KR102011684B1 - Extracts of the mori folium having immunity enhancing activity and a method for producing the same - Google Patents
Extracts of the mori folium having immunity enhancing activity and a method for producing the same Download PDFInfo
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- KR102011684B1 KR102011684B1 KR1020170016363A KR20170016363A KR102011684B1 KR 102011684 B1 KR102011684 B1 KR 102011684B1 KR 1020170016363 A KR1020170016363 A KR 1020170016363A KR 20170016363 A KR20170016363 A KR 20170016363A KR 102011684 B1 KR102011684 B1 KR 102011684B1
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- extract
- macrophages
- upper leaf
- lactobacillus
- enhancing
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Abstract
본 발명은 상엽 추출물을 포함하는 면역증강용 조성물 및 그 제조방법에 관한 것이다.
본 발명에 일 실시예에 따른 면역증강용 조성물은 상엽 추출물을 유효성분으로 포함하는 것일 수 있다.
본 발명에 따른 상엽 추출물을 포함하는 면역증강용 조성물을 사용하는 경우 대식세포가 증가 내지 활성화 되어 면역활성을 증진시킬 수 있고, 이에 따라 면역관련 질환을 예방 또는 개선하는 효과를 낼 수 있다.The present invention relates to a composition for immuno-enhancing comprising the extract of the upper lobe and a method for producing the same.
Immunity-enhancing composition according to an embodiment of the present invention may be to include the upper leaf extract as an active ingredient.
When using the composition for immuno-enhancement comprising the extract of the upper lobe according to the present invention, macrophages can be increased or activated to enhance immune activity, thereby preventing or improving immune-related diseases.
Description
본 발명은 상엽 추출물을 포함하는 면역증강용 조성물 및 그 제조방법에 관한 것이다. 보다 상세하게는 본 발명은 상엽 추출물의 면역증진 활성을 확인하고 면역 활성 증진을 위한 조성물 및 그 제조방법은 제공하기 위한 것이다.The present invention relates to a composition for immuno-enhancing comprising the extract of the upper lobe and a method for producing the same. More specifically, the present invention is to provide a composition for confirming the immunopromoting activity of the upper leaf extract and to enhance the immunological activity and a method for producing the same.
면역 반응은 생체 자기 방어기작 중의 하나로 감염성 질환으로부터의 보호와, 외부물질과 자신을 생리적으로 인식하여 체내에 유입된 유해 물질의 중화 또는 제거하는 현상이다. 생체의 항상성 유지를 위하여, 면역에 관여하는 기관들이 면역계를 구성하며 이들이 서로 협동하고 집합적으로 작용하여 면역반응을 일으키며, 면역반응의 이상으로 인한 면역시스템에 불균형은 각종 난치성 질환의 발병과 진행의 원인이 된다(Ghazal, P., Dickinson, P. and Smith, C. L. 2013. Early life response to infection. Curr Opin Infect Dis 26, 213-218. , Sirisinha, S. 2011. Insight into the mechanisms regulating immune homeostasis in health and disease. Asian Pac J Allergy Immunol 29, 1-14.)The immune response is one of biological self-defense mechanisms to protect against infectious diseases and to neutralize or eliminate harmful substances introduced into the body by physiologically recognizing foreign substances and themselves. In order to maintain the homeostasis of the living body, the organs involved in immunity make up the immune system, and they cooperate with each other and collectively cause an immune response, and the imbalance in the immune system due to the abnormality of the immune response causes the development and progress of various intractable diseases. (Ghazal, P., Dickinson, P. and Smith, CL 2013.Early life response to infection.Cur Opin Infect Dis 26, 213-218., Sirisinha, S. 2011. Insight into the mechanisms regulating immune homeostasis in health and disease.Asian Pac J Allergy Immunol 29, 1-14.)
면역 기능을 증강시키기 위한 목적으로는 일차적으로 대식세포의 활성화를 촉진하는 물질 및 이의 활성기작에 대한 연구들이 진행되어오고 있다. 활성화된 대식세포는 항원의 흡수 및 세포독성의 기능이 현저히 향상될 뿐 아니라 인터루킨-1(Interlukin-1)과 같은 각종 물질을 분비하여 다른 면역 세포의 활성화를 촉진함으로써 면역계 전반의 활성화를 유도하게 된다.For the purpose of enhancing immune function, studies have been conducted on substances that promote macrophage activation and their activation mechanisms. Activated macrophages not only significantly improve the function of antigen uptake and cytotoxicity, but also stimulate the activation of other immune cells by releasing various substances such as Interlukin-1 to induce activation of the immune system. .
면역 시스템과 관련된 세포 중에서 대식세포는 거의 모든 조직에 존재하며, 면역반응에 매우 중요한 역할을 수행한다.( Kudrin A, Ray A. Cunning factor: macrophage migration inhibitory factor as a redox-regulated target. Immunol Cell Biol 2008;86:232-8., Krneta T, Gillgrass A, Ashkar AA. The influence of macrophages and the tumor microenvironment on natural killer cells. Curr Mol Med 2013;13:68-79.)Among the cells involved in the immune system, macrophages are present in almost all tissues and play a very important role in immune responses (Kudrin A, Ray A. Cunning factor: macrophage migration inhibitory factor as a redox-regulated target.Immunol Cell Biol 2008; 86: 232-8., Krneta T, Gillgrass A, Ashkar AA.The influence of macrophages and the tumor microenvironment on natural killer cells.Cur Mol Med 2013; 13: 68-79.)
골수로부터 생산되는 대식세포(macrophage)는 크게 3가지 기능을 한다. 첫째, 대식세포는 외부로부터 들어오는 이물질을 직접 흡입하는 식작용(phagocytosis)을 하며, 둘째로 함입된 병원체를 활성산소(superoxide)를 이용하여 분해 및 제거하며, 셋째로 외부 항원을 변형시킨 후 이를 세포표면으로 표현하여 T세포로 항원을 인식하도록 하고, 궁극적으로 항체를 형성시킨다. 또한 활성화된 대식세포는 T세포, B세포를 포함한 다른 여러 세포를 자극하며 새로운 반응을 유도한다.Macrophage produced from the bone marrow has three functions. First, phagocytes induce phagocytosis by directly inhaling foreign substances from the outside, and secondly, by breaking down and removing the embedded pathogens by using superoxide. To recognize antigens by T cells and ultimately to form antibodies. Activated macrophages also stimulate other cells, including T cells and B cells, and induce new responses.
대식세포의 식작용은 세포막에 존재하는 수용체 단백질들을 매개로 하여 이루어진다. 수용체 단백질로는 IgG를 인지하는 Fc 수용체와 보체(complement)를 인지하는 보체 수용체가 가장 잘 알려져 있으며(Aderem A and Underhill DM. Mechanisms of phagocytosis in macrophage. Annu Rev Immunol 1999;17:593-623), 세포 내에 엑틴 필라멘트를 조절하는 작은 GTPase들 또한 중요한 역할을 하는 것으로 알려져 있다(Caron E and Hall A.Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases. Science 1998;282:1717-21).Phagocytosis of macrophages is mediated by receptor proteins present in the cell membrane. Receptor proteins are best known as Fc receptors that recognize IgG and complement receptors that recognize complement (Aderem A and Underhill DM. Mechanisms of phagocytosis in macrophage.Annu Rev Immunol 1999; 17: 593-623), Small GTPases that regulate actin filaments in cells are also known to play an important role (Caron E and Hall A. Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases. Science 1998; 282: 1717-21).
대식세포는 림포카인, 엔도톡신 또는 RNA와 같은 인터페론 유도물질을 포함하는 외부자극에 의하여 활성화된다.Macrophages are activated by external stimuli that include interferon inducers such as lymphokines, endotoxins or RNA.
활성화된 대식세포는 식작용과 종양세포 파괴기능이 항진되며, 세포의 크기가 증가하고 여러 가지의 물질을 다량 분비한다. 또한 활성화된 대식세포의 표면에는 클라스 I MHC 단백질, Mac-1 또는 Fc 수용체와 같은 표면 단백질이 증가한다.Activated macrophages promote phagocytosis and tumor cell destruction, increase cell size and secrete large quantities of various substances. The surface of activated macrophages also increases surface proteins such as class I MHC proteins, Mac-1 or Fc receptors.
또한 대식세포는 활성산소를 이용하여 세포 내 함입된 병원체를 분해한다. 따라서 대식세포의 활성산소 생성능이 감소 또는 소실되는 경우 심각한 면역질환이 발생될 수 있다. 뿐만 아니라 대식세포는 종양세포와 접촉하여 H2O2나 OH 라디칼을 분비하여 종양세포를 제거한다. 활성산소는 NADPH-옥시데이즈라는 단백질 복합체인 효소작용에 의해 생성되며, 대식세포가 활성화되면 도 1과 같이 세포내의 단백질인 Rac, p67phox, p47phox 및 p40phox이 막단백질인 Rap, p22 및 gp91 단백질들과 결합하여 활성산소를 생성시킨다(Babior BM. NADPH oxidase: anupdate. Blood 1999;93:1464-76).Macrophages also use free radicals to break down pathogens embedded in cells. Therefore, severe immune diseases can occur when the free radical production capacity of macrophages is reduced or lost. In addition, macrophages remove tumor cells by contacting tumor cells to secrete H 2 O 2 or OH radicals. Free radicals are produced by enzymatic action, a protein complex called NADPH-Oxidases. When macrophages are activated, the intracellular proteins Rac, p67phox, p47phox and p40phox are membrane proteins Rap, p22 and gp91 proteins as shown in FIG. Bind to produce free radicals (Babior BM. NADPH oxidase: anupdate. Blood 1999; 93: 1464-76).
또한 대식세포는 상기에 언급한 바와 같이 생체 내에서 양성적인 작용을 하지만 때로는 음성적인 작용을 하기도 한다. 즉 대식세포는 동맥혈관의 손상된 부위에 축적된 산화성 저밀도 지단백질(low density lipoprotein, LDL)을 탐식하여 거품세포로 전환되어 축적되게 된다. 거품세포들의 축적에 의하여 형성된 지질층은 평활근 세포(smooth muscle cell)의 이동을 초래하고, 이들은 단핵세포-주화성 단백질-1(monocyte-chemotactic protein-1: MCP-1)의 분비를 촉진하여 대식세포들의 소집을 증가시킨다. 거품세포들은 결국 자유 라디칼이나 산화질 단백질의 세포독성으로 인하여 지질을 방출하고, 혈관 손상 부위 내에 세포괴사 지질 핵을 형성시킨다. 이러한 과정이 반복되면서 동맥은 좁아지게 되고, 이로 인하여 혈액 응고, 심장질환 및 뇌졸증 등이 발생할 가능성이 증가되게 된다(Li AC,Glass CK. The macrophage foam cell as a target for therapeutic intervention. Nat Med 2002;8:1235-42).Macrophages also act positively in vivo, as mentioned above, but sometimes negatively. In other words, the macrophages phagocytosed oxidized low density lipoprotein (LDL) accumulated in the damaged areas of the arterial vessels, and converted to foam cells and accumulated. The lipid layer formed by the accumulation of foam cells causes the migration of smooth muscle cells, which promote the secretion of monocyte-chemotactic protein-1 (MCP-1) Increase their call. Foam cells eventually release lipids due to the cytotoxicity of free radicals or oxidative proteins, and form cell necrotic lipid nuclei within the site of blood vessel damage. As this process is repeated, the arteries become narrower, which increases the chance of blood coagulation, heart disease, and stroke (Li AC, Glass CK. The macrophage foam cell as a target for therapeutic intervention. Nat Med 2002; 8: 1235-42).
이에 본 발명자들은 상엽 추출물이 면역 활성 증가에 미치는 영향을 연구하여 상엽 추출물의 대식세포에 대한 활성증가를 확인하고 이를 기초로 상기 상엽 추출물에서 면역 활성 효과를 극대화시키고 기타 부작용의 문제가 없는 조성물을 제공하기 위하여 본 발명을 완성하였다.In this regard, the present inventors have investigated the effect of the upper leaf extract on the increase of immune activity, confirming the increase of the activity of the upper leaf extract on macrophages, and based on this, maximize the effect of the immune activity on the upper leaf extract and provide a composition without problems of other side effects. In order to complete the present invention.
선행기술 1(KR 10-2005-0021026 A, 2005년 3월 7일 공개)은 장생도라지 추출물을 포함하는 면역계 질환의 예방 및 치료용 조성물에 관한 것으로 면역계의 대식세포의 활성화를 통하여 각종 면역 관련 사이토카인의 생성을 증진하는 효과를 개시하고 있다.Prior art 1 (KR 10-2005-0021026 A, published 7 March 2005) relates to a composition for the prevention and treatment of diseases of the immune system, including Jangsaeng Doraji extract. Disclosed is an effect of enhancing the production of caine.
선행기술 2(KR 10-2012-0092871 A, 2012년 8월 22일 공개)는 석이버섯을 이용한 세포면역 증강용 조성물에 관한 것으로, 대식세포를 포함하는 세포의 면역 증진효과를 개시하고 있다.Prior art 2 (KR 10-2012-0092871 A, published on August 22, 2012) relates to a composition for enhancing cell immunity using a fungus, and discloses an immune enhancing effect of cells including macrophages.
본 발명의 목적은 부작용이 없으면서도 대식세포를 증가 내지 활성화시켜 면역체계를 강화 또는 증진시키는 상엽 추출물을 제공하는 것이다. 특히, 상엽 추출물의 대식세포에 대한 활성화 효과가 극대화 되는 추출물 및 그 사용범위를 제공한다.It is an object of the present invention to provide an upper leaf extract that enhances or enhances the immune system by increasing or activating macrophages without side effects. In particular, it provides an extract and its use range is maximized the activation effect on the macrophage of the upper leaf extract.
본 발명의 다른 목적은 상기 상엽 추출물을 이용하여 제조한 식품 조성물 또는 약제 조성물을 제공하는 것이다.It is another object of the present invention to provide a food composition or a pharmaceutical composition prepared using the extract of the upper leaf.
또한 본 발명은 상기 상엽 추출물의 제조방법을 제공하기 위한 것이다.In another aspect, the present invention is to provide a method for preparing the upper leaf extract.
상기 목적을 달성하기 위하여, 본 발명의 일 실시예에 따른 면역증강용 조성물은 상엽 추출물을 유효성분으로 포함하는 것이다.In order to achieve the above object, the composition for immuno-enhancement according to an embodiment of the present invention is to include the upper leaf extract as an active ingredient.
상기 상엽 추출물은 물, 탄소수 1 내지 6의 알코올 및 이들의 혼합 용매로 구성되는 군으로부터 선택된 어느 하나를 용매로 하여 추출되는 것일 수 있다.The upper leaf extract may be extracted by using any one selected from the group consisting of water, an alcohol having 1 to 6 carbon atoms and a mixed solvent thereof.
상기 면역증강용 조성물은 대식세포의 활성을 촉진시키는 것일 수 있다.The immunopotentiating composition may be to promote the activity of macrophages.
상기 상엽은 락토바실러스 카제이(Lactobacillus casei), 락토바실러스 람노수스(Lactobacillus rhamnosus), 비피도 박테리윰 비피듐(Bifidobacterium bifidum) 비피더스균, 비피도 박테리움 브레브(Bifidobacterium breve) 비피더스균, 및 락토바실러스 아시도 필루스(Lactobacillus acidophilus)로 이루어진 군에서 선택되는 하나 이상을 접종하여 발효된 것일 수 있다.The upper leaves are Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium bifidum, Bifidobacterium, Bifidobacterium breve, Bifidobacterium breve and Lactobacillus, It may be fermented by inoculating one or more selected from the group consisting of Bacillus adophyllus (Lactobacillus acidophilus).
본 발명의 다른 일 실시예에 따른 면역증강용 약학 조성물은 상기 면역증강용 조성물을 포함하는 것이다.Immunity-enhancing pharmaceutical composition according to another embodiment of the present invention is to include the immuno-enhancing composition.
본 발명의 또 다른 일 실시예에 따른 면역증강용 식품 조성물은 상기 면역증강용 조성물을 포함하는 것이다.Immunity-enhancing food composition according to another embodiment of the present invention is to include the immuno-enhancing composition.
본 발명의 또 다른 일실시예에 따른 면역증강용 조성물의 제조방법은 상엽을 건조하는 상엽 건조단계; 상기 상엽 건조단계를 거쳐 건조된 상엽을 물, 탄소수 1 내지 6의 알코올 및 이들의 혼합 용매로 구성되는 군으로부터 선택된 어느 하나를 사용하여 추출하는 상엽 추출단계; 상기 상엽 추출단계를 거친 상엽 추출물을 냉각하는 상엽 추출물 냉각단계; 상기 추출물 냉각단계를 거진 상엽 추출물을 2 내지 8배로 농축하는 상엽 추출물 농축단계; 상기 추출물 농축 단계를 거친 상엽 추출물을 1 내지 9℃에서 12 내지 48 시간 동안 정치(定置)하는 상엽 추출물 정치단계; 상기 정치단계를 거친 상엽 추출물에서 침전물을 회수하는 상엽 침전물 회수단계 및 상기 상엽 침전물 회수단계로 회수한 침전물을 건조하여 분말화 하는 상엽 추출물 분말 제조 단계를 포함하는 것일 수 있다.Method for producing a composition for immuno-enhancing according to another embodiment of the present invention comprises an upper leaf drying step of drying the upper leaf; An upper leaf extraction step of extracting the upper leaf dried through the upper leaf drying step using any one selected from the group consisting of water, an alcohol having 1 to 6 carbon atoms, and a mixed solvent thereof; An upper leaf extract cooling step of cooling the upper leaf extract having undergone the upper leaf extracting step; Upper leaf extract concentration step of concentrating the extract of the upper leaf having undergone the
상기 면역증강용 조성물의 제조방법은 상기 상엽 건조단계 및 상엽 추출단계 사이에, 상기 상엽 건조단계를 거친 건조된 상엽에 락토바실러스 카제이(Lactobacillus casei), 락토바실러스 람노수스(Lactobacillus rhamnosus), 비피도 박테리윰 비피듐(Bifidobacterium bifidum) 비피더스균, 비피도 박테리움 브레브(Bifidobacterium breve) 비피더스균, 및 락토바실러스 아시도 필루스(Lactobacillus acidophilus)로 이루어진 군에서 선택되는 하나 이상의 미생물을 접종하고, 배양하는 상엽 발효단계를 더 포함하는 것일 수 있다.The method for preparing the composition for immuno-enhancing is between the upper leaf drying step and the upper leaf extracting step, Lactobacillus casei, Lactobacillus rhamnosus (Lactobacillus rhamnosus), Bifido in the dried upper leaf after the upper leaf drying step Inoculating and culturing at least one microorganism selected from the group consisting of Bifidobacterium bifidum bifidus, Bifidobacterium breve bifidus, and Lactobacillus acidophilus It may be to further include an upper leaf fermentation step.
이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명의 일 실시예에 따른 면역증강용 조성물은 상엽 추출물을 유효성분으로 포함하는 것이다.Immunity-enhancing composition according to an embodiment of the present invention is to include the upper leaf extract as an active ingredient.
본 발명에서 있어서 "면역증강용"이란 사전적 의미 외에 면역조절효과를 포함하는 것이고, 면역 조절 인자의 활성을 높여 면역 체계의 활성을 높이고 신생 혈관 생성 억제, 병원균에 대한 억지력 증가 등으로 면역 관련 질병을 예방하는 효과를 내는 것이고, 특히 대식세포의 활성을 높여 외부 병원균에 대한 방어, 사멸세포 제거 및 면역매개체의 분비 등의 효과를 내는 것으로 정의한다.In the present invention, "immune augmentation" is meant to include immunomodulatory effects in addition to the dictionary meaning, to increase the activity of immune regulatory factors to increase the activity of the immune system, inhibit the formation of neovascularization, increase the deterrent to pathogens, immune-related diseases It is to have the effect of preventing, in particular to increase the activity of macrophages to define the effects of defense against external pathogens, removal of dead cells and secretion of immune media.
본 발명에 있어서, "예방"은 상기 조성물의 투여에 의해 IL-1β에 의한 질환을 억제하거나 발병을 지연시키는 모든 행위를 의미하며, '치료'는 상기 조성물의 투여에 의해 IL-1β에 의한 질환에 의한 증세가 호전되거나 이롭게 변경하는 행위를 의미한다.In the present invention, "prevention" refers to any action that inhibits or delays the onset of the disease caused by IL-1β by administration of the composition, and 'treatment' refers to the disease caused by IL-1β by administration of the composition. Means an improvement or beneficial change in symptoms.
본 발명에서 말하는 상엽은 뽕나무 Morus alba Linne 또는 산뽕나무 Morus bombycis Koidzumi(뽕나무과 Moraceae)의 잎이다. 일명 동상엽(冬桑葉)이라고도 한다. 잎으로 달걀모양 또는 넓은 달걀모양이고 3 내지 5개로 갈라진 것도 있으며 길이 8 내지 15cm, 너비 7 내지 13cm이다. 윗면은 황록색 또는 연한 황갈색이다. 잎 끝은 뾰족하고 아랫부분은 심장형으로 되어 있으며, 잎 가장자리는 거치가 있다. 아랫면에는 엽맥이 돌출되어 있고 작은 엽맥은 그물 모양이며 그 위에 털이 나 있다. 질은 부스러지기 쉽고 가볍다. 냄새가 거의 없고 맛은 담담하나 약간 쓰며 떫다.The upper leaf of the present invention is a leaf of the mulberry Morus alba Linne or wild mulberry Morus bombycis Koidzumi (Moraceae Moraceae). Also known as frostbite (엽 葉). Leaves are egg-shaped or broad egg-shaped, 3-5 divided, 8-15 cm long, 7-13 cm wide. The upper side is yellowish green or light yellowish brown. The tip of the leaf is pointed, the lower part is heart shaped, and the leaf edge is hanged. The lower side has protruding leaf veins. The small leaf veins are reticulated and have hairs on them. The quality is fragile and light. Almost no smell and tastes light but bitter and steamed.
상기 상엽 추출물은 물, 탄소수 1 내지 6의 알코올 및 이들의 혼합 용매로 구성되는 군으로부터 선택된 어느 하나를 용매로 하여 추출되는 것일 수 있다. 본 발명에 있어서, 상기 상엽 추출물은 물, 탄소수 1 내지 6의 알코올 및 이들의 혼합 용매로 구성되는 군으로부터 선택된 용매로 추출한 것일 수 있다. The upper leaf extract may be extracted by using any one selected from the group consisting of water, an alcohol having 1 to 6 carbon atoms and a mixed solvent thereof. In the present invention, the upper leaf extract may be extracted with a solvent selected from the group consisting of water, alcohol having 1 to 6 carbon atoms and mixed solvents thereof.
본 발명에 있어서, 상기 “상엽 추출물”이란 상엽을 추출하여 수득한 추출물을 의미한다. In the present invention, the "leaf extract" means an extract obtained by extracting the leaf.
상기 상엽 추출물은 상엽 분쇄물을 물, 에탄올, 메탄올 등과 같은 탄소수(C1) 내지 (C6)의 알코올과 같은 극성 용매, 또는 알코올과 물의 1:0.1 내지 1:10의 혼합비를 갖는 혼합 용매로 용출할 수 있으며, 바람직하게는 1: 3 내지 5 의 에탄올과 물의 혼합비를 가지는 혼합 용매로 용출할 수 있다. 이 때, 추출 온도는 10℃ 내지 100℃, 바람직하게는 실온에서, 추출 기간은 12시간 내지 4일, 바람직하게는 24시간 동안 추출한 추출물 일 수 있다The upper leaf extract may be used as a mixed solvent having a mixed ratio of a mixture of alcohol and water in a polar solvent such as alcohol having a carbon number of (C 1 ) to (C 6 ) such as water, ethanol, methanol, or the like. It can be eluted, and preferably can be eluted with a mixed solvent having a mixing ratio of 1: 3 to 5 ethanol and water. At this time, the extraction temperature may be an extract extracted from 10 ℃ to 100 ℃, preferably room temperature, the extraction period is 12 hours to 4 days, preferably 24 hours
여과된 추출물을 진공 회전 농축기로 감압 농축하여 수득한 결과물일 수 있으나, 본 발명의 면역증강 효과를 나타낼 수 있는 상엽 추출물인 한, 이에 제한되지 않고, 추출액, 추출액의 희석액 또는 농축액, 추출액을 건조하여 얻어지는 건조물, 또는 이의 조정제물 또는 정제물을 모두 포함한다.The filtered extract may be a result obtained by concentrating the vacuum extract under reduced pressure with a vacuum rotary concentrator, but the present invention is not limited thereto, as long as it is an upper leaf extract capable of exhibiting the immuno-enhancing effect of the present invention, and extracts, dilutions or concentrates of the extracts, and dried extracts. It includes all the dried products obtained, or the adjusted or purified products thereof.
바람직하게는 상기 에탄올은 12 내지 22 중량%인 것일 수 있다. 상기 범위에 의한 추출물은 유효성분의 수득에 가장 유리할 수 있다.Preferably the ethanol may be 12 to 22% by weight. The extract according to the above range may be most advantageous for obtaining an active ingredient.
상기 상엽은 뽕나무의 줄기, 뿌리, 열매 등에 비하여 대식세포의 활성을 높일 수 있으므로 면역 증가 활성 효과가 우수하다. 따라서, 바람직하게는 상엽에 대한 에탄올 추출물을 사용하는 것일 수 있다.The upper leaf can increase the activity of the macrophages compared to the stem, root, fruit, etc. of the mulberry tree is excellent in the immune increasing activity effect. Therefore, it may be preferable to use an ethanol extract for the upper leaf.
상기 면역증강용 조성물은 대식세포의 활성을 촉진시키는 것일 수 있다.The immunopotentiating composition may be to promote the activity of macrophages.
상기 상엽은 락토바실러스 카제이(Lactobacillus casei), 락토바실러스 람노수스(Lactobacillus rhamnosus), 비피도 박테리윰 비피듐(Bifidobacterium bifidum) 비피더스균, 비피도 박테리움 브레브(Bifidobacterium breve) 비피더스균, 및 락토바실러스 아시도 필루스(Lactobacillus acidophilus)로 이루어진 군에서 선택되는 하나 이상을 접종하여 발효된 것일 수 있다.The upper leaves are Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium bifidum, Bifidobacterium, Bifidobacterium breve, Bifidobacterium breve and Lactobacillus, It may be fermented by inoculating one or more selected from the group consisting of Bacillus adophyllus (Lactobacillus acidophilus).
상기 발효에 사용하는 미생물로는 락토바실러스 살리바리우스(Lactobacillus salivarius), 락토바실러스 아시도필루스(Lactobacillus acidophilus), 락토바실러스 브레비스(Lactobacillus brevis), 락토바실러스 람노수스(Lactobacillus rhamnosus), 락토바실러스 플랜타룸(Lactobacillus plantarum), 락토바실러스 헬베티쿠스(Lactobacillus helveticus), 락토바실러스 퍼멘툼(Lactobacillus fermentum), 락토바실러스 파라카세이(Lactobacillus paracasei), 락토바실러스 카세이(Lactobacillus casei), 락토바실러스 델브루에키(Lactobacillus delbrueckii), 락토바실러스 레우테리(Lactobacillus reuteri), 락토바실러스 부츠네리(Lactobacillus buchneri), 락토바실러스 가세리(Lactobacillus gasseri), 락토바실러스 존스니(Lactobacillus johonsonii), 락토바실러스 케피르(Lactobacillus kefir) 등과 같은 유산 바실리, 락토코코스 락티스(Lactococcus lactis), 락토코코스 플랜타룸(Lactococcus plantarum), 락토코코스 라피노락티스(Lactococcus raffinolactis), 엔테로코코스파에칼리스(Enterococcus faecalis), 엔테로코코스 파에시늄(Enterococcus faecium), 스트렙토코코스 터모필리우스(Streptococcus thermophilus), 류코노스톡락티스(Leuconostoc lactis), 류코노스톡 메센테로이드(Leuconostoc mesenteroides) 등과 같은 유산 콕사이 및 비피도박테리움 애닐멀스(Bifidobacterium animals), 비피도 박테리움 비피듐(Bifidobacterium bifidum),비피도박테리움 브레브(Bifidobacterium breve), 비피도박테리움 인판티스(Bifidobacterium infantis), 비피도바테리움 롱굼(Bifidobacterium longum), 비피도박테리움 수도롱굼(Bifidobacterium pseudolongum), 비피도박테리움 터모필룸(Bifidobacterium themophilum), 비피도박테리움 아돌센티스(Bifidobacterium adolescentis) 등과 같은 비피도박테리아를 포함할 수 있으며, 더욱 바람직하게는 락토바실러스 카제이(Lactobacillus casei), 락토바실러스 람노수스(Lactobacillus rhamnosus), 비피도 박테리윰 비피듐(Bifidobacterium bifidum) 비피더스균, 비피도 박테리움 브레브(Bifidobacterium breve) 비피더스균, 및 락토바실러스 아시도 필루스(Lactobacillus acidophilus)로 이루어진 군에서 선택되는 하나 이상인 것일 수 있다.The microorganisms used for the fermentation include Lactobacillus salivarius, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus lamnosus, Lactobacillus rhamnosus and Lactobacillus plantarum. Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus del brui del bruii Lactobacillus kefir, Lactobacillus kefir, Lactobacillus keuter, Lactobacillus buchneri, Lactobacillus gasseri, Lactobacillus johonsonii, Lactobacillus kefir, etc. Lactococcus lactis, lactoco Lactococcus plantarum, Lactococcus raffinolactis, Enterococcus faecalis, Enterococcus faecium, Streptococcus thermophilus, Streptococcus thermophilus Lactic acid cocci and Bifidobacterium animals, Bifidobacterium bifidum, Bifidobacterium, such as Leuconostoc lactis, Leuconostoc mesenteroides Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium pseudolongum, Bifidobacterium turmofilum , Bifidobacterium adolescentis, and the like, and may also contain Bifidobacterium adolescentis. Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium bifidum, Bifidobacterium, Bifidobacterium breve, Bifidobacterium, and Lactobacillus casei Bacillus adophyllus (Lactobacillus acidophilus) may be one or more selected from the group consisting of.
다만 상기 범위에 의하는 경우 대식세포의 활성을 높여 우수한 면역 증강효과를 낼 수 있다.However, according to the above range can increase the activity of the macrophages can produce an excellent immune boosting effect.
바람직하게 상기 면역증강용 조성물은 병조희풀 추출물을 더 포함하는 것일 수 있다.Preferably the composition for immuno-enhancement may be further comprising a bottle extract of the bottle.
본 명세서에서 말하는 '병조희풀'은 낙엽 관목으로 미나리아재빗과 에 속한다. 그 잎은 마주나고 3개의 작은 잎으로 구성되는 3출 겹잎이다. 작은 잎은 넓은 달걀꼴이며 3개 중 옆에 달린 2개는 작다. 잎은 위로 올라갈수록 커지는데 끝이 뾰족하고 거칠며 가장자리에 약간 붉은빛이 도는 거친 톱니가 드문드문 있으나 흔히 3개로 얕게 갈라진다.As used herein, the term "herbaceous herb" is a deciduous shrub belonging to the genus Butaria javital. The leaf is a three-folded double leaf that is opposite and consists of three small leaves. Small leaves are broad egg-shaped, 2 of 3 are small. The leaves grow larger as they go up, with a sharp, rough tip and sparse coarse teeth slightly reddish at the edges, but are often shallowly divided into three.
상기 병조희풀을 사용하는 경우 상엽 추출물과 혼합되어 면역 활성을 진증시킬 수 있으며 그 향미를 높이는 효과를 가진다. 다만 상기 병조희풀은 자체의 독성이 있으므로 발효과정을 통하여 독성을 제거할 필요가 있다.In the case of using the bottled grass, it can be mixed with the extract of the upper leaf to increase the immune activity and increase the flavor. However, the bottled grass has its own toxicity, so it is necessary to remove the toxicity through the fermentation process.
바람직하게 상기 병조희풀 추출물은 발효 추출법에 의해 제조된 병조희풀 발효 추출물인 것일 수 있다. 상기 병조희풀은 약한 독성이 있으므로 발효과정을 통하여 독성을 제거할 필요가 있다.Preferably, the bottle of the dianthus extract may be that of the bottle distillation fermented extract prepared by the fermentation extraction method. The bottled grass has a weak toxicity, so it is necessary to remove the toxicity through the fermentation process.
본 발명에서 말하는 병조희풀 발효 추출물은 쌀뜨물 100 부피부에 대하여, 1 내지 3 부피부의 당류 및 0.05 내지 0.25 부피부의 천일염을 첨가하고, 0.5 내지 2.0 부피부의 미생물액을 혼합한 혼합물에 병조희풀 분쇄물을 10 내지 30 부피비로 부피부로 침지시킨 후, 공기와 차단된 상온 분위기에서 3 내지 15일 발효시켜 수득할 수 있다.The fermented herb extract according to the present invention is added to 1 to 3 parts by weight of sugar and 0.05 to 0.25 parts by weight of sea salt with respect to 100 parts by weight of rice water, and to a mixture of 0.5 to 2.0 parts by weight of microbial fluid. After dipping the pulverized pulverized powder to 10 parts by volume in a volume ratio, it can be obtained by fermentation for 3 to 15 days in a room temperature atmosphere blocked with air.
상기 미생물은 락토바실러스 카제이(Lactobacillus casei), 락토바실러스 람노수스(Lactobacillus rhamnosus), 비피도 박테리윰 비피듐(Bifidobacterium bifidum) 비피더스균, 비피도 박테리움 브레브(Bifidobacterium breve) 비피더스균, 및 락토바실러스 아시도 필루스(Lactobacillus acidophilus)로 이루어진 군에서 선택된 어느 하나 이상인 것일 수 있다.The microorganisms include Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium bifidum, Bifidobacterium, Bifidobacterium breve, Bifidobacterium bacterium, and Lactobacillus casei. Bacillus adophyllus (Lactobacillus acidophilus) may be any one or more selected from the group consisting of.
바람직하게 상기 면역증강용 조성물은 노간주나무 열수추출물을 더 포함하는 것일 수 있다.Preferably the composition for immuno-enhancing may further comprise juniper hot water extract.
본 발명에서 사용하는 노간주나무는 두송(杜松)이라고도 불리우며, 측백나뭇과에 속한다. 잎은 좁고 가는 선형의 바늘잎이 한 마디에서 3개씩 돌려나는데 길이는 12 내지 20mm이고 끝이 뾰족하며 표면의 가운데에 흰색의 좁은 홈이 있다.The juniper tree used in the present invention is also called Dosong (杜松) and belongs to the cypress tree family. Leaves are narrow, thin linear needle leaves, 3 to 3 in a row, 12 to 20 mm long, with sharp tips, with white narrow grooves in the center of the surface.
바람직하게 상기 노간주나무는 상기 병조희풀과 동일한 방법으로 발효된 것일 수 있다. 상기 노간주나무를 발효시키는 경우 상엽이 가지는 고유한 텁텁한 향을 제거하는 효과가 높고 향미가 증진될 수 있도록 하는 장점을 가진다.Preferably the juniper may be fermented by the same method as the bottled grass. When fermenting the juniper has the advantage of having a high effect of removing the inherent thick fragrance of the upper leaf and the flavor can be enhanced.
본 발명에서 사용하는 '낭아초'는 장미과에 속한 여러해살이풀. 높이는 30~100센티미터 정도이고, 굵은 뿌리줄기에서 줄기가 나와 자라며 전체에 털이 있다. 잎은 어긋나고 깃꼴 겹잎이며 작은 잎은 5 내지 7개이다. 6 내지 8월에 노란 꽃이 총상(總狀) 꽃차례로 피고, 열매에는 가시 모양의 털이 있어 다른 것에 잘 붙는다.'Nanchocho' used in the present invention is a perennial plant belonging to the rosaceae. Its height is about 30 ~ 100 centimeters, and stems come out from the coarse rhizome. Leaves are alternate, pinnate double leaves, 5 to 7 small leaves. Yellow flowers bloom in inflorescence in June-August. Fruits have spiny hairs that adhere well to others.
본 발명의 다른 일 실시예에 따른 면역증강용 약학 조성물은 상기 면역증강용 조성물을 포함하는 것이다.Immunity-enhancing pharmaceutical composition according to another embodiment of the present invention is to include the immuno-enhancing composition.
본 발명의 약학 조성물은 약학적으로 허용 가능한 담체를 포함할 수 있다. 약학적으로 허용 가능한 담체를 포함하는 조성물은 경구 또는 비경구의 여러 가지 제형일 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구 투여를 위한 고형제제에는 정제환제, 산제, 과립제, 캡슐제 등이 포함될 수 있으며, 이러한 고형제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로오스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제들도 사용될 수 있다. 경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들어 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결 건조제제, 좌제가 포함될 수 있다. 비수성용제, 현탁용제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테로 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다. 또한, 본 발명의 약학 조성물은 정제, 환제, 산제, 과립제, 캡슐제, 현탁제, 내용액제, 유제, 시럽제, 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제 및 좌제로 이루어진 군으로부터 선택되는 어느 하나의 제형을 가질 수 있다.The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. Compositions comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. When formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used. Solid form preparations for oral administration may include tablet pills, powders, granules, capsules, and the like, which may include at least one excipient such as starch, calcium carbonate, sucrose or lactose (at least one compound). lactose), gelatin and the like can be mixed. In addition to the simple excipients, lubricants such as magnesium stearate, talc and the like can also be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used. In addition, the pharmaceutical composition of the present invention is from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories It may have any one formulation selected.
본 발명의 상엽 추출물은 예로부터 식용 및 약용으로 사용되어 온 것으로 그 투여용량에 특별한 제약은 없고, 체내 흡수도, 체중, 환자의 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율, 질환의 중증도 등에 따라 변화될 수 있다. 일반적으로 상엽 추출물은 바람직하게는 체중 1kg당 10 내지 1000 mg 정도를 투여할 수 있으며, 보다 바람직하게는 체중 1 kg당 50 내지 500 mg 정도 투여할 수 있다. 본 발명의 상엽 추출물을 유효성분으로 포함하는 약학 조성물은 유효량 범위를 고려하여 제조하도록 하며, 이렇게 제형화된 단위 투여형 제제는 필요에 따라 약제의 투여를 감시하거나 관찰하는 전문가의 판단과 개인의 요구에 따라 전문화된 투약법을 사용하거나 일정 시간 간격으로 수회 투여할 수 있다.The leaf extract of the present invention has been used for food and medicinal use since ancient times, and there is no particular restriction on its dosage, and the body absorbency, weight, age of the patient, sex, health condition, diet, administration time, administration method, excretion rate And the severity of the disease. In general, the upper leaf extract may preferably be administered about 10 to 1000 mg per kg of body weight, more preferably about 50 to 500 mg per kg of body weight. A pharmaceutical composition comprising the extract of the present invention as an active ingredient is to be prepared in consideration of the effective amount range, and the unit dosage form formulated in this way is the expert's judgment and individual's request to monitor or observe the administration of the drug as needed. Depending on the specific dosage regimen, it may be administered several times at regular time intervals.
본 발명의 또 다른 일 실시예에 따른 면역증강용 식품 조성물은 상기 면역증강용 조성물을 포함하는 것이다.Immunity-enhancing food composition according to another embodiment of the present invention is to include the immuno-enhancing composition.
본 발명에 있어서, 상기 식품조성물은 일반식품, 건강기능성 식품, 식품첨가제, 기능성 식품 첨가제를 포함하는 것으로 정의한다.In the present invention, the food composition is defined as containing a general food, health functional food, food additives, functional food additives.
본 발명의 상엽 추출물을 식물조성물로 사용할 경우, 상기 상엽 추출물을 식품에 그대로 첨가하거나 다른 일반 식품, 기능성 식품 또는 기능성 성분과 혼합 또는 첨가하여 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용할 수 있다. 유효성분의 혼합량은 사용 목적에 따라 적합하게 결정될 수 있다.When the upper leaf extract of the present invention is used as a plant composition, the upper leaf extract may be added to a food as it is, or mixed or added with other general foods, functional foods or functional ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the intended use.
본 발명의 식품 조성물의 종류는 특별한 제한은 없다. 상기 면역증강용 조성물의 첨가될 수 있는 일반식품 또는 건강기능식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 등이 있고, 통상적인 의미에서의 건강기능식품을 모두 포함할 수 있으며, 동물을 위한 사료로 이용되는 식품을 포함할 수 있다. 상기 외에 본 발명의 식품 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 천연 과일쥬스, 과일쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다.The type of food composition of the present invention is not particularly limited. Examples of general foods or health functional foods that may be added to the immune enhancing composition include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various Soup, beverages, tea, drinks, alcoholic beverages and vitamin complexes, and the like, may include all of the health functional foods in a conventional sense, and may include foods used as feed for animals. In addition to the above, the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols. And carbonation agents used in carbonated beverages. Others may contain pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks.
본 발명의 또 다른 일실시예에 따른 면역증강용 조성물의 제조방법은 상엽을 건조하는 상엽 건조단계; 상기 상엽 건조단계를 거쳐 건조된 상엽을 물, 탄소수 1 내지 6의 알코올 및 이들의 혼합 용매로 구성되는 군으로부터 선택된 어느 하나를 사용하여 추출하는 상엽 추출단계; 상기 상엽 추출단계를 거친 상엽 추출물을 냉각하는 상엽 추출물 냉각단계; 상기 추출물 냉각단계를 거진 상엽 추출물을 2 내지 8배로 농축하는 상엽 추출물 농축단계; 상기 추출물 농축 단계를 거친 상엽 추출물을 1 내지 9℃에서 12 내지 48 시간 동안 정치(定置)하는 상엽 추출물 정치단계; 상기 정치단계를 거친 상엽 추출물에서 침전물을 회수하는 상엽 침전물 회수단계 및 상기 상엽 침전물 회수단계로 회수한 침전물을 건조하여 분말화 하는 상엽 추출물 분말 제조 단계를 포함하는 것일 수 있다.Method for producing a composition for immuno-enhancing according to another embodiment of the present invention comprises an upper leaf drying step of drying the upper leaf; An upper leaf extraction step of extracting the upper leaf dried through the upper leaf drying step using any one selected from the group consisting of water, an alcohol having 1 to 6 carbon atoms, and a mixed solvent thereof; An upper leaf extract cooling step of cooling the upper leaf extract having undergone the upper leaf extracting step; Upper leaf extract concentration step of concentrating the extract of the upper leaf having undergone the cooling
상기 면역증강용 조성물의 제조방법은 상기 상엽 건조단계 및 상엽 추출단계 사이에, 상기 상엽 건조단계를 거친 건조된 상엽에 락토바실러스 카제이(Lactobacillus casei), 락토바실러스 람노수스(Lactobacillus rhamnosus), 비피도 박테리윰 비피듐(Bifidobacterium bifidum) 비피더스균, 비피도 박테리움 브레브(Bifidobacterium breve) 비피더스균, 및 락토바실러스 아시도 필루스(Lactobacillus acidophilus)로 이루어진 군에서 선택되는 하나 이상의 미생물을 접종하고, 배양하는 상엽 발효단계를 더 포함하는 것일 수 있다.The method for preparing the composition for immuno-enhancing is between the upper leaf drying step and the upper leaf extracting step, Lactobacillus casei, Lactobacillus rhamnosus (Lactobacillus rhamnosus), Bifido in the dried upper leaf after the upper leaf drying step Inoculating and culturing at least one microorganism selected from the group consisting of Bifidobacterium bifidum bifidus, Bifidobacterium breve bifidus, and Lactobacillus acidophilus It may be to further include an upper leaf fermentation step.
본 발명에 따른 상엽 추출물을 포함하는 면역증강용 조성물을 사용하는 경우 대식세포가 증가 내지 활성화 되어 면역활성을 증진시킬 수 있고, 이에 따라 면역관련 질환을 예방 또는 개선하는 효과를 낼 수 있다.When using the composition for immuno-enhancement comprising the extract of the upper lobe according to the present invention, macrophages can be increased or activated to enhance immune activity, thereby preventing or improving immune-related diseases.
특히 본 발명의 상엽 추출물 및 그 유효범위에 의하는 경우 대식세포가 증가 내지 활성화가 극대화 될 수 있다. In particular, by the upper leaf extract of the present invention and its effective range, the increase or activation of macrophages can be maximized.
본 발명에 의하는 경우 부작용 없이 면역 증진 효과를 낼 수 있으므로 면역 증진 등을 목적으로 하는 건강기능식품, 면역질환 개선을 위한 의약품 등으로 제공될 수 있다.If the present invention can produce an immune enhancing effect without side effects, it can be provided as a health functional food for the purpose of enhancing immunity, medicines for improving immune diseases and the like.
도 1은 WEMF 처리에 따른 RAW 264.7 대식세포의 활성화 시험에 관한 것이다.
도 2는 WEMF 처리에 따른 RAW 264.7 대식세포의 활성화 시험에 관한 것이다.
도 3은 RAW 264.7 대식세포에서 WEMF 처리에 따른 PEG2 및 TNF-α의 생성 증가 시험에 관한 것이다.
도 4는 RAW 264.7 대식세포에서 WEMF 처리에 따른 PEG2 및 TNF-α의 생성 증가 시험에 관한 것이다.
도 5는 RAW 264.7 대식세포에서 NO와 PGE 2 생성에 미치는 WEMF의 영향에 관한 것이다.
도 6은 RAW 264.7 대식세포에서 다양한 cytokine의 발현에 미치는 WEMF의 영향에 관한 것이다.
도 7은 RAW 264.7 대식세포에서 다양한 cytokine의 발현에 미치는 WEMF의 영향에 관한 것이다.
도 8은 WEMF의 RAW 264.7 대식세포에 대한 탐식 능력을 향상 효과에 관한 것이다.1 relates to the activation test of RAW 264.7 macrophages following WEMF treatment.
Figure 2 relates to the activation test of RAW 264.7 macrophages according to WEMF treatment.
3 relates to an increase in production of PEG2 and TNF-α following WEMF treatment in RAW 264.7 macrophages.
4 relates to an increase in production of PEG2 and TNF-α following WEMF treatment in RAW 264.7 macrophages.
5 relates to the effect of WEMF on NO and
Figure 6 relates to the effect of WEMF on the expression of various cytokine in RAW 264.7 macrophages.
Figure 7 relates to the effect of WEMF on the expression of various cytokine in RAW 264.7 macrophages.
8 relates to the effect of improving the phagocytic ability of WEMF for RAW 264.7 macrophages.
본 발명은 다양한 변환을 가할 수 있고 여러 가지 실시예를 가질 수 있는 바, 특정 실시예들을 도면에 예시하고 상세한 설명에 상세하게 설명하고자 한다. 본 발명의 효과 및 특징, 그리고 그것들을 달성하는 방법은 도면과 함께 상세하게 후술되어 있는 실시예들을 참조하면 명확해질 것이다. 그러나 본 발명은 이하에서 개시되는 실시예들에 한정되는 것이 아니라 다양한 형태로 구현될 수 있다. 이하의 실시예에서, 제1, 제2 등의 용어는 한정적인 의미가 아니라 하나의 구성 요소를 다른 구성 요소와 구별하는 목적으로 사용되었다. 또한, 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다. 또한, 포함하다 또는 가지다 등의 용어는 명세서상에 기재된 특징, 또는 구성요소가 존재함을 의미하는 것이고, 하나 이상의 다른 특징들 또는 구성요소가 부가될 가능성을 미리 배제하는 것은 아니다. 또한, 도면에서는 설명의 편의를 위하여 구성 요소들이 그 크기가 과장 또는 축소될 수 있다. 예컨대, 도면에서 나타난 각 구성의 크기 및 두께는 설명의 편의를 위해 임의로 나타내었으므로, 본 발명이 반드시 도시된 바에 한정되지 않는다.As the invention allows for various changes and numerous embodiments, particular embodiments will be illustrated in the drawings and described in detail in the written description. Effects and features of the present invention, and methods of achieving them will be apparent with reference to the embodiments described below in detail together with the drawings. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various forms. In the following embodiments, the terms first, second, etc. are used for the purpose of distinguishing one component from other components rather than a restrictive meaning. Also, the singular forms “a”, “an” and “the” include plural forms unless the context clearly indicates otherwise. In addition, the terms including or have means that the features or components described in the specification are present, and does not preclude the possibility of adding one or more other features or components. In addition, in the drawings, components may be exaggerated or reduced in size for convenience of description. For example, the size and thickness of each component shown in the drawings are arbitrarily shown for convenience of description, and thus the present invention is not necessarily limited to the illustrated.
이하, 첨부된 도면을 참조하여 본 발명의 실시예들을 상세히 설명하기로 하며, 도면을 참조하여 설명할 때 동일하거나 대응하는 구성 요소는 동일한 도면부호를 부여하고 이에 대한 중복되는 설명은 생략하기로 한다.Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings, and the same or corresponding components will be denoted by the same reference numerals, and redundant description thereof will be omitted. .
[[ 제조예Production Example : : 상엽Upper lobe 추출물의 제조] Preparation of Extracts]
1. One. 상엽의Upper lobe 준비 Ready
뽕나무(M. alba L)의 잎인 상엽(Mori folium)은 바이오-포트 코리사사(부산, 한국)로부터 입수하였고, 동의대 한의대(부산, 한국), 생화학과의 S.H. Hong 교수에 의해서 인증받았다. 건조된 잎은 작은 조각으로 절단하고, 미세한 분말로 만든 후에, 3시간 동안 증류수(50g/500㎖)에서 가열하였다. 구체적인 방법은 본 발명에 기재된 면역증강용 조성물의 제조방법에 따라 제조하였다. The leaf of the mulberry (M. alba L) (Mori folium) was obtained from Bio-Port Colisasa (Busan, Korea), S.H. Certified by Professor Hong. The dried leaves were cut into small pieces, made into fine powder, and then heated in distilled water (50 g / 500 mL) for 3 hours. Specific method was prepared according to the method for producing an immuno-enhancing composition described in the present invention.
추출액은 불용성 물질을 제거하기 위해서 와트만 3호 여과지를 통하여 두 번 여과하고, 여액은 동결건조시킨 다음, 미세한 분말로 분쇄하였다. 상엽 추출물(WEMF)은 증류수로 100㎎/㎖의 농도로 용해시키고, 저장액(stock solution)은 사용하기 전에 목적하는 농도로 배지에 희석하였다.The extract was filtered twice through Whatman 3 filter paper to remove insoluble matters, the filtrate was lyophilized and then ground to a fine powder. Upper leaf extract (WEMF) was dissolved in distilled water at a concentration of 100 mg / ml, and the stock solution was diluted in the medium to the desired concentration before use.
또한, 락토바실러스 카세이(Lactobacillus casei)를 접종하여 5일간 발효시킨 상엽을 사용하여 상기 상엽 추출물의 제조방법과 동일하게 추출하여 발효상엽 추출물을 제조하였다.In addition, the fermented leaf extract was prepared by extracting the same as the method of preparing the leaf extract using the upper leaf inoculated with Lactobacillus casei fermented for 5 days.
[시약 및 항체]Reagents and Antibodies
세포배양을 위한 Dulbecco's modified eagle's medium (DMEM), bovine serum (FBS), penicillin/streptomycin은 WELGENE (대구, 한국)에서 구입하였고, Escherichia coli 유래 lipopolysaccaride (LPS), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT), Griess reagent, sodium nitrite는 Sigma-Aldrich Chemical Co. (St. Lousi, MO, USA)에서 구입하였다. TNF-α 측정용 enzyme-linked immunosorbent assay (ELISA) kit는 R&D Systems (Minneapolis, MN, USA)에서, PGE2 ELISA kit와 cyclooxygenase-2 (COX-2) 항체는 Cayman Chemical (Ann Arbor, MI, USA)에서, extracellular signal-regulated kinase (ERK) 및 p38 mitogen-activated protein kinase (MAPK) 항체는 Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA)에서 구입하였다. c-Jun N-terminal kinase (JNK), phospho-JNK, phospho-ERK, phosphor-p38 MAPK 항체는 Cell Signaling (Beverly, MA, USA)에서 구입하였다. 대식세포주 RAW 264.7은 한국 세포주 은행(서울, 한국)에서 구입하였고 5v% 미만의 CO2, 37 에서 DMEM 배지에서 보관하였다.Dulbecco's modified eagle's medium (DMEM), bovine serum (FBS) and penicillin / streptomycin for cell culture were purchased from WELGENE (Daegu, Korea), and lipopolysaccaride (LPS) derived from Escherichia coli (LPS), 3- (4,5-Dimethylthiazol-2 -yl) -2,5-diphenyl tetrazolium bromide (MTT), Griess reagent, sodium nitrite are manufactured by Sigma-Aldrich Chemical Co. (St. Lousi, MO, USA). The enzyme-linked immunosorbent assay (ELISA) kit for measuring TNF-α is available from R & D Systems (Minneapolis, MN, USA), and the PGE2 ELISA kit and cyclooxygenase-2 (COX-2) antibody are derived from Cayman Chemical (Ann Arbor, MI, USA). In addition, extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) antibodies were synthesized by Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). c-Jun N-terminal kinase (JNK), phospho-JNK, phospho-ERK and phosphor-p38 MAPK antibodies were purchased from Cell Signaling (Beverly, MA, USA). Macrophage line RAW 264.7 was purchased from Bank of Korea Cell Line (Seoul, Korea) and stored in DMEM medium at less than 5v% CO 2 , 37.
[[ 실험예Experimental Example 1: One: 상엽Upper lobe 추출물의 Of extract 대식세포에 대한 활성증가Increased activity against macrophages ]]
1. RAW 264.7 대식세포에서 NO 1.NO in RAW 264.7 Macrophages 생성능Generating ability 측정 Measure
Griess 시약을 사용하여 배양 상등액에서 NO의 생성능을 분석 하였다. 요약하면, 세포를 24 시간 동안 다양한 농도의 WEMF 또는 0.5 ng/ml LPS로 처리 하였다. 그 후, 상등액을 수집하고 어두운 곳에서 실온에서 10분 동안 동일한 부피의 Griess reagent와 혼합하였다. 흡광도는 마이크로 플레이트 판독기를 사용하여 540 nm에서 측정하였다. 또한, NO의 농도를 아질산 나트륨의 공지 concen trations 의해 생성 된 표준 곡선을 참조(Lee H, Pyo MJ, Bae SK, Heo Y, Kim CG, Kang C, et al. Improved therapeutic profiles of PLA2-free bee venom prepared by ultrafiltration method. Toxicol Res 2015;31:33-40.)하여 산출 하였다. 한편, 신선한 배지를 모든 실험에서 블랭크로서 사용 하였다.The production of NO in the culture supernatant was analyzed using Griess reagent. In summary, cells were treated with various concentrations of WEMF or 0.5 ng / ml LPS for 24 hours. The supernatant was then collected and mixed with the same volume of Griess reagent for 10 minutes at room temperature in the dark. Absorbance was measured at 540 nm using a micro plate reader. In addition, refer to the standard curve generated by known concen trations of sodium nitrite concentrations (Lee H, Pyo MJ, Bae SK, Heo Y, Kim CG, Kang C, et al. Improved therapeutic profiles of PLA2-free bee venom prepared by ultrafiltration method.Toxicol Res 2015; 31: 33-40. On the other hand, fresh medium was used as a blank in all experiments.
2. RAW 264.7 대식세포에서 2. In RAW 264.7 Macrophages PGEPGE -2, -2, TNFTNF -α and IL--α and IL- 1β1β 생성능Generating ability 측정 Measure
PGE-2, TNF-α, IL-1β 및 IL-10의 생산을 측정하기 위해, 세포를 위 NO 측정 분석과 같은 조건으로 배양하였다. PGE-2, TNF-α, IL-1β, IL-10의 생성능은 제조업체의 지침(Wang L, Xu ML, Liu J, Wang Y, Hu JH, Wang MH. Sonchus asper extract inhibits LPS-induced oxidative stress and pro-inflammatory cytokine production in RAW264.7 macrophages. Nutr Res Pract 2015;9:579-85)에 따라 선택적 ELISA 키트로 측정 하였다.To measure the production of PGE-2, TNF-α, IL-1β and IL-10, cells were cultured under the same conditions as the above NO measurement assay. The ability to produce PGE-2, TNF-α, IL-1β, and IL-10 was determined by the manufacturer's instructions (Wang L, Xu ML, Liu J, Wang Y, Hu JH, Wang MH.Sonchus asper extract inhibits LPS-induced oxidative stress and Pro-inflammatory cytokine production in RAW264.7 macrophages.Nutr Res Pract 2015; 9: 579-85) was measured with a selective ELISA kit.
3. RNA의 분리 및 3. Isolation of RNA and 역전사Reverse transcription 중합효소의 연쇄 반응(RT- Chain Reaction of Polymerase (RT- PCRPCR ))
RNA는 제조업체의 지침에 따라 TRIzol reagent를 사용하여 배양세포로부터 분리 한 후 cDNA를 생성하기 위해 M-MLV 역전사 키트를 사용하여 역전사를 진행하였다. 또한 PCR을 사용하여 RT-generated cDNA의 엔코딩을 진행하였다.RNA was isolated from the culture cells using TRIzol reagent according to the manufacturer's instructions, and then reverse transcription was performed using the M-MLV reverse transcription kit to generate cDNA. PCR was also used to encode RT-generated cDNA.
4. 단백질의 분리와 Western blot 분석4. Protein Isolation and Western Blot Analysis
WEMF 처리에 따른 번역 수준에서의 유전자 발현 변화의 관찰을 위하여 준비된 세포들을 250 mM NaCl, 25 mM Tris-HCl (pH 7.5), 10 mM EDTA, 1% NP-40 와 0.1mM phenyl-methylsulfonylfluoride, protease inhibitor 등이 함유된 lysis buffer를 사용하여 용해시켰다. 분리된 단백질들의 농도를 측정한 후, Western blot analysis를 위해 동량의 단백질들을 sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis를 이용하여 분리하고 nitrocellulose membrane (Schleicher & Schuell, Keene, NH, USA)으로 전이시켰다. 각각의 membrane을 적정 항체 및 enhanced chemiluminescence (ECL, Amersham Corp. Arlington Heights, IL, USA) 용액을 이용하여 단백질들의 발현 변화를 조사하였다. Cells prepared for observation of changes in gene expression at the translational level following WEMF treatment were treated with 250 mM NaCl, 25 mM Tris-HCl (pH 7.5), 10 mM EDTA, 1% NP-40 and 0.1 mM phenyl-methylsulfonylfluoride, a protease inhibitor. It was dissolved using a lysis buffer containing the back. After measuring the concentration of the isolated proteins, the same amount of proteins were separated by sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis for Western blot analysis and transferred to nitrocellulose membrane (Schleicher & Schuell, Keene, NH, USA). . Each membrane was examined for expression changes of proteins using titration antibody and enhanced chemiluminescence (ECL, Amersham Corp. Arlington Heights, IL, USA) solution.
5. 면역 형광 측정5. Immunofluorescence Measurement
RAW 264.7 세포의 탐식 능력은 제조업체의 지침에 따라 Phagocytosis Assay Kit (IgG-FITC) (Cayman Chemical Co, Ann Arbor, MI, USA)를 사용하여 검출하였다. 한편 phagocytosis의 범위는 620nm에서의 흡광도를 측정함으로써 결정 하였다.The phagocytosis of RAW 264.7 cells was detected using the Phagocytosis Assay Kit (IgG-FITC) (Cayman Chemical Co, Ann Arbor, MI, USA) according to the manufacturer's instructions. The range of phagocytosis was determined by measuring the absorbance at 620 nm.
6. 통계처리6. Statistical Processing
모든 실험 결과는 평균 ± 표준편차로 표시하였고 SigmaPlot (Systat Software Inc., San Jose, CA, USA)을 이용하여 Student t-test를 이용하여 통계적 유의성을 얻었다.All experimental results were expressed as mean ± standard deviation and statistical significance was obtained using Student t-test using SigmaPlot (Systat Software Inc., San Jose, CA, USA).
[실험결과 1: [Experimental Result 1: 상엽Upper lobe 추출물의 Of extract 대식세포에 대한 활성증가Increased activity against macrophages ]]
1. One. WEMFWEMF 처리에 따른 RAW 264.7 대식세포의 활성화 Activation of RAW 264.7 Macrophages Following Treatment
LPS는 그람 음성 박테리아의 세포 외막에 존재함으로써, iNOS, COX-2, macrophage-related cytokines와 같은 유전자의 전사를 자극하는 세포 신호 전달 경로의 활성화를 통하여 대식세포를 활성화시킨다(Ivashkiv LB. Inflammatory signaling in macrophages: transitions from acute to tolerant and alternative activation states. Eur J Immunol 2011;41:2477-481., Morris MC, Gilliam EA, Li L. Innate immune programing by endotoxin and its pathological consequences. Front Immunol 2015;5:680.).LPS is present in the outer membrane of Gram-negative bacteria, thereby activating macrophages through activation of cell signaling pathways that stimulate transcription of genes such as iNOS, COX-2 and macrophage-related cytokines (Ivashkiv LB. Inflammatory signaling in macrophages: transitions from acute to tolerant and alternative activation states.Eur J Immunol 2011; 41: 2477-481., Morris MC, Gilliam EA, Li L. Innate immune programing by endotoxin and its pathological consequences.Front Immunol 2015; 5: 680 .).
이에 LPS를 양성 물질(positive substance)로 사용하여 대시세포를 활성화 시키고, WEMF에 의한 RAW 264.7 대식세포 활성화 여부의 조사를 위한 실험 조건 설정을 위하여 다양한 농도의 WEMF가 함유된 배지에서 배양된 RAW 264.7 세포를 대상으로 MTT assay를 실시하였다. 그 결과 도 1의 결과에서 알 수 있듯이, WEMF를 처리한 범위 특히 0.5 내지 2.0 ng/ml의 범위에서 세포독성이 없다는 것을 확인하였다. 한편, WEMF은 세포독성 없을 뿐만 아니라 도 2를 참조하면, WEMF가 대식세포의 활성화에 기여한다는 것을 확인할 수 있다.Thus, LPS was used as a positive substance to activate dashed cells, and RAW 264.7 cells cultured in medium containing WEMF in order to set experimental conditions for investigating RAW 264.7 macrophage activation by WEMF. MTT assay was performed on the subjects. As a result, as can be seen in the results of Figure 1, it was confirmed that there is no cytotoxicity in the range treated with WEMF, particularly in the range of 0.5 to 2.0 ng / ml. On the other hand, WEMF is not only cytotoxic, referring to Figure 2, it can be seen that WEMF contributes to the activation of macrophages.
2. RAW 264.7 대식세포에서 2. In RAW 264.7 Macrophages WEMFWEMF 처리에 따른 According to treatment PEG2PEG2 및 And TNFTNF -α의 생성 증가increased production of -α
대식세포의 활성화에는 다양한 세포 내 신호전달계가 관여하고 있음은 여러 선행 연구에서 확인되었으며, 이들 신호계의 제어 현상에 대한 이해는 대식세포 활성 촉진 물질의 발굴 및 그들의 세포 내 역할에 대한 많은 정보를 제공하여 준다. 이러한 세포 내 신호전달계 중, MAPKs는 대식세포 활성을 포함한 세포 활성물질의 생성 및 다양한 생물학적기능을 조절하며(Rao, K. M. 2001. MAP kinase activation in macrophages. J Leukoc Biol 69, 3-10., Schorey, J. S. and Cooper, A. M. 2003. Macrophage signalling upon mycobacterial infection: the MAP kinases lead the way. Cell Microbiol 5, 133-142.), 세포의 증식 및 성장에 관여한다고 알려진 phosphatidylinositol-3-kinase (PI3K)/Akt 경로 역시 대식세포의 활성에 핵심적인 역할을 하는 것으로 알려져 있다(Ha, T., Liu, L., Kelley, J., Kao, R., Williams, D. and Li, C. 2011. Toll-like receptors: new players in myocardial ischemia/reperfusion injury. Antioxid Redox Signal 15, 1875-1893., Ha, T., Liu, L., Kelley, J., Kao, R., Williams, D. and Li, C. 2011. Toll-like receptors: new players in myocardial ischemia/reperfusion injury. Antioxid Redox Signal 15, 1875-1893.)Several previous studies have shown that various intracellular signaling systems are involved in the activation of macrophages, and understanding of the control phenomena of these signaling systems provides a great deal of information on the discovery of macrophage activation promoters and their intracellular roles. give. Among these intracellular signaling systems, MAPKs regulate the production of cell activators including macrophage activity and various biological functions (Rao, KM 2001. MAP kinase activation in macrophages. J Leukoc Biol 69, 3-10., Schorey, JS and Cooper, AM 2003. Macrophage signaling upon mycobacterial infection: the MAP kinases lead the way.Cell Microbiol 5, 133-142.), A phosphatidylinositol-3-kinase (PI3K) / Akt pathway known to be involved in cell proliferation and growth. It is also known to play a key role in macrophage activity (Ha, T., Liu, L., Kelley, J., Kao, R., Williams, D. and Li, C. 2011. Toll-like receptors : new players in myocardial ischemia / reperfusion injury.Antioxid Redox Signal 15, 1875-1893., Ha, T., Liu, L., Kelley, J., Kao, R., Williams, D. and Li, C. 2011 Toll-like receptors: new players in myocardial ischemia / reperfusion injury.Antioxid Redox Signal 15, 1875-1893.)
대식세포가 다양한 조직에 존재하고 염증성 자극을 포함한 인자들에 대한 생체방어 기능을 한다는 점에서 이러한 인자들에 의하여 활성화된 대식세포는 PEG2와 같은 염증성 매개인자나 TNF-α를 포함하는 cytokine의 생성을 촉진한다(Guslandi, M. 1998. Nitric oxide and inflammatory bowel diseases. Eur J Clin Invest 28, 904-907., Ritchlin, C. T., Haas-Smith, S. A., Li, P., Hicks, D. G. and Schwarz, E. M. 2003. Mechanisms of TNF-alpha- and RANKL-mediated osteoclastogenesis and bone resorption in psoriatic arthritis. J Clin Invest 111, 821-831.). 따라서 상기에서 WEMF 처리에 따른 RAW 264.7 세포의 형태적 변화가 대식세포 활성화와 직접 연관성이 있는지를 조사하기 위하여 PEG2 및 TNF-α의 생성에 미치는 WEMF의 영향을 조사하였다. 도 3 및 도 4의 결과에서 알 수 있듯이 LPS 처리군과 비교하여 WEMF의 처리에 따라 RAW 264.7 대식세포의 PEG2의 생성이 유의적으로 증가하였음을 알 수 있었으며, TNF-α의 생성 또한 WEMF의 처리 농도 의존적으로 증가된다는 것을 확인할 수 있다.Since macrophages are present in various tissues and act as bioprotective agents for factors including inflammatory stimuli, macrophages activated by these factors are responsible for the production of cytokine containing TNF-α or inflammatory mediators such as PEG2. (Guslandi, M. 1998. Nitric oxide and inflammatory bowel diseases.Eur J Clin Invest 28, 904-907., Ritchlin, CT, Haas-Smith, SA, Li, P., Hicks, DG and Schwarz, EM 2003 Mechanisms of TNF-alpha- and RANKL-mediated osteoclastogenesis and bone resorption in psoriatic arthritis.J Clin Invest 111, 821-831.). Therefore, the effects of WEMF on the production of PEG2 and TNF-α were investigated to investigate whether the morphological changes of RAW 264.7 cells following WEMF treatment were directly related to macrophage activation. As can be seen from the results of FIGS. 3 and 4, the production of PEG2 in RAW 264.7 macrophages was significantly increased according to the treatment of WEMF compared to the LPS treatment group, and the production of TNF-α was also treated with WEMF. It can be seen that the concentration increases.
3. RAW 264.7 대식세포에서 NO와 3.NO and NO in RAW 264.7
WEMF에 의해 NO와 PGE 2 생성에 미치는 영향을 알아보기 위하여 RT-PCR 및 웨스턴 블롯 분석을 적용하여, iNOS 및 COX-2의 발현의 조절과 관련이 있는지 여부를 조사 하였다.To determine the effect of WEMF on NO and
그 결과 도 5를 참조하면, WEMF가 효과적으로 WEMF가 코딩하는 유전자의 발현을 유도하여 NO와 PGE-2의 생성을 증가시킨다는 것을 알 수 있다.As a result, referring to FIG. 5, it can be seen that WEMF effectively induces expression of a gene encoded by WEMF and increases production of NO and PGE-2.
4. RAW 264.7 대식세포에서 다양한 4. Diverse in RAW 264.7 Macrophages cytokine의cytokine 발현에 미치는 On expression WEMF의Of WEMF 영향 effect
한편 다양한 종류의 cytokine이 대식세포의 활성화에 관여하며, cytokine의 종류에 따라 다양한 면역반응을 매개함으로서 대식세포의 운명도 달라진다. 그러나 대식세포의 활성과 면역체계에 연관된 cytokine 신호체계 기전은 여전히 많은 부분이 알려져 있지 않다. 본 연구에서는 WEMF가 함유된 배지에서 배양된 RAW 264.7 세포에서 분비되는 cytokine에 대한 기초 정보를 확립하기 위하여 cytokine array를 실시하였으며, 이를 LPS 처리군과 비교하였다. On the other hand, various types of cytokine are involved in the activation of macrophages, and the fate of macrophages also varies by mediating various immune responses depending on the type of cytokine. However, much of the cytokine signaling mechanisms involved in macrophage activity and immune system are still unknown. In this study, cytokine array was performed to establish basic information on cytokine secreted from RAW 264.7 cells cultured in WEMF-containing medium, and compared with LPS-treated group.
도 6을 참조하면, TNF-α 및 IL (IL-1β, IL-6 및 IL-10)의 분비는 WEMF 및 LPS 처리 후 유의하게 증가되었다는 것을 알 수 있다. 예를 들어, 2.0 mg/ml WFRG 및 LPS 자극 세포의 배지에서 TNF-α의 농도는 각각 미처리 대조군 세포의 것보다 약 9.5배, 7.5 배 더 높았다. 또한 도 7를 참조하면, 사이토 카인 생산에서 얻어진 결과와 일치하게 TNF-α 및 IL의 mRNA 및 단백질 수준도 WEMF 처리 농도에 의존적으로 증가되는 것을 알 수 있었다. 이러한 결과는 WEMF가 RAW 264.7 세포에서 전사 및 번역 수준에서 사이토 카인 생산을 양적으로 조절한다는 것을 의미하는 것으로 볼 수 있다.Referring to Figure 6, it can be seen that the secretion of TNF-α and IL (IL-1β, IL-6 and IL-10) was significantly increased after the WEMF and LPS treatment. For example, the concentration of TNF-α in the medium of 2.0 mg / ml WFRG and LPS stimulating cells was about 9.5 and 7.5 times higher than that of untreated control cells, respectively. Referring to FIG. 7, the mRNA and protein levels of TNF-α and IL were also increased depending on the concentration of WEMF, consistent with the results obtained in cytokine production. These results can be seen as meaning that WEMF quantitatively regulates cytokine production at the level of transcription and translation in RAW 264.7 cells.
5. 5. WEMF의Of WEMF RAW 264.7 RAW 264.7 대식세포에 대한 탐식 능력을Phagocytosis of macrophages 향상 효과 Enhancement effect
식균 작용은 다양한 종류의 항균 및 세포 독성 반응을 일으키는 대 식세포의 주요 기능이므로(Schultze JL, Schmidt SV. Molecular features of macrophage activation. Semin Immunol 2015;27:416-23., McCabe A, MacNamara KC. Macrophages: Key regulators of steady-state and demand-adapted hematopoiesis. Exp Hematol 2016;44:213-22.) 우리는 대 식세포 식균 작용에 대한 WEMF의 영향을 조사하였다.Phagocytosis is a major function of macrophages that cause various types of antibacterial and cytotoxic responses (Schultze JL, Schmidt SV. Molecular features of macrophage activation.Semin Immunol 2015; 27: 416-23., McCabe A, MacNamara KC.Macrophages : Key regulators of steady-state and demand-adapted hematopoiesis.Exp Hematol 2016; 44: 213-22.) We investigated the effects of WEMF on macrophage phagocytosis.
하기의 도 8을 참조하면, WEMF를 처리한 경우 RAW 264.7 대식세포의 식세포 성질이 극적으로 활성화 된다는 것을 알 수 있다.Referring to FIG. 8, it can be seen that the phagocytic properties of RAW 264.7 macrophages are dramatically activated when WEMF is treated.
[[ 실험예Experimental Example 2: 복합추출물의 제조] 2: Preparation of Complex Extracts]
상기 상엽 추출물 A 및 락토바실러스 카세이(Lactobacillus casei)로 발효시킨 발효 상엽 추출물 B를 제조하였다.Fermented leaf extract B was fermented with the leaf extract A and Lactobacillus casei.
또한 이를 쌀뜨물로 발효시킨 노간주나무에 대한 열수추출물(추출물 C), 이를 쌀뜨물로 발효시킨 병조희풀에 대한 열수추출물(추출물 D), 낭아초 열수추출물(추출물 E), 녹두에 대한 열수추출물(추출물 F)를 제조하였다.In addition, hot water extracts (extract C) on juniper trees fermented with rice water, hot water extracts (Extract D) for bottled grasses fermented with rice water, Nyacho hot water extracts (Extract E), and hot water extracts for mung beans ( Extract F) was prepared.
이후 상기 추출물을 하기의 [표 1]에 따라 혼합하여 실시예를 제조하였다.Thereafter, the extract was mixed according to the following [Table 1] to prepare an example.
(단위: 중량부)(Unit: parts by weight)
[실험결과 2: 복합추출물의 [Experimental Result 2: Complex Extract 관능성Sensuality 평가] evaluation]
위 복합추출물 T1 내지 T10을 음료로 제조하고, 성인남녀 20인에 대한 관능성 평가를 수행하였다. 관능성 평가는 향과 맛을 기초로 기호성에 따라 1 내지 10의 지수로 평가하여 평균지수로 구분하였고 그 결과를 [표 2]에 나타내었다.(하기의 지수는 그 숫자가 높을수록 기호성이 높은 것이다)Gastric complex extracts T1 to T10 were prepared as beverages, and sensory evaluation was performed on 20 adult men and women. Sensory evaluation was evaluated by the index of 1 to 10 according to the palatability and taste based on the flavor and taste and divided into the average index and the results are shown in [Table 2]. will be)
(단위: 지수)(Unit: exponent)
상기 표 2를 참조하면, 상엽 추출물은 텁텁한 맛이 있고 기호성 있는 향이 나지 않는다. 발효에 의하는 경우 세포독성이 저감되는 장점이 있으나 향미가 높아지는 것은 아니라는 것을 확인할 수 있다.Referring to Table 2, the upper leaf extract has a rich taste and does not taste palatable. When fermentation has the advantage of reducing cytotoxicity, but it can be seen that the flavor does not increase.
또한 상기 발효된 상엽에 대한 향은 발효된 병조희풀만으로는 제거되지 않았고, 발효된 노간주나무 열수추출물을 함께 사용하는 경우 거의 나지 않는다는 것을 확인할 수 있었다.In addition, the fragrance for the fermented upper leaf was not removed only by fermented bottled grass, it was confirmed that the fermented juniper hot water extract is rarely used together.
또한, 낭아초 및 녹두 추출물을 사용하는 경우 음료의 풍미가 높아지면서 높은 기호성을 가지는 것을 확인할 수 있었다. In addition, it was confirmed that having a high palatability as the flavor of the beverage is increased when using the Nyacho and mung bean extract.
따라서 상기 복합 추출물에 의하는 경우 상엽 추출물을 포함하여 IL-1β에 의한 관절염 예방 또는 개선 효과를 낼 수 있으면서도, 기호성이 높아 보급하기 용이한 식품 또는 기능성 식품으로 제공될 수 있다.Therefore, the complex extract may be provided as a food or functional food that is easy to spread while having high palatability while having an effect of preventing or improving arthritis by IL-1β including the upper leaf extract.
또한, 설명한 본 발명의 상세한 설명에서는 본 발명의 바람직한 실시 예를 참조하여 설명하였지만, 해당 기술 분야의 숙련된 당업자 또는 해당 기술분야에 통상의 지식을 갖는 자라면 후술할 특허청구범위에 기재된 본 발명의 사상 및 기술 영역으로부터 벗어나지 않는 범위 내에서 본 발명을 다양하게 수정 및 변경시킬 수 있음을 이해할 수 있을 것이다. 따라서, 본 발명의 기술적 범위는 명세서의 상세한 설명에 기재된 내용으로 한정되는 것이 아니라 특허청구범위에 의해 정하여져야만 할 것이다.In addition, in the detailed description of the present invention described with reference to a preferred embodiment of the present invention, those skilled in the art or those skilled in the art having ordinary knowledge of the present invention described in the claims to be described later It will be understood that various modifications and variations can be made in the present invention without departing from the spirit and scope of the art. Therefore, the technical scope of the present invention should not be limited to the contents described in the detailed description of the specification but should be defined by the claims.
Claims (8)
쌀뜬물로 발효시킨 노간주나무에 대한 열수 추출물;
쌀뜬물로 발효시킨 병조희풀에 대한 열수추출물;
낭아초 열수 추출물; 및
녹두 열수 추출물을 유효성분으로 포함하는
면역 증강용 식품 조성물.Fermented lettuce extract;
Hot water extract for juniper tree fermented with rice water;
Hot water extracts for bottled grasses fermented with rice floats;
Cannabis Hot Water Extract; And
Contains mung bean hot water extract as an active ingredient
Immunity-enhancing food composition.
상기 발효 상엽 추출물은 물, 탄소수 1 내지 6의 알코올 및 이들의 혼합 용매로 구성되는 군으로부터 선택된 어느 하나를 용매로 하여 추출되는
면역 증강용 식품 조성물.The method of claim 1,
Fermentation The upper leaf extract is extracted by using any one selected from the group consisting of water, alcohols having 1 to 6 carbon atoms and mixed solvents thereof
Immunity-enhancing food composition.
상기 면역 증강용 조성물은 대식세포의 활성을 촉진시키는 것인
면역 증강용 식품 조성물.The method of claim 1,
The immune enhancing composition is to promote the activity of macrophages
Immunity-enhancing food composition.
상기 발효 상엽은 락토바실러스 카제이(Lactobacillus casei), 락토바실러스 람노수스(Lactobacillus rhamnosus), 비피도 박테리윰 비피듐(Bifidobacterium bifidum) 비피더스균, 비피도 박테리움 브레브(Bifidobacterium breve) 비피더스균, 및 락토바실러스 아시도 필루스(Lactobacillus acidophilus)로 이루어진 군에서 선택되는 하나 이상을 접종하여 발효된 것인
면역 증강용 식품 조성물.The method of claim 1,
The fermented upper leaves are Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium bifidum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium breve, and Bifidobacterium. It is fermented by inoculating one or more selected from the group consisting of Lactobacillus acidophilus
Immunity-enhancing food composition.
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| KR20210070546A (en) | 2019-12-05 | 2021-06-15 | 농업회사법인 주식회사 삼원네이처 | A composition for immune enhancement comprising narrow-leaf erecta fig extract |
| WO2024117770A1 (en) * | 2022-12-01 | 2024-06-06 | 한국 한의학 연구원 | Composition including colored rice extract as active ingredient for enhancing immunity |
| WO2024172497A1 (en) * | 2023-02-15 | 2024-08-22 | 주식회사 고바이오랩 | Lacticaseibacillus rhamnosus strain and immunity enhancement uses thereof |
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| KR102223310B1 (en) * | 2019-05-20 | 2021-03-04 | 동의대학교 산학협력단 | Functional feed composition containing oil cake |
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| KR20050021026A (en) | 2001-06-15 | 2005-03-07 | 주식회사 장생도라지 | Pharmaceutical composition for prevention and treatment of disease causing from abnormality of immune system comprising old platycodon extracts |
| KR20120092871A (en) | 2011-02-14 | 2012-08-22 | 충북대학교 산학협력단 | Composition for enhancing immuneresponse comprising extract from umbilicaria esculenta |
| KR20150051624A (en) * | 2013-11-05 | 2015-05-13 | 전북대학교산학협력단 | Mulberry leaves extract with enhanced antioxidant activity and flavonol fermented by lactic acid bacteria and food composition containing the same |
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| KR20210070546A (en) | 2019-12-05 | 2021-06-15 | 농업회사법인 주식회사 삼원네이처 | A composition for immune enhancement comprising narrow-leaf erecta fig extract |
| WO2024117770A1 (en) * | 2022-12-01 | 2024-06-06 | 한국 한의학 연구원 | Composition including colored rice extract as active ingredient for enhancing immunity |
| WO2024172497A1 (en) * | 2023-02-15 | 2024-08-22 | 주식회사 고바이오랩 | Lacticaseibacillus rhamnosus strain and immunity enhancement uses thereof |
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