KR20180038090A - Composition for immune enhancement comprising black rice extracts as active ingredient - Google Patents
Composition for immune enhancement comprising black rice extracts as active ingredient Download PDFInfo
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- KR20180038090A KR20180038090A KR1020160128123A KR20160128123A KR20180038090A KR 20180038090 A KR20180038090 A KR 20180038090A KR 1020160128123 A KR1020160128123 A KR 1020160128123A KR 20160128123 A KR20160128123 A KR 20160128123A KR 20180038090 A KR20180038090 A KR 20180038090A
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- South Korea
- Prior art keywords
- black rice
- extract
- rice bran
- ccl2
- composition
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Abstract
Description
본 발명은 흑미 미강 추출물을 유효성분으로 포함하는 면역증강용 조성물에 관한 것이다. The present invention relates to a composition for enhancing immunity comprising a black rice microgranular extract as an active ingredient.
흑미는 특유의 색과 향으로 인하여 다양한 형태의 식품으로 가공되고 있고 그 소비가 점차 증가하고 있다. 흑미의 향은 에탄디올(ethanediol), 구아야콜(guaiacol)과 같은 알코올 성분과 헥사노데카노인산(hexadecanoic acid), 헥산알(hexanal), 아세트산(acetic acid)과 같은 케톤, 알데하이드 및 유기산에 기인하며, 색소성분은 시아니딘-3-글루코시드(cyanidin-3-glucoside)와 페오니딘-3-글루코시드(peonidin-3-glucoside)와 같은 배당체가 주된 성분이라고 보고되고 있다.Black rice is processed into various forms of food due to its unique color and flavor, and its consumption is gradually increasing. The aroma of black rice is derived from alcohol components such as ethanediol and guaiacol and ketones such as hexadecanoic acid, hexanal and acetic acid, aldehydes and organic acids. , And the pigment component is reported to be mainly composed of glycosides such as cyanidin-3-glucoside and peonidin-3-glucoside.
특히, 흑미의 색소성분은 다양한 구조와 분자량을 지니는 폴리페놀 화합물을 함유하고 있으며, 이러한 폴리페놀 화합물은 항산화성, 항균성, 항암성 등의 생리활성을 갖는 것으로 확인되고 있다. 또한, 흑미는 현미로 보급되며 일반 현미보다 식이섬유 함량이 높고 독특한 향미와 단백질, 비타민 B, 무기질 함량도 많다.In particular, the pigment component of black rice contains polyphenol compounds having various structures and molecular weights, and these polyphenol compounds have been confirmed to have physiological activities such as antioxidant, antimicrobial and anticancer properties. In addition, black rice is supplied in brown rice, has higher dietary fiber content than ordinary brown rice, has unique flavor, protein, vitamin B, and mineral content.
면역이란 동물체 내에 존재하는 자기방어 체계로서, 외부로부터 침입해오는 각종 물질이나 생명체를 자기자신과 구별해내어 이 침입자를 제거하는 생물학적 현상이다. 이러한 자기방어를 위한 감시체계는 선천성 면역과 적응성 면역(adaptive immunity)으로 대별할 수 있다.Immunity is a biological phenomenon that is a self-defense system in an animal. It is a biological phenomenon that distinguishes various substances or living things invading from the outside from oneself and removes this intruder. This monitoring system for self defense can be divided into congenital immunity and adaptive immunity.
적응성 면역은 B 세포나 T 세포에 의한 면역반응으로 일단 체내로 침입한 항원에 대하여 반응을 하되, 반드시 같은 종류의 항원이 계속 존재하거나 반복 침입해왔을 경우에 작용하는 면역체계이다. 따라서, 이러한 면역반응은 특정 항원에 대한 특이한 반응이다. 이에 반해, 선천성 면역은 체내에는 어떤 항원에 대해 노출된 경험이 없는 경우라도 직접적으로 반응하여 공격세포를 파괴하는 비특이적(non-specific) 면역반응으로, 단핵세포/대식세포(monocytes/macrophages) 및 중성구(neutrophils)와 같은 식세포(phagocyte) 등이 관여하여 공격대상 세포의 종류에 별로 구애됨이 없이 다양한 기능을 발휘하는 것이 특징이며, 진화적으로 보다 오래된 자기방어 체제이다. Adaptive immunity is an immune system that reacts to an antigen that enters the body through an immune response by a B cell or a T cell, but only when the same kind of antigen is present or repeatedly invaded. Thus, such an immune response is a specific response to a particular antigen. In contrast, congenital immunity is a non-specific immune response that directly attacks and destroys attack cells, even in the absence of exposure to certain antigens in the body, and is associated with monocytes / macrophages and neutrophils (phagocyte) such as neutrophils are involved, and it is characterized by exhibiting various functions without being restricted to kinds of cells to be attacked. It is an evolutionarily older self defense system.
대식세포(Macrophage)는 다양한 기능을 가진 세포로 산화적 스트레스 상황에서 여러 가지 사이토카인(cytokine)과 일산화산소(NO)를 생성하여 면역체계에서 중요한 역할을 한다. 특히 대식세포에서 LPS(Lipopolysaccharide), 사이토카인, TNF-α와 같은 자극에 의해 발현되는 iNOS는 장시간 동안 다량의 NO를 생산하여, NFκB 활성을 촉진시키는 것으로 알려져 있다. 활성화된 대식세포에 의한 슈퍼옥사이드 음이온(superoxide anion, O2-), 과산화수소(hydrogen peroxide, H2O2)와 같은 활성산소종(reactive oxygen species; ROS) 및 일산화질소(nitric oxide, NO)의 생산은 비특이적 면역에 있어서 중요한 세포독성 및 세포활성 억제기작이다. 대식세포에 의한 ROS 및 NO의 생성에 어떤 천연화합물이 영향을 미치는지 많은 연구들이 수행되어 왔다. 대식세포는 항원을 제시하거나(antigen-presenting), 종양을 없애거나(tumoricidal) 미생물세포를 죽이는(microbicidal) 세포로서, 세포매개(cell-mediated) 또는 체액성 면역(humoral immunity)에 중심적인 역할을 하는 조절세포로, 활성화된 대식세포에서 생산되는 NO는 비특이적 숙주방어기작인 대식작용, 그리고 세균 및 암세포의 증식억제활성을 보인다(Dawson T.M., et al., Annu. Rev. Med, 47, pp.219-227, 1996).Macrophage is a multifunctional cell that plays an important role in the immune system by producing various cytokines and NO in the context of oxidative stress. In particular, iNOS expressed by stimuli such as Lipopolysaccharide (LPS), cytokine, and TNF-α in macrophages is known to produce large amounts of NO over a long period of time to promote NFκB activity. Production of reactive oxygen species (ROS) and nitric oxide (NO) such as superoxide anion (O2-), hydrogen peroxide (H2O2) by activated macrophages is not nonspecific Which is an important cytotoxic and cellular activity inhibitory mechanism. A number of studies have been conducted on which natural compounds affect the production of ROS and NO by macrophages. Macrophages play a central role in cell-mediated or humoral immunity as antigen-presenting, tumoricidal, or microbicidal cells. (Dawson TM, et al., Annu. Rev. Med., 47, pp. 219-219). In addition, it has been shown that the NO produced by activated macrophages is a nonspecific host defense mechanism, -227, 1996).
대식세포는 많은 라이소솜을 가지고 있고 이들은 산성가수분해효소와 과산화효소를 함유하고 있다. 또한, 유리면과 플라스틱 표면에 강하게 부착하는 성질이 있으며 미생물이나 종양세포 등을 활발하게 탐식한다. 상기 세포는 IFN-γ등의 사이토카인 수용체를 가지고 있다. 이들은 보체성분, 인터페론, 인터루킨-1 및 종양괴사인자 같은 사이토카인을 생산하며 T-세포로부터 생산되는 여러 가지 사이토카인에 의해 기능이 증강될 수 있다(Richard A. Goldsby, et al., KUBY Immunology. 2000).Macrophages contain many lysosomes, which contain acid hydrolytic enzymes and peroxidase. In addition, it strongly attaches to glass surface and plastic surface, and actively digests microorganisms and tumor cells. The cell has a cytokine receptor such as IFN-y. They produce cytokines such as complement components, interferons, interleukin-1 and tumor necrosis factor, and can be augmented by various cytokines produced from T-cells (Richard A. Goldsby, et al., KUBY Immunology. 2000).
면역증강제란 숙주의 특이 또는 비특이, 세포성 및 체액성 면역반응을 상승시키는 모든 물질을 말하는데, 최근에 감염이나 종양의 치료에 면역강화가 이용되면서 많은 관심을 불러일으키고 있다. 부작용이 없으면서 유용한 면역증강 효과를 나타내는 물질의 개발은 대단히 중요한 의미를 가져, 그 개발이 요구되고 있는 실정이다. Immune enhancers are all substances that increase the host's specific or nonspecific, cellular and humoral immune response. Recently, immune enrichment has been used in the treatment of infections and tumors, which has attracted much attention. The development of a substance which shows useful immune enhancing effect without side effects is of great importance and development is required.
본 발명의 목적은 흑미 미강 추출물을 유효성분으로 포함하는 면역증강용 약학 조성물을 제공하는 것이다. It is an object of the present invention to provide a pharmaceutical composition for enhancing immunity comprising an extract of black rice microgranules as an active ingredient.
본 발명의 또 다른 목적은 흑미 미강 추출물을 유효성분으로 포함하는 면역증강용 건강기능식품을 제공하는 것이다. It is still another object of the present invention to provide a health functional food for immunity enhancement comprising a black rice bran extract as an active ingredient.
본 발명의 또 다른 목적은 감마오리자놀을 유효성분으로 포함하는 면역증강용 조성물을 제공하는 것이다. Another object of the present invention is to provide a composition for enhancing immunity comprising gamma-orizanol as an active ingredient.
본 발명의 또 다른 목적은 (a) 단핵구(Monocyte)에서 CCL2 또는 CXCL8을 발현하는 세포주를 구축하는 단계; (b) 상기 (a)단계에서 구축된 세포주에 시험물질을 접촉시키고 CCL2 또는 CXCL8의 발현량을 측정하는 단계; 및 (c) 상기 (b) 단계에서 측정된 CCL2 또는 CXCL8의 발현량이 대조군보다 증가된 시험물질을 선별하는 단계;를 포함하는 것을 특징으로 하는 면역증강제 스크리닝 방법을 제공하는 것이다. Yet another object of the present invention is to provide a method for producing a cell line, comprising: (a) constructing a cell line expressing CCL2 or CXCL8 in a monocyte; (b) contacting the test substance with the cell line constructed in the step (a) and measuring the expression level of CCL2 or CXCL8; And (c) selecting a test substance whose expression level of CCL2 or CXCL8 measured in step (b) is higher than that of the control group.
상기 목적을 달성하기 위하여, 본 발명은 흑미 미강 추출물을 유효성분으로 포함하는 면역증강용 조성물을 제공한다. In order to achieve the above object, the present invention provides a composition for enhancing immunity comprising an extract of black rice microgranules as an active ingredient.
본 발명의 일실시예에 있어서, 상기 흑미 미강 추출물은 물 또는 유기용매를 이용하여 수득한 추출물인 것일 수 있다. In one embodiment of the present invention, the black rice microgranular extract may be an extract obtained by using water or an organic solvent.
본 발명의 일실시예에 있어서, 상기 유기용매는 메탄올, 에탄올, 프로판올, 이소프로판올, 부탄올, 아세톤, 에테르, 벤젠, 클로로포름, 에틸아세테이트, 메틸렌클로라이드, 헥산 또는 시클로헥산인 것일 수 있다. In one embodiment of the present invention, the organic solvent may be methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane or cyclohexane.
본 발명의 일실시예에 있어서, 상기 흑미 미강 추출물은 흑미 100 중량부에 대하여 에탄올 85 내지 95 중량부를 첨가하여 음지에서 3일 동안 정치하여 추출하는 것일 수 있다. In one embodiment of the present invention, the black rice bran extract may be prepared by adding 85 to 95 parts by weight of ethanol to 100 parts by weight of black rice, and allowing the mixture to stand for 3 days on a shade.
본 발명의 일실시예에 있어서, 상기 흑미 미강 추출물의 농도는 10 내지 200 μg/ml 인 것일 수 있고, 바람직하게는 50 내지 100 μg/ml인 것일 수 있고, 더욱 바람직하게는 100 ㎍/ml 인 것일 수 있다. In one embodiment of the present invention, the concentration of the black rice bran extract may be 10-200 μg / ml, preferably 50-100 μg / ml, more preferably 100 μg / ml Lt; / RTI >
본 발명의 일실시예에 있어서, 상기 면역증강은 비특이적인 선천성 면역반응에 대한 활성을 증가시키는 것일 수 있다. In one embodiment of the invention, the immunostimulation may be to increase the activity against a nonspecific congenital immune response.
또한, 본 발명은 흑미 미강 추출물을 유효성분으로 포함하는 면역증강용 건강기능식품을 제공한다. In addition, the present invention provides a health functional food for immunity enhancement comprising a black rice bran extract as an active ingredient.
본 발명의 일실시예에 있어서, 상기 건강기능식품은 캡슐, 정제, 분말, 과립, 액상, 환, 편상, 페이스트상, 시럽, 겔, 젤리 또는 바인 것일 수 있다. In one embodiment of the present invention, the health functional food may be a capsule, a tablet, a powder, a granule, a liquid, a ring, a flake, a paste, a syrup, a gel, a jelly or a bar.
또한, 본 발명은 감마오리자놀 복합체를 유효성분으로 포함하는 면역증강용 조성물을 제공한다. The present invention also provides a composition for enhancing immunity comprising gamma-oryzanol complex as an active ingredient.
본 발명의 일실시예에 있어서, 상기 감마오리자놀 복합체는 (a) 흑미 미강 추출물을 감압여과기에서 여과시키고 감압농축기로 여과액을 농축시킨 후 300ml의 아세톤으로 재용해하는 단계; (b) 60℃ 오븐에서 1시간 정치하여 맑은 용액(clear solution)을 획득한 후 2℃ 냉장고에서 24시간 정치하여 고체상과 액체상을 분리시키는 단계; (c) 상기 분리된 고체상은 버리고(dewazing), 상기에서 획득한 액체상의 여과액을 -5℃에서 다시 24시간 정치한 후 분리된 하층액을 버리고(degumming), 상기에서 획득한 상층액을 감압농축시키는 단계; 및 (d) 상기 농축된 상층액은 400ml의 발효주정:아세톤(7:3, v/v) 용매로 재용해하고 -60℃에서 48시간 이상 정치시켜 고체상과 액체상으로 분리하킨 후 여과하여 감마오리자놀 복합체를 획득하는 단계;를 포함하는 것일 수 있다. In one embodiment of the present invention, the gamma-oryzanol complex is prepared by: (a) filtering a black rice bran extract with a vacuum filter, concentrating the filtrate with a vacuum concentrator, and redissolving the filtrate with 300 ml of acetone; (b) standing in a 60 ° C oven for 1 hour to obtain a clear solution, and then standing in a refrigerator at 2 ° C for 24 hours to separate the solid phase and the liquid phase; (c) dewazing the separated solid phase, allowing the filtrate of the liquid phase obtained above to stand at -5 DEG C for another 24 hours, then removing the separated lower layer liquid, Concentrating; And (d) the concentrated supernatant is redissolved in 400 ml fermentation alcohol: acetone (7: 3, v / v), and the mixture is left at -60 캜 for at least 48 hours to separate into solid and liquid phases. And acquiring the complex.
본 발명의 일실시예에 있어서, 상기 감마오리자놀의 농도는 10 내지 100 μg/ml 인 것일 수 있고, 바람직하게는 50 내지 100 μg/ml인 것일 수 있고, 더욱 바람직하게는 50 μg/ml인 것일 수 있다. In one embodiment of the present invention, the concentration of gamma oryzanol may be 10-100 μg / ml, preferably 50-100 μg / ml, more preferably 50 μg / ml .
본 발명의 일실시예에 있어서, 상기 면역증강은 비특이적인 선천성 면역반응에 대한 활성을 증가시키는 것일 수 있다. In one embodiment of the invention, the immunostimulation may be to increase the activity against a nonspecific congenital immune response.
본 발명은 (a) 단핵구(Monocyte)에서 CCL2(chemokine (C-C motif) ligand 2) 또는 IL-8(Interleukin 8)을 발현하는 세포주를 구축하는 단계; (b) 상기 (a)단계에서 구축된 세포주에 후보물질을 접촉시키고 CCL2 또는 IL-8의 발현량을 측정하는 단계; 및 (c) 상기 (b)단계에서 측정된 CCL2 또는 IL-8의 발현량이 대조군보다 증가된 후보물질을 선별하는 단계;를 포함하는 것일 수 있다. (A) constructing a cell line expressing CCL2 (chemokine (C-C motif) ligand 2) or IL-8 (Interleukin 8) in a monocyte; (b) contacting a candidate substance with the cell line constructed in the step (a) and measuring the expression level of CCL2 or IL-8; And (c) selecting candidate substances for which the expression level of CCL2 or IL-8 measured in step (b) is higher than that of the control group.
본 발명의 일실시예에 있어서, 상기 면역증강은 비특이적인 선천성 면역반응에 대한 활성을 증가시키는 것일 수 있다. In one embodiment of the invention, the immunostimulation may be to increase the activity against a nonspecific congenital immune response.
본 발명은 (a) 흑미 미강 추출물을 감압여과기에서 여과시키고 감압농축기로 여과액을 농축시킨 후 300ml의 아세톤으로 재용해하는 단계; (b) 60℃ 오븐에서 1시간 정치하여 맑은 용액(clear solution)을 획득한 후 2℃ 냉장고에서 24시간 정치하여 고체상과 액체상을 분리시키는 단계; (c) 상기 분리된 고체상은 버리고(dewazing), 상기에서 획득한 액체상의 여과액을 -5℃에서 다시 24시간 정치한 후 분리된 하층액을 버리고(degumming), 상기에서 획득한 상층액을 감압농축시키는 단계; 및 (d) 상기 농축된 상층액은 400ml의 발효주정:아세톤(7:3, v/v) 용매로 재용해하고 -60℃에서 48시간 이상 정치시켜 고체상과 액체상으로 분리하킨 후 여과하여 감마오리자놀 복합체를 획득하는 단계;를 포함하는 감마오리자놀 복합체의 제조방법을 제공한다. (A) filtering the black rice bran extracts in a vacuum filter, concentrating the filtrate with a vacuum concentrator, and redissolving the filtrate with 300 ml of acetone; (b) standing in a 60 ° C oven for 1 hour to obtain a clear solution, and then standing in a refrigerator at 2 ° C for 24 hours to separate the solid phase and the liquid phase; (c) dewazing the separated solid phase, allowing the filtrate of the liquid phase obtained above to stand at -5 DEG C for another 24 hours, then removing the separated lower layer liquid, Concentrating; And (d) the concentrated supernatant is redissolved in 400 ml fermentation alcohol: acetone (7: 3, v / v), and the mixture is left at -60 캜 for at least 48 hours to separate into solid and liquid phases. To obtain a complex; and a process for producing a gamma oriazolol complex.
본 발명에 따른 흑미 미강 추출물 또는 감마오리자놀은 면역활성과 관련된 유전자인 CD14 및 TLR4 유전자의 발현량을 증가시키고, TNFα, IL-1β, IL-8, CCL2 및 CCL5 등의 사이토카인 유전자의 발현량도 증가시키며, CCL2, CCL3/4 및 CCL5 사이토카인의 분비량을 현저히 증가시키고, 특히, 대식세포의 탐식작용을 증가시킴으로써, 비특이적인 선천성 면역반응의 활성을 유도할 수 있어 면역증강제로 유용하게 사용할 수 있다.The amount of expression of cytokine genes such as TNFα, IL-1β, IL-8, CCL2 and CCL5 is increased by increasing the expression level of CD14 and TLR4 genes, which are genes related to immunological activity, And can increase the secretion amount of CCL2, CCL3 / 4 and CCL5 cytokines, and in particular, can increase the phagocytic activity of macrophages, thereby inducing the activity of a nonspecific congenital immune response, thus being useful as an immunity enhancer .
도 1은 흑미 미강으로부터 추출된 감마오리자놀 복합체의 분말을 나타낸 것이다.
도 2는 HPLC를 이용하여 흑미 미강 추출물에서 감마오리자놀을 분석한 결과이다.
도 3은 흑미 미강 추출물을 THP-1 단핵구 세포에 처리한 후, 세포생존능을 측정하여 흑미 미강 추출물에 대한 세포독성 여부를 확인한 결과이다.
도 4는 흑미 미강 추출물을 THP-1 세포에 처리한 후 면역활성 관련 유전자에 대한 mRNA 발현량을 RT-PCR로 확인한 결과이다. (A)는 CD14 및 TLR4 유전자의 mRNA 발현량을 확인한 것이고, (B)는 TNFα, IL-1β, IL-8, CCL2 및 CCL5 유전자의 mRNA 발현량을 확인한 결과이다.
도 5는 흑미 미강 추출물을 THP-1 세포에 처리한 후 IL-8과 CCL2 유전자에 대한 mRNA 발현량을 RT-PCR로 확인한 결과이다. (A)는 RT-PCR 결과를 1% 한천겔에서 전기영동한 결과이다. (B)는 (A)의 결과를 바탕으로 IL-8 RT-PCR 밴드 결과를 ImageJ 프로그램을 이용하여 GAPDH 밴드 수치로 보정한 것을 도식화한 것이고, (C)는 (A)의 CCL2 RT-PCR 밴드 결과를 ImageJ 프로그램을 이용하여 GAPDH 밴드 수치로 보정한 것을 도식화한 것이다.
도 6은 흑미 미강 추출물을 THP-1 세포에 처리한 후 배양액으로 분비되는 사이토카인의 종류 및 분비량을 항체어레이를 이용하여 확인한 결과이다. (A)는 사이토카인 항체가 점적된 어레이 모식도이고, (B)는 흑미 미강 추출물을 처리한 경우 배양액으로 분비된 사이토카인을 확인한 결과이며, (C)는 상기 (B)의 결과를 바탕으로 ImageJ 프로그램을 이용하여 정량한 결과이다.
도 7은 흑미 미강 추출물을 RAW 264.7 세포에 처리한 후 형광물질이 부착된 라텍스-비드의 탐식능을 확인한 결과이다. (A)는 대식세포인 RAW 264.7 세포에 의해 탐식된 비드를 형광현미경으로 관찰한 것이고, (B)는 형광 라텍스-비드를 탐식한 대식세포의 수를 측정한 결과이다.
도 8은 감마오리자놀을 THP-1 세포에 처리한 후 IL-8과 CCL2 유전자에 대한 mRNA 발현량을 RT-PCR로 확인한 결과이다. (A)는 RT-PCR 결과를 1% 아가로오스 겔에서 전기영동한 결과이다. (B)는 (A)의 IL-8에 대한 밴드 결과를 ImageJ 프로그램을 이용하여 수치화한 결과이고, (C)는 (A)의 CCL2에 대한 밴드 결과를 ImageJ 프로그램을 이용하여 수치화한 결과이다.
도 9는 흑미 미강 추출물, 감마오리자놀 및 IL-8을 RAW 264.7 세포에 처리한 후 형광물질이 부착된 라텍스-비드의 탐식능을 확인한 결과이다. (A)는 대식세포인 RAW 264.7 세포에 의해 탐식된 비드를 형광현미경으로 관찰한 것이고, (B)는 형광 라텍스-비드를 탐식한 대식세포의 수를 측정한 결과이다.Fig. 1 shows the powder of gamma oriazolol complex extracted from black rice bran.
FIG. 2 shows the results of analysis of gamma oryzanol in black rice microcarpa extract using HPLC.
FIG. 3 shows the results of determining the cytotoxicity of the black rice extract of rice bran by measuring the cell viability after treatment of the black rice bran extract with THP-1 mononuclear cells.
FIG. 4 shows the results of RT-PCR analysis of the amount of mRNA expression in the immunoreactivity-related gene after treatment of black rice microbial extract with THP-1 cells. (A) shows the mRNA expression levels of the CD14 and TLR4 genes, and (B) shows the mRNA expression levels of the TNFα, IL-1β, IL-8, CCL2 and CCL5 genes.
FIG. 5 shows the results of RT-PCR analysis of mRNA expression levels of IL-8 and CCL2 genes after treatment of THP-1 cells in black rice extract. (A) is the result of RT-PCR with 1% agarose gel electrophoresis. (B) is a graphical representation of the results of IL-8 RT-PCR bands calibrated to GAPDH band values using the ImageJ program based on the results of (A), (C) And the result is calibrated to the GAPDH band value using the ImageJ program.
FIG. 6 shows the results of confirming the type and amount of secretion of cytokine secreted from culture broth of THP-1 cells after treatment of black rice microbial extract with an antibody array. (B) shows the result of confirming the cytokine secreted by the culture broth when the black rice bran extract was treated, (C) shows the result of (B) The results are quantified using a program.
FIG. 7 shows the results of confirming the ability of the latex-beads to attach the fluorescent substance to the RAW 264.7 cells treated with the black rice bran extract. (A) is a fluorescence microscopic observation of beads fished by RAW 264.7 cells, which are macrophages, and (B) is a result of measurement of the number of macrophages that have fluorescence latex-beads.
FIG. 8 shows the results of RT-PCR analysis of mRNA expression levels of IL-8 and CCL2 genes after treatment of gamma oryzanol with THP-1 cells. (A) shows the result of RT-PCR on 1% agarose gel electrophoresis. (B) is a result of quantifying the band result for IL-8 in (A) using the ImageJ program, and (C) is a result of quantizing the band result for CCL2 in (A) using the ImageJ program.
FIG. 9 shows the results of confirming the ability of the latex-bead to adsorb the fluorescent substance after treatment of RAW 264.7 cells with the black rice extract of rice bran, gamma oryzanol, and IL-8. (A) is a fluorescence microscopic observation of beads fished by RAW 264.7 cells, which is a macrophage, and (B) is a result of measuring the number of macrophages that have been photographed with a fluorescent latex-bead.
상기 흑미으로부터 추출물을 추출하기 위한 적절한 용매로는 이에 제한되지 않으나, 물 또는 유기용매를 사용할 수 있으며, 예를 들어, 정제수, 메탄올(methanol), 에탄올(ethanol), 프로판올(propanol), 이소프로판올(isopropanol), 부탄올(butanol) 등을 포함하는 탄소수 1 내지 4의 알코올, 아세톤(acetone), 에테르(ether), 벤젠(benzene), 클로로포름(chloroform), 에틸아세테이트(ethyl acetate), 메틸렌클로라이드(methylene chloride), 헥산(hexane) 및 시클로헥산(cyclohexane) 등의 각종 용매를 단독으로 혹은 혼합하여 사용할 수 있다. 바람직하게는 에탄올(주정), n-헥산 및 에틸아세테이트 용매를 사용할 수 있다.Suitable solvents for extracting the black rice include, but are not limited to, water or an organic solvent. For example, purified water, methanol, ethanol, propanol, isopropanol Acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, and the like, which have a carbon number of 1 to 4, and include butanol, , Hexane, and cyclohexane, may be used alone or in combination. Preferably, ethanol (alcohol), n-hexane and ethyl acetate solvents can be used.
추출 방법으로는 열수추출법, 냉침추출법, 환류냉각추출법, 용매추출법, 수증기증류법, 초음파추출법, 용출법, 압착법 등의 방법 중 어느 하나를 선택하여 사용할 수 있다. 또한, 목적하는 추출물은 추가로 통상의 분획 공정을 수행할 수도 있으며, 통상의 정제 방법을 이용하여 정제될 수도 있다. 본 발명의 흑미 미강 추출물의 제조방법에는 제한이 없으며, 공지되어 있는 어떠한 방법도 이용될 수 있다.As the extraction method, any one of the methods such as hot water extraction method, cold extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method and compression method can be selected and used. In addition, the desired extract may be further subjected to a conventional fractionation process or may be purified using a conventional purification method. There is no limitation on the method for producing the black rice bran extract of the present invention, and any known method can be used.
예를 들면, 본 발명의 조성물에 포함되는 흑미 미강 추출물은 상기한 열수 추출 또는 용매 추출법으로 추출된 1차 추출물을, 감압 증류 및 동결 건조 또는 분무 건조 등과 같은 추가적인 과정에 의해 분말 상태로 제조할 수 있다. 또한, 상기 1차 추출물을 실리카겔 컬럼 크로마토그래피(silica gel column chromatography), 박층 크로마토그래피(thin layer chromatography), 고성능 액체 크로마토그래피(high performance liquid chromatography) 등과 같은 다양한 크로마토그래피를 이용하여 추가로 정제된 분획을 얻을 수도 있다. 따라서, 본 발명에 있어서, 흑미 미강 추출물은 추출, 분획 또는 정제의 각 단계에서 얻어지는 모든 추출액, 분획 및 정제물, 그들의 희석액, 농축액 또는 건조물을 모두 포함하는 개념이다.For example, the black rice extract of rice bran contained in the composition of the present invention can be prepared into a powder state by an additional process such as vacuum distillation, freeze drying, or spray drying, have. In addition, the primary extract can be further fractionated using a variety of chromatographies, such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, . Therefore, in the present invention, the black rice bran extract is a concept that includes all the extracts, fractions and tablets obtained in each step of extraction, fractionation or purification, their diluted solutions, concentrates or dried products.
본 발명에 따른 추출물을 함유하는 약학조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있으며, 추출물을 포함하는 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.The pharmaceutical composition containing the extract according to the present invention may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions Examples of the carrier, excipient and diluent which can be contained in the composition containing the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, Gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.
본 발명의 추출물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나 바람직한 효과를 위해서, 본 발명의 추출물은 1일 0.0001 내지 100㎎/㎏으로, 바람직하게는 0.001 내지 10㎎/㎏으로 투여하는 것이 좋다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The preferred dosage of the extract of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg per day, preferably 0.001 to 10 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.
본 발명의 추출물은 쥐, 생쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁내 경막 또는 뇌혈관내(intracerebroventricular) 주사에 의해 투여될 수 있다.The extract of the present invention can be administered to mammals such as rats, mice, livestock, humans and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.
또한, 상기 흑미 미강 추출물을 유효성분으로 포함하는 본 발명의 조성물은 식품 조성물로 사용될 수 있다. 특히, 본 발명에 따른 흑미 미강 추출물은 세포에 독성을 유도하지 않음을 일실시예를 통해 확인할 수 있었다. 따라서 본 발명의 식품 조성물은 유효성분인 흑미 미강 추출물을 함유하는 것 외에 통상의 식품 조성물과 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다.In addition, the composition of the present invention containing the above-described black rice microgranular extract as an active ingredient can be used as a food composition. In particular, it was confirmed by one embodiment that the black rice extract of rice bran according to the present invention does not induce toxicity to cells. Therefore, the food composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient as well as ordinary food compositions, in addition to containing the black rice extract of rice bran as an effective ingredient.
상기 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상기 향미제는 천연 향미제 (타우마틴), 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제 (사카린, 아스파르탐 등)를 유리하게 사용할 수 있다.Examples of such natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. The flavors may be advantageously used as natural flavors (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.).
본 발명의 식품 조성물은 상기 약제학적 조성물과 동일한 방식으로 제제화되어 기능성 식품으로 이용하거나, 각종 식품에 첨가할 수 있다. 본 발명의 조성물을 첨가할 수 있는 식품으로는 예를 들어, 음료류, 육류, 초코렛, 식품류, 과자류, 피자, 라면, 기타 면류, 껌류, 사탕류, 아이스크림류, 알코올 음료류, 비타민 복합제 및 건강보조식품류 등이 있다.The food composition of the present invention can be formulated in the same manner as the above pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolates, foods, confectionery, pizza, ram noodles, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, .
또한, 상기 식품 조성물은 유효성분인 흑미 미강 추출물 외에 여러 가지 영양제, 비타민, 광물 (전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제 (치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 식품 조성물은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다.In addition, the food composition may further contain various additives such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and flavors such as natural flavors, coloring agents and aging agents (cheese, chocolate, etc.) Alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.
실시예Example 1. One. 흑미Black rice 미강Rice bran 추출물 및 The extract and 감마오리자놀Gamma oruzanol 복합체의 제조 Manufacture of Composites
흑미 미강 추출물은 흑미 10kg을 9L의 발효주정을 유리병에 넣은 후 음지에서 3일간 정치하여 추출하였고, 이로 부터 500g의 미강 분말(rice bran powder)을 얻었다.The rice bran extract of black rice was prepared by adding 10 kg of black rice into a glass bottle of 9 L fermented juice, and then letting it stand for 3 days in a shade to obtain 500 g of rice bran powder.
감마오리자놀 복합체는 상기 흑미 미강 추출물을 감압여과기에서 여과시키고 감압농축기로 여과액을 농축시킨 후 300ml의 아세톤으로 재용해하여 비커에 담았다. 60℃ 오븐에서 1시간 정치하여 맑은 용액(clear solution)을 획득한 후 2℃ 냉장고에서 24시간 정치하여 고체상과 액체상을 분리한 후, 분리된 고체상은 버리고(dewazing), 획득한 액체상의 여과액을 -5℃에서 다시 24시간 정치한 후 분리된 하층액을 버리고(degumming), 획득한 상층액을 감압농축하였다. 농축된 상층액은 400ml의 발효주정:아세톤(7:3, v/v) 용매로 재용해하고 -60℃에서 48시간 이상 정치시켜 고체상과 액체상으로 분리시킨 후 여과하여 감마오리자놀 복합체를 획득하였다(도 1). The gamma-oryzanol complex was filtered through a vacuum filter, and the filtrate was concentrated using a vacuum concentrator, followed by re-dissolving in 300 ml of acetone. The solution was allowed to stand in an oven at 60 ° C for 1 hour to obtain a clear solution. The solution was allowed to stand in a refrigerator at 2 ° C for 24 hours to separate the solid phase from the liquid phase. The separated solid phase was discarded, After standing for 24 hours at -5 DEG C, the separated lower layer was removed, and the obtained supernatant was concentrated under reduced pressure. The concentrated supernatant was redissolved in 400 ml of fermentation alcohol: acetone (7: 3, v / v) and allowed to stand at -60 ° C for at least 48 hours, separated into solid and liquid phases and then filtered to obtain gamma oriazolol complex 1).
실시예Example 2. 2. HPLCHPLC (high-performance liquid chromatography)를 이용한 (high-performance liquid chromatography) 감마오Gamma-o 리자놀 복합체 성분의 분석Analysis of Lysanol Complex Component
감마오리자놀 복합체의 성분은 YMC PAKC ODA-AM (4.6×250 mm I.D., 4 μm; YMC Co., Ltd., Japan) 역상 컬럼과 2998 photodiode array detector (PDA)를 장착한 Alliance e2695 HPLC system (Waters Co. Milford,MA, USA)을 이용하여 분석하였다. 검출 파장은 250 내지 400 nm (대표파장 325 nm)으로 설정하였으며, 오븐 온도는 30℃, 유속은 1.4 ml/min 이었다. 이동상은 MeOH : ACN : MC : acetic acid (50:44:3:3, v/v/v/v)으로 50분간 일정하게 흘려주었다. 총 10개의 감마오리자놀(1. Δ7-stigmastenyl ferulate; 2. stigamsteryl ferulate; 3. cycloartenyl ferulate; 4. 24-methylenecycloartanyl ferulate; 5. Δ7-campestenyl ferulate; 6. campesteryl ferulate; 7. Δ7-sitostenyl ferulate; 8. sitosteryl ferulate; 9. campestanyl ferulate; 및 10. sitostanyl ferulate)을 확인하였으며(도 2), cycloartenyl ferulate (CAF) 및 24-methylenecycloartanyl ferulate 등이 주요(major) 성분으로 확인되었다.The components of the gamma oryzanol complex were Alliance e2695 HPLC system (Waters Co., Ltd.) equipped with a reversed phase column of YMC PAKC ODA-AM (4.6 x 250 mm ID, 4 μm; YMC Co., Ltd., Japan) and a 2998 photodiode array detector Milford, Mass., USA). The detection wavelength was set to 250 to 400 nm (typical wavelength: 325 nm). The oven temperature was 30 ° C and the flow rate was 1.4 ml / min. The mobile phase was flowed constantly for 50 minutes with MeOH: ACN: MC: acetic acid (50: 44: 3: 3, v / v / v / v). A total of 10 gamma oryzanol (1.7-stigmasthenyl ferulate; 2. stigamasteryl ferulate; 3. cycloheterenyl ferulate; 4. 24-methylenecycloartanyl ferulate; 5. Δ7-campestenyl ferulate; 6. campesteryl ferulate; 7. Δ7-sitostenyl ferulate; (CAST) and 24-methylenecycloartanyl ferulate were identified as the major constituents of the cells (Fig. 2).
실시예Example 3. 흑미 미강 추출물에 대한 세포독성 분석 3. Cytotoxicity analysis of rice black rice extract
흑미 미강 추출물에 대한 세포독성 여부를 CCK-8 키트(Cell Counting Kit-8; Dojindo Molecular Technologies, Gaithersburg, MD, USA)를 이용하여 분석하였다. THP-1(human Leukemic monocyte, 인간 단핵구 세포주)는 ATCC(American Type Culture Collection, Manassas, VA, MD, 미국)에서 구입하였다. THP-1 세포는 10% FBS(Fetal Bovine Serum, Thermo Scientific Hyclone, USA)가 포함된 RPMI-1640 배양액을 사용하여 배양되었고, 96-well 플레이트에 1 x 104의 세포수를 분주한 후, 37℃, 5% CO2 인큐베이터에서 24시간 동안 배양하였다. 배양 후 배양액을 제거하고 0, 10, 50, 100 및 200 ㎍/mL 농도의 흑미 미강 추출물을 첨가한 후, 다시 24시간 동안 배양하였다. 10 ㎕의 CCK-8 용액을 첨가하여 2시간 반응시킨 후, 마이크로플레이트 리더를 이용하여 450nm에서 흡광도를 측정하였다. 통계적 유의성은 GraphPad Prism version 7.0 software(GraphPad Software, La Jolla, CA, USA)를 이용하여 분석하였다.Cytotoxicity of rice bran extract was analyzed by CCK-8 kit (Dojindo Molecular Technologies, Gaithersburg, MD, USA). THP-1 (human leukemic monocyte, human monocytic cell line) was purchased from ATCC (American Type Culture Collection, Manassas, Va., MD, USA). THP-1 cells were cultured in RPMI-1640 medium supplemented with 10% FBS (Fetal Bovine Serum, Thermo Scientific Hyclone, USA). Cells were seeded at 1 × 10 4 cells in a 96- Lt; 0 > C, 5% CO 2 incubator for 24 hours. After culturing, the culture broth was removed, and black rice bran extracts at 0, 10, 50, 100 and 200 ㎍ / mL were added and cultured again for 24 hours. 10 μl of CCK-8 solution was added and allowed to react for 2 hours. Absorbance was measured at 450 nm using a microplate reader. Statistical significance was analyzed using GraphPad Prism version 7.0 software (GraphPad Software, La Jolla, CA, USA).
그 결과, 흑미 미강 추출물은 200 ㎍/㎖ 농도까지 처리하여도 세포 생존능에 아무런 변화를 주지 않았으며, 아무 처리도 하지 않은 대조 세포군과 비교하였을 때 통계적으로 유의할만한 차이가 나타나지 않았다(도 3). 따라서, 흑미 미강 추출물은 THP-1 세포에 대하여 세포독성이 나타나지 않음을 알 수 있었다.As a result, there was no statistically significant difference in comparison with the control cell group in which no treatment was performed, no change was observed in the cell viability even after treatment with the black rice microbial extract at a concentration of 200 μg / ml (FIG. 3). Therefore, it was found that the black rice extract of rice bran was not cytotoxic to THP-1 cells.
실시예Example 4. 흑미 미강 추출물에 의한 면역활성 관련 유전자의 발현량 분석 4. Analysis of the expression level of immunological activity-related genes by rice bran extract
CD14(cluster of differentiation 14) 단백질은 주로 대식세포나 호중구 과립 백혈구(neutrophil), 수지상세포(dendritic cell) 표면에 발현되어 LPS(lipopolysaccharide) 등과 같은 박테리아 톡신을 감지하여 TLR4(toll-like receptor 4)를 활성화시키는 TLR4의 공동-수용체(co-receptor)이다. 따라서, CD14와 TLR4 유전자 발현량이 증가하면, 외부 병원균 감염에 따른 염증 반응을 조절하는 중추적 사이토카인인 TNFα(tumor necrosis factor alpha)와 IL-1(interleukin-1) 및 IL-6(interleukin-6) 생성이 증가하여 염증병소에서의 면역반응이 촉진되는 것으로 알려져 있다. CD14 (cluster of differentiation 14) proteins are expressed on the surface of macrophages, neutrophils, and dendritic cells to detect TLR4 (toll-like receptor 4) by detecting bacterial toxins such as LPS (lipopolysaccharide) Lt; RTI ID = 0.0 > TLR4 < / RTI > Therefore, when the expression levels of CD14 and TLR4 genes are increased, tumor necrosis factor alpha (TNFα), interleukin-1 (IL-1), and interleukin-6 (IL-6), which are central cytokines that regulate the inflammatory response to external pathogen infection, It is known that the increase of the production promotes the immune response in inflammatory lesions.
흑미 미강 추출물이 CD14와 TLR4 유전자의 발현량을 촉진시키는지 알아보기 위하여, THP-1 세포에 100 ㎍/mL의 흑미 미강 추출물을 처리한 후, 24시간 후에 세포를 수확하여 RT-PCR(Reverse Transcription-Polymerase Chain Reaction, 역전사-중합효소연쇄반응)을 수행하여 CD14와 TLR4 mRNA 발현량을 측정하였다. 양성대조 약물로는 LPS (0.1 ㎍/㎖)를 사용하였다. To investigate whether the extracts of black rice microbes promoted the expression of CD14 and TLR4 gene, THP-1 cells were treated with 100 μg / mL of black rice bran extract, and after 24 hours, the cells were harvested and subjected to RT-PCR -Polymerase Chain Reaction, and reverse transcription-polymerase chain reaction) to measure the expression levels of CD14 and TLR4 mRNA. LPS (0.1 μg / ml) was used as a positive control.
RT-PCR은 TRIzol RNA Isolation Reagents(라이프테크놀로지스 코리아 회사)를 이용하여 총 RNA(total RNA)를 추출하였고, iScript cDNA synthesis kit(Bio-Rad, Hercules, CA, USA)를 사용하여 제조회사의 권장하는 방법에 의하여 cDNA(상보적 DNA)를 합성하였다. PCR(polymerase chain reaction)을 수행하기 위한 프라이머 서열은 하기 표 1과 같다. 대조군으로는 항존유전자(housekeeping gene)인 GAPDH(glyceraldehyde-3-phosphate dehydrogenase)를 사용하였다. PCR의 조건은 95℃, 5분에서 변성시켰고, 94℃, 1분; 65℃, 2분; 및 70℃, 1분에서 30회 반복하였다. PCR 산물은 1% 아가로오스 겔에서 전기영동하였고, EtBr(ethidium bromide)로 염색하여 확인하였다. RT-PCR was performed using the iScript cDNA synthesis kit (Bio-Rad, Hercules, Calif., USA) using TRIzol RNA Isolation Reagents (Life Technologies, CDNA (complementary DNA) was synthesized by the method. Primer sequences for performing PCR (polymerase chain reaction) are shown in Table 1 below. GAPDH (glyceraldehyde-3-phosphate dehydrogenase), a housekeeping gene, was used as a control. The PCR conditions were denaturation at 95 ° C for 5 min, 94 ° C for 1 min; 65 DEG C, 2 min; And 70 < [deg.] ≫ C for 30 minutes. The PCR product was electrophoresed on 1% agarose gel and stained with EtBr (ethidium bromide).
RT-PCR을 수행한 결과, THP-1 세포에 흑미 미강 추출물을 처리한 경우 양성 대조군인 LPS와 유사한 정도로 CD14 및 TLR4 유전자에 대한 mRNA 발현량이 증가되었음을 확인하였다 (도 4A). As a result of RT-PCR, it was confirmed that mRNA expression levels of CD14 and TLR4 genes were increased in THP-1 cells similar to that of positive control LPS (Fig. 4A).
또한, 흑미 미강 추출물에 의한 면역활성능을 평가하기 위하여 면역반응의 활성과 관련된 사이토카인 유전자의 발현량을 측정하였다. THP-1 세포에 100 ㎍/ml의 흑미 미강 추출물을 처리한 후 24시간 동안 배양하고, TNFα, IL-1β, IL-8(interleukin 8 또는 chemokine (C-X-C motif) ligand 8(CXCL8)), CCL2(chemokine (C-C motif) ligand 2) 및 CCL5(chemokine (C-C motif) ligand 5)을 포함하는 사이토카인의 mRNA 발현량을 측정하기 위하여 RT-PCR을 수행하였으며, 프라이머 서열은 표 1과 같다. The expression level of the cytokine gene related to the activity of the immune response was measured in order to evaluate the surface role performance of the black rice extract of rice bran. TNFα, IL-1β, IL-8 (
그 결과, 흑미 미강 추출물은 TNFα, IL-1β 및 IL-8의 mRNA 발현량을 양성대조군인 LPS (0.1 ㎍/㎖)와 유사한 정도로 증가시켰고, CCL2 및 CCL5의 mRNA 발현량은 대조군에 비해 현저하게 증가시켰음을 확인하였다 (도 4B).As a result, the amount of mRNA expression of TNFα, IL-1β and IL-8 was increased to a similar level to that of positive control LPS (0.1 μg / ml), and the amount of mRNA expression of CCL2 and CCL5 was significantly (Fig. 4B).
실시예Example 5. 흑미 미강 추출물의 농도 의존적 면역활성 사이토카인 분비량 분석 5. Concentration-dependent immunoreactive cytokine secretion analysis of rice black rice extract
농도에 따른 흑미 미강 추출물의 효과를 알아보기 위하여, THP-1 세포에 다양한 농도의 흑미 미강 추출물 (0, 10, 50, 및 100 ㎍/ml)을 24시간 처리한 후, 실시예 4에서와 동일한 방법으로 RT-PCR을 수행하여 IL-8과 CCL2 사이토카인의 mRNA 발현량을 측정하였다(프라이머 서열은 표 1과 같음). 양성대조 약물로는 LPS (0.1 ㎍/ml)를 사용하였다. In order to examine the effect of the black rice bran extracts according to the concentration, THP-1 cells were treated with various concentrations of black rice bran extract (0, 10, 50, and 100 / / ml) for 24 hours, RT-PCR was performed to measure the mRNA expression levels of IL-8 and CCL2 cytokines (primer sequences are as shown in Table 1). LPS (0.1 ㎍ / ml) was used as a positive control.
그 결과, 흑미 미강 추출물은 농도 의존적으로 IL-8과 CCL2 mRNA 발현을 증가시켰음을 확인하였다 (도 5A). 특히, 10, 50, 및 100 ㎍/ml 농도의 흑미 미강 추출물에 의해서 IL-8 mRNA 발현량은 각각 3.2±0.30, 5.7±0.76, 6.9±0.51 배로 증가하였으며 (도 5B), CCL2 mRNA 발현량은 각각 1.5±0.25, 3.0±0.47, 6.8±0.30 배 증가하였다 (도 5C). 100 ㎍/ml의 흑미 미강 추출물은 양성 대조군(LPS)과 유사한 수준으로 IL-8 과 CCL2 mRNA 발현량을 증가시킨 것으로 확인되었다 (LPS의 경우 IL-8 과 CCL2 mRNA 발현량은 각각 8.7±0.85 배와 8.0±0.75 배 증가됨). As a result, it was confirmed that the black rice rice bran extract increased IL-8 and CCL2 mRNA expression in a concentration-dependent manner (FIG. 5A). In particular, the amount of IL-8 mRNA expression was increased by 3.2 ± 0.30, 5.7 ± 0.76, and 6.9 ± 0.51-fold, respectively, by the 10, 50, and 100 μg / 1.5 ± 0.25, 3.0 ± 0.47, and 6.8 ± 0.30 times, respectively (FIG. 5C). It was also found that the 100 μg / ml black rice extract of the rice bran increased IL-8 and CCL2 mRNA expression levels at a level similar to that of the positive control (LPS). IL-8 and CCL2 mRNA expression levels were 8.7 ± 0.85 And 8.0 +/- 0.75 fold, respectively).
따라서, 흑미 미강 추출물은 10 내지 100 ㎍/ml의 농도에서 단핵구와 같은 면역세포에 작용하여 선천성 면역반응(innate immunity)에 중요한 CCL2 및 IL-8 등과 같은 사이토카인 유전자 발현을 촉진함으로써, 생체 면역 활성을 유도시킨다는 것을 알 수 있었다. Therefore, the black rice extract of rice bran acts on the immune cells such as monocytes at a concentration of 10 to 100 / / ml to promote cytokine gene expression such as CCL2 and IL-8, which are important for innate immunity, . ≪ / RTI >
실시예Example 6. 6. 흑미Black rice 미강Rice bran 추출물에 대한 면역활성 사이토카인의 분비량 분석 Analysis of secretion of immune-active cytokines to extracts
흑미 미강 추출물에 의한 면역활성 사이토카인의 생성을 단백질 수준에서 분석하였다. 실시예 3에서와 같이, THP-1 세포에 100 ㎍/ml 농도의 흑미 미강 추출물을 처리하여 24시간 동안 배양한 후, 배양액으로 분비되는 사이토카인의 종류를 분석하였다. 세포가 분비하는 사이토카인의 종류는 36종류의 사이토카인 항체가 점적된 Proteome Profiler Antibody Arrays(Bio-Techne, Minneapolis, MN, USA)를 이용하여 분석되었다 (표 2 및 도 6A). 표 2는 사이토카인 어레이에 점적된 항체의 종류를 나타낸 것이다. The production of immunologically active cytokines by rice bran extract was analyzed at the protein level. As in Example 3, the THP-1 cells were treated with a 100 μg / ml black rice bran extract and cultured for 24 hours. Then, the types of cytokines secreted into the culture medium were analyzed. The cytokine secreted by the cells was analyzed using Proteome Profiler Antibody Arrays (Bio-Techne, Minneapolis, MN, USA) in which 36 types of cytokine antibodies were spotted (Table 2 and FIG. 6A). Table 2 shows the types of antibodies that are scattered in the cytokine array.
그 결과, 흑미 미강 추출물은 CCL2(spot #4)와 CCL3/CCL4(spot #5), CCL5(spot #6) 및 IL-8(spot #8)을 포함하는 사이토카인의 분비량을 증가시켰음을 확인하였다 (도 6B 및 도 6C). As a result, it was confirmed that black rice bran extract increased the secretion of cytokines including CCL2 (spot # 4), CCL3 / CCL4 (spot # 5), CCL5 (spot # 6) and IL-8 (Fig. 6B and Fig. 6C).
실시예Example 7. 7. 흑미Black rice 추출액에 의한 대식세포 Macrophage by extract 탐식능Pickling 촉진 효과 Facilitating effect
흑미 미강 추출물의 면역 활성능을 확인하기 위하여 대식세포(macrophage)의 탐식작용(phagocytosis, 식세포작용)을 조사하였다. 대식세포주인 RAW 264.1 세포는 ATCC(American Type Culture Collection, Manassas, VA, MD, USA)에서 구입하여 사용되었고, 10% FBS가 포함된 DMEM 배양액에서 배양되었다. 형광물질이 부착된 라텍스-비드(Latex-bead, Sigma, USA)는 10% FBS와 1시간 동안 반응시켜 옵소닌화(opsonization) 시킨 후, 아무 처리도 하지 않은 대조군과 100 ㎍/ml 농도의 흑미 미강 추출물이 처리된 RAW 264.1 세포에 첨가하였다. 양성 대조군으로는 LPS를 사용하였다. 형광라텍스-비드를 첨가하고 2시간 후에 탐식되지 않은 라텍스-비드를 PBS(phosphate buffered saline) 완충액으로 씻어낸 후, 형광현미경을 이용하여 대식세포가 탐식한 비드를 측정하였다.The phagocytosis (phagocytosis) of macrophages was investigated in order to determine the immunoreactivity of the black rice extract. RAW 264.1 cells, macrophages, were purchased from ATCC (American Type Culture Collection, Manassas, Va., MD, USA) and cultured in DMEM medium supplemented with 10% FBS. Latex-bead (Sigma, USA) with a fluorescent substance was subjected to opsonization by reacting with 10% FBS for 1 hour. Then, a control group in which no treatment was performed and a control group of 100 μg / The rice bran extract was added to the treated RAW 264.1 cells. LPS was used as a positive control. Fluorescent latex-beads were added and after 2 hours, unlabeled latex-beads were rinsed with PBS (phosphate buffered saline) buffer, and macrophage-phagocytosed beads were measured using a fluorescence microscope.
그 결과, 흑미 미강 추출물을 처리한 경우 대조군에 비해 다량의 라텍스-비드가 대식세포 내에서 관찰되었고, 이는 LPS를 처리한 경우와 비슷한 수준이었다 (도 7).As a result, a larger amount of latex-beads was observed in macrophages than in the control group when black rice bran extract was treated, which was similar to that of LPS treatment (FIG. 7).
따라서, 흑미 미강 추출물은 대식세포를 활성화시켜 식세포작용(phagocytosis)을 촉진시킴으로써 선천성 면역반응(innate immunity)의 활성을 유도하는 효과가 있음을 알 수 있었다.Therefore, it was found that the extracts of black rice microgranules stimulate the phagocytosis by activating macrophages, thereby inducing the activity of innate immunity.
실시예 8. 감마오리자놀에 의한 면역활성 사이토카인 유전자 발현 촉진 효과Example 8: Gamma oryzanol stimulated immunostimulatory cytokine gene expression
도 2에서 나타낸 바와 같이, 흑미 미강 추출물의 주요 성분인 감마오리자놀이 선천성 면역반응에 중요한 사이토카인 유전자 발현을 촉진하는지 조사하였다. THP-1 세포에 다양한 농도의 감마오리자놀 (0, 10, 50, 및 100 ㎍/mL)을 24 시간 처리한 후, 상기 실시예 3과 동일한 방법으로 RT-PCR을 수행하여 IL-8과 CCL2 사이토카인의 mRNA 발현량을 측정하였다(프라이머 서열은 표 1과 같음). 양성대조 약물로는 LPS (0.1 ㎍/㎖)를 사용하였다. As shown in FIG. 2, gamma oruzanol, which is a major component of black rice extract of rice bran, was examined to promote cytokine gene expression important for innate immune response. RT-PCR was performed on THP-1 cells in the same manner as in Example 3, after treating various concentrations of gamma oryzanol (0, 10, 50, and 100 쨉 g / The amount of mRNA expression of cine was measured (primer sequences are as shown in Table 1). LPS (0.1 μg / ml) was used as a positive control.
그 결과, 감마오리자놀은 10 ㎍/mL 이상의 농도에서 IL-8과 CCL2 mRNA 발현을 증가시켰다 (도 8A). 감마오리자놀을 10, 20 및 50 ㎍/mL 농도로 처리하면 아무 처리도 하지 않은 대조군에 비해 IL-8 mRNA 발현은 각각 3.4±0.28, 4.3±0.32, 5.6±0.92 배 증가하였으며 (도 8B), CCL2 mRNA 발현은 각각 3.3±0.17, 3.5±0.20, 4.8±0.42 배 증가하였다 (도 8C). As a result, gamma oryzanol increased IL-8 and CCL2 mRNA expression at concentrations of 10 / / mL or more (Fig. 8A). Treatment of gamma oryzanol at 10, 20 and 50 ㎍ / mL concentrations resulted in 3.4 ± 0.28, 4.3 ± 0.32, and 5.6 ± 0.92 fold increase in IL-8 mRNA expression, respectively, compared to the control group without any treatment (FIG. 8B) mRNA expression increased 3.3 ± 0.17, 3.5 ± 0.20, and 4.8 ± 0.42 fold, respectively (FIG. 8C).
따라서, 흑미 미강 추출물에 함유되어 있는 감마오리자놀은 10 내지 100 ㎍/mL 농도에서 CCL2와 IL-8 등과 같은 사이토카인 유전자 발현을 유도시킨다는 것을 알 수 있었다. Therefore, it was found that gamma oryzanol contained in the black rice extract of rice bran extracts cytokine gene expression such as CCL2 and IL-8 at a concentration of 10 to 100 μg / mL.
실시예 9. 감마오리자놀과 IL-8에 의한 대식세포 탐식능 촉진 효과Example 9. Promotion of Macrophage Phagocytosis by Gamma Orzanol and IL-8
흑미 미강 추출물의 주요 성분인 감마오리자놀이 대식세포 탐식능을 촉진하는지 조사하였다. 대식세포주인 RAW 264.1 세포에 아무 처리도 하지 않은 대조군과 100 ㎍/mL 농도의 감마오리자놀, 20 nM 농도의 IL-8을 각각 처리한 후 상기 실시예 7과 동일한 방법을 이용하여 대식세포의 탐식능력을 조사하였다. 양성 대조군으로는 100 ㎍/mL 농도의 흑미 미강 추출물을 사용하였다. 형광라텍스-비드를 첨가하고 2시간 후에 탐식되지 않은 라텍스-비드를 PBS(phosphate buffered saline) 완충액으로 씻어낸 후, 형광현미경을 이용하여 대식세포가 탐식한 비드를 측정하였다.We investigated whether Gamma oryzanol, which is a major component of black rice extract, stimulates macrophage colonization. RAW 264.1 cells, macrophage cell line, were treated with a control group that had no treatment, gamma oruzolol at a concentration of 100 μg / mL, and IL-8 at a concentration of 20 nM respectively, and then the phagocytic ability Respectively. As a positive control, black rice rice bran extract of 100 ㎍ / mL concentration was used. Fluorescent latex-beads were added and after 2 hours, unlabeled latex-beads were rinsed with PBS (phosphate buffered saline) buffer, and macrophage-phagocytosed beads were measured using a fluorescence microscope.
그 결과, 감마오리자놀을 처리한 경우 아무 처리도 하지 않은 대조군에 비해 다량의 라텍스-비드가 대식세포 내에서 관찰되었다 (도 9A). 라텍스-비드를 탐식한 대식세포수는 흑미 미강 추출물을 처리한 경우와 비슷한 수준이었다 (도 9B).As a result, a large amount of latex-beads was observed in macrophages when treated with gamma oryzanol compared with the control group without any treatment (Fig. 9A). The number of macrophages that were latex-bead-digested was similar to that of black rice bran extract (FIG. 9B).
따라서, 흑미 미강 추출물에 함유되어있는 주요 성분인 감마오리자놀은 대식세포를 활성화시켜 탐식능을 촉진시킴으로써 선천성 면역반응(innate immunity)의 활성을 유도하는 효과가 있음을 알 수 있었다.Therefore, it was found that gamma oryzanol, which is a major component contained in the black rice extract of rice bran, activates macrophages to promote the phagocytosis, thereby inducing the activity of innate immunity.
<110> Industry-Academic Cooperation Foundation of Konkuk University <120> Composition for immune enhancement comprising black rice extracts as active ingredient <130> PN1608-296 <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 1 cgaggaccta aagataaccg gc 22 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 2 gttgcagctg agatcgagca c 21 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 3 acagaagctg gtggctgtg 19 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 4 tctttaaatg cacctggttg g 21 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 5 gagtgacaag cctgtagccc atgttgt 27 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 6 gcaatgatcc caaagtagac ctgcccagac 30 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 7 aaacagatga agtgctcctt ccagg 25 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 8 tggagaacac cacttgttgc tcca 24 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 9 ccactgtgcc ttggtttc 18 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 10 tcttgcacaa atatttgatg c 21 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 11 cttctgtgcc tgctgctcat ag 22 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 12 ctggacaagc aaacccaaac 20 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 13 ccccgtgccc acatcaagga gtattt 26 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 14 cgtccagcct ggggaaggtt tttgta 26 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 15 acccactcct ccacctttg 19 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> artificial sequence <400> 16 ctcttgtgct cttgctggg 19 <110> Industry-Academic Cooperation Foundation of Konkuk University <120> Composition for immune enhancement comprising black rice extracts as active ingredient <130> PN1608-296 <160> 16 <170> KoPatentin 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 1 cgaggaccta aagataaccg gc 22 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 2 gttgcagctg agatcgagca c 21 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 3 acagaagctg gtggctgtg 19 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 4 tctttaaatg cacctggttg g 21 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 5 gagtgacaag cctgtagccc atgttgt 27 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 6 gcaatgatcc caaagtagac ctgcccagac 30 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 7 aaacagatga agtgctcctt ccagg 25 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 8 tggagaacac cacttgttgc tcca 24 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 9 ccactgtgcc ttggtttc 18 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 10 tcttgcacaa atatttgatg c 21 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 11 cttctgtgcc tgctgctcat ag 22 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 12 ctggacaagc aaacccaaac 20 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 13 ccccgtgccc acatcaagga gtattt 26 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 14 cgtccagcct ggggaaggtt tttgta 26 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 15 acccactcct ccacctttg 19 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 16 ctcttgtgct cttgctggg 19
Claims (14)
상기 흑미 미강 추출물은 물 또는 유기용매를 이용하여 수득한 추출물인 것을 특징으로 하는 조성물. The method according to claim 1,
Wherein the black rice microgranular extract is an extract obtained by using water or an organic solvent.
상기 유기용매는 메탄올, 에탄올, 프로판올, 이소프로판올, 부탄올, 아세톤, 에테르, 벤젠, 클로로포름, 에틸아세테이트, 메틸렌클로라이드, 헥산 및 시클로헥산으로 이루어진 그룹에서 선택되는 어느 하나 이상인 것을 특징으로 하는 조성물. 3. The method of claim 2,
Wherein the organic solvent is at least one selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane.
상기 흑미 미강 추출물은 흑미 100 중량부에 대하여 에탄올 85 내지 95 중량부를 첨가하여 음지에서 3일 동안 정치하여 추출하는 것을 특징으로 하는 조성물.The method according to claim 1,
Wherein the black rice bran extract is prepared by adding 85 to 95 parts by weight of ethanol to 100 parts by weight of black rice and allowing to stand for 3 days on a shade.
상기 흑미 미강 추출물의 농도는 10 내지 100 μg/ml 인 것을 특징으로 하는 조성물.The method according to claim 1,
Wherein the concentration of the black rice bran extract is from 10 to 100 μg / ml.
상기 면역증강은 비특이적인 선천성 면역반응에 대한 활성을 증가시키는 것을 특징으로 하는 조성물.The method according to claim 1,
Wherein said immune enhancement increases activity against nonspecific congenital immune response.
상기 건강기능식품은 캡슐, 정제, 분말, 과립, 액상, 환, 편상, 페이스트상, 시럽, 겔, 젤리 및 바로 이루어진 그룹에서 선택되는 어느 하나인 것을 특징으로 하는 건강기능식품.8. The method of claim 7,
Wherein the health functional food is any one selected from the group consisting of capsules, tablets, powders, granules, liquids, pills, flakes, pastes, syrups, gels, jellies and flours.
상기 감마오리자놀 복합체는
(a) 흑미 미강 추출물을 감압여과기에서 여과시키고 감압농축기로 여과액을 농축시킨 후 300ml의 아세톤으로 재용해하는 단계;
(b) 60℃ 오븐에서 1시간 정치하여 맑은 용액(clear solution)을 획득한 후 2℃ 냉장고에서 24시간 정치하여 고체상과 액체상을 분리시키는 단계;
(c) 상기 분리된 고체상은 버리고(dewazing), 상기에서 획득한 액체상의 여과액을 -5℃에서 다시 24시간 정치한 후 분리된 하층액을 버리고(degumming), 상기에서 획득한 상층액을 감압농축시키는 단계; 및
(d) 상기 농축된 상층액은 400ml의 발효주정:아세톤(7:3, v/v) 용매로 재용해하고 -60℃에서 48시간 이상 정치시켜 고체상과 액체상으로 분리하킨 후 여과하여 감마오리자놀 복합체를 획득하는 단계;를 포함하는 것을 특징으로 하는 조성물.10. The method of claim 9,
The gamma oryzanol complex
(a) filtering the black rice bran extracts through a vacuum filter, concentrating the filtrate with a vacuum concentrator, and redissolving the filtrate with 300 ml of acetone;
(b) standing in a 60 ° C oven for 1 hour to obtain a clear solution, and then standing in a refrigerator at 2 ° C for 24 hours to separate the solid phase and the liquid phase;
(c) dewazing the separated solid phase, allowing the filtrate of the liquid phase obtained above to stand at -5 DEG C for another 24 hours, then removing the separated lower layer liquid, Concentrating; And
(d) The concentrated supernatant was redissolved in 400 ml of fermentation alcohol: acetone (7: 3, v / v) and allowed to stand at -60 ° C for at least 48 hours to separate into solid and liquid phases. ≪ / RTI > of the composition.
상기 감마오리자놀의 농도는 10 내지 100 μg/ml 인 것을 특징으로 하는 조성물.10. The method of claim 9,
Wherein the concentration of gamma oryzanol is from 10 to 100 [mu] g / ml.
상기 면역증강은 비특이적인 선천성 면역반응에 대한 활성을 증가시키는 것을 특징으로 하는 조성물.10. The method of claim 9,
Wherein said immune enhancement increases activity against nonspecific congenital immune response.
(b) 상기 (a)단계에서 구축된 세포주에 후보물질을 접촉시키고 CCL2 또는 IL-8의 발현량을 측정하는 단계; 및
(c) 상기 (b)단계에서 측정된 CCL2 또는 IL-8의 발현량이 대조군보다 증가된 후보물질을 선별하는 단계;를 포함하는 것을 특징으로 하는 면역증강제 스크리닝 방법. (a) constructing a cell line expressing CCL2 (chemokine (CC motif) ligand 2) or IL-8 (Interleukin 8) in a monocyte;
(b) contacting a candidate substance with the cell line constructed in the step (a) and measuring the expression level of CCL2 or IL-8; And
(c) selecting candidate substances whose expression level of CCL2 or IL-8 measured in step (b) is higher than that of the control group.
상기 면역증강은 비특이적인 선천성 면역반응에 대한 활성을 증가시키는 것을 특징으로 하는 방법.
14. The method of claim 13,
Wherein said immune enhancement increases activity against a nonspecific congenital immune response.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111171103A (en) * | 2020-01-17 | 2020-05-19 | 杭州益品新五丰药业有限公司 | Method for extracting oryzanol by multiple solvents |
| JP2020202835A (en) * | 2020-07-15 | 2020-12-24 | 株式会社神明きっちん | Food composition for skin moisture retention and method for ingestion of food composition for skin moisture retention |
| KR20210051474A (en) | 2019-10-30 | 2021-05-10 | 샘표식품 주식회사 | Lactobacillus Plantarum with excellent 4-vinylguaiacol productivity and a natural flavor compositin comprising the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20210051474A (en) | 2019-10-30 | 2021-05-10 | 샘표식품 주식회사 | Lactobacillus Plantarum with excellent 4-vinylguaiacol productivity and a natural flavor compositin comprising the same |
| CN111171103A (en) * | 2020-01-17 | 2020-05-19 | 杭州益品新五丰药业有限公司 | Method for extracting oryzanol by multiple solvents |
| CN111171103B (en) * | 2020-01-17 | 2021-07-06 | 杭州益品新五丰药业有限公司 | Method for extracting oryzanol by multiple solvents |
| JP2020202835A (en) * | 2020-07-15 | 2020-12-24 | 株式会社神明きっちん | Food composition for skin moisture retention and method for ingestion of food composition for skin moisture retention |
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