KR20190019367A - Composition and Method for Stimulating Maturation of Immature Dendritic Cell Comprising Extract of pine tree leaf - Google Patents
Composition and Method for Stimulating Maturation of Immature Dendritic Cell Comprising Extract of pine tree leaf Download PDFInfo
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- KR20190019367A KR20190019367A KR1020170104144A KR20170104144A KR20190019367A KR 20190019367 A KR20190019367 A KR 20190019367A KR 1020170104144 A KR1020170104144 A KR 1020170104144A KR 20170104144 A KR20170104144 A KR 20170104144A KR 20190019367 A KR20190019367 A KR 20190019367A
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- dendritic cells
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- immature dendritic
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Abstract
Description
본 발명은 미성숙수지상세포의 성숙화유도용 조성물에 대한 것으로, 보다 구체적으로는 솔잎추출물을 이용한 미성숙수지상세포의 성숙화유도용 조성물 및 성숙화유도방법에 관한 것이다. The present invention relates to a composition for inducing maturation of immature dendritic cells, and more particularly, to a composition for inducing maturation of immature dendritic cells using a pine leaf extract and a matured induction method.
소나무(Pinus desiflora Siebold et Zuccarini)는 우리 나라의 식물군집 중 가장 광범위하게 분포하고 있는 목본식물로 예로부터 구황작물 뿐 아니라 약용식물로도 널리 이용되어 왔다. 우리나라에서 자생하고 있는 소나무과 중에서 잣나무잎은 민간요법에서는 임질과 매독의 치료약으로 사용되고 있으나 {御影雅辛 외 3명 (1991) } 국내부존자원 중 쉽게 얻을 수 있는 솔잎의 약리작용에 대해서는 완전히 밝혀지지 않고 있다. 한의서와 민간요법에 따르면 솔잎은 간장 질환, 비뇨생식기계질환, 위장질환, 신경계질환, 순환기계질환, 피부질환 등에 효과가 있다고 보고되어있다.{Kang, Y. H. (1995)}. 성분분석결과 솔잎에는 엽록소, 비타민 A와 비타민 K가 함유되어 있고, 그 외에도 단백질, 지방, 인, 철, 효소, 정유(식물성 휘발류, 테르펜계열), 미네랄, 비타민 C가 함유되어 있으며, 체내의 노폐물을 배출시켜 신진대사를 활발하게 하는 성분들이 함유되어 있다 {上原美鈴(1995)}. Pine (Pinus desiflora Siebold et Zuccarini) is the most widely distributed woody plant in the country. It has been widely used as a medicinal plant as well as a wild plant. Among the pine trees native to Korea, the pine tree leaves are used as a remedy for gonorrhea and syphilis in folk remedies (3 御 影 雅 辛 et al. (1991)), but the pharmacological action of pine needles have. According to the Korean consent and folk medicine, pine needle is reported to be effective for liver diseases, genitourinary diseases, gastrointestinal diseases, neurological diseases, circulatory diseases and skin diseases {Kang, Y. H. (1995)}. The composition analysis showed that the pine leaves contained chlorophyll, vitamin A and vitamin K and also contained proteins, fats, phosphorus, enzymes, essential oils (vegetable volatiles, terpene family), minerals and vitamin C, (1995)). In the present study, we have investigated the effects of the antioxidant enzymes on the metabolism.
수지상세포는 1868년 랑겔한스 (Langerhans)에 의해 피부에서 처음 발견되었으며 1973년 칸 (Cohn)과 스테인만(Steinmann)에 의해 면역보조 세포로 기능이 알려지게 되었다. 그리고 1990년대에는 강력한 항원제시세포(professional antigen presenting cells; APC)로 기능이 밝혀지면서 면역유도 및 면역조절에서의 중요한 역할을 함을 알게 되었다. 인체 내의 수지상세포는 전체 순환 백혈구의 단지 약 0.3%를 차지할 뿐이지만 대식세포와는 구별되는 표현형을 지닌 이질적인 집단으로 구성되어 있다. 수지상세포는 비교적 약한 항원제시능력을 지닌 B세포나 대식세포와는 달리 강력한 항원제시세포라는 점에서 차별화된다. 수지상세포는 항원과 접한 적이 없는 원시 T세포(naive T cell)를 자극시킬 수 있는 일차면역반응 (primary immune response) 유도 능력을 지니며, 또한 면역기억을 유도할 수 있는 특성을 갖는 유일한 면역세포이다. 수지상세포가 강력한 면역반응 활성화 기능을 할 수 있는 것은 항원제시세포 (APC, Antigen Presenting Cell)로서 세포 표면에 MHC 분자 (I/II)뿐만 아니라, 보조자극분자 (co-stimulatory molecules) 예컨대 CD 80, CD 86와 부착 분자 (adhesion molecule) 예컨대 ICAM-1들을 고농도로 발현하고 있으며, 여러 가지의 사이토카인 (cytokine, IFN-alpha, IL-12, IL-18 등)을 분비하기 때문인 것으로 알려져 있다. 수지상세포는 세포 표면에 항원제시분자들 (MHC 분자와 보조자극분자들)을 많이 발현하고 있고, IFN-알파, IL-12 등 여러 가지 사이토카인을 분비하기 때문에 항원 특이 세포살해 T 세포의 생성, Th1 세포의 증식 및 활성화를 유도할 수 있다고 알려져 있어 면역요법에 유용하게 적용 가능하다.Dendritic cells were first found in skin by Langerhans in 1868, and in 1973 Cohn and Steinmann became known as immune-assisted cells. In the 1990s, it became known that it functions as a powerful antigen presenting cell (APC) and plays an important role in immune regulation and immune regulation. The dendritic cells in the human body are composed of heterogeneous populations with phenotypes distinct from macrophages, but only about 0.3% of total circulating white blood cells. Dendritic cells differ from B cells or macrophages with relatively weak antigen presentation ability in that they are powerful antigen presenting cells. Dendritic cells are the only immune cells with the ability to induce a primary immune response that can stimulate naive T cells that have never been contacted by the antigen, and are also capable of inducing immune memory . In addition to MHC molecules (I / II) on the cell surface, co-stimulatory molecules such as CD 80, Is known to express CD86 and adhesion molecules such as ICAM-1 at high concentrations and secrete various cytokines (IFN-alpha, IL-12, IL-18, etc.). Dendritic cells express many antigen presenting molecules (MHC molecules and co-stimulatory molecules) on the cell surface, secrete various cytokines such as IFN-alpha and IL-12, Th1 cell proliferation and activation, which is useful for immunotherapy.
따라서, 수지상세포(dendritic cell)는 가장 강력한 항원 전달 세포 (Antigen presenting cells)로서, 생체 내 극히 적은 수로 존재하지만 T 세포 면역을 조절하는 기능이 매우 뛰어나기 때문에 암 또는 병원균의 특이 항원에 대한 면역을 유도하기 위한 임상 연구에서 세포 백신으로서 응용할 수 있다.Therefore, dendritic cells are the most powerful antigen-presenting cells. Although they are present in very small numbers in vivo, they have immune-tolerance to specific antigens of cancer or pathogens because they are highly capable of controlling T-cell immunity. Can be used as a cell vaccine in clinical studies to induce apoptosis.
하지만, 수지상세포를 항암면역치료에 활용하기 위해서는, 체외에서 수지상세포를 분화 제조하는 기술과 T 세포 면역을 효과적으로 유도하기 위한 성숙화 기술이 필수적이다. 체외에서 단핵구로부터 미성숙 수지상세포를 제조하는 방법이나 성숙화 과정은 전 세계적으로 아직 표준화 되어있지 못하다. 특히 분화 제조된 미성숙 수지상세포의 성숙화는 수지상세포를 림프절로 이동시켜 임파절의 원시 T 세포에 항원을 제시하여 Th1 면역반응을 유도할 수 있는 세포로 전환시키는 과정으로, 완전히 성숙한 수지상세포는 미숙한 수지상세포에 비해 세포 표면에 MHC I형 및 II형 분자, 및 T 세포 보조자극인자, 즉 CD80 및 CD86 등을 고농도로 발현한다. 또한, 성숙한 수지상세포는 다량의 사이토카인을 발현하며, 이들 사이토카인은 T 세포 면역반응 유도에 직접적으로 관여한다. 성숙화를 통한 이러한 변화에 의해 수지상세포의 T 세포 면역반응 유도능이 크게 증가하게 된다.However, in order to utilize dendritic cells for anticancer immunotherapy, it is necessary to develop a technique for differentiating dendritic cells from the outside of the body and a maturation technique for effectively inducing T cell immunity. The method of producing immature dendritic cells from mononuclear cells in vitro or the process of maturation is not yet standardized around the world. In particular, the maturation of immature dendritic cells produced by differentiation is a process of transferring dendritic cells to lymph nodes and presenting antigens to lymph node primitive T cells to transform them into cells capable of inducing a Th1 immune response. The fully mature dendritic cells are immature dendritic cells MHC type I and type II molecules, and T cell-assisted stimulating factors, that is, CD80 and CD86, are expressed at higher concentrations on the cell surface than the cells. In addition, mature dendritic cells express large amounts of cytokines, which are directly involved in inducing T cell immune responses. These changes through maturation greatly increase the T cell immune response inducing capacity of dendritic cells.
수지상세포 성숙화 기술로, 단핵구배양유래 배지(MCM: Monocyte Conditioned Media)를 사용하는 기술이 알려져 있다. 상기 MCM은 단핵구를 시험관에 배양하면서 수거한 배지가 성숙화 인자의 공급원으로서 사용된다. 그러나, MCM을 이용하는 방법의 경우, 외부의 위험 신호를 인식하여 단핵구가 분비하는 염증유발 사이토카인(proinflammatory cytokine)의 분비량이 사람마다 변동 폭이 크기 때문에, 결과적으로 동일한 성숙화 조건의 제조공정을 유지하기 힘들 뿐 아니라, MCM 제조를 위하여 다량의 말초혈액단핵세포를 채취하여 사용해야 하는 매우 큰 단점이 있다. 이에, MCM을 대체할 수 있는 방법으로, MCM의 성분 중 중요한 사이토카인들을 조합하여 만든 cytokine cocktail (IL-1β, IL-6, TNF-α, PGE2)을 사용하는 기술이 개발되었다. Cytokine cocktail 을 사용할 경우 MCM의 문제점을 해결할 수 있을 뿐 아니라 기능면에서도 비교 우위를 보이는 수지상세포를 안정적으로 제조할 수 있다. 그러나 이들 성숙화 cytokine cocktail 만으로는 수지상세포의 성숙화가 충분히 유도되지 않는다.As a dendritic cell maturation technique, a technique using a monocyte conditioned medium (MCM) is known. The MCM is used as a source of the maturation factor when the collected mononuclear cells are cultured in a test tube. However, in the case of the method using MCM, since the secretion amount of proinflammatory cytokine secreted by monocytes is recognized by recognizing an external danger signal, it is possible to maintain the manufacturing process of the same mature condition as a result In addition to being difficult, there is a great disadvantage that a large amount of peripheral blood mononuclear cells must be collected and used for MCM production. Therefore, a technique using a cytokine cocktail (IL-1β, IL-6, TNF-α, PGE2), which is a combination of important cytokines among components of MCM, has been developed as a substitute for MCM. Cytokine cocktail not only solves the problems of MCM but also can stably produce dendritic cells with comparative advantage in function. However, these mature cytokine cocktails do not induce the maturation of dendritic cells.
이와 같이 적응면역반응을 일으키는데 있어서는 수지상세포의 성숙화가 필수적으로 요구되지만, 종양미세환경 등에서는 종양세포의 다양한 세포반응으로 인하여 수지상세포의 표현형이 미성숙 상태로 존재하는 경우가 많으며 성숙화반응이 억제되어 있어 항종양면역반응을 일으키는데 어려움을 겪게되어 면역 관용 또는 면역반응이 결여되는 상태로 존재하게 된다. 따라서 종양미세환경과 같은 수지상세포의 면역반응을 저해하는 상태에서 다시 수지상세포의 성숙화를 유도하는 것은 암 면역치료 등 다양한 면역학적 치료방법을 개발하는데 중요한 방법으로 제시될 수 있다.In order to induce an adaptive immune response, dendritic cells must be maturated. However, in the tumor microenvironment, the phenotype of dendritic cells is often immature due to various cellular reactions of tumor cells, and the maturation reaction is suppressed It is difficult to cause an antitumor immune response and thus the immune tolerance or immune response is lacking. Therefore, inducing the maturation of dendritic cells in a state of inhibiting the immune response of dendritic cells such as tumor microenvironment may be suggested as an important method for developing various immunotherapeutic methods such as cancer immunotherapy.
본 발명자들은 솔잎추출물의 새로운 용도를 발견함으로써 본 발명을 완성하였다.The present inventors have found a new use of pine leaf extract to complete the present invention.
따라서, 본 발명의 목적은 솔잎추출물을 유효성분으로 포함하여 미성숙수지상세포의 성숙화를 효과적으로 유도할 수 있는 미성숙수지상세포의 성숙화유도용 조성물, 유도방법 및 건강기능성식품을 제공하는 것이다. Accordingly, an object of the present invention is to provide a composition for inducing maturation of immature dendritic cells, an induction method, and a health functional food, which can effectively induce maturation of immature dendritic cells by containing pine needle extract as an active ingredient.
본 발명의 목적들은 이상에서 언급한 목적들로 제한되지 않으며, 언급되지 않은 또 다른 목적들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.The objects of the present invention are not limited to the above-mentioned objects, and other objects not mentioned can be clearly understood by those skilled in the art from the following description.
상술된 본 발명의 목적을 달성하기 위해, 본 발명은 솔잎추출물을 유효성분으로 포함하는 미성숙수지상세포의 성숙화유도용 조성물을 제공한다.In order to accomplish the object of the present invention described above, the present invention provides a composition for inducing maturation of immature dendritic cells comprising the pine needle extract as an active ingredient.
바람직한 실시예에 있어서, 상기 추출물은 솔잎의 수용성추출물이다.In a preferred embodiment, the extract is an aqueous extract of pine needle.
바람직한 실시예에 있어서, 상기 추출물은 CD80, Cd86, MHC class I, MHC class II의 발현을 증가시킨다.In a preferred embodiment, the extract increases the expression of CD80, Cd86, MHC class I, MHC class II.
바람직한 실시예에 있어서, 상기 추출물은 IL-12p40 및 IL-12/p70 유전자 발현을 증가시킨다.In a preferred embodiment, the extract increases IL-12p40 and IL-12 / p70 gene expression.
또한, 본 발명은 생체로부터 분리된 미성숙 수지상 세포에 솔잎추출물을 처리하는 단계를 포함하는 미성숙 수지상세포의 성숙화 유도방법을 제공한다.The present invention also provides a method for inducing maturation of immature dendritic cells comprising treating the immature dendritic cells isolated from a living body with a pine needle extract.
바람직한 실시예에 있어서, 상기 추출물은 솔잎의 수용성추출물이다. In a preferred embodiment, the extract is an aqueous extract of pine needle.
바람직한 실시예에 있어서, 상기 추출물은 CD80, Cd86, MHC class I, MHC class II의 발현을 증가시킨다. In a preferred embodiment, the extract increases the expression of CD80, Cd86, MHC class I, MHC class II.
바람직한 실시예에 있어서, 상기 추출물은 IL-12p40 및 IL-12/p70 유전자 발현을 증가시킨다.In a preferred embodiment, the extract increases IL-12p40 and IL-12 / p70 gene expression.
또한, 본 발명은 솔잎추출물을 유효성분으로 포함하는 미성숙수지상세포의 성숙화유도용 건강기능성식품을 제공한다.The present invention also provides a health functional food for inducing maturation of immature dendritic cells comprising the pine needle extract as an active ingredient.
바람직한 실시예에 있어서, 상기 추출물은 솔잎의 수용성추출물이다. In a preferred embodiment, the extract is an aqueous extract of pine needle.
바람직한 실시예에 있어서, 분말, 과립, 정제, 캡슐 및 음료 중 어느 하나의 제형을 갖는다.In a preferred embodiment, it has a formulation of either powder, granules, tablets, capsules or beverages.
본 발명의 솔잎추출물은 세포독성을 가지지 않는 농도에서 수지상세포의 세포표면분자인 CD80, CD86, MHC class I, MHC class II의 발현을 증가시키는 효능을 가지므로, 이를 이용하여 다양한 항암치료시 면역반응을 증강시키는 특성을 갖는 미성숙수지상세포 성숙화유도용 조성물, 그 유도방법 및 기능성 식품소재로 유용하게 이용될 수 있다.The pine needle extract of the present invention has an effect of increasing the expression of CD80, CD86, MHC class I, and MHC class II, which are cell surface molecules of dendritic cells, at a concentration not having cytotoxicity, , A method for inducing the maturation of the immature dendritic cell, and a functional food material.
도 1은 본 발명의 실시예에 따른 솔잎추출물이 처리된 마우스 미성숙수지상세포의 세포독성측정 결과 그래프이다.
도 2는 본 발명의 실시예에 따른 솔잎추출물이 처리된 미성숙수지상세포의 마커인 CD11c와 성숙화 마커인 CD80, CD86, MHC class I, MHC class II를 함께 염색하여 측정한 결과그래프다.
도 3a 및 도 3b는 본 발명의 실시예에 따른 솔잎추출물이 수지상세포의 사이토카인 발현에 미치는 효과를 나타낸 결과그래프이다.
도 4는 본 발명의 실시예에 따른 솔잎추출물이 수지상세포의 항원포식능에 미치는 효과를 나타낸 결과그래프이다.1 is a graph showing cytotoxicity measurement results of mouse immature dendritic cells treated with pine needle extract according to an embodiment of the present invention.
FIG. 2 is a graph showing staining of CD11c, a marker of immature dendritic cells treated with pine needle extract according to an embodiment of the present invention, and CD80, CD86, MHC class I, and MHC class II as maturation markers.
FIGS. 3A and 3B are graphs showing the effect of the pine needle extract according to the present invention on the expression of cytokines of dendritic cells. FIG.
FIG. 4 is a graph showing the effect of the pine needle extract according to the present invention on the antigen-presenting ability of dendritic cells.
본 발명에서 사용되는 용어는 가능한 현재 널리 사용되는 일반적인 용어를 선택하였으나, 특정한 경우는 출원인이 임의로 선정한 용어도 있는데 이 경우에는 단순한 용어의 명칭이 아닌 발명의 상세한 설명 부분에 기재되거나 사용된 의미를 고려하여 그 의미가 파악되어야 할 것이다.Although the terms used in the present invention have been selected as general terms that are widely used at present, there are some terms selected arbitrarily by the applicant in a specific case. In this case, the meaning described or used in the detailed description part of the invention The meaning must be grasped.
이하, 첨부한 도면 및 바람직한 실시예들을 참조하여 본 발명의 기술적 구성을 상세하게 설명한다.Hereinafter, the technical structure of the present invention will be described in detail with reference to the accompanying drawings and preferred embodiments.
그러나, 본 발명은 여기서 설명되는 실시예에 한정되지 않고 다른 형태로 구체화 될 수도 있다. 명세서 전체에 걸쳐 본 발명을 설명하기 위해 사용되는 동일한 참조번호는 동일한 구성요소를 나타낸다.However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Like reference numerals used to describe the present invention throughout the specification denote like elements.
본 발명의 기술적 특징은 솔잎추출물이 수지상세포의 세포표면분자인 CD80, CD86, MHC class I, MHC class II의 발현을 증가시키는 효능을 갖는다는 점에서 착안된 것으로 솔잎추출물의 새로운 용도인 미성숙수지상세포의 성숙화유도활성을 이용하여 다양한 항암치료시 면역반응을 증강시키는 특성을 갖는 미성숙수지상세포 성숙화유도용 조성물, 그 유도방법 및 건강기능성식품을 제공하는 것이다. The technical feature of the present invention is that the pine needle extract has an effect of increasing the expression of CD80, CD86, MHC class I, and MHC class II, the cell surface molecules of dendritic cells. A method for inducing immature dendritic cell maturation, a method for inducing immature dendritic cell maturation, and a health functional food.
즉, 수지상세포는 골수에서 유래된 전구체 세포에서 분화를 하여 높은 항원포식능을 가지는 미성숙한 수지상세포의 상태로 존재를 하다가 외부의 자극 및 항원을 인식하게 되면 미성숙 수지상세포의 성숙화가 진행되어 세포 표면 분자의 발현이 증가되고 T 림프구 밀집지역으로 이동하여 적응면역반응을 개시하게 되므로, 적응면역반응을 일으키는데 있어서는 수지상세포의 성숙화가 필수적으로 요구되지만, 종양미세환경 등에서는 종양세포의 다양한 세포반응으로 인하여 수지상세포의 표현형이 미성숙 상태로 존재하는 경우가 많으며 성숙화반응이 억제되어 있어 항종양면역반응을 일으키는데 어려움이 있으므로, 종양미세환경과 같은 수지상세포의 면역반응을 저해하는 상태에서 다시 수지상세포의 성숙화를 유도하는 방법을 개발하는 것은 암 면역치료 등 다양한 면역학적 치료방법을 개발하는데 중요하기 때문이다.In other words, dendritic cells differentiate from precursor cells derived from bone marrow and are present in the state of immature dendritic cells having high antigen-repelling ability. When external stimuli and antigens are recognized, immature dendritic cells are matured, In order to induce adaptive immune response, maturation of dendritic cells is essential to increase the expression of molecules and to initiate adaptive immune response by moving to the T lymphocyte dense region. However, due to various cell reactions of tumor cells in the tumor microenvironment, Since the dendritic cell phenotype is often immature and inhibits the maturation reaction, it is difficult to induce antitumor immune response. Therefore, the dendritic cell maturation is further inhibited in the dendritic cell immune response such as tumor microenvironment Developing a method to induce cancer Such treatment station because it is important to develop a variety of immunological treatment.
따라서, 본 발명의 미성숙수지상세포의 성숙화유도용 조성물은 솔잎추출물을 유효성분으로 포함한다. Accordingly, the composition for inducing maturation of immature dendritic cells of the present invention contains pine needle extract as an active ingredient.
상술된 바와 같이 솔잎추출물이 세포독성을 가지지 않는 농도에서 수지상세포의 성숙화 마커로 알려진 CD80, Cd86, MHC class I, MHC class II의 발현을 농도의존적으로 증가시키기 때문이다. 그 결과 본 발명의 약학조성물을 경구 또는 비경구적으로 투여하여 인간을 포함한 포유류의 항암치료 등 다양한 면역학적 치료시 면역반응을 증강시키기 위해 사용될 수 있다. Cd86, MHC class I, and MHC class II, which are known as maturation markers of dendritic cells, at a concentration that does not have cytotoxicity, as described above. As a result, the pharmaceutical composition of the present invention can be administered orally or parenterally to enhance the immune response in a variety of immunological treatments such as chemotherapy of mammals including humans.
여기서, 솔잎 추출물은 천연물로부터 추출물을 추출하는 당업계에 공지된 통상적인 방법에 따라, 즉, 통상적인 온도, 압력의 조건 하에서 통상적인 추출 용매를 사용하여 분리할 수 있다. 추출 용매로는 추출공정에서 일반적으로 사용할 수 있는 용매를 사용할 수 있는데, 물, 탄소수 1-4개의 무수 또는 함수 저급 알코올, 아세톤, 에틸아세테이트, 부틸아세테이트, 디클로로메탄 및 1,3-부틸렌 글리콜로 구성된 군으로부터 선택되는 용매를 사용하여 추출될 수 있다. 일 구현예로서 솔잎 추출물은 솔잎의 수용성추출물, 특히 실시예에 기재된 바와 같이 에탄올추출물일 수 있다.Herein, the pine leaf extract can be separated by using conventional extracting methods in accordance with a conventional method known in the art for extracting an extract from a natural product, that is, under ordinary temperature and pressure conditions. As the extraction solvent, there can be used a solvent generally used in the extraction process, such as water, an anhydrous or a lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, butyl acetate, dichloromethane and 1,3-butylene glycol And can be extracted using a solvent selected from the group consisting of. In one embodiment, the pine leaf extract may be an aqueous extract of pine needle, particularly an ethanol extract as described in the Examples.
본 발명의 약학조성물은 솔잎추출물을 단독으로 포함할 수 있으며, 이외 제형, 사용방법 및 사용목적에 따라 약리학적으로 허용가능한 담체 또는 부형제를 더 포함할 수 있다. 혼합물로 제공되는 경우, 유효성분인 솔잎추출물은 약학조성물에 0.001 내지 99 중량%로 포함될 수 있다. The pharmaceutical composition of the present invention may contain the pine needle extract alone, and may further include a pharmacologically acceptable carrier or excipient depending on the formulation, method of use, and purpose of use. When provided as a mixture, the active ingredient pine leaf extract may be included in the pharmaceutical composition in an amount of 0.001 to 99% by weight.
담체 또는 부형제로는 물, 덱스트린, 칼슘카보네이드, 락토스, 프로필렌글리콜, 리퀴드 파라핀, 생리식염수, 덱스트로스, 수크로즈, 솔비톨, 만니톨, 자이리톨, 에리스리톨, 말티톨, 전분, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 폴리비닐피롤리돈, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유가 있으며, 이들은 1종이상 사용될 수 있으나, 이에 한정되는 것은 아니며 통상의 담체 및 부형제는 모두 사용가능하다. Examples of the carrier or excipient include water, dextrin, calcium carbonate, lactose, propylene glycol, liquid paraffin, physiological saline, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. These may be used alone or in combination, but not limited thereto, All excipients are available.
또한 본 발명의 약학조성물을 약제화하는 경우, 통상의 충진제, 증량제, 결합제, 붕해제, 계면활성제, 항응집제, 윤활제, 습윤제, 향료, 유화제 또는 방부제 등을 더 포함할 수 있다. 본 발명의 약학조성물은 경구 또는 비경구로 투여할 수 있고, 비경구로 투여되는 경우, 피부에 국소적으로 도포, 정맥내 주입, 피하 주입, 근육 주입, 복강 주입, 경피 투여 등으로 투여할 수 있다. When the pharmaceutical composition of the present invention is made into a pharmaceutical composition, it may further contain conventional fillers, extenders, binders, disintegrators, surfactants, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives. The pharmaceutical composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be administered by local application to the skin, intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration or the like.
개별 투약 형태에서 이의 유효성분인 솔잎추출물의 함량은 1회에 투여되는 양으로, 예컨대 통상 1일 투여량의 전부, 1/2, 1/3 또는 1/4배일 수 있다. 본 발명의 약학조성물의 투여량은 환자의 연령, 성별, 상태, 체내에서 활성 성분의 흡수도, 불활성율 및 병용되는 약물을 고려하여 결정하는 것이 좋으며, 예컨대 1회 유효성분을 기준으로 하였을 때 60 ㎍/ml 이하의 농도로, 1일 1 내지 5회 투여할 수 있다. 한편, 본 발명의 약제학적 조성물의 경구 투여량은 바람직하게는 1일 당 0.00001-100 mg/kg(체중)일 수 있다. 본 발명의 조성물에 포함되는 유효성분의 농도는 치료 목적, 환자의 상태, 필요기간 등을 고려하여 결정할 수 있으며 특정 범위의 농도로 한정되지 않는다.The content of the pine needle extract, which is an active ingredient thereof in the individual dosage form, may be an amount administered in one administration, for example, the full daily dose, 1/2, 1/3 or 1/4 times the usual daily dose. The dosage of the pharmaceutical composition of the present invention is preferably determined in consideration of the age, sex, condition, the degree of absorption of the active ingredient in the patient, the inactivation rate of the active ingredient, and the drug to be used together. For example, Gt; mg / ml, < / RTI > 1 to 5 times per day. On the other hand, the oral dosage amount of the pharmaceutical composition of the present invention may preferably be 0.00001-100 mg / kg (body weight) per day. The concentration of the active ingredient contained in the composition of the present invention can be determined in consideration of the purpose of treatment, the condition of the patient, the period of time required, and the like, and is not limited to a specific range of concentration.
다음으로, 본 발명의 미성숙수지상세포의 성숙화유도용 건강기능성식품은 상술된 약학조성물에 포함되는 솔잎추출물을 유효성분으로 포함함으로써 미성숙수지상세포의 성숙화를 유도하여 수지상세포의 세포표면분자인 CD80, CD86, MHC class I, MHC class II의 발현을 증가시키는 활성을 갖게 되므로 항암치료 등 다양한 면역학적 치료시 면역반응을 증강시키기 위한 목적으로 사용될 수 있다.Next, the health-functional food for inducing the maturation of immature dendritic cells of the present invention comprises the pine needle extract contained in the above-mentioned pharmaceutical composition as an active ingredient, thereby inducing the maturation of immature dendritic cells, and the cell surface molecules CD80, CD86 , MHC class I, and MHC class II, and thus can be used for enhancing the immune response in various immunotherapeutic treatments such as chemotherapy.
본 발명에서 '건강기능성식품'이란 영양소를 한 가지 또는 그 이상 함유하고 있는 천연물 또는 가공품을 의미하며, 바람직하게는 식품에 물리적, 생화학적, 생물공학적 수법 등을 이용하여 해당 식품의 기능을 특정 목적에 작용, 발현하도록 부가가치를 부여한 식품군이나 식품 조성이 갖는 생체방어리듬조절, 질병방지와 회복 등에 관한 체조절기능을 생체에 대하여 충분히 발현하도록 설계하여 가공한 식품을 의미한다. 건강기능성식품에는 식품학적으로 허용 가능한 식품 보조 첨가제를 포함할 수 있으며, 건강기능성 식품의 제조에 통상적으로 사용되는 적절한 담체, 부형제 및 희석제를 더욱 포함할 수 있다.In the present invention, the term 'health functional food' means a natural product or a processed product containing one or more nutrients. Preferably, the function of the food is determined by physical, biochemical, biotechnological, Means a food group that has been imparted with added value to function and express, and a food which is designed and manufactured so that the body control function related to regulation of bio-defense rhythm of food composition, prevention and recovery of disease, etc. is sufficiently expressed in living body. The health functional food may include a pharmaceutically acceptable food supplementary additive and may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of health functional foods.
본 발명의 솔잎추출물을 첨가할 수 있는 건강 기능성 식품으로는 예를 들어, 각종 식품류, 음료, 껌, 차, 비타민 복합제 등이 있다. 추가로, 특수영양식품(예, 조제유류, 영,유아식 등), 건강보조식품, 과자류(예, 스넥류), 유가공품(예, 발효유, 치즈 등), 기타 가공식품, 음료(예, 과실,채소류 음료, 두유류, 발효음료류 등) 등을 포함하나 이에 한정되지 않는다. 상술된 식품, 음료 또는 식품첨가제는 통상의 제조방법으로 제조될 수 있다.The health functional food to which the pine needle extract of the present invention can be added includes, for example, various foods, beverages, gums, tea, and vitamin complex. In addition, it is also possible to use nutritional products such as special nutritious foods (eg crude oil, milk, baby food), health supplements, confectionery (eg snacks), dairy products (eg fermented milk, Beverages, two-oil, fermented beverages, etc.), but are not limited thereto. The food, beverage or food additives described above can be produced by a conventional production method.
본 발명의 건강 기능성 식품은 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, 보호성콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 물, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 이러한 성분을 독립적으로 또는 조합하여 사용할 수 있다. The health functional food of the present invention can be used as a nutritional supplement, a vitamin, a mineral (electrolyte), a flavoring agent such as a synthetic flavor agent and a natural flavor agent, a colorant and an aging agent (cheese, chocolate etc.), a pectic acid and its salt, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, water, carbonating agents used in carbonated beverages and the like. These components can be used independently or in combination.
이와 같이 본 발명의 건강기능성식품은 상술된 바와 같이 다양한 제형을 갖는데, 특히 분말, 과립, 정제, 캡슐 및 음료 중 어느 하나의 제형을 가질 수 있다.Thus, the health functional food of the present invention has various formulations as described above, and may have any one of powder, granule, tablet, capsule and beverage.
일 구현예로서, 본 발명의 건강기능성식품을 음료로 구현하는 경우 필수 성분으로서 본 발명의 솔잎추출물을 함유하는 외에는 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 천연 탄수화물의 예는 모노사카라이드, 예를 들어 포도당, 과당 등 디사카라이드, 예를 들어 말토스, 수크로스 등 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상기한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. In one embodiment, when the health functional food of the present invention is implemented as a beverage, there are no particular restrictions on other components other than those containing the pine needle extract of the present invention as an essential ingredient, and various flavors or natural carbohydrates, And may be contained as an additional component. Examples of natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrins, cyclodextrins and the like, and xylitol, Sorbitol, and erythritol. Natural flavors (tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above .
또한, 미성숙수지상세포의 성숙화를 유도하기 위한 것을 목적으로 하는 건강기능성식품에서도 유효성분인 솔잎추출물의 함량은 60 ㎍/ml 이하의 농도로 포함될 수 있을 것이다. 경우에 따라서는 1일 당 0.00001-100 mg/kg(체중)일 수 있다. 본 발명의 기능성식품에 포함되는 유효성분의 농도는 복용목적, 복용자의 상태, 필요기간 등을 고려하여 결정할 수 있으며 특정 범위의 농도로 한정되지 않는다.In addition, in the health functional food intended to induce maturation of immature dendritic cells, the content of pine needle extract as an active ingredient may be contained at a concentration of 60 ㎍ / ml or less. In some cases, it may be 0.00001-100 mg / kg (body weight) per day. The concentration of the active ingredient contained in the functional food of the present invention can be determined in consideration of the purpose of taking, the condition of the user, the period of time required, etc., and is not limited to a specific range of concentration.
실시예 1Example 1
솔잎은 대한민국의 전라남도 고성 해발 350미터 이상 고산지대에 서식하는 적송에서 채취를 하였으며, 증류수를 이용하여 3회 세척 후 탈수과정, 그 다음 서늘한 그늘에서 자연건조 하여 원료를 확보하였다. 이렇게 확보한 원료를 90℃ 건조기에서 24시간 동안 건조하여 수분을 제거한 후, 믹서기를 이용하여 가루 상태로 만들었다. 솔잎 가루 100 g을 80% 에탄올 900 ml에 넣고 교반하며 2일째에 3MM 필터용지에 걸러서 솔잎 추출물을 수거하였다. 위 방법을 3번 반복하여 총 6일간 솔잎을 추출한 후 획득한 솔잎 추출물은 감압농축기를 이용하여 농축한 후 동결건조기를 이용하여 용매를 모두 제거하고 가루 상태의 솔잎 추출물을 획득하였다. 획득한 솔잎 추출물을 멸균된 3차 증류수에 20㎍/ml 농도로 녹여 약학조성물1(20 ㎍/ml)을 제조하였다. The pine leaves were collected from the shrimp inhabited in the high mountains of more than 350 meters from the Goseong Sea, Jeollanam - do, Republic of Korea. The pine leaves were washed three times with distilled water, dehydrated, and then dried naturally in the cool shade to obtain the raw materials. The thus obtained raw materials were dried in a drier at 90 캜 for 24 hours to remove moisture, and then powdered using a blender. 100 g of pine leaf powder was added to 900 ml of 80% ethanol and stirred. On the second day, the pine leaf extract was collected by filtering on 3MM filter paper. After repeating the above method 3 times for 6 days, the pine leaf extract was concentrated by using a vacuum concentrator, and then the solvent was removed using a freeze dryer to obtain a pine needle extract powder. The obtained pine leaf extract was dissolved in sterilized tertiary distilled water at a concentration of 20 μg / ml to prepare pharmaceutical composition 1 (20 μg / ml).
실시예 2Example 2
솔잎추출물의 농도를 40㎍/ml 로 한 것을 제외하면 실시예1과 동일한 방법으로 약학조성물2(40 ㎍/ml)를 제조하였다.Pharmaceutical composition 2 (40 占 퐂 / ml) was prepared in the same manner as in Example 1, except that the concentration of pine leaf extract was changed to 40 占 퐂 / ml.
실시예 3Example 3
솔잎추출물의 농도를 60 ㎍/ml 로 한 것을 제외하면 실시예1과 동일한 방법으로 약학조성물3(60 ㎍/ml)을 제조하였다.Pharmaceutical composition 3 (60 ㎍ / ml) was prepared in the same manner as in Example 1, except that the concentration of pine leaf extract was changed to 60 ㎍ / ml.
실험예 1Experimental Example 1
1. 세포배양1. Cell culture
재조합 마우스 GM-CSF는 R & D Systems (Minneapolis, MN, USA)에서 구입 하였다. Sigma-Aldrich (St. Louis, MO, USA)로부터 Dextran-FITC (분자량 40,000) 및 LPS (Escherichia li 055 : B5 유래)를 구입하였다. 골수 유래 마우스 수지상세포를 다음과 같이 배양하였다. Recombinant mouse GM-CSF was purchased from R & D Systems (Minneapolis, MN, USA). Dextran-FITC (molecular weight 40,000) and LPS (from Escherichia li 055: B5) were purchased from Sigma-Aldrich (St. Louis, Mo., USA). Bone marrow-derived mouse dendritic cells were cultured as follows.
골수를 C57BL / 6수컷 쥐에서 대퇴부의 경골을 발골 한 뒤 흘려서 염화 암모늄으로 적혈구를 고갈시켰다. 열로 보체를 제거한 소태아 혈청(10 % FBS), 2 mM L-glutamine, 100 U / ml penicillin로 보충 된 RPMI1640 (Welgene, KOREA)에 6 웰 배양 판 (2*106/3ml세포, 10 mM HEPES (pH 7.4), 20 ng / ml, rmGM-CSF로37 ℃, 5 % CO2에서 처리 하였다. 배양 2 일째, 4일째 부유 세포를 부드럽게 제거하고 새로운 배지를 첨가 하였다. 배양 6 일째, 비 부착 성 세포 및 부착하는 증식 수지상세포 응집체를 분석하기 위해 수확하거나 일부 실험에서 60 mm 접시 (106/5ml세포/ dish)에 배양하였다. 6 일째, 비 부착 성 세포의 80 % 이상이 CD11c를 발현 하였다. The bone marrow was shed in the tibia of the thigh in C57BL / 6 male rats and depleted of red blood cells with ammonium chloride. Fetal bovine serum (10% FBS) to remove the heat of complement, 2 mM L-glutamine, 6 wells in a RPMI1640 (Welgene, KOREA) supplemented with 100 U / ml penicillin culture plates (2 * 10 6 / 3ml cells, 10 mM HEPES (pH 7.4), 20 ng / ml, rmGM-CSF at 37 ° C and 5% CO2. On the second day of culture, the floating cells were gently removed and the new medium was added on the fourth day. and attaching proliferation cultured dendritic cells harvested from 60 mm plates or some experiments to analyze the aggregate (10 6 / 5ml cells / dish) to. 80% or more of 6 days, non-adherent cells were expressing CD11c.
이와 같이 6일째에 비-부착성 수지상세포 및 약하게 부착된 수지상세포를 미성숙 수지상세포로 이용하였다.On day 6, non-adherent dendritic cells and weakly adherent dendritic cells were used as immature dendritic cells.
2. 세포독성능 평가2. Cytotoxic performance evaluation
상술된 바와 같이 골수 유래 전구체로부터 GM-CSF를 사용하여 미성숙 수지상세포를 유도한 후, 6일째에 실시예1 내지 3에서 얻어진 약학조성물1 내지 3(20, 40, 60 ㎍/ml)을 처리한 후 24시간 동안 배양하였다. 24시간 후, 배양된 수지상세포는 Annexin/PI staining 을 통하여 세포독성을 판별하였고, 그 결과를 도 1에 나타내었다. 데이터는 4번의 개별반복 실험 결과를 대표한다.GM-CSF was used to induce immature dendritic cells from bone marrow-derived precursors as described above, followed by treatment with the pharmaceutical compositions 1 to 3 (20, 40, and 60 占 퐂 / ml) obtained in Examples 1 to 3 on the 6th day Lt; / RTI > for 24 hours. After 24 hours, the cultured dendritic cells were subjected to Annexin / PI staining to determine cytotoxicity, and the results are shown in Fig. The data represent the results of four individual repeat experiments.
도 1에 도시된 바와 같이, 솔잎추출물의 농도가 60 ㎍/ml인 약학조성물3의 경우에도 수지상세포에 대하여 세포독성을 나타내지 않는 것을 확인하였다. As shown in Fig. 1, it was confirmed that the pharmaceutical composition 3 having a pine needle extract concentration of 60 占 퐂 / ml did not show cytotoxicity on dendritic cells.
실험예 2Experimental Example 2
실시예1 내지 3에서 얻어진 솔잎추출물을 유효성분으로 포함하는 약학조성물1 내지 3에 의해 수지상세포의 표현형이 달라지는지 여부를 확인하기 위하여, 하기와 같이 약학조성물 1 내지 3(20, 40, 60 ㎍/ml)을 처리한 후 수지상세포의 표현형을 분석하고 그 결과를 도 2에 나타내었다.In order to confirm whether the phenotype of dendritic cells was differentiated by the pharmaceutical compositions 1 to 3 containing the pine needle extract obtained in Examples 1 to 3 as an active ingredient, the pharmaceutical compositions 1 to 3 (20, 40, 60 μg / ml), the phenotype of dendritic cells was analyzed, and the results are shown in Fig.
수지상세포의 표현형은 세포표면분자를 유세포 분석방법을 이용하여 분석하였다. 6일째에 약학조성물1 내지 3 및 LPS (양성대조군)을 처리한 세포를 24시간 배양 후 파이펫팅을 하여 회수한다. 회수된 세포는 차가운 PBS를 이용하여 2회 세척을 해준다. 세척된 세포에 FITC가 부착된 항-CD11c항체(수지상세포의 마커)와 PE가 부착된 항-CD80, CD86, MHC class I, MHC class II 항체(성숙화마커)를 조합하여 처리한 후, 4℃ 냉장고에서 30분간 반응시킨다. 염색된 세포는 다시 차가운 PBS를 이용하여 2회 세척한 뒤, 4% paraformaldehyde 이용하여 고정하였다. 고정된 세포는 FC500 (Beckman coulter)기기를 이용하여 측정한 후, kaluza software(Beckman coulter)를 이용하여 분석하였다. 데이터는 4번의 개별반복 실험 결과를 대표한다.The phenotype of dendritic cells was analyzed by flow cytometry. On day 6, the cells treated with the pharmaceutical compositions 1 to 3 and LPS (positive control) were cultured for 24 hours and then recovered by pipetting. The recovered cells are washed twice with cold PBS. The washed cells were treated with a combination of anti-CD11c antibody (dendritic cell marker) with FITC attached and anti-CD80, CD86, MHC class I, and MHC class II antibody (maturation marker) React in the refrigerator for 30 minutes. The stained cells were washed twice with cold PBS and fixed with 4% paraformaldehyde. Fixed cells were analyzed using a FC500 (Beckman coulter) instrument and analyzed using the Kaluza software (Beckman coulter). The data represent the results of four individual repeat experiments.
도 2에 도시된 바와 같이 약학조성물1 내지 3(20, 40, 60 ㎍/ml)은 수지상세포의 성숙화 마커로 알려진 공동 자극 분자 B7-1 (CD80), B7-2 (CD86) 및 MHC 클래스 I 및 II의 단백질 발현 수준을 결정하였는데, 다양한 농도를 가진 솔잎추출물이 CD11c + 수지상세포(DC)에서 CD80, Cd86, MHC class I, MHC class II의 발현을 농도의존적으로 증가시킴을 확인할 수 있다. 이러한 결과는 약학조성물1 내지 3에 포함된 솔잎 추출물이 수지상세포의 세포표현형을 성숙화 상태로 유도한다는 것을 나타낸다.As shown in Fig. 2, the pharmaceutical compositions 1 to 3 (20, 40, 60 쨉 g / ml) contain co-stimulatory molecules B7-1 (CD80), B7-2 (CD86) and MHC class I And II were determined. The pine needle extracts with various concentrations showed a concentration-dependent increase in CD80, Cd86, MHC class I, and MHC class II expression in CD11c + dendritic cells (DC). These results indicate that the pine leaf extract contained in the pharmaceutical compositions 1 to 3 induces the cell phenotype of dendritic cells to a mature state.
실험예 3Experimental Example 3
솔잎추출물을 유효성분으로 포함하는 약학조성물이 수지상세포의 기능적 성숙화에 미치는 영향을 확인하기 위하여 다음과 같이 실험하였다. 미성숙 수지상세포는 LPS 존재 또는 부재 하에서 약학조성물 3(60㎍/ml)에 의해 24시간 동안 자극되었다. 수지상 세포의 표면분자인 CD11c와 세포의 IL-12와 IL-10의 발현야을 확인하기 위하여 더블염색법으로 생산량을 확인하고, 그 결과를 도 3a 및 도 3b에 나타내었다. 이 때, 본 발명의 약학조성물 3(60㎍/ml)이 처리된 수지상세포의 염색은 FITC로 라벨 된 CD11c로 염색을 진행하고, PE 표지 된 항체 마우스 IL-12p40/p70, IL-10 세포 내의 염색을 진행하였다.In order to confirm the effect of the pharmaceutical composition containing the pine needle extract as an active ingredient on the functional maturation of dendritic cells, the following experiment was conducted. Immature dendritic cells were stimulated with pharmaceutical composition 3 (60 [mu] g / ml) for 24 hours in the presence or absence of LPS. To confirm the expression level of CD11c, a surface molecule of dendritic cells, and IL-12 and IL-10 in cells, the production amount was confirmed by double staining, and the results are shown in FIGS. 3A and 3B. At this time, the dendritic cells treated with the pharmaceutical composition 3 (60 μg / ml) of the present invention were stained with FITC labeled CD11c, and stained with PE-labeled antibody mouse IL-12p40 / p70, And stained.
이미 알려진 바와 같이 수지상 세포, 대식세포, 단핵세포는 IL-12와 같은 전 염증성 분자의 원천으로 기능한다는 가설이 세워졌으므로, 본 발명의 약학조성물3이 처리된 수지상세포(DC)가 전 염증성 사이토카인을 생성하는 능력을 조사하였다. 즉, IL-12 발현은 수지상 세포의 활성 및 T 헬퍼 타입 1 (Th1) 면역 반응의 특이적 마커로 확인되었으며, 수지상 세포 성숙에 중요한 마커로 간주되어 Th1 우성 보조제를 선택하는 데 사용될 수 있기 때문이다. It has been hypothesized that dendritic cells, macrophages, and mononuclear cells function as a source of proinflammatory molecules such as IL-12 as already known. Therefore, dendritic cells (DCs) treated with the pharmaceutical composition 3 of the present invention are proinflammatory cytokines Was investigated. That is, IL-12 expression has been identified as a specific marker of dendritic cell activity and T helper type 1 (Th1) immune response, and may be considered as an important marker for dendritic cell maturation and may be used to select Thl dominant adjuvant .
따라서, 본 발명의 약학조성물이 처리된 수지상세포에서 IL-12p40/70 및IL-10 활성 생산량을 확인한 결과, 도 3a 및 도 3b에 나타난 바와 같이, LPS와 본 발명의 약학조성물3이 처리된 수지상 세포는 LPS가 단독으로 처리된 수지상 세포와 비교하였을 때, LPS와 본 발명의 약학조성물3이 같이 처리된 수지상 세포에서의 IL-12p40/p70 발현이 높게 나타남을 확인 할 수 있었으며, 반면에 IL-10의 발현은 변화가 나타나지 않았다 이러한 결과는 본 발명의 약학조성물의 유효성분인 솔잎 추출물이 수지상세포의 기능적 성숙화를 유도한다는 것을 보여준다.Therefore, as shown in FIGS. 3A and 3B, when LPS and the pharmaceutical composition 3 of the present invention were treated with the dendritic cells of the present invention, as shown in FIGS. 3A and 3B, IL-12p40 / 70 and IL- The cells were found to show high expression of IL-12p40 / p70 in dendritic cells treated with LPS and the pharmaceutical composition 3 of the present invention, compared to dendritic cells treated with LPS alone, while IL- 10 expression did not change. These results show that the pine needle extract, which is an active ingredient of the pharmaceutical composition of the present invention, induces functional maturation of dendritic cells.
실험예 4Experimental Example 4
미성숙 수지상세포는 항원을 포식하기 위하여 엔도사이토시스(endocytosis)의 기능이 높게 나타나지만 성숙된 수지상세포에서는 이러한 항원포식능이 감소되어 나타나는 것이 특징이다. 수지상세포의 항원포식능에 솔잎추출물이 미치는 영향을 확인하기 위하여 다음과 같이 배양 6일째의 수지상세포에 약학조성물 3(60 ㎍/ml)과 LPS(양성대조군)을 처리한 후 FITC-dextran으로 처리하고 수지상세포 마커인 CD11c를 염색하여 수지상세포가 포식한 FITC-dextran의 양을 측정했고 그 결과를 도 4에 나타내었다. Immature dendritic cells are characterized by high endocytosis function to predispose to the antigen, but in the matured dendritic cells, the ability to suppress the antigen is reduced. After treatment a pharmaceutical composition 3 (60 ㎍ / ml) and LPS (positive control) to the dendritic cells of the culture on day 6 as follows to determine the effect of antigen predation neunge pine needle extract of dendritic cells on treatment with FITC-dextran coming And the dendritic cell marker CD11c was stained to measure the amount of FITC-dextran predated by dendritic cells. The results are shown in FIG.
수지상세포의 항원포식능을 분석하기 위해 미성숙 수지상세포를 4℃와 37℃에서 1 mg/ml의 FITC-dextran(sigma)를 처리하여 40분간 반응시켰다. 반응이 끝난 세포는 4% paraformaldehyde를 이용하여 고정을 하고 고정된 세포는 PE가 결합된 항-CD11c항체로 염색하였다. 염색된 세포는 유세포 분석기로 측정한 후 kaluza software를 이용하여 분석하였다. 실험 결과는 4번은 개별반복 실험을 대표한다.Immunoreactive dendritic cells were treated with 1 mg / ml FITC-dextran (Sigma) at 4 ℃ and 37 ℃ for 40 min. The cells were fixed with 4% paraformaldehyde and the fixed cells were stained with PE-conjugated anti-CD11c antibody. The stained cells were analyzed by flow cytometry and analyzed with kaluza software. The results of the experiment represent the individual repeat experiments.
도 4에 도시된 바와 같이 약학조성물 3(60 ㎍/ml)을 처리한 수지상세포에서 항원포식능이 낮아졌음을 확인하였고 양성대조군인 LPS를 처리한 군에서도 항원포식능이 감소하였음을 확인하였다. Dextran과 수지상세포의 비특이적 결합을 배제하기 위하여 4℃에서 동일한 실험을 시행하였고, 낮은 온도에서 수지상세포는 dextran에 대하여 반응하지 않음을 확인하였다. 그 결과, 수지상세포의 엔도사이토시스(endocytosis)는 항원포식능의 활성화 반응에 의한 것을 알 수 있었다. 이러한 결과는 솔잎 추출물을 유효성분으로 포함하는 본 발명의 약학조성물이 수지상세포의 성숙화를 유도한다는 것을 나타냄을 알 수 있다.As shown in FIG. 4, the dendritic cells treated with the pharmaceutical composition 3 (60 μg / ml) showed decreased antigen-phagocytic activity and the antigen-predominant ability was also decreased in the LPS-treated group. The same experiment was carried out at 4 ° C to exclude nonspecific binding of Dextran and dendritic cells, and it was confirmed that dendritic cells did not react with dextran at low temperature. As a result, endocytosis of dendritic cells was found to be due to the activation reaction of the antigen-trapping ability. These results indicate that the pharmaceutical composition of the present invention containing the pine needle extract as an active ingredient induces maturation of dendritic cells.
이상의 실험결과들을 통해 본 발명에서는 솔잎추출물이 미성숙수지상세포에 처리되면 수지상세포의 성숙화 마커로 알려진 CD80, CD86, MHC class I, MHC class II의 발현을 농도의존적으로 증가시킬 뿐만 아니라, IL-12p40/p70 유전자 발현을 농도의존적으로 증가시켜 성숙화를 유도하는 것을 증명하였다.In the present invention, when pine needle extract is treated with immature dendritic cells, expression of CD80, CD86, MHC class I, and MHC class II, which are known as maturation markers of dendritic cells, is increased in a concentration-dependent manner, p70 gene expression in a dose-dependent manner, leading to maturation.
따라서, 본 발명의 솔잎추출물이 미성숙수지상세포의 성숙화유도활성을 가지므로 이들이 항암치료 등 면역학적 치료시 면역반응을 증강시키는 새로운 약학조성물은 물론 기능성 식품 소재로 사용될 수 있음을 알 수 있다. Therefore, the pine needle extract of the present invention has an activity of inducing the maturation of immature dendritic cells, so that they can be used as a functional food material as well as a novel pharmaceutical composition for enhancing the immune response upon immunotherapy such as chemotherapy.
본 발명은 이상에서 살펴본 바와 같이 바람직한 실시 예를 들어 도시하고 설명하였으나, 상기한 실시 예에 한정되지 아니하며 본 발명의 정신을 벗어나지 않는 범위 내에서 당해 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 다양한 변경과 수정이 가능할 것이다.While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be taken by way of limitation, Various changes and modifications will be possible.
Claims (11)
A composition for inducing maturation of immature dendritic cells comprising pine needle extract as an active ingredient.
상기 추출물은 솔잎의 수용성추출물인 것을 특징으로 하는 미성숙수지상세포의 성숙화유도용 조성물.
The method according to claim 1,
Wherein the extract is a water-soluble extract of pine needle.
상기 추출물은 CD80, CD86, MHC class I, MHC class II의 발현을 증가시키는 것을 특징으로 하는 미성숙수지상세포의 성숙화유도용 조성물.
3. The method according to claim 1 or 2,
Wherein the extract enhances the expression of CD80, CD86, MHC class I, and MHC class II.
상기 추출물은 IL-12p40 및 IL-12/p70 유전자 발현을 증가시키는 것을 특징으로 하는 미성숙수지상세포의 성숙화유도용 조성물.
3. The method according to claim 1 or 2,
Wherein the extract enhances IL-12p40 and IL-12 / p70 gene expression.
A method for inducing maturation of immature dendritic cells comprising treating the immature dendritic cells isolated from a living body with a pine needle extract.
상기 추출물은 솔잎의 수용성추출물인 것을 특징으로 하는 미성숙수지상세포의 성숙화 유도방법.
6. The method of claim 5,
Wherein the extract is a water-soluble extract of pine needle.
상기 추출물은 CD80, CD86, MHC class I, MHC class II의 발현을 증가시키는 것을 특징으로 하는 미성숙수지상세포의 성숙화 유도방법.
The method according to claim 5 or 6,
Wherein the extract promotes the expression of CD80, CD86, MHC class I, and MHC class II.
상기 추출물은 IL-12p40 및 IL-12/p70 유전자 발현을 증가시키는 것을 특징으로 하는 미성숙수지상세포의 성숙화 유도방법.
The method according to claim 5 or 6,
Wherein said extract promotes IL-12p40 and IL-12 / p70 gene expression.
A health functional food for inducing maturation of immature dendritic cells comprising pine needle extract as an active ingredient.
상기 추출물은 솔잎의 수용성추출물인 것을 특징으로 하는 미성숙수지상세포의 성숙화유도용 건강기능성식품.
10. The method of claim 9,
Wherein said extract is a water-soluble extract of pine needle. 26. A health functional food for inducing maturation of immature dendritic cells.
분말, 과립, 정제, 캡슐 및 음료 중 어느 하나의 제형을 갖는 것을 특징으로 하는 미성숙수지상세포의 성숙화유도용 건강기능성식품.11. The method according to claim 9 or 10,
Wherein the composition has any one of a powder, a granule, a tablet, a capsule and a drink.
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