cell lines treated with a mixture of 1 μg / ml LPS and 200, 400 and 800 μg / ml water extract of horseradish leaves (WJL-W), respectively, were incubated at 37 ° C. for 24 hours. Total RNA was isolated from the cultured cells using the method presented below, and iNOS mRNA was analyzed by RT-PCR method.
RNA 분리는 TRIzol을 이용하여 분리하였다. 구체적으로, 상기 배양한 RAW 264.7 세포에서 iNOS mRNA를 분리하기 위하여 0.1% DEPC(Diethyl pyrocarbonate)가 첨가된 PBS로 세포를 3회 세척 후, TRIzol 900 ㎕를 첨가하여 균질화시켰다. 여기에 클로로포름 100 ㎕를 넣고 15분 동안 얼음에서 정치시켰다. 그 후 4℃, 12,000 rpm에서 15분 동안 원심 분리하고 상층을 조심스럽게 취하였다. 동량의 이소프로판올을 첨가하여 -20℃에서 45분 정치한 후 원침하고, 70% DEPC-에탄올로 1회 세척하였다. 분리한 RNA를 실온에서 건조시킨 후, DEPC가 첨가된 증류수로 일정량 희석하여 스펙트로포토미터에서 농도를 결정하였다. RNA isolation was performed using TRIzol. Specifically, in order to separate iNOS mRNA from the cultured RAW 264.7 cells, cells were washed three times with PBS to which 0.1% DEPC (Diethyl pyrocarbonate) was added, followed by homogenization by adding 900 μl of TRIzol. 100 µl of chloroform was added thereto and allowed to stand on ice for 15 minutes. It was then centrifuged at 4 ° C., 12,000 rpm for 15 minutes and the upper layer was taken carefully. The same amount of isopropanol was added, left at −20 ° C. for 45 minutes, then immersed, and washed once with 70% DEPC-ethanol. After the isolated RNA was dried at room temperature, a predetermined amount was diluted with distilled water to which DEPC was added to determine the concentration on a spectrophotometer.
5 ×RT(Reverse transcription) buffer 2 ㎕, 10 mM dATP 0.25 ㎕, 10 mM dGTP 0.25 ㎕, 10 mM dTTP 0.25 ㎕, 10 mM dCTP 0.25 ㎕, MMLV 역전사효소(200 U/㎕) 0.25 ㎕, RNase 저해제 (28 U/㎕) 0.25 ㎕, 50 μM 올리고 dT 프라이머 0.5 ㎕, DEPC-DW 4㎕를 PCR 시험관에 넣어 RT-혼합물을 만들고, 여기에 상기 분리한 전 체 RNA를 첨가하였다. 이 시험관을 PCR 기계(PTC-100TM Programmable Thermal Controllar; MJResearch, Inc.)에 넣고, 42℃에서 60분 동안 열처리하여 역전사 반응을 완료하였다. PCR은 먼저 10 ×PCR buffer 3 ㎕, 25 mM MgCl2 1.8 ㎕, 10 mM dATP 0.3 ㎕, 10 mM dGTP 0.3 ㎕, 10 mM dTTP 0.3 ㎕, 10 mM dCTP 0.3 ㎕, 50 μM 센스 및 안티센스 프라이머 0.25 ㎕, Taq 폴리머레이즈(5 U/㎕, Promega Co.) 0.25 ㎕를 혼합하고, 여기에 증류수를 넣어 최종 용액량이 20 ㎕되게 하여 PCR 혼합물을 제조하였다. 상기 PCR 혼합물을 PCR 시험관에 넣고, 여기에 역전사 반응물 5 ㎕를 넣고 혼합한 뒤 PCR 기기에 넣어 다음의 조건으로 PCR을 실시하였다. PCR 반응은 94℃에서 3분 동안 1회 반응 후, 94℃에서 45초, 57℃에서 45초, 72℃에서 45초간 35회 반응시켰으며, 72℃에서 10분 동안 연장(extension)을 시행한 후 반응을 완료시켰다. 증폭된 산물은 1.2% 아가로즈 겔에 전기 영동하여 UV 자외선 투과기(UV transilluminater)를 이용하여 분리된 DNA 밴드를 확인하였다. 2 μl 5 × RT (Reverse transcription) buffer, 0.25 μl 10 mM dATP, 0.25 μl 10 mM dGTP, 0.25 μl 10 mM dTTP, 0.25 μl 10 mM dCTP, 0.25 μl MMLV reverse transcriptase (200 U / μl), RNase inhibitor ( 28 U / μL) 0.25 μL, 50 μM oligo dT primer 0.5 μL, and 4 μL DEPC-DW were placed in a PCR tube to make an RT-mixture, to which the whole RNA isolated was added. This test tube was placed in a PCR machine (PTC-100 ™ Programmable Thermal Controllar; MJResearch, Inc.) and heat treated at 42 ° C. for 60 minutes to complete the reverse transcription reaction. PCR was first performed with 3 μl of 10 × PCR buffer, 1.8 μl of 25 mM MgCl 2 , 0.3 μl of 10 mM dATP, 0.3 μl of 10 mM dGTP, 0.3 μl of 10 mM dTTP, 0.3 μl of 10 mM dCTP, 0.25 μl of 50 μM sense and antisense primer, 0.25 μl of Taq polymerase (5 U / μl, Promega Co.) was mixed and distilled water was added thereto so that the final solution amount was 20 μl to prepare a PCR mixture. The PCR mixture was placed in a PCR test tube, 5 μl of reverse transcription reactant was added thereto, mixed, and placed in a PCR device, and PCR was performed under the following conditions. PCR reaction was performed once for 3 minutes at 94 ° C, then for 45 seconds at 94 ° C, 45 seconds at 57 ° C, 45 times at 72 ° C, and extended at 72 ° C for 10 minutes. After the reaction was completed. The amplified product was electrophoresed on a 1.2% agarose gel to identify the separated DNA bands using a UV transilluminater.
LPS 1㎍/㎖만 단독으로 처리한 RAW 264.7 세포주, LPS 1㎍/㎖와 고추냉이 잎의 물 추출물의 혼합물을 처리하지 않은 RAW 264.7 세포주에 대해서도 상기와 동일한 방법을 실시하였다. The same method was performed for the RAW 264.7 cell line treated with only LPS of 1 μg / ml, and the RAW 264.7 cell line not treated with the mixture of 1 μg / ml of LPS and water extract of horseradish leaves.
크기 마커(size marker)인 대조 DNA 밴드로 G3PDH를 사용하여 상기와 동일한 방법으로 전기 영동하였다. Electrophoresis was performed in the same manner as above using G3PDH as a control DNA band as a size marker.
그 결과를 도 2에 나타내었다.The results are shown in FIG.
도 2에서 M은 대조 DNA 밴드로 G3PDH를 의미한다.In Figure 2, M means G3PDH as the control DNA band.
도 2에서 보여지는 바와 같이, LPS만 단독으로 처리한 RAW 264.7 세포주와 LPS 및 본 발명에 따른 고추냉이 잎의 물 추출물의 혼합물을 처리한 RAW 264.7 세포주로부터 분리한 iNOS mRNA를 각각 PCR 반응시키고, 생성된 DNA를 전기 영동하여 비교하였을 때, LPS 및 본 발명에 따른 고추냉이 잎의 물 추출물의 혼합물을 처리한 RAW 264.7 세포주로부터 생성된 iNOS mRNA가 감소하였음을 알 수 있다. 즉, LPS 자극에 의해 iNOS mRNA의 발현 및 iNOS 발현이 강하게 유도되었으나, LPS와 고추냉이 잎의 물 추출물의 혼합물을 처리함으로써 iNOS mRNA의 발현이 고추냉이 잎의 물 추출물의 농도 의존적으로 감소됨을 알 수 있다. As shown in FIG. 2, PCR reaction was performed for iNOS mRNA isolated from a RAW 264.7 cell line treated with LPS alone and a RAW 264.7 cell line treated with a mixture of LPS and wasabi leaves according to the present invention, respectively. Compared by electrophoresis, the iNOS mRNA generated from the RAW 264.7 cell line treated with the mixture of LPS and the water extract of horseradish leaves according to the present invention was reduced. In other words, the expression of iNOS mRNA and iNOS were strongly induced by LPS stimulation, but the expression of iNOS mRNA was reduced depending on the concentration of water extract of horseradish leaf by treating the mixture of LPS and wasabi leaf. have.
고추냉이가 대식 세포에서 LPS 자극에 의한 iNOS 단백질 발현에 미치는 영향을 알아보기 위하여 하기와 같은 실험을 수행하였다. To investigate the effect of horseradish on iNOS protein expression by LPS stimulation in macrophages, the following experiment was performed.
LPS 1㎍/㎖와 고추냉이 잎의 물 추출물(WJL-W) 200, 400 및 800 ㎍/㎖의 혼합물을 각각 처리한 RAW 264.7 세포주를 37℃에서 24시간 동안 배양하였다. 하기에 제시되어 있는 방법을 이용하여 배양한 세포로부터 전체 단백질을 분리하고, 웨스턴 블롯(Western blotting) 방법에 의해 iNOS 단백질을 분석하였다. RAW 264.7 cell lines treated with a mixture of 1 μg / ml LPS and 200, 400 and 800 μg / ml water extract of horseradish leaves (WJL-W), respectively, were incubated at 37 ° C. for 24 hours. Total protein was isolated from the cultured cells using the method shown below, and iNOS protein was analyzed by Western blotting method.
세포에서의 단백질 분리는 lysis buffer(50mM Tris-Cl, 25mM EDTA, 650mM NaCl, 5% Triton X-100, 100 X PMSF, a 100X protease inhibitor coctail, 5X lysis buffer)를 이용하여 분리하였다. 추출된 단백질을 95℃에서 5분간 가열한 후 12% SDS-폴리아크릴아미드 겔에 전기영동 후 니트로셀룰로우스 막(Protran®, nitrocellulose membrane)에 전이시켰다. 10% 스킴 밀크(skim milk)를 함유한 PBST(0.1% Tween 20 in PBS)를 이용하여 배양(incubation)하면서 비특이적인 단백질에 대한 블록킹(blocking)을 실시하고, anti-iNOS 항체와 함께 배양한 후 ECL 용액을 적용시킨 다음 X-ray 필름에 감광시켜 iNOS 단백질 발현을 조사하였다. 대조 단백질 밴드로 HSP70을 사용하여 상기와 동일한 방법으로 관찰하였다.Protein separation was performed using lysis buffer (50mM Tris-Cl, 25mM EDTA, 650mM NaCl, 5% Triton X-100, 100X PMSF, a 100X protease inhibitor coctail, 5X lysis buffer). The extracted protein was heated at 95 ° C. for 5 minutes, followed by electrophoresis on a 12% SDS-polyacrylamide gel, and then transferred to a nitrocellulose membrane (Protran®, nitrocellulose membrane). Blocking of nonspecific proteins while incubating with PBST (0.1
그 결과를 도 3에 나타내었다.The results are shown in FIG.
도 3에서 M은 대조 DNA 밴드로 HSP70를 의미한다.In Figure 3, M means HSP70 as the control DNA band.
도 3에서 보여지는 바와 같이, LPS만 단독으로 처리한 RAW 264.7 세포주와 LPS 및 본 발명에 따른 고추냉이 잎의 물 추출물의 혼합물을 처리한 RAW 264.7 세포주로부터 분리한 iNOS 단백질을 전기 영동하여 비교하였을 때, LPS 및 본 발명에 따른 고추냉이 잎의 물 추출물의 혼합물을 처리한 RAW 264.7 세포주로부터 생성된 iNOS 발현이 감소하였음을 알 수 있다. 즉, LPS 자극에 의해 iNOS 발현이 강하게 유도되었으나, LPS와 고추냉이 잎의 물 추출물의 혼합물을 처리함으로써 iNOS의 발현이 고추냉이 잎의 물 추출물의 농도 의존적으로 감소됨을 알 수 있다. As shown in FIG. 3, iNOS protein isolated from a RAW 264.7 cell line treated with LPS alone and a RAW 264.7 cell line treated with a mixture of LPS and wasabi leaves according to the present invention was subjected to electrophoresis. It can be seen that iNOS expression generated from RAW 264.7 cell line treated with a mixture of LPS and water extracts of horseradish leaves according to the present invention was reduced. That is, although iNOS expression was strongly induced by LPS stimulation, it was found that iNOS expression was reduced depending on the concentration of water extract of horseradish leaves by treating a mixture of LPS and wasabi leaves.
따라서, 본 발명에 따른 고추냉이를 함유한 기능성 식품은 iNOS mRNA의 발현 및 iNOS 발현을 억제하여 산화 질소 생성을 감소시킬 수 있다.Therefore, the functional food containing wasabi according to the present invention can suppress the expression of iNOS mRNA and iNOS expression to reduce the nitric oxide production.
<실험예 5>Experimental Example 5
고추냉이의 잔틴 산화제 저해 효과Xanthine oxidant inhibitory effect of horseradish
고추냉이가 생체내에서 슈퍼옥사이드 라디칼을 생성하는 잔틴 산화제의 활성을 억제하는 효과가 있는지 알아보기 위하여 하기와 같은 실험을 수행하였다.In order to determine whether horseradish has an effect of inhibiting the activity of xanthine oxidant that generates superoxide radicals in vivo, the following experiment was performed.
대한 바이오 링크(충북, 음성)로부터 웅성 랫트(Sprague-Dawley계, 체중 200g)를 분양받아 실험 전 1 주일동안 순화시키고 실험에 사용하였다. 사육 기간 동안 물과 사료는 자유로이 섭취시키고, 사육실의 온도는 22 ±2℃, 습도는 55 ±2% 로 유지하였으며, 명암은 12 시간 주기로 조명하였다.Male rats (Sprague-Dawley system, body weight 200g) were distributed from Daehan Biolink (Chungbuk, Negative) and purified for 1 week before the experiment and used for the experiment. During the breeding period, water and feed were freely ingested, the room temperature was maintained at 22 ± 2 ° C, the humidity at 55 ± 2%, and the contrast was illuminated at 12 hour intervals.
상기 웅성 랫트에 콜레스테롤 함량이 높은 식이를 4주일 동안 투여하고, 정상 식이로 투여시에 고추냉이 잎을 5% 첨가하여 2 주일 동안 투여하였다(WJL5군). 이로부터 간 조직을 분리하고, 잔틴 기질을 첨가한 후 30℃에서 10분 동안 반응시켜 생성된 요산의 함량을 292nm에서 측정함으로써, 간 조직에 함유되어 있는 잔틴 산화제의 활성도를 측정하였다(Stripe와 Della Corte의 방법을 이용하였음). 잔틴 산화제의 활성도는 간 조직에 함유되어 있는 잔틴 산화제 1mg이 1분 동안 잔틴과 반응하여 생성한 요산의 양을 nmol 농도로 표시하였다.A high cholesterol diet was administered to the male rats for 4 weeks, and when administered as a normal diet, 5% of horseradish leaves were added for 2 weeks (WJL5 group). From this, the liver tissue was separated, and after the addition of the xanthine substrate, the reaction was carried out at 30 ° C. for 10 minutes to determine the content of the produced uric acid at 292 nm, thereby measuring the activity of the xanthine oxidant contained in the liver tissue (Stripe and Della). Using the Corte method). Xanthine oxidant activity was expressed in nmol concentration of the amount of uric acid produced by reacting xantine for 1 minute with 1 mg of xanthine oxidant contained in liver tissue.
상기에서 고추냉이 잎 대신에 고추냉이 뿌리를 5% 첨가하여 정상 식이와 함께 2 주일 동안 투여하고(WJR5군), 상기와 동일한 방법으로 잔틴 산화제의 활성도를 측정하였다.Instead of wasabi leaves was added 5% horseradish root for 2 weeks with a normal diet (WJR5 group), the activity of xanthine oxidant was measured in the same manner as above.
대조군으로서 콜레스테롤 함량이 높은 식이를 4주일 동안 투여하고, 정상 식이만 2 주일 동안 투여한 웅성 랫트에 대해서도 상기와 동일한 방법으로 잔틴 산화제의 활성도를 측정하였다. As a control, the high cholesterol content was administered for 4 weeks, and the normal rats were administered for 2 weeks, and the activity of xanthine oxidant was measured in the same manner as above.
또한, 정상군으로서 정상 식이를 6 주일 동안 투여한 웅성 랫트에 대해서도 상기와 동일한 방법으로 잔틴 산화제의 활성도를 측정하였다.In addition, the activity of the xanthine oxidant was measured in the same manner as above for the male rats that received the normal diet for 6 weeks as a normal group.
그 결과를 표 4에 나타내었다.The results are shown in Table 4.
상기 표 4에서 보여지는 바와 같이, 대조군의 경우 잔틴 산화제의 활성도가 1.91 ±0.12 nmol/mg 단백질/min 이었으나, 본 발명에서 사용되는 고추냉이의 잎 또는 뿌리를 정상 식이와 함께 투여한 경우 잔틴 산화제의 활성도가 1.46 ±0.04 nmol/mg 단백질/min 또는 1.34 ±0.02 nmol/mg 단백질/min로 감소하였다. 이를 통해 고추냉이가 잔틴 산화제의 활성도를 억제할 수 있음을 알 수 있다. 따라서, 본 발명에 따른 고추냉이를 함유하는 기능성 식품은 잔틴 산화제의 활성을 억제하여 슈퍼옥사이드 라디칼의 생성을 감소시킴으로써 항산화 효과를 가지고 있다.As shown in Table 4, the activity of the xanthine oxidant in the control group was 1.91 ± 0.12 nmol / mg protein / min, but when the leaves or roots of horseradish used in the present invention were administered with a normal diet, Activity was reduced to 1.46 ± 0.04 nmol / mg protein / min or 1.34 ± 0.02 nmol / mg protein / min. This shows that wasabi can inhibit the activity of xanthine oxidant. Therefore, the functional food containing wasabi according to the present invention has an antioxidant effect by reducing the production of superoxide radicals by inhibiting the activity of xanthine oxidant.
<실험예 6>Experimental Example 6
고추냉이의 항고지혈증 효과Antihyperlipidemic Effect of Wasabi
(1) 고지혈증 유발 및 실험 동물의 처리(1) hyperlipidemia induction and treatment of experimental animals
대한 바이오 링크(충북, 음성)로부터 웅성 랫트(Sprague-Dawley계, 체중 200g)를 분양받아 실험 전 1 주일동안 순화시키고 실험에 사용하였다. 사육 기간 동안 물과 사료는 자유로이 섭취시키고, 사육실의 온도는 22 ±2℃, 습도는 55 ±2% 로 유지하였으며, 명암은 12 시간 주기로 조명하였다.Male rats (Sprague-Dawley system, body weight 200g) were distributed from Daehan Biolink (Chungbuk, Negative) and purified for 1 week before the experiment and used for the experiment. During the breeding period, water and feed were freely ingested, the room temperature was maintained at 22 ± 2 ° C, the humidity at 55 ± 2%, and the contrast was illuminated at 12 hour intervals.
상기 웅성 랫트를 군당 5 마리씩 4 개의 군으로 나누어 실험을 실시하였는데, 구체적으로는 고지혈증을 유발시키고 정상 식이를 투여한 대조군, 고지혈증을 유발시키고 정상 식이와 고추냉이 잎을 함께 투여한 WJL5군, 고지혈증을 유발시키고 정상 식이와 고추냉이 뿌리를 함께 투여한 WJR5군 및 고지혈증을 유발시키지 않고 정상 식이만을 투여한 정상군으로 나누어 실험을 실시하였다.The male rats were divided into four groups of five rats per group, and the experiments were performed. The experiment was conducted by dividing the WJR5 group, which was administered with the normal diet and wasabi root, and the normal group, which was administered only the normal diet without causing hyperlipidemia.
또한, 정상 식이 투여시에 고추냉이 잎 또는 뿌리를 함께 투여하면서 체중 변화를 알아보기 위하여 실험을 실시하기 전에 랫트의 평균 체중(A)을 측정하였다.In addition, the average body weight (A) of the rat was measured before the experiment to determine the weight change while administering wasabi leaves or roots at the time of normal diet.
고지혈증 유발은 노 등의 방법에 준하여 식이를 제조하고 실시하였다. 구체적으로 고지혈증 유발 식이는 일반 사료 100g에 콜레스테롤 1g, 콜린 산(cholic acid)0.25g, 올리브 오일(olive oil)2.5g으로 구성되어 있다. 상기 웅성 랫트 15 마리에는 상기에서 제조한 고지혈증 유발 식이를 4 주일 동안 투여하였다. 고지혈증 유발 유무를 확인하고 채혈한 후, 채혈한 혈액을 2 시간 동안 냉장 상태에서 방치시키고 3,000 rpm에서 15분 동안 원심 분리하여 혈청을 분리하였다. 분리한 혈청을 혈청 지질 함량 측정용 키트(아산 제약 주식회사)를 사용하여 총 콜레스테롤, 트리글리세라이드 및 고밀도 지단백질-콜레스테롤을 측정하였다. 또한, 저밀도 지단백질-콜레스테롤은 상기 측정치 및 하기 수학식 3에 의해 그 값을 산출하였다.Induction of hyperlipidemia was prepared and carried out according to the method of the furnace. Specifically, the hyperlipidemia-induced diet is composed of 100 g of general feed, 1 g of cholesterol, 0.25 g of cholic acid, and 2.5 g of olive oil. The male 15 rats were administered the hyperlipidemia induced diet prepared above for 4 weeks. After confirming whether hyperlipidemia was induced and collecting blood, the collected blood was left in a cold state for 2 hours and centrifuged at 3,000 rpm for 15 minutes to separate serum. The separated serum was measured for total cholesterol, triglycerides and high density lipoprotein-cholesterol using a kit for measuring serum lipid content (Asan Pharmaceutical Co., Ltd.). In addition, the low density lipoprotein-cholesterol was calculated by the above measured value and the following equation (3).
이로부터 총 콜레스테롤(total cholesterol, TC), 저밀도 지단백질-콜레스테롤(LDL), 고밀도 지단백질-콜레스테롤(HDL) 및 트리글리세라이드를 얻었다. 그 결과는 표 5에 나타내었다. From this, total cholesterol (TC), low density lipoprotein-cholesterol (LDL), high density lipoprotein-cholesterol (HDL) and triglycerides were obtained. The results are shown in Table 5.
또한, 정상군 5 마리에게는 정상 식이를 4 주일 동안 투여하고, 상기와 동일한 방법으로 총 콜레스테롤, 저밀도 지단백질-콜레스테롤, 고밀도 지단백질-콜레스테롤 및 트리글리세라이드를 얻었다. 그 결과는 표 5에 나타내었다.In addition, 5 normal groups were administered a normal diet for 4 weeks, and total cholesterol, low density lipoprotein-cholesterol, high density lipoprotein-cholesterol, and triglyceride were obtained in the same manner as above. The results are shown in Table 5.
상기 표 5에서 보여지는 바와 같이, 고지혈증에 걸린 웅성 랫트의 총 콜레스테롤 및 저밀도 지단백질-콜레스테롤은 정상군에 비하여 증가하였고, 고밀도 지단백질-콜레스테롤은 감소하였다. As shown in Table 5, the total cholesterol and low density lipoprotein-cholesterol of male rats with hyperlipidemia increased compared to the normal group, and the high density lipoprotein-cholesterol decreased.
상기 결과를 참고로 하여 고지혈증에 걸린 웅성 랫트를 WJL5군, WJR5군 및 대조군으로 나누고 실험을 진행하였다. Referring to the above results, male rats suffering from hyperlipidemia were divided into WJL5 group, WJR5 group, and control group, and the experiment was conducted.
WJL5군은 정상 식이와 세절한 고추냉이 잎 5%를 혼합하여 2 주일 동안 투여하며, WJR5군은 정상 식이와 세절한 고추냉이 뿌리 5%를 혼합하여 2 주일 동안 투여하였다. 대조군은 정상 식이를 2주일 동안 투여하였다. 그리고 나서, WJL5군, WJR5군 및 대조군에 대하여 12시간 동안 절식시키고 실험에 사용하였다.WJL5 group was administered for 2 weeks by mixing 5% of normal diet and sliced wasabi leaves, and WJR5 group was administered for 2 weeks by mixing 5% of normal diet and 5% wasabi root. The control group received a normal diet for 2 weeks. Then, fasting for 12 hours for the WJL5 group, WJR5 group and the control group and used in the experiment.
또한, 정상군은 정상 식이를 2 주일 동안 투여한 후 12시간 동안 절식시키고 실험에 사용하였다.In addition, the normal group was fasted for 12 hours after the normal diet was administered for 2 weeks and used in the experiment.
(2) 체중 변화, 식이 효율 및 장기 무게 측정(2) weight change, dietary efficiency and long-term weight measurement
상기 고지혈증 유발 및 실험 동물의 처리를 거친 WJL5군, WJR5군, 대조군 및 정상군을 실험 대상으로 하여 랫트의 체중 변화, 식이 효율 및 장기 무게를 측정하였다.Weight change, dietary efficiency and organ weight of rats were measured using the WJL5 group, the WJR5 group, the control group, and the normal group, which were treated with the hyperlipidemia-induced and treated animals.
구체적으로, 상기 처리를 완료한 후 평균 체중(B)을 측정함으로써 체중 변화(A-B)를 구함으로써 랫트의 체중 변화를 얻었다. 그 결과는 표 6에 나타내었다.Specifically, the weight change of the rat was obtained by obtaining the weight change (A-B) by measuring the average body weight (B) after completing the treatment. The results are shown in Table 6.
상기 체중 변화(A-B, g/일)를 같은 기간 동안 섭취한 식이 섭취량(g/일)으로 나누어 랫트의 식이 효율(Food Efficacy Ratio, FER)을 구하였다. 그 결과는 표 6에 나타내었다.The weight change (A-B, g / day) was divided by the dietary intake (g / day) ingested during the same period to determine the rat's food efficiency ratio (Food Efficacy Ratio, FER). The results are shown in Table 6.
상기 표 6에서 보여지는 바와 같이, 실험 기간 동안 정상군, 대조군, WJL5군 및 WJR5군에서 체중 변화 및 식이 효율에는 차이가 없었다. As shown in Table 6, there was no difference in body weight change and dietary efficiency in the normal group, control group, WJL5 group and WJR5 group during the experimental period.
상기 평균 체중(B)을 측정하고 랫트를 에테르로 마취시킨 후, 21 게이지(gauge)의 일회용 주사기를 사용하여 채혈하였다. 채혈한 혈액은 2 시간 동안 냉장 상태에서 방치시켜 하기 혈청 지질 성분의 측정에 사용하였다. 마취시킨 랫트로부터 간, 비장 및 신장을 구하였다. 간은 생리 식염수로 문맥을 관루, 탈혈한 다음 여과지로 물기를 제거하여 무게를 측정하였고, 비장 및 신장은 생리 식염수에 한 번 세척한 후 물기를 제거하여 무게를 측정함으로써 랫트의 장기 무게를 얻었다. 그 결과는 표 7에 나타내었다.The average body weight (B) was measured and rats were anesthetized with ether and blood was collected using a 21 gauge disposable syringe. The collected blood was left in refrigerated state for 2 hours and used for the measurement of the following serum lipid components. Liver, spleen and kidney were obtained from anesthetized rats. The liver was weighed by passing through the portal vein with physiological saline, bleeding, and then draining with filter paper. The spleen and kidney were washed once with physiological saline, and then drained to obtain the organ weight of the rat. The results are shown in Table 7.
상기 표 7에서 보여지는 바와 같이, 고지혈증에 걸린 WJL5군, WJR5군 및 대조군의 간장 및 신장의 무게는 고지혈증에 걸리지 않은 정상군에 비하여 유의성 있게 높게 관찰되었다. 또한 비장의 경우에는 모든 실험군에서 차이를 관찰할 수 없었다.As shown in Table 7, the hepatic and kidney weights of the WJL5 group, the WJR5 group, and the control group with hyperlipidemia were significantly higher than those of the normal group without hyperlipidemia. In addition, no difference was observed in the spleen in all experimental groups.
(3) 고추냉이의 항고지혈증 효과(3) Antihyperlipidemic Effect of Wasabi
상기 랫트로부터 채혈한 혈액을 2시간 동안 냉장 상태에서 방치시킨 후, 3,000 rpm에서 15분 동안 원심 분리하여 혈청을 분리하였다. 분리한 혈청을 혈청 지질 함량 측정용 키트(아산 제약 주식회사)를 사용하여 총 콜레스테롤, 트리글리세라이드 및 고밀도 지단백질-콜레스테롤을 측정하였다. 또한, 저밀도 지단백질-콜레스테롤은 상기 측정치 및 하기 수학식 4에 의해 그 값을 산출하였다.The blood collected from the rats was left in a cold state for 2 hours, and then serum was separated by centrifugation at 3,000 rpm for 15 minutes. The separated serum was measured for total cholesterol, triglycerides and high density lipoprotein-cholesterol using a kit for measuring serum lipid content (Asan Pharmaceutical Co., Ltd.). In addition, low-density lipoprotein-cholesterol was calculated by the above measured value and the following equation (4).
상기 측정한 총 콜레스테롤은 도 4에 나타내었다. 상기 측정한 고밀도 지단백질-콜레스테롤은 도 5에 나타내었다. 상기 측정한 저밀도 지단백질-콜레스테롤은 도 6에 나타내었다.The measured total cholesterol is shown in FIG. 4. The measured high density lipoprotein-cholesterol is shown in FIG. 5. The measured low density lipoprotein-cholesterol is shown in FIG. 6.
도 4에서 보여지는 바와 같이 고추냉이 잎 또는 뿌리를 정상 식이와 함께 투여한 WJL5군 또는 WJR5군에서 총 콜레스테롤의 함량은 대조군에 비하여 감소하였다. As shown in FIG. 4, the total cholesterol content in the WJL5 group or the WJR5 group in which horseradish leaves or roots were administered with a normal diet was reduced compared to the control group.
도 5에서 보여지는 바와 같이 고추냉이 잎 또는 뿌리를 정상 식이와 함께 투여한 WJL5군 또는 WJR5군에서 고밀도 지단백질-콜레스테롤의 함량은 대조군에 비하여 유의성 있게 증가하였다.As shown in FIG. 5, the content of high density lipoprotein-cholesterol was significantly increased in the WJL5 group or the WJR5 group in which wasabi leaves or roots were administered with a normal diet.
도 6에서 보여지는 바와 같이 고추냉이 잎 또는 뿌리를 정상 식이와 함께 투여한 WJL5군 또는 WJR5군에서 저밀도 지단백질-콜레스테롤의 함량은 대조군에 비하여 유의성 있게 감소되었다.As shown in Figure 6, the content of low-density lipoprotein-cholesterol in the WJL5 group or the WJR5 group in which wasabi leaves or roots were administered with a normal diet was significantly reduced compared to the control group.
따라서, 본 발명에 따른 고추냉이를 함유하는 기능성 식품은 고밀도 지단백질-콜레스테롤의 함량은 증가시키는데 비하여, 총 콜레스테롤 및 저밀도 지단백질-콜레스테롤의 함량은 감소시킴으로써 항고지혈증 효과를 가지고 있다.Therefore, the functional food containing wasabi according to the present invention has an antihyperlipidemic effect by reducing the content of high cholesterol and low density lipoprotein-cholesterol, while increasing the content of high density lipoprotein-cholesterol.
상기 기술한 바와 같이, 본 발명에서 사용되는 고추냉이는 DPPH에 대한 수소 공여능이 뛰어나고, 직접적으로 산화 질소 생성을 억제하며, iNOS mRNA 발현 및 iNOS 발현을 억제하여 간접적으로 산화 질소 생성을 억제하고, 잔틴 산화제의 활성을 저해하여 슈퍼옥사이드 라디칼의 생성을 억제하는 항산화 효과를 가지고 있다. 또한, 본 발명에서 사용되는 고추냉이는 고밀도 지단백질-콜레스테롤의 함량은 증가시키고, 총 콜레스테롤 및 저밀도 지단백질-콜레스테롤의 함량은 감소시키는 항고지혈증 효과를 가지고 있다. 본 발명에서는 이러한 효과를 가지고 있는 고추냉이를 주성분으로 또는 부성분으로 함유하는 기능성 식품을 제공함으로써 지방 대사와 관련된 질병을 예방 또는 치료하는 데 사용할 수 있도록 하였다.As described above, horseradish used in the present invention is excellent in hydrogen donating ability to DPPH, directly inhibits nitric oxide production, indirectly inhibits nitric oxide production by inhibiting iNOS mRNA expression and iNOS expression, xanthine It has an antioxidant effect that inhibits the activity of the oxidant to inhibit the production of superoxide radicals. In addition, wasabi used in the present invention has an antihyperlipidemic effect of increasing the content of high-density lipoprotein-cholesterol and reducing the content of total cholesterol and low-density lipoprotein-cholesterol. In the present invention, by providing a functional food containing horseradish as a main component or a minor component having such an effect can be used to prevent or treat diseases related to fat metabolism.
Claims (4)
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| KR100814774B1 (en) | 2006-11-15 | 2008-03-19 | 동해식품 주식회사 | Method for preparation of soy sauce containing horseradish components |
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