KR20240000077A - Anti-imflammatory composition comprising velvet bean extract and sword bean extract - Google Patents
Anti-imflammatory composition comprising velvet bean extract and sword bean extract Download PDFInfo
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- KR20240000077A KR20240000077A KR1020220076573A KR20220076573A KR20240000077A KR 20240000077 A KR20240000077 A KR 20240000077A KR 1020220076573 A KR1020220076573 A KR 1020220076573A KR 20220076573 A KR20220076573 A KR 20220076573A KR 20240000077 A KR20240000077 A KR 20240000077A
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Abstract
본 발명은 벨벳콩 추출물 및 작두콩 추출물을 유효성분으로 포함하는 항염증용 조성물에 관한 것이다. 본 발명의 일 측면인 항염증용 조성물은, NO와 같은 염증인자의 발생을 억제함으로써 체내 염증을 근본적으로 해결할 수 있다. 또한, 본 발명의 조성물은 인체 독성이 없고, 항염 효과가 우수하여 식품·화장품·의약품 등 다양한 분야에 활용할 수 있다.The present invention relates to an anti-inflammatory composition containing velvet bean extract and pea extract as active ingredients. The anti-inflammatory composition, which is one aspect of the present invention, can fundamentally solve inflammation in the body by suppressing the generation of inflammatory factors such as NO. In addition, the composition of the present invention is not toxic to the human body and has excellent anti-inflammatory effects, so it can be used in various fields such as food, cosmetics, and pharmaceuticals.
Description
본 발명은 벨벳콩 추출물 및 작두콩 추출물을 유효성분으로 포함하는 항염증용 조성물에 관한 것이다.The present invention relates to an anti-inflammatory composition containing velvet bean extract and pea extract as active ingredients.
염증반응은 외상이나 세균 감염 등의 외부 자극에 대한 우리 몸의 방어 작용으로써, 조직의 구조와 기능을 정상적으로 회복하기 위한 필수적인 생체 반응이다.염증반응은 발병기간과 원인 물질 등에 의해 급성 염증과 만성 염증으로 분류된다. The inflammatory response is a defense function of our body against external stimuli such as trauma or bacterial infection, and is an essential biological reaction to restore normal tissue structure and function. The inflammatory response can be divided into acute inflammation and chronic inflammation depending on the onset period and causative agent. It is classified as
이 중 급성 염증은 주로 대식세포, 수지상 세포, 조직구, 쿠퍼세포 등의 면역세포가 자극을 인식함에 따라 면역 시스템이 활성화된다. 자극을 받은 세포들의 표면에는 병원체연관분자유형(pathogen-associated molecular pattern, PAMP), 손상연관분자유형(Damage-Associated Molecular Pattern, DAMP)이 있는데, 조직이 손상을 받으면 이와 같은 분자를 면역세포가 인식하면서 NF-κB 등의 전사인자를 통한 신호전달경로가 시작된다. 그 결과 종양괴사인자(tumor necrosis factor-α, TNF-α), 인터루킨-6(interleukin-6, IL-6), 인터루킨-1(interleukin-1, IL-1), 인터루킨-8(interleukin-8, IL-8) 등 염증성 사이토카인이 분비되고 이어 염증 증상을 유발하는 염증전달물질들의 분비가 이루어진다. 특히, 대식세포에 의해 산화질소(nitric oxide, NO) 및 프로스타글란딘 E2(prostaglandin E2, PGE 2)와 같은 염증인자가 만들어지는데 염증반응이 과도하게 일어나는 경우에는 이와 같은 기전에 의해 많은 양의 iNOS가 세포들에서 생성되고 이어서 NO량이 증가한다. 이와 같이 과도하게 생산된 NO 는 DNA 구조 손상, 신경 손상, 부종 또는 상피세포암 등의 문제를 유발하는 것으로 알려져 있다.Among these, acute inflammation mainly activates the immune system as immune cells such as macrophages, dendritic cells, histiocytes, and Kupffer cells recognize the stimulus. On the surface of stimulated cells, there are pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). When tissue is damaged, immune cells recognize these molecules. As this happens, a signal transduction pathway through transcription factors such as NF-κB begins. As a result, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1 (IL-1), interleukin-8 (interleukin-8) , IL-8) and other inflammatory cytokines are secreted, followed by the secretion of inflammatory transmitters that cause inflammatory symptoms. In particular, inflammatory factors such as nitric oxide (NO) and prostaglandin E2 (PGE 2) are produced by macrophages, and when an inflammatory response occurs excessively, a large amount of iNOS is absorbed into cells by this mechanism. It is produced in the field and then the amount of NO increases. This excessively produced NO is known to cause problems such as DNA structural damage, nerve damage, edema, or epithelial cell cancer.
또한, 염증 반응이 있는 경우에는 염증 관련 인자와 함께 자유라디칼이 생성되는데, 일반적으로는 세포의 분화, 성장, 생존 등의 항상성 유지에 관여하지만, 자유라디칼 중 활성 산소종(reactive oxygen species, ROS)은 호흡과 면역반응에 의해 산소의 산화와 환원 과정을 거쳐 지속적으로 생성되면서 그 반응성으로 인해 몸에 유해한 작용을 할 수 있기 때문에 체내 항산화 과정을 거쳐 그 제거가 이루어지도록 한다. 그러나 이러한 생성과 제거의 균형이 무너지면 산화적 스트레스가 발생하면서 염증, 노화, 조직손상, 암 등의 문제가 발생할 수 있다.In addition, when there is an inflammatory reaction, free radicals are generated along with inflammation-related factors. Generally, they are involved in maintaining homeostasis such as cell differentiation, growth, and survival, but among free radicals, reactive oxygen species (ROS) Silver is continuously produced through the oxidation and reduction process of oxygen due to respiration and immune reactions, and can have harmful effects on the body due to its reactivity, so it is removed through the body's antioxidant process. However, when the balance between production and removal is disrupted, oxidative stress occurs, which can lead to problems such as inflammation, aging, tissue damage, and cancer.
이에, 염증을 적절한 수준으로 컨트롤할 수 있는 항염증 약물이 필요하나 현재 알려진 대부분의 염증 치료제는 인체 부작용 이슈가 있는 인공합성물들이다. 따라서, 인체 부작용으로부터 자유로운 천연물을 활용한 항염증제에 대한 연구가 수년째 계속되고 있으나, 아직까지 만족할 만한 효과를 얻은 항염증제의 개발사례는 알려진 바 없다.Accordingly, anti-inflammatory drugs that can control inflammation to an appropriate level are needed, but most of the currently known inflammation treatments are artificial compounds that have side effects on the human body. Accordingly, research on anti-inflammatory drugs using natural products that are free from side effects on the human body has been ongoing for several years, but there are no known cases of development of anti-inflammatory drugs that have achieved satisfactory effects.
일 측면에서 본 발명의 목적은 염증을 근본적으로 해결할 수 있는 조성물을 제공하는 것이다.In one aspect, the purpose of the present invention is to provide a composition that can fundamentally solve inflammation.
일 측면에서 본 발명의 목적은 자연 유래 물질을 활용하여 염증을 효율적으로 제거하는 것이다.In one aspect, the purpose of the present invention is to efficiently remove inflammation using natural substances.
일 측면에서 본 발명의 목적은 벨벳콩 및 작두콩에 함유된 유효성분을 효과적으로 추출하는 것이다.In one aspect, the purpose of the present invention is to effectively extract the active ingredients contained in velvet beans and black beans.
일 측면에서 본 발명의 목적은 항염증 효과가 있는 유효성분을 다량 포함한 벨벳콩 추출물 및 작두콩 추출물을 제공하는 것이다.In one aspect, the purpose of the present invention is to provide velvet bean extract and pea extract containing a large amount of active ingredients with anti-inflammatory effects.
상기 목적을 달성하기 위하여, 본 발명은, 벨벳콩(Mucuna pruriens) 추출물 및 작두콩(Canavalia gladiata) 추출물을 유효성분으로 포함하는 항염증용 조성물을 제공한다.In order to achieve the above object, the present invention provides an anti-inflammatory composition containing velvet bean ( Mucuna pruriens ) extract and small bean ( Canavalia gladiata ) extract as active ingredients.
상기 벨벳콩 추출물은, 작두콩 추출물 100 중량부를 기준으로, 50~200 중량부로 포함될 수 있다.The velvet bean extract may be included in an amount of 50 to 200 parts by weight, based on 100 parts by weight of the velvet bean extract.
상기 조성물은 NO(Nitric oxide)의 발생을 억제 또는 감소시킬 수 있다.The composition can inhibit or reduce the generation of NO (Nitric oxide).
상기 벨벳콩 추출물 및 작두콩 추출물의 추출용매는, 물, 유기 용매 또는 이들의 혼합물을 포함할 수 있다.The extraction solvent for the velvet bean extract and the pea extract may include water, an organic solvent, or a mixture thereof.
구체적으로, 상기 벨벳콩 추출물의 추출용매는 물을 포함할 수 있으며, 상기 작두콩 추출물의 추출용매는 알코올을 포함할 수 있다.Specifically, the extraction solvent of the velvet bean extract may include water, and the extraction solvent of the velvet bean extract may include alcohol.
또한, 상기 알코올의 농도는 50~90%(w/w)일 수 있다.Additionally, the concentration of the alcohol may be 50 to 90% (w/w).
또한, 식품 조성물, 약학적 조성물 또는 화장료 조성물을 포함할 수 있다.Additionally, it may include a food composition, pharmaceutical composition, or cosmetic composition.
본 발명의 일 측면인 항염증용 조성물은, NO와 같은 염증인자의 발생을 억제함으로써 체내 염증을 근본적으로 해결할 수 있다. 또한, 본 발명의 조성물은 인체 독성이 없고, 항염 효과가 우수하여 식품·화장품·의약품 등 다양한 분야에 활용할 수 있다.The anti-inflammatory composition, which is one aspect of the present invention, can fundamentally solve inflammation in the body by suppressing the generation of inflammatory factors such as NO. In addition, the composition of the present invention is not toxic to the human body and has excellent anti-inflammatory effects, so it can be used in various fields such as food, cosmetics, and pharmaceuticals.
도 1은 본 발명의 일 측면에 따른 벨벳콩 추출물과 작두콩 추출물의 혼합물의 세포 독성을 확인한 도이다(MW : 벨벳콩 열수 추출물, CE : 70% 에탄올로 추출한 작두콩 추출물).
도 2는 본 발명의 일 측면에 따른 벨벳콩 추출물과 작두콩 추출물의 혼합물의 NO 저해 활성을 확인한 도이다(MW : 벨벳콩 열수 추출물, CE : 70% 에탄올로 추출한 작두콩 추출물, N: LPS 무처리군, C: LPS 처리군, 50/75/100/300 : 처리된 제조예 3의 농도).
도 3은 본 발명의 일 측면에 따른 벨벳콩 추출물과 작두콩 추출물의 혼합물(MW+CE)의 iNOS 발현 저해능을 확인한 도이다(MW : 벨벳콩 열수 추출물, CE : 70% 에탄올로 추출한 작두콩 추출물, N: LPS 무처리군, C: LPS 처리군, 50/75/100/300 : 처리된 제조예 3의 농도).Figure 1 is a diagram confirming the cytotoxicity of a mixture of velvet bean extract and pea extract according to one aspect of the present invention (MW: velvet bean hot water extract, CE: pea extract extracted with 70% ethanol).
Figure 2 is a diagram confirming the NO inhibition activity of a mixture of velvet bean extract and pea extract according to one aspect of the present invention (MW: velvet bean hot water extract, CE: pea extract extracted with 70% ethanol, N: LPS untreated group , C: LPS treated group, 50/75/100/300: concentration of treated Preparation Example 3).
Figure 3 is a diagram confirming the ability of a mixture of velvet bean extract and pea extract (MW+CE) according to one aspect of the present invention to inhibit iNOS expression (MW: velvet bean hot water extract, CE: pea extract extracted with 70% ethanol, N : LPS untreated group, C: LPS treated group, 50/75/100/300: concentration of treated Preparation Example 3).
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
이하의 설명은 하나의 예시에 불과할 뿐, 본 발명의 권리범위가 다음 내용에 의해 제한되지 아니한다.The following description is only an example, and the scope of the present invention is not limited by the following.
또한, 본 발명의 명세서 및 청구범위에 사용된 용어 또는 단어는 통상적이거나 사전적인 의미로 한정 해석되지 아니하며, 발명자는 그 자신의 발명을 가장 최선의 방법으로 설명하기 위해 용어의 개념을 적절하게 정의할 수 있다는 원칙에 입각하여 본 발명의 기술적 사상에 부합하는 의미와 개념으로 해석되어야만 한다.In addition, the terms or words used in the specification and claims of the present invention are not to be construed as limited to their ordinary or dictionary meanings, and the inventor may appropriately define the concept of terms in order to explain his/her invention in the best way. It must be interpreted with meaning and concept consistent with the technical idea of the present invention based on the principle that it can be done.
본 발명의 명세서 전체에 있어서, 어떤 부분이 어떤 구성 요소를 "포함" 한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성 요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.Throughout the specification of the present invention, when a part is said to "include" a certain component, this means that it does not exclude other components but may further include other components unless specifically stated to the contrary. .
본 발명은 일 측면에서, 벨벳콩(Mucuna pruriens) 추출물 및 작두콩(Canavalia gladiata) 추출물을 유효성분으로 포함하는 항염증용 조성물이다.In one aspect, the present invention is an anti-inflammatory composition comprising velvet bean ( Mucuna pruriens ) extract and black bean ( Canavalia gladiata ) extract as active ingredients.
상기 "벨벳콩"은, 콩과의 덩굴성 관목으로, 예로부터 파킨슨 병에 효과가 있는 것으로 알려져 있다.The "velvet bean" is a climbing shrub of the legume family and has long been known to be effective in treating Parkinson's disease.
상기 "작두콩"은, 콩과의 한해살이 덩굴풀로, 콩깍지의 생김새가 작두와 닮았다하여 작두콩이라고 불린다. 작두콩은 예로부터 치질, 축농증 만성 설사, 월경불순 등에 도움이 된다고 알려져 약용으로 널리 사용되었다.The "jakdu bean" is an annual vine of the legume family, and is called jakdu bean because the shape of the bean pod resembles a pod. Peanuts have long been known to help with hemorrhoids, sinusitis, chronic diarrhea, and menstrual irregularities, and have been widely used for medicinal purposes.
본 명세서에서 "항염증"이란 염증의 발생을 억제하는 기능뿐만 아니라 발생한 염증을 개선하는 기능을 포함하는 광의로 해석된다.As used herein, “anti-inflammatory” is interpreted broadly to include not only the function of suppressing the occurrence of inflammation but also the function of improving the inflammation that has occurred.
본 명세서에서 "염증"은 생체조직에서 항원 등의 염증 유발물질에 의해 발생하는 복합적인 면역반응의 일종을 의미한다. 구체적으로, 염증 반응은 생체나 조직에 물리적 작용이나 화학적 물질, 세균감염 등의 어떠한 기질적 변화를 가져오는 침습이 가해질 때, 그 손상 부위를 회복하려는 재생 기전을 의미할 수 있다. 상기 염증은, 급성 염증과 만성 염증을 포함할 수 있다.As used herein, “inflammation” refers to a type of complex immune response caused by inflammatory substances such as antigens in biological tissues. Specifically, the inflammatory response may refer to a regenerative mechanism that attempts to restore the damaged area when an invasion that causes any organic change, such as a physical action, chemical substance, or bacterial infection, is applied to the living body or tissue. The inflammation may include acute inflammation and chronic inflammation.
본 명세서에서, "추출물"은 추출 방법, 추출 용매, 추출된 성분 또는 추출물의 형태를 불문하고, 천연물의 성분을 뽑아냄으로써 얻어진 물질을 모두 포함하는 것이다. 또한, 천연물의 성분을 뽑아내어 얻어진 물질을 추출 후 다른 방법으로 가공 또는 처리하여 얻어질 수 있는 물질을 모두 포함하는 광범위한 개념으로, 구체적으로 상기 가공 또는 처리는 추출물을 추가적으로 발효, 또는 효소처리 하는 것일 수 있다. 따라서 본 명세서에서 추출물은 발효물, 농축물, 건조물을 포함하는 광범위한 개념이다.In this specification, “extract” includes all substances obtained by extracting components of natural products, regardless of the extraction method, extraction solvent, extracted component, or form of the extract. In addition, it is a broad concept that includes all materials that can be obtained by extracting the components of natural products and processing or treating them by other methods. Specifically, the processing or treatment refers to additional fermentation or enzyme treatment of the extract. You can. Therefore, in this specification, extract is a broad concept including fermented product, concentrate, and dried product.
상기와 같은 측면에서, 상기 추출물의 추출용매는, 물, 유기용매 또는 이들의 혼합물을 포함할 수 있다.In the above aspect, the extraction solvent for the extract may include water, an organic solvent, or a mixture thereof.
상기 유기용매는, 헥산, 메틸렌클로라이드, 알코올 등이 이용될 수 있으나, 이에 한정되는 것은 아니다. 상기에서 물은 증류수 또는 정제수를 포함하고, 유기 용매는 C1~C5의 저급 알코올을 예로 들 수 있는 알코올, 아세톤, 에테르, 에틸아세테이트, 디에틸에테르, 메탄올, 에틸메틸케톤 및 클로로포름으로 이루어진 군에서 선택된 하나 이상을 포함하나, 이에 제한되는 것은 아니다.The organic solvent may be hexane, methylene chloride, alcohol, etc., but is not limited thereto. In the above, water includes distilled water or purified water, and the organic solvent is a group consisting of alcohol including lower alcohols of C 1 to C 5 , acetone, ether, ethyl acetate, diethyl ether, methanol, ethyl methyl ketone, and chloroform. Includes, but is not limited to, one or more selected from.
상기 추출 용매가 물일 때, 가열 추출, 냉침 추출, 환류냉각 추출, 또는 초음파 추출 등이 이용될 수 있으며, 바람직하게는 열수를 사용하는 가온 추출이 이용될 수 있고, 바람직하게는 약 40 내지 120℃(도씨) 더욱 바람직하게는 90~100℃, 구체적으로는 약 100℃의 온도에서 추출할 수 있으나, 이에 한정되는 것은 아니다. 추출시간은 바람직하게는 약 1 내지 4시간, 더욱 바람직하게는 약 2.5~3.5 시간 동안 수행할 수 있으나 이에 한정되는 것은 아니다. 또한, 상기 추출은 동일한 조건에서 1~5회, 바람직하게는 2~4회 수행할 수 있다.When the extraction solvent is water, heating extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction can be used, preferably heating extraction using hot water can be used, preferably at about 40 to 120°C. (degree Celsius) More preferably, extraction can be performed at a temperature of 90 to 100°C, specifically about 100°C, but is not limited thereto. The extraction time is preferably about 1 to 4 hours, more preferably about 2.5 to 3.5 hours, but is not limited thereto. Additionally, the extraction can be performed 1 to 5 times, preferably 2 to 4 times, under the same conditions.
일 구현예에서, 상기 유기 용매는 알코올일 수 있고, 상기 알코올은, 에탄올일 수 있다.In one embodiment, the organic solvent may be alcohol, and the alcohol may be ethanol.
상기 알코올의 농도는 30~99%(w/w), 또는 50~99%(w/w), 또는 50~80%(w/w), 또는 60~80%(w/w)일 수 있으며, 바람직하게는 65~75%(w/w)일 수 있다.The concentration of the alcohol may be 30 to 99% (w/w), or 50 to 99% (w/w), or 50 to 80% (w/w), or 60 to 80% (w/w), , preferably 65 to 75% (w/w).
일 측면에서, 상기 벨벳콩 추출물은 물 추출물을 포함할 수 있고, 일 구현예에서 열수 추출물을 포함할 수 있다. 일 구현예에서, 상기 벨벳콩 추출물은 약 90~110℃(도씨)의 열수를 이용하여 추출한 것을 포함할 수 있다.In one aspect, the velvet bean extract may include a water extract, and in one embodiment, it may include a hot water extract. In one embodiment, the velvet bean extract may include extraction using hot water at about 90 to 110 degrees Celsius.
상기와 같은 공정을 통해 약 15% 이상의 수율로 벨벳콩 추출물을 수득할 수 있다.Through the above process, velvet bean extract can be obtained with a yield of about 15% or more.
또한, 일 측면에서, 상기 작두콩 추출물은 작두콩의 알코올 추출물을 포함할 수 있다. 구체적으로, 일 구현예에서 상기 작두콩 추출물은 작두콩의 에탄올 추출물을 포함할 수 있고, 예컨대, 약 60~80%(w/w)의 에탄올을 이용하여 작두콩을 추출한 것을 포함할 수 있다.Additionally, in one aspect, the pea extract may include an alcohol extract of pea beans. Specifically, in one embodiment, the pea extract may include an ethanol extract of pea beans, for example, may include extracting pea beans using about 60 to 80% (w/w) ethanol.
상기와 같은 공정을 통해 약 5% 이상의 수율로 작두콩 추출물을 수득할 수 있다.Through the above process, a pea extract can be obtained with a yield of about 5% or more.
일 측면에서, 상기 벨벳콩 추출물은, 작두콩 추출물 100 중량부를 기준으로 50~200 중량부로 포함될 수 있다. 구체적으로, 상기 벨벳콩 추출물은, 작두콩 추출물 100 중량부를 기준으로, 50~180 중량부, 또는 60~180 중량부, 또는 60~160 중량부, 또는 60~150 중량부, 또는 70~150 중량부, 또는 70~130 중량부, 또는 80~120 중량부, 또는 90~110 중량부로 포함될 수 있다.In one aspect, the velvet bean extract may be included in an amount of 50 to 200 parts by weight based on 100 parts by weight of the pea extract. Specifically, the velvet bean extract is 50 to 180 parts by weight, or 60 to 180 parts by weight, or 60 to 160 parts by weight, or 60 to 150 parts by weight, or 70 to 150 parts by weight, based on 100 parts by weight of the pea extract. , or 70 to 130 parts by weight, or 80 to 120 parts by weight, or 90 to 110 parts by weight.
일 측면에서, 상기 조성물은 NO(Nitric oxide)의 발생을 억제할 수 있다. In one aspect, the composition can inhibit the generation of NO (Nitric oxide).
일산화질소(nitric oxide)는, 1980년 Furchgott와 Zawadzki에 의하여 endothelial-derived relaxing factor(EDRF)로 처음 생체 내에서 그 존재가 알려진 물질이다. 일산화질소는 혈관 내 항상성 유지(vascular homeostasis), 신경전달(neurotransmission), 혈액 응고, 항암작용(anti-tumor activity) 및 세포독성(cell toxicity) 등 다양한 생물학적 활성을 나타내는 것으로 알려져 있다. 구체적으로, 일산화질소는 유리기 상태로 심장과 혈관, 폐, 신장, 뇌, 췌장, 위장관 및 면역계 등에서 생성되며, 이들은 장기의 각종 병태 생리적 상황에 관여하는 것으로 알려져 있다. 일반적으로 적정 수준의 일산화질소의 형성은 박테리아를 죽이거나 종양을 제거하는 등의 신체에 유익한 역할을 하지만, 과도하게 과량 발생한 일산화질소는, 염증을 유발하고, 조직의 손상, 유전자 변이 및 신경 손상 등의 다양한 문제를 초래한다. Nitric oxide is a substance first known to exist in vivo by Furchgott and Zawadzki in 1980 as endothelial-derived relaxing factor (EDRF). Nitric oxide is known to exhibit various biological activities such as maintaining vascular homeostasis, neurotransmission, blood coagulation, anti-tumor activity, and cell toxicity. Specifically, nitric oxide is produced in a free radical state in the heart, blood vessels, lungs, kidneys, brain, pancreas, gastrointestinal tract, and immune system, and is known to be involved in various pathophysiological conditions of organs. In general, the formation of an appropriate level of nitric oxide plays a beneficial role in the body, such as killing bacteria or removing tumors, but excessive amounts of nitric oxide cause inflammation, tissue damage, genetic mutation, and nerve damage. causes various problems.
일산화질소는 외부 항원의 자극으로 활성화된 대식세포에서 NO synthase (NOS)에 의해 L-arginine으로부터 생성된다. 구체적으로, NOS는 물리 화학적 성상에 따라 type I, Ⅱ 및 III 등 3종류으로 분류된다. Type I(neuronal NOS, nNOS)과 type III(endothelial NOS, eNOS)는 세포 내에 상시 존재하기 때문에 구성 NOS (constitutive NOS)로 분류하며, type II는 상대적으로 일부 세포에서 LPS, cytokine 및 박테리아 같은 특수한 자극제에 노출되는 경우에만 발현되어 유도형 NOS(iNOS)로 분류된다. 이러한 NOS들은 L-arginine을 L-citrulline으로 전환시키는 과정에서 NO를 형성한다. 일산화질소의 생산량은 NOS 중 iNOS에 의한 생산량이 절대적으로 많다.Nitric oxide is produced from L-arginine by NO synthase (NOS) in macrophages activated by external antigen stimulation. Specifically, NOS is classified into three types, including types I, II, and III, depending on their physical and chemical properties. Type I (neuronal NOS, nNOS) and type III (endothelial NOS, eNOS) are classified as constitutive NOS (constitutive NOS) because they are always present in cells. Type II is relatively irritated by special stimulants such as LPS, cytokines, and bacteria in some cells. It is expressed only when exposed to and is classified as inducible NOS (iNOS). These NOS form NO in the process of converting L-arginine to L-citrulline. Among NOS, the production of nitrogen monoxide is absolutely higher due to iNOS.
일 측면에서, 본 발명은 iNOS의 발현을 억제함으로써, 일산화질소의 발생을 억제할 수 있다.In one aspect, the present invention can suppress the generation of nitric oxide by suppressing the expression of iNOS.
일 측면에서, 본 발명의 조성물은, 대조군 대비 약 70% 이상의 iNOS 발현 억제능을 나타낼 수 있고, 구체적으로 약 75% 이상의 iNOS 발현 억제능, 또는 약 78% 이상의 iNOS 발현 억제능을 나타낼 수 있으며, 약 70~80%의 iNOS 발현 억제능을 나타낼 수 있다. In one aspect, the composition of the present invention may exhibit an iNOS expression inhibition ability of about 70% or more compared to the control group, and specifically may exhibit an iNOS expression inhibition ability of about 75% or more, or an iNOS expression inhibition ability of about 78% or more, and may exhibit an iNOS expression inhibition ability of about 70-70% or more. It can show an 80% inhibition of iNOS expression.
또한, 본 발명의 조성물은, 대조군 대비 약 40% 이상의 NO 저해능을 나타낼 수 있고, 구체적으로 약 45% 이상의 NO 저해능, 또는 47% 이상의 NO 저해능을 나타낼 수 있으며, 일 구현예에서 약 48~55%의 NO 저해능을 나타낼 수 있다.In addition, the composition of the present invention may exhibit a NO inhibition ability of about 40% or more compared to the control group, and specifically may exhibit a NO inhibition ability of about 45% or more, or a NO inhibition ability of 47% or more, and in one embodiment, about 48 to 55%. can exhibit NO inhibition ability.
일 측면에서, 상기 조성물은, 식품 조성물, 약학적 조성물 또는 화장료 조성물을 포함할 수 있다.In one aspect, the composition may include a food composition, pharmaceutical composition, or cosmetic composition.
상기 식품 조성물은, 건강 기능식품을 포함할 수 있다. 건강 기능식품은, 일상 식사에서 결핍되기 쉬운 영양소나 인체에 유용한 기능을 가진 원료나 성분(기능성원료)을 사용하여 제조한 것으로, 인체의 정상적인 기능을 유지하거나 생리기능 활성화를 통하여 건강을 유지하고 개선하는 식품을 의미할 수 있으나, 이에 제한되지 않는다. The food composition may include health functional foods. Health functional foods are manufactured using nutrients that are easily deficient in daily diet or raw materials or ingredients (functional raw materials) with useful functions for the human body. They maintain and improve health by maintaining the normal function of the human body or activating physiological functions. It may mean food that does, but is not limited to this.
상기 건강식품은 정제, 캡슐, 분말, 과립, 액상, 환 등의 형태로 제조, 가공될 수 있으나, 이에 한정되지 않으며 법률에 따라 어떤 형태로든지 제조, 가공될 수 있다.The above health foods may be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., but are not limited thereto and may be manufactured and processed in any form in accordance with the law.
유효 성분의 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조시에 본 발명의 상기 각각의 추출물은 원료에 대하여 각각 15 중량% 이하, 바람직하게는 10 중량%이하의 양으로 첨가된다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when producing food or beverages, each of the extracts of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw materials. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.
상기 식품의 종류에는 특별한 제한은 없다. 본 발명의 추출물을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농 제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강 식품을 모두 포함한다.There are no special restrictions on the types of foods above. Examples of foods to which the extract of the present invention can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, There are drinks, alcoholic beverages and vitamin complexes, and it includes all health foods in the conventional sense.
상기 외에 본 발명의 추출물은 다양한 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. In addition to the above, the extract of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonic acid. It may contain carbonating agents used in beverages.
본 발명의 조성물은 통상적인 방법에 따라 약학 제형으로 제조될 수 있다. 제형의 제조에 있어서, 활성 성분을 담체와 함께 혼합 또는 희석하거나, 용기 형태의 담체 내에 봉입시키는 것이 바람직할 수 있다. 담체가 희석제로 사용되는 경우에는 활성 성분에 대한 담체, 부형제 또는 매질(medium)로 작용하는 고형, 반고형 또는 액상의 물질일 수 있다. 따라서, 제형은 정제, 환제, 분제, 사쉐, 엘릭시르, 현탁제, 유제, 용액제, 시럽제, 에어로졸, 연질 또는 경질 젤라틴 캅셀제, 멸균 주사제, 멸균 분제 등의 형태일 수 있다.The composition of the present invention can be prepared into a pharmaceutical formulation according to conventional methods. In preparing a dosage form, it may be desirable to mix or dilute the active ingredient with a carrier or enclose it in a carrier in the form of a container. When a carrier is used as a diluent, it may be a solid, semi-solid, or liquid substance that acts as a carrier, excipient, or medium for the active ingredient. Therefore, the dosage form may be in the form of tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injections, sterile powders, etc.
적합한 담체, 부형제 및 희석제의 예로는, 락토로스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제형은 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제, 방부제 등을 추가로 포함할 수 있다. 본 발명의 조성물은 포유동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 잘 알려진 방법을 사용하여 제형화될 수 있다.Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propyl. Examples include hydroxybenzoate, talc, magnesium stearate, and mineral oil. The formulation may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. Compositions of the present invention can be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.
본 발명의 약학 조성물은 경구, 경피, 피하, 정맥, 복강, 근육, 국소도포, 첩포 및 이온토포레시스(iontophoresis)를 포함한 여러 경로를 통해 투여될 수 있고, 이 중에서 국소 적용 및 경구투여가 바람직하다.The pharmaceutical composition of the present invention can be administered through several routes, including oral, transdermal, subcutaneous, intravenous, peritoneal, intramuscular, topical application, patch, and iontophoresis, of which topical application and oral administration are preferred. do.
사람의 경우, 상기 조성물의 통상적인 1일 투여량은 1 내지 100mg/kg체중, 바람직하게는 5 내지 70mg/kg체중의 범위일 수 있고, 1회 또는 수회로 나누어 투여할 수 있다. 그러나, 조성물의 실제 투여량은 치료할 질환, 투여 경로, 환자의 연령, 성별 및 체중, 및 질환의 중증도 등의 여러 관련 인자에 비추어 결정되어야 하는 것으로 이해되어야 하며, 따라서, 상기 투여량은 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.For humans, a typical daily dosage of the composition may range from 1 to 100 mg/kg body weight, preferably 5 to 70 mg/kg body weight, and may be administered once or in divided doses. However, it should be understood that the actual dosage of the composition should be determined in light of several relevant factors such as the disease to be treated, the route of administration, the patient's age, sex and weight, and the severity of the disease, and therefore, the dosage is determined by any method. It does not limit the scope of the present invention.
화장료 조성물로는 예를 들어, 모발용 화장료, 바디용 화장료, 기초 화장료, 메이크업 화장료 등이 있을 수 있고, 그 제형은 특별히 제한되지 않으며, 목적하는 바에 따라 적절히 선택할 수 있다. 예를 들면, 상기 화장료 조성물은 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클린싱, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이 등으로 제형화될 수 있으나, 이에 한정되는 것은 아니다. 보다 상세하게는, 샴푸, 린스, 바디클렌저 등의 세정료, 헤어토닉, 젤 또는 무스 등의 정발제, 양모제 또는 염모제 등의 모발용 화장료 조성물, 유연화장수, 영양화장수, 로션, 바디로션, 영양 크림, 마사지 크림, 모이스처 크림, 핸드크림, 에센스, 아이 크림, 클렌징 크림, 클렌징 폼, 클렌징 워터, 팩, 젤, 패치, 수중유(O/W)형, 유중수(O/W)형 등의 기초 화장료로 제형화 될 수 있다. Cosmetic compositions may include, for example, hair cosmetics, body cosmetics, basic cosmetics, makeup cosmetics, etc. The formulation is not particularly limited and can be appropriately selected depending on the purpose. For example, the cosmetic composition can be formulated as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, and spray. It may be possible, but it is not limited to this. More specifically, cleaning agents such as shampoo, conditioner, and body cleanser, hair tonics, hair straighteners such as gel or mousse, hair cosmetic compositions such as hair tonics or hair dyes, softening lotion, nourishing lotion, lotion, body lotion, nutritious cream, Basic cosmetics such as massage cream, moisture cream, hand cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, gel, patch, oil-in-water (O/W) type, water-in-oil (O/W) type, etc. It can be formulated as:
상기 화장료 조성물은 화장품학적으로 허용가능한 매질 또는 기제를 함유한다. 이는 국소적용에 적합한 모든 제형으로, 예를 들면 용액, 겔, 고체 또는 반죽 무수 생성물, 수상에 유상을 분산시켜 얻은 에멀젼, 현탁액, 마이크로에멀젼, 마이크로캡슐, 미세과립구 또는 이온형(리포좀) 및/또는 비이온형의 소낭 분산제의 형태로, 또는 크림, 스킨, 로션, 파우더, 연고, 스프레이 또는 콘실 스틱의 형태로 제공될 수 있다. 이들 조성물은 당해 분야의 통상적 방법에 따라 제조될 수 있다.The cosmetic composition contains a cosmetically acceptable medium or base. These are all formulations suitable for topical application, such as solutions, gels, solid or pasty anhydrous products, emulsions obtained by dispersing the oil phase in the water phase, suspensions, microemulsions, microcapsules, microgranules or ionic forms (liposomes) and/or It may be provided in the form of a non-ionic vesicular dispersion, or in the form of a cream, skin, lotion, powder, ointment, spray or concealer stick. These compositions can be prepared according to conventional methods in the art.
본 발명의 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용해화제 또는 유탁화제가 이용될 수 있고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌 글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 있다.When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent, or emulsifying agent may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol. , 1,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan.
본 발명의 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다.When the formulation of the present invention is a suspension, the carrier ingredients include water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, and microcrystals. Cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.
본 발명의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier ingredient. You can.
본 발명의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다. When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient. In particular, when the formulation is a spray, chlorofluorohydrocarbon and propane may be used as carrier ingredients. /May contain propellants such as butane or dimethyl ether.
본 발명의 일 구현예에서, 상기 화장료 조성물은 추가적으로 점증제를 함유할 수 있다. 본 발명의 화장료 조성물에 포함되는 점증제는 메틸 셀룰로스, 카르복시 메틸 셀룰로스, 카르복시 메틸 하이드록시 구아닌, 하이드록시 메틸 셀룰로스, 하이드록시에틸셀룰로스, 카르복시 비닐 폴리머, 폴리쿼터늄, 세테아릴 알콜, 스테아릭산, 카라기난 등이 있다. In one embodiment of the present invention, the cosmetic composition may additionally contain a thickener. The thickening agent included in the cosmetic composition of the present invention includes methyl cellulose, carboxy methyl cellulose, carboxy methyl hydroxy guanine, hydroxy methyl cellulose, hydroxyethyl cellulose, carboxy vinyl polymer, polyquaternium, cetearyl alcohol, stearic acid, Carrageenan, etc.
본 발명의 일 측면에서 상기 화장료 조성물은 필요에 따라 적절한 각종의 기제와 첨가제를 함유할 수 있으며, 이들 성분의 종류와 양은 발명자에 의해 용이하게 선정될 수 있다. 필요에 따라 허용 가능한 첨가제를 함유할 수 있으며, 예를 들면, 당업계에 통상적인 방부제, 색소, 첨가제 등의 성분을 추가로 포함할 수 있다.In one aspect of the present invention, the cosmetic composition may contain various appropriate bases and additives as needed, and the types and amounts of these ingredients can be easily selected by the inventor. If necessary, it may contain acceptable additives. For example, it may additionally contain ingredients such as preservatives, colorants, and additives that are common in the art.
일 측면에서 본 발명의 상기 조성물의 투여량은 약 400μg/mL 이하일 수 있고, 구체적으로 약 350μg/mL 이하, 또는 약 330μg/mL 이하, 또는 약 320μg/mL 이하, 또는 약 310μg/mL 이하일 수 있다. 또한, 상기 조성물의 투여량은 약 50μg/mL 이상일 수 있고, 또는 70μg/mL 이상, 또는 100μg/mL 이상, 또는 130μg/mL 이상, 또는 170μg/mL 이상, 또는 190μg/mL 이상, 또는 210μg/mL 이상, 또는 230 μg/mL 이상, 또는 250μg/mL 이상, 또는 270μg/mL 이상일 수 있다. 상기 조성물은 상기와 같은 범위 내에서 세포 독성은 없이 우수한 항염증 효과를 나타낼 수 있다.In one aspect, the dosage of the composition of the present invention may be about 400 μg/mL or less, specifically about 350 μg/mL or less, or about 330 μg/mL or less, or about 320 μg/mL or less, or about 310 μg/mL or less. . Additionally, the dosage of the composition may be at least about 50 μg/mL, or at least 70 μg/mL, or at least 100 μg/mL, or at least 130 μg/mL, or at least 170 μg/mL, or at least 190 μg/mL, or at least 210 μg/mL. It may be more than 230 μg/mL, or more than 250 μg/mL, or more than 270 μg/mL. The composition can exhibit excellent anti-inflammatory effects without cytotoxicity within the above range.
이하, 제조예 및 실시예를 통하여 본 발명에 대해 구체적으로 설명하기로 한다. 이들 예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 예에 의해 제한되는 것은 아니다.Hereinafter, the present invention will be described in detail through preparation examples and examples. These examples are merely for illustrating the present invention, and the scope of the present invention is not limited by these examples.
[제조예] 벨벳콩 추출물 및 작두콩 추출물의 혼합물 제조[Preparation example] Preparation of a mixture of velvet bean extract and black bean extract
[제조예 1] 벨벳콩 추출물의 제조[Preparation Example 1] Preparation of velvet bean extract
추출물 제조에 사용된 벨벳콩은 미래 영농으로부터 구매하여 사용하였다. 벨벳콩 열수 추출물은 분쇄한 벨벳콩 100 g을 삼각 플라스크에 담아 시료 중량의 약 10배에 해당하는 증류수를 가한 후, 99℃의 수욕 상에서 3시간 동안 중탕하는 과정을 3회 반복하였다. 그 다음 추출물을 여과지(No. 20 filter paper, Hyundai micro Co., Ltd., Korea)를 사용하여 여과한 후 감압 농축 및 동결 건조하여 분말 상태의 벨벳콩 열수 추출물 시료를 제조하였다. 벨벳콩 추출물의 수율은 15.1%로 측정되었다. 시료는 -20℃에서 보관하여 하기 실험에 사용하였다.The velvet beans used to prepare the extract were purchased from Mirae Farming. For the velvet bean hot water extract, 100 g of crushed velvet beans were placed in an Erlenmeyer flask, distilled water equivalent to about 10 times the weight of the sample was added, and the process of boiling in a water bath at 99°C for 3 hours was repeated three times. Next, the extract was filtered using filter paper (No. 20 filter paper, Hyundai micro Co., Ltd., Korea), then concentrated under reduced pressure and freeze-dried to prepare a powdered velvet bean hot water extract sample. The yield of velvet bean extract was measured at 15.1%. The sample was stored at -20°C and used in the following experiment.
[제조예 2] 작두콩 추출물의 제조[Preparation Example 2] Preparation of small bean extract
추출물 제조에 사용된 작두콩은 미래 영농으로부터 구매하여 사용하였다. 작두콩 70% ethanol 추출물은, 분쇄한 작두콩 100 g을 삼각 플라스크에 담아 시료 중량의 약 10배에 해당하는 70% ethanol을 침지시킨 후, 실온에서 24시간 동안 추출하는 과정을 3회 반복하였다. 그 다음 추출물을 여과지(No. 20 filter paper, Hyundai micro Co., Ltd., Korea)를 사용하여 여과한 후 감압 농축 및 동결 건조하여 분말 상태의 작두콩 70% ethanol 추출물 시료를 제조하였다. 작두콩 추출물의 수율은 5.23%로 측정되었으며, 시료는 -20℃에서 보관하여 하기 실험에 사용하였다.The soybeans used to prepare the extract were purchased from Mirae Farming. For the 70% ethanol extract of small soybeans, 100 g of pulverized small soybeans were placed in an Erlenmeyer flask, immersed in 70% ethanol equivalent to about 10 times the weight of the sample, and the extraction process was repeated three times for 24 hours at room temperature. Next, the extract was filtered using filter paper (No. 20 filter paper, Hyundai micro Co., Ltd., Korea), concentrated under reduced pressure, and freeze-dried to prepare a powdered 70% ethanol extract sample of black bean. The yield of the pea extract was measured to be 5.23%, and the sample was stored at -20°C and used in the following experiment.
[제조예 3] 벨벳콩 추출물 및 작두콩 추출물의 혼합물 제조[Preparation Example 3] Preparation of a mixture of velvet bean extract and black bean extract
제조예 1 및 2에서 제조된 벨벳콩 추출물과 작두콩 추출물을 약 1:1의 비율로 혼합하여, 혼합물을 제조하였다.A mixture was prepared by mixing the velvet bean extract and the black bean extract prepared in Preparation Examples 1 and 2 in a ratio of about 1:1.
[실험예][Experimental example]
[실험예 1] 세포 생존율 측정[Experimental Example 1] Measurement of cell viability
가. 세포배양go. cell culture
macrophage RAW 264.7 cell에 상기 추출물을 투여하여 세포 생존율(독성)을 측정하였다. 실험에 사용된 macrophage RAW 264.7 cell의 배양에는 10% fetal bovine serum(FBS), 1% penicillin-streptomycin을 첨가한 Dulbeco's modified eagle's medium (DMEM)을 사용하였으며, 37℃, 5% CO2 incubator에 적응시켜 계대 배양하였다.The extract was administered to macrophage RAW 264.7 cells and cell viability (toxicity) was measured. To culture the macrophage RAW 264.7 cells used in the experiment, Dulbeco's modified eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin was used, and the cells were adapted to a 37°C, 5% CO 2 incubator. Subculture was carried out.
나. MTT assay를 통한 세포 생존율 측정me. Measurement of cell viability through MTT assay
세포 생존율 측정은 Carmichael 등(1987)의 방법에 따라 실험을 진행하였다. 96well plate에 RAW 264.7 cell을 5Х103 cells/well이 되도록 180 μL 분주하고, 농도별로 제조한 시료를 20μL씩 첨가한 후 37℃, 5% CO2 incubator에서 24시간 동안 배양하였다. 배양한 다음 MTT(5 mg/mL) 용액20 μL를 첨가하여 2시간 동안 반응시켰다. 반응 후 배양액을 제거하고 각 well당 dimethyl sulfoxide(DMSO) 100μL를 가하여 실온에서 10분간 반응 시킨 다음 540nm에서 흡광도를 측정하였다. 세포 생존율 측정은 시료 용액의 첨가군과 무첨가군의 흡광도 감소율로 나타내었다.Cell viability was measured according to the method of Carmichael et al. (1987). 180 μL of RAW 264.7 cells were dispensed into a 96-well plate to make 5Х10 3 cells/well, and 20 μL of samples prepared at each concentration were added and incubated at 37°C in a 5% CO 2 incubator for 24 hours. After culturing, 20 μL of MTT (5 mg/mL) solution was added and reacted for 2 hours. After reaction, the culture medium was removed, 100 μL of dimethyl sulfoxide (DMSO) was added to each well, reacted at room temperature for 10 minutes, and absorbance was measured at 540 nm. Cell viability was measured by the rate of decrease in absorbance of the group with and without the addition of the sample solution.
<식 1><Equation 1>
RAW 264.7 cell에 대한 제조예 3의 혼합물 독성을 MTT assay를 통해 측정한 결과, 도 1에 나타낸 바와 같이 최고 농도인 500μg/mL에서 세포 독성을 나타내었다. 따라서 세포 독성 또는 변성으로 인한 cell population 저하에 우려가 없도록 90% 이상의 세포 생존율을 나타낸 300μg/mL 이하의 농도를 세포 실험에 적용하여 진행하였다(도 1).As a result of measuring the toxicity of the mixture of Preparation Example 3 to RAW 264.7 cells through MTT assay, it showed cytotoxicity at the highest concentration of 500 μg/mL, as shown in Figure 1. Therefore, to avoid concerns about cell population deterioration due to cytotoxicity or degeneration, cell experiments were conducted at a concentration of 300 μg/mL or less, which showed a cell survival rate of more than 90% (Figure 1).
[실험예 2] 항염증 효과 확인 : NO 저해능 측정[Experimental Example 2] Confirmation of anti-inflammatory effect: Measurement of NO inhibition ability
가. Nitric oxide(NO) inhibition activity 측정go. Nitric oxide (NO) inhibition activity measurement
NO 저해능 측정은 Grayand 등(1975)의 방법을 이용하여 실험을 진행하였다. RAW 264.7 cell의 상청액에 존재하는 NO의 양을 nitrite and nitrate로서 측정하였으며, nitrite and nitrate의 검출·정량에 사용되는 시약인 griess reagent를 사용하였다. 96 well plate에 5Х104 cells/well이 되도록 RAW 264.7 cell을 분주한 후 confluence가 80%일 때, 무혈청 배지로 교체한 다음 농도별로 제조한 시료(제조예 3) 20μL와 1μg/mL 농도의 lipopolysacchride (LPS)를 무처리군을 제외한 각 well에 20μL씩 첨가하여 24시간 동안 자극하였다. NO 생성량은 상층액 100μL와 griess regent 100μL를 실온에서 15분간 반응시킨 다음 540nm에서 흡광도를 측정하였다.The NO inhibition ability was measured using the method of Grayand et al. (1975). The amount of NO present in the supernatant of RAW 264.7 cells was measured as nitrite and nitrate, and griess reagent, a reagent used for detection and quantification of nitrite and nitrate, was used. After distributing RAW 264.7 cells to 5Х10 4 cells/well in a 96 well plate, when the confluence was 80%, replaced with serum-free medium, samples prepared by concentration (Preparation Example 3) lipopolysacchride at concentrations of 20μL and 1μg/mL (LPS) was added at 20 μL to each well except for the untreated group and stimulated for 24 hours. The amount of NO produced was measured by reacting 100 μL of supernatant with 100 μL of griess regent for 15 minutes at room temperature and then measuring the absorbance at 540 nm.
그 결과, LPS를 처리하지 않은 Normal group에서는 20% 미만의 NO가 생성되었고, LPS와 제조예 3의 시료를 처리한 후 측정한 NO 생성량을 LPS만 처리한 대조군과 비교 확인한 결과, 최고 농도인 300μg/mL에서 52.33%의 NO 저해 활성을 나타내었다(도 2).As a result, less than 20% of NO was produced in the Normal group that was not treated with LPS, and when the NO production amount measured after treating the sample of Preparation Example 3 with LPS was compared with the control group treated only with LPS, the highest concentration was 300 μg. /mL showed 52.33% NO inhibition activity (Figure 2).
나. western blot을 이용한 단백질 발현양상 측정me. Measurement of protein expression pattern using western blot
RAW 264.7 cell를 6well cell culture plate에 5Х105 cells/well이 되도록 분주하고, confluence가 80% 일 때, DMEM을 무혈청 배지로 교체한 다음 농도별로 희석한 시료 200μL와 LPS(1μg/mL) 200μL를 처리하여 24시간 동안 배양하였다. 상청액을 제거한 후 PBS로 2회 세척한 다음 lysis buffer를 첨가하여 cell을 용해시키고, scrapper로 수확하여 4℃, 12,000rpm에서 15분 동안 원심 분리하였다. 원심 분리하여 얻은 단백질은 bovine serum albumin(BSA)을 표준물질로 하여 Bicinchoninic acid(BCA) assay로 정량하였으며, 10% SDS-polyacrylamide gel을 이용하여 100 V에서 2시간 동안 전기영동하였다. 전기영동이 끝난 후 0.25A에서 2시간 이상 transfer를 실시하여 단백질을 멤브레인으로 옮겨준 다음 5% skim milk로 2시간 동안 blocking하여 background를 제거하였다. 그 다음 1차 antibody (1:1000)를 4℃에서 overnight하여 반응시킨 후, 1ХTBST 용액으로 3회 이상 세척한 다음 2차 antibody (1:1000)를 1시간 30분동안 반응시켰다. 반응 시킨 후 1 Х TBST로 3회 이상 세척을 반복한 다음 western imaging system을 이용하여 단백질 발현양상을 측정하였다. 양성 대조군으로는 세포의 조건에서도 발현 정도의 차이가 거의 없는 하우스 키핑 유전자인 β-Actin을 사용하였다.Dispense RAW 264.7 cells into a 6-well cell plate culture at 5Х10 5 cells/well, and when confluence is 80%, replace DMEM with serum-free medium and add 200μL of sample diluted by concentration and 200μL of LPS (1μg/mL). Treated and cultured for 24 hours. After removing the supernatant, the cells were washed twice with PBS, then lysis buffer was added to lyse the cells, harvested with a scraper, and centrifuged at 4°C and 12,000 rpm for 15 minutes. The protein obtained by centrifugation was quantified by Bicinchoninic acid (BCA) assay using bovine serum albumin (BSA) as a standard, and electrophoresed at 100 V for 2 hours using a 10% SDS-polyacrylamide gel. After electrophoresis was completed, transfer was performed at 0.25A for more than 2 hours to transfer the protein to the membrane, and then blocked with 5% skim milk for 2 hours to remove the background. Next, the primary antibody (1:1000) was reacted overnight at 4°C, washed more than three times with 1ХTBST solution, and then the secondary antibody (1:1000) was reacted for 1 hour and 30 minutes. After reaction, washing was repeated at least three times with 1 Х TBST, and protein expression patterns were measured using a western imaging system. As a positive control, β-Actin, a housekeeping gene with little difference in expression level even under cell conditions, was used.
그 결과, 최고 농도인 300 μg/mL에서 제조예 3은 78.88%의 우수한 iNOS 단백질 억제 효능을 나타내었다(도 3).As a result, Preparation Example 3 showed excellent iNOS protein inhibition efficacy of 78.88% at the highest concentration of 300 μg/mL (Figure 3).
Claims (7)
상기 벨벳콩 추출물은, 작두콩 추출물 100 중량부를 기준으로, 50~200 중량부로 포함되는, 항염증용 조성물.According to paragraph 1,
An anti-inflammatory composition comprising 50 to 200 parts by weight of the velvet bean extract, based on 100 parts by weight of the velvet bean extract.
상기 조성물은,
NO(Nitric oxide) 발생을 억제하는, 항염증용 조성물.According to paragraph 1,
The composition is,
An anti-inflammatory composition that inhibits the generation of NO (Nitric oxide).
상기 벨벳콩 추출물 및 작두콩 추출물의 추출용매는,
물, 유기 용매 또는 이들의 혼합물을 포함하는, 항염증용 조성물.According to paragraph 1,
The extraction solvent for the velvet bean extract and the pea extract is,
An anti-inflammatory composition comprising water, an organic solvent, or a mixture thereof.
상기 벨벳콩 추출물의 추출용매는 물을 포함하고,
상기 작두콩 추출물의 추출용매는 알코올을 포함하는, 항염증용 조성물.According to paragraph 1,
The extraction solvent of the velvet bean extract includes water,
An anti-inflammatory composition wherein the extraction solvent of the pea extract contains alcohol.
상기 알코올의 농도는 50~90%(w/w)인, 항염증용 조성물.According to clause 5,
An anti-inflammatory composition wherein the alcohol concentration is 50 to 90% (w/w).
상기 조성물은,
식품 조성물, 약학적 조성물 또는 화장료 조성물을 포함하는, 항염증용 조성물.According to any one of claims 1 to 6,
The composition is,
An anti-inflammatory composition, including a food composition, pharmaceutical composition, or cosmetic composition.
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