KR101484021B1 - A Composition Comprising the extract of combined herbs including Curcuma Longa L. for immuno-stimulating activity - Google Patents
A Composition Comprising the extract of combined herbs including Curcuma Longa L. for immuno-stimulating activity Download PDFInfo
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- KR101484021B1 KR101484021B1 KR20130052794A KR20130052794A KR101484021B1 KR 101484021 B1 KR101484021 B1 KR 101484021B1 KR 20130052794 A KR20130052794 A KR 20130052794A KR 20130052794 A KR20130052794 A KR 20130052794A KR 101484021 B1 KR101484021 B1 KR 101484021B1
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Abstract
본 발명은 울금포함 복합물 추출물을 함유하는 면역저하증 보호용 조성물에 관한 것으로, 본 발명의 울금포함 복합물 추출물은 LP-BM5 leukemia retrovirus로 면역파괴 유발시킨 동물모델에서 복합물 식이 제공이 사이토카인, T/B 세포의 증식, 항산화 효소 활성 등을 측정해 본 결과, 면역증진 효과가 탁월함을 확인하고 면역 저하증의 치료 및 예방용 식품, 건강기능식품, 의약품, 의약외품 등의 조성물로 유용하게 이용될 수 있음을 확인하였다.The present invention relates to a composition for the protection of immunodeficiency comprising an extract of Composition containing Ulvae, wherein the Composition Extract of Ulvae of the present invention is a combination of cytokine, T / B cell And antioxidant enzyme activity. As a result, it was confirmed that the immunity enhancing effect was excellent, and it was confirmed that the composition could be usefully used as a composition for treating and preventing hypnotics, health functional foods, medicines and quasi-drugs .
Description
본 발명은 울금포함 복합물 추출물을 함유한 면역증강용 조성물에 관한 것이다.The present invention relates to a composition for enhancing immunity containing a complex extract of Ulvae.
[문헌 1] Immunology. 49, 1992; Dig dis Sci. 52, pp 1890-1896, 2007; J Life Sci. 19, pp 479-485, 2009; Biol Pharm Bull. 27, pp 617-620, 2004[Literature 1] Immunology. 49, 1992; Dig dis Sci. 52, pp 1890-1896, 2007; J Life Sci. 19, pp 479-485, 2009; Biol Pharm Bull. 27, pp 617-620, 2004
[문헌 2] J Allergy clin immunol. 9, pp 616, 1993; Immunology. 47, pp 75, 1982[Document 2] J Allergy clin immunol. 9, pp 616, 1993; Immunology. 47, pp 75, 1982
[문헌 3] Life Sci. 44, pp 3-15, 1989[Literature 3] Life Sci. 44, pp 3-15, 1989
[문헌 4] J Korean Soc Food Sci Nutr. 35 pp 1329-1335, 2006; J Korean Soc Food Sci Nutr 38, pp 423-429 2009; Korean J Plant Res 24, pp 105-112, 2011; J Korean Soc Food Sci Nutr 40, pp 379-384, 2011[Literature 4] J Korean Soc Food Sci Nutr. 35 pp 1329-1335, 2006; J Korean Soc Food Sci Nutr 38, pp 423-429 2009; Korean J Plant Res 24, pp 105-112, 2011; J Korean Soc Food Sci Nutr 40, pp 379-384, 2011
[문헌 5] 김창민, 신민교 외, 정담 도서출판, 중약대사전, 7권, pp3282-3287, 1997[Literature 5] Kim Chang-min, Shin Min-kyo et al., Jongdam Publishing, Chinese Medicine Dictionary, Vol.7, pp3282-3287, 1997
[문헌 6] J Agric. Food Chem. 51, pp 628-633, 2003; J Nat. Prod. 62, p 802, 1999; [Literature 6] J Agric. Food Chem. 51, pp 628-633, 2003; J Nat. Prod. 62, p 802, 1999;
[문헌 7] J Med. Food. 4 pp 211-218, 2001[Literature 7] J Med. Food. 4 pp 211-218, 2001
[문헌 8] J Agric Food Chem. 49, pp 1948-1951, 2001[Literature 8] J Agric Food Chem. 49, pp 1948-1951, 2001
[문헌 9] Cancer Lett. in press pp 1-12, 2005[Document 9] Cancer Lett. in press pp 1-12, 2005
[문헌 10] 중약대사전, 김창민 외, 1997, pp2175-2176[Literature 10] Encyclopedia of Chinese medicine, Kim Chang Min et al., 1997, pp 2175-2176
[문헌 11] J Korean Soc Food Sci Nutr. 35, pp 1329-1335, 2006[Document 11] J Korean Soc Food Sci Nutr. 35, pp 1329-1335, 2006
[문헌 12] Immunopharmacology. 29, pp 225-233, 1995[Literature 12] Immunopharmacology. 29, pp 225-233, 1995
[문헌 13] Biosci. Biotechnol. Biochem. 75, pp 1234-1239, 2011[Literature 13] Biosci. Biotechnol. Biochem. 75, pp 1234-1239, 2011
[문헌 14] J Korean Soc Food Sic Nutr. 40, pp 525-535, 2011[Document 14] J Korean Soc Food Sic Nutr. 40, pp 525-535, 2011
[문헌 15]J Korean Soc Food Sic Nutr. 38, pp 423-429, 2009
[Literature 15] J Korean Soc Food Sic Nutr. 38, pp 423-429, 2009
인체는 항상성을 유지하려고 하는 특징을 가지고 있으며 이는 면역 시스템에서도 작용을 하게 된다. “면역”이란 생체가 자기 성분 이외의 물질 등이 생체의 항상성을 깨뜨리거나 자기를 위협하는 것을 배제하기 위해 일어나는 일련의 생체 방어 반응을 의미하며 피부, 소화관, 호흡기 등을 통해 침입한 유해물질로부터 신체를 보호하는 방어체계로 외부 자극물질을 제거하는 대식 작용을 하거나, 상처에 발생한 심각한 염증을 줄이는 작용을 통한 항상성 유지 활동이라고 할 수 있다. 체내 병원균의 유입으로 인해 활성화 되었던 염증 반응을 포함한 모든 면역작용은 병원균의 제거가 이루어지고 나면 정상의 상태로 돌아오게 되지만 면역 조절에 어려움이 있는 경우, 면역 반응이 정상인 사람에 비해 현저히 높거나 낮게 발생한다. 면역기능이 결핍되거나 저하된 상태를 ‘면역부전’이라고 하며 이 상태에서는 면역반응이 제대로 활성화 되지 못하여 체내의 이물질에 대한 반응을 제대로 못하여 감염을 일으키게 된다. 반대로 ‘면역과민반응’은 일부의 면역반응이 과하게 반응하여 면역체계가 불균형을 이루어 결과적으로 알레르기 반응을 일으키는 것이다 (Immunology. 49, 1992; Dig dis Sci. 52, pp 1890-1896, 2007; J Life Sci. 19, pp 479-485, 2009; Biol Pharm Bull. 27, pp 617-620, 2004). The human body has the characteristic of trying to maintain homeostasis, and it also works in the immune system. "Immunity" means a series of biological defense reactions that occur when a substance other than a magnetic component destroys the homeostasis of a living body or threatens the self. The term "immune" refers to a series of biological defense reactions that occur through the skin, digestive tract, respiratory apparatus, It is a defensive system that protects the external stimulant substances to remove the action or to reduce serious inflammation of the wound is a homeostasis activity by action. All immune responses, including the inflammatory response activated by influx of pathogens in the body, return to normal after removal of the pathogen, but when the immune response is difficult, immune responses are significantly higher or lower than those of normal individuals do. Immune deficiency or deficiency state is called 'immunodeficiency' in this state, the immune reaction is not activated properly, the body does not respond properly to foreign substances, causing the infection. In contrast, 'immune hypersensitivity' is a condition in which some immune responses are overreacted, resulting in an imbalance of the immune system resulting in an allergic reaction (Immunology. 49, 1992; Dig dis Sci., 52, pp 1890-1896, 2007; Sci., 19, pp 479-485, 2009; Biol Pharm Bull. 27, pp 617-620, 2004).
면역 체계가 정상적으로 조절되지 못하고 불균형을 이루게 되면 체내 사이토카인의 불균형, T/B 세포의 증식 불균형, 항산화 영양소 고갈 등의 원인으로 면역 조절에 장애가 발생하여 염증성 물질 분비가 증가하여 세포와 조직에 손상을 입히게 되며 병원체 유입에 대처하지 못하여 염증의 심화가 이루어질 수 있다. 따라서 인체의 면역반응은 균형을 이루어 조절이 정상적으로 되어야 건강을 유지할 수 있으므로 면역 조절 능력은 질병 예방과 치료에 중요시 된다 (J Allergy clin immunol. 9, pp 616, 1993; Immunology. 47, pp 75, 1982). If the immune system is not properly regulated and becomes unbalanced, imbalance of cytokine in the body, unbalance of T / B cell proliferation, depletion of antioxidant nutrients may cause impaired immune regulation and increase inflammatory substance secretion, And it can not cope with influx of pathogens, so that inflammation can be intensified. Therefore, the immune response of the human body is balanced and the control can be maintained normally, so that the immune control ability is important for the prevention and treatment of disease (J Allergy Clin Immunol., 9, pp 616, 1993; ).
비장에서는 면역조절에 관여하며 interferon (IFN)-γ, tumor necrosis factor (TNF)-α과 interleukin (IL)-1β, IL-5, IL-6, PGE2 등의 사이토카인을 분비한다. 이런 사이토카인은 면역반응의 실행단계에서 작용을 나타내며, 면역 세포와 염증계간의 신호 전달 과정에서도 중요한 역할을 한다.In the spleen, it is involved in immune regulation and secretes cytokines such as interferon (IFN) -γ, tumor necrosis factor (TNF) -α and interleukin (IL) -1β, IL-5, IL-6 and PGE2. These cytokines act in the execution phase of the immune response and play an important role in the signal transduction between the immune cells and the inflammatory system.
B 세포는 표면에 면역글로불린(Ig) 수용체를 발현하는 골수 유래성 림프구이다. 항원자극 및 T세포를 매개로 하는 자극에 따라 항체생산세포로 성숙하여 IgM, IgG, IgA, IgE의 항체를 생산하고 분비한다. 또한 항원제시세포로서 Ig수용체를 매개로 하여 세포 내에 항원을 수습, 분단하고, 항원유래 펩티드를 조직적 합성 항원과 더불어 세포 표면에 제시하여 T세포를 활성화하는 역할을 한다. 체액성 면역에는 B 세포에 의하여 이루어지는데, 항원특이성 분자인 항체에 의해 이루어지고 세포보다는 혈청 내에 존재하며 신체 각 부위에서 전달되고 이러한 항체는 T 세포의 도움을 받아 B 세포에 의해 생성되므로 B cell proliferation assay로 인하여 체액성 면역반응을 판단 할 수 있다. T 세포와는 달리 B 세포의 경우 세포성 면역체계가 아닌 항체를 생성하는 면역체계임을 감안할 때 직접적인 감염의 영향은 적을 것으로 생각된다. 하지만 LP-BM5 retrovirus의 감염으로 인해 그 활성은 마찬가지로 감소하는 것으로 알려져 있다 (Life Sci. 44, pp 3-15, 1989).B cells are bone marrow-derived lymphocytes that express immunoglobulin (Ig) receptors on the surface. It produces and secretes IgM, IgG, IgA, and IgE antibodies by mating to antibody producing cells following antigen stimulation and stimulation mediated by T cells. In addition, as an antigen presenting cell, an antigen is separated and separated into cells through an Ig receptor, and a peptide derived from an antigen is displayed on the cell surface together with a synthetic synthetic antigen to activate T cells. Humoral immunity is mediated by B cells, which are made up of antibodies that are antigen-specific molecules. They are present in the serum rather than the cells. They are delivered from each part of the body. These antibodies are produced by B cells with the help of T cells. assay can be used to determine the humoral immune response. In contrast to T cells, B cells are thought to be less affected by direct infection, given that they are immune systems that produce antibodies that are not cellular immune systems. However, the activity of LP-BM5 retrovirus has been reported to decrease as a result of infection (Life Sci. 44, pp 3-15, 1989).
LP-BM5 leukemia retrovirus에 감염된 쥐 AIDS 모델은 사람 AIDS 모델과 유사하게 면역 반응을 유발하는 것으로 알려져 동물모델에서 면역 평가를 위하여 많이 사용된다. AIDS에 감염될 경우 Th1 type cytokines과 Th2 type cytokines의 균형이 파괴되면서 정상적인 면역 반응이 일어나지 않고 면역 시스템이 파괴되어 질병에 대한 면역력을 저하시키게 된다. 또한 활성산소가 과잉 생성되어 산화적 스트레스를 유발하게 되며 DNA 및 세포막, 단백질 등을 손상시켜 암 뿐만 아니라 성인병, 노화를 유발시켜 심하게는 조기사망을 일으킨다는 보고가 있다 (J Korean Soc Food Sci Nutr. 35 pp 1329-1335, 2006; J Korean Soc Food Sci Nutr 38, pp 423-429 2009; Korean J Plant Res 24, pp 105-112, 2011; J Korean Soc Food Sci Nutr 40, pp 379-384, 2011).
The rat AIDS model infected with LP-BM5 leukemia retrovirus is known to induce an immune response similar to the human AIDS model, and is widely used for immunological evaluation in animal models. When AIDS is infected, the balance between Th1 type cytokines and Th2 type cytokines is destroyed, and normal immune response does not occur, and the immune system is destroyed and the immunity against the disease is lowered. In addition, there is a report that excessive oxygen production causes oxidative stress, damages DNA, cell membranes and proteins, causing cancer, as well as adult diseases and aging, resulting in premature death (J Korean Soc Food Sci Nutr. J Korean Soc Food Sci Nutr 40, pp 379-384, 2011; J Plant Res 24, pp 105-112, 2011; .
울금은 생강과의 식물인 울금 (Curcuma Longa L.)의 덩이뿌리를 그대로 말린 것을 말한다. 중국에서는 울금 및 생강과의 동속식물인 광서아출 (Curcuma kwangsiensis SG Lee & CF Liang:廣西莪朮), 온욱금(Curcuma wenyujin YH Chen & C. Ling:溫郁金), 봉아출(Curcuma phaeocaulis Valeton:蓬莪朮)의 뿌리줄기를 말하며, 일본에서는 가을 울금(Curcuma longa Linn)의 덩이뿌리를 말한다. 이는 강황 (Curcuma longa Linn)과 유사한 외형을 하고 있으나 잎의 양면이 모두 매끈한 특징을 갖는다. 중국 남부와 인도, 오키나와를 비롯한 동남아시아지역에서 자생, 재배되며 우리나라의 중남부지역에서도 재배된다. 술과 함께 섞으면 누렇게 금처럼 되어 붙여진 이름이며 모양이 아술(莪)과 비슷하고 말의 질병을 치료하므로 마술(馬)이라 하기도 하였다 (문헌 1: 김창민, 신민교 외, 정담 도서출판, 중약대사전, 7권, pp3282-3287, 1997).Curcuma is a ginger plant, Curcuma Longa L.) root of the root is dried. In China, curcuma kwangsiensis SG Lee & CF Liang, Curcuma wenyujin YH Chen & C. Ling, Curcuma phaeocaulis Valeton, And in Japan it refers to the roots of the root of Curcuma longa Linn. It has a similar appearance to Curcuma longa Linn, but both sides of the leaves are smooth. It grows naturally in Southeast Asia including southern China, India, and Okinawa, and is cultivated in the southern part of Korea. It is called "magic" because it is similar to a spoon and it is called a magic (horse) because it treats the disease of the horse (1) Pp. 3282-3287, 1997).
한편, 자색고구마 (자미고구마; Purple-Fleshed sweet potato)는 1993년 가공용 다수성 품종 건미를 모본으로하고 자색색소를 함유한 야마까와무라사끼를 부본으로 인공 교배하여 그 후대인 실생 개체선발 및 계통선발시험에서 색소용으로 선발된 계통(MI9308-11, 색소원료용 자색고구마의 평가방법과 육종 (1994. Kinnosuke Odake 등 8인, 일본) 기술보고 41(4), 287~293)으로, 생산력검정시험과 지역적응시험(목포29호)에서 다수성이 확인되어 품종교배를 통해 지난 1998년 자미, 2000년 보라미, 2001년에 신자미 등 3가지 신품종으로 육성되었다. 이들 신품종은 야생 고구마의 고유 성분은 모두 간직하고 있으면서 크기는 일반 고구마와 비슷한 반면 수확량은 야생보다 2배 이상 많고, 고구마 고유의 당질과 섬유소를 가지고 있다는 장점을 지니고 있다.Purple-Fleshed sweet potato was used in 1993 as a model for the cultivated multi-species varieties, and it was used for the artificial crossing of Yamakawamura-sacci containing purple pigment as a duplicate, (MI9308-11, evaluation method and breeding of purplish sweet potato for raw materials of coloring materials (8 persons, Kinnosuke Odake et al., Japan) 41 (4), 287 ~ 293) Many varieties were identified in the test and local adaptation test (Mokpo 29), and they were cultivated in three new varieties, namely, Jami in 1998, Boram in 2000, and Sinja in 2001. These new varieties have all of the native ingredients of wild sweet potatoes, similar in size to regular sweet potatoes, but harvest more than twice as much as the wild ones, and have the advantage of having sweet potatoes' own saccharides and fibrin.
이러한 자색고구마의 주요 활성성분은 플라보노이드계의 색소인 안토시아닌으로 항산화작용(J Agric. Food Chem. 51, pp 628-633, 2003; J Nat. Prod. 62, p 802, 1999; J Med. Food. 4 pp 211-218, 2001), 항 당뇨(J Agric Food Chem. 49, pp 1948-1951, 2001), 항암작용(Cancer Lett. in press pp 1-12, 2005) 효과 등이 있는 것으로 보고되어 있다. 자색고구마의 색소추출물은 생체 중량대비 약 3.8 %를 함유하고 있어 표피층뿐만 아니라 육질 전체가 진한 자색을 띠고 있는 데, 이 기능성 색소함량이 크린베리 보다는 10-60배, 포도보다는 5-7배 높아 천연 기능성 색소 원뿐만 아니라 기능성 식품소재로서 가치가 우수하다.The main active ingredient of these purple sweet potatoes is antioxidant, which is an anthocyanin of flavonoid system (J Agric. Food Chem. 51, pp 628-633, 2003; J Nat. Prod. 62, p 802, 1999; 4 pp 211-218, 2001), antidiabetic activity (J Agric Food Chem. 49, pp 1948-1951, 2001) and anticancer activity (Cancer Lett. In press pp 1-12, 2005) . The color pigment extract of purple sweet potato contains about 3.8% of the weight of living body, and not only the skin layer but also the whole meat color is deep purple color. The functional pigment content is 10-60 times higher than clean berries and 5-7 times higher than grapes, It is not only a functional pigment source but also a functional food material.
상엽은 뽕나무과 식물 상백나무 (Morus alba L.)의 건조된 잎이다. 대부분의 말린 잎몸은 쪼그라들어 둥글고 부서져 있다. 형태가 완전한 것은 달걀 모양 또는 넓은 달걀 모양이며 길이는 8~13 cm, 너비는 7~11 cm이다. 끝은 뾰족하고 가장자리는 톱니 모양이거나 때로는 불규칙하게 갈라지며 기부는 절단형이거나 원형 또는 심장형이다. 윗면은 황록색이고 조금 광택이 있으며 잎맥을 따라 가늘고 작은 부드러운 털이 있다. 아랫면은 색이 약간 엷고 잎맥이 돌출되어 있으며 작은 잎맥은 망상으로 교차되고 가는 털이 밀생한다. 질은 취약하고 부서지기 쉽다. 냄새가 조금 있으며 맛은 싱겁고 조금 쓰고 떫다. 잎몸이 완전하고 크고 두껍고 황록색이며 질이 취약하며 불순물이 없는 것을 양품으로 한다. 일반적으로 10~11월에 서리가 내린 후에 채집하여 이물질을 제거하고 햇볕에 말린 후 약재로 사용한다 (중약대사전, 김창민 외, 1997, pp2175-2176).The leaves are Morus alba L.). Most dried leaf blossoms are squashed and round and broken. The complete shape is ovate or wide ovate, 8 to 13 cm in length, and 7 to 11 cm in width. The tip is pointed, the edges are sawtooth or sometimes irregularly split, and the base is truncated or circular or heart shaped. The upper surface is yellowish green, a little shiny, and along the veins are thin, small soft hairs. The lower surface is slightly thin in color and the leaf vein is protruding. The small vein is intersecting with delusions and the thin hair grows densely. Quality is vulnerable and fragile. There is a little bit of smell, and the taste is light and little bitter. Leaves are perfect, large, thick yellowish green, weak in quality and free from impurities. Generally, it collects after frost down in October ~ November, removes foreign matter, and is used as a medicinal product after drying in the sun (Chinese medicinal herb, Kim Chang Min et al., 1997, pp2175-2176).
이에 본 발명자들은 지난 연구에서 면역조절 효능에 대한 기능성이 입증된 울금, 자색고구마, 뽕잎을 이용하여 제작한 복합물의 면역조절 효능을 평가하기위하여 LP-BM5 leukemia retrovirus로 면역파괴 유발 시킨 동물모델에서 복합물 식이 제공하여 사이토카인, T/B 세포의 증식, 항산화 효소 활성 등을 측정해 본 결과, 면역증진 효과가 탁월함을 확인하고 면역 저하증의 치료 및 예방용 식품, 의약품, 의약외품 등의 조성물로 유용하게 이용될 수 있음을 확인하여, 본 발명을 완성하였다.
Therefore, in order to evaluate the immunomodulatory effect of the complex prepared by using Ulgum, Purple Sweet Potato, and Mulberry Leaf which have been proved as functional in immuno-modulating efficacy in the past, the present inventors have found that, in an animal model induced by immunodeficiency with LP-BM5 leukemia retrovirus, As a result of measuring the cytokine, T / B cell proliferation, antioxidant enzyme activity and the like provided by the diet, it is confirmed that the immunity enhancing effect is excellent, and it is usefully used as a composition for the treatment and prevention of immunosuppression, medicines and quasi-drugs The present invention has been completed.
상기 목적을 수행하기 위하여, 본 발명은 울금, 자색고구마 및 뽕잎으로 구성된 복합물 추출물을 유효성분으로 함유하는 면역저하증의 치료 및 예방용 약학 조성물을 제공한다.In order to accomplish the above object, the present invention provides a pharmaceutical composition for the treatment and prevention of hypo-immunodeficiency comprising, as an active ingredient, a complex extract composed of ganoderma, purple sweet potato and mulberry leaf.
본원에서 정의되는 추출물은 정제수를 포함한 물, 탄소수 1 내지 4의 저급알코올 또는 이들의 혼합용매 바람직하게는 물에 가용한 추출물을 포함한다.The extracts defined herein include water containing purified water, lower alcohols having 1 to 4 carbon atoms, or mixtures thereof, preferably water-soluble extracts.
본원에서 정의되는 상기 울금, 자색고구마 및 뽕잎으로 구성된 복합물 추출물은 건조 중량 대비 상대적 중량 배합비가 1-10:0.1-20:0.1-20(w/w), 바람직하게는 1-10:1-10:1-10(w/w)로 배합된 복합물을 포함한다.The complex extract composed of the herbaceous, purple sweet potatoes and mulberry leaves defined in the present application has a relative weight ratio to dry weight of 1-10: 0.1-20: 0.1-20 (w / w), preferably 1-10: 1-10 : 1-10 (w / w).
상기 울금, 자색고구마 및 뽕잎으로 구성된 복합물 추출물의 약학조성물은 총 중량에 대하여 0.1 내지 50 중량%로 사용이 가능하다.
The pharmaceutical composition of the complex extract consisting of the above-described herb, purple sweet potato and mulberry leaf can be used in an amount of 0.1 to 50% by weight based on the total weight.
본원에서 정의되는 면역저하증은 화학요법 및 방사선요법과 같은 항암요법에 의한 면역기능의 저하 또는 골수이식 후 면역저하로 인한 질환, 면역계손상으로 인한 에이즈 및 면역기능의 저하로 인한 암질환 등과 같은 면역저하증, 바람직하게는 화학요법 및 방사선요법과 같은 항암요법에 의한 면역기능의 저하 또는 골수이식 후 면역저하로 인한 질환을 포함한다.
The immunosuppressive conditions defined herein include those caused by a decrease in immune function by chemotherapy or radiation therapy or a disease caused by immune reduction after bone marrow transplantation, a disease caused by immune system damage and a cancer disease caused by a decrease in immune function, , Preferably diseases caused by a decrease in immune function by chemotherapy or chemotherapy such as chemotherapy or radiotherapy or a decrease in immunity after bone marrow transplantation.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 혼합 추출물은 하기와 같이 수득될 수 있다. The mixed extract of the present invention can be obtained as follows.
예를 들어, 건조 상태의 울금, 자색고구마 및 뽕잎 각각에 시료 중량의 약 1 내지 100배(v/w)의 물, C1 내지 C4의 저급 알콜 또는 이들의 혼합용매를 가하여 시료를 추출하기 1일 내지 2일전에 약재를 담가 1차 추출하는 제 1단계; 1차 추출후에 30 내지 150℃, 바람직하게는 50 내지 120℃에서 1 내지 24시간, 바람직하게는 2 내지 12시간동안 냉침추출, 열수추출, 초음파 추출, 환류냉각추출 또는 가열추출법, 바람직하게는, 환류 추출법으로 2차 추출하는 제 2단계; 상기 수득한 시료를 여과하여 30 내지 150℃, 바람직하게는 50 내지 120℃에서 중탕 및 농축시키는 제 3단계: 상기 농축된 추출물을 2 내지 4일, 바람직하게는 12시간 내지 2일 동안에 -150℃ 내지 0℃, 바람직하게는 -50 내지 -100℃에서 냉각시키고 20 내지 -100℃, 바람직하게는 -20 내지 -60℃로 동결건조시켜 분말을 수득하는 제 4단계; 울금, 자색고구마 및 뽕잎 추출분말을 바람직하게는 상대적 중량 배합비가 1-10:0.1-20:0.1-20(w/w), 바람직하게는 1-10:1-10:1-10(w/w)로 배합하는 제 5단계를 포함하는 공정을 통하여 본원 발명의 혼합추출물을 제조가능하다.
For example, a sample is extracted by adding water, about 1 to 100 times (v / w) water, C 1 to C 4 lower alcohols or a mixed solvent thereof, to each of the dried corn, purple sweet potato and mulberry leaves A first step of immersing and firstly extracting a medicinal substance one to two days before; Hot extraction, ultrasonic extraction, reflux cooling extraction or heat extraction method at 30 to 150 ° C, preferably 50 to 120 ° C, for 1 to 24 hours, preferably 2 to 12 hours after the first extraction, A second step of secondary extraction using a reflux extraction method; The obtained sample is filtered and concentrated at a temperature of 30 to 150 ° C, preferably 50 to 120 ° C. Step 3: The concentrated extract is filtered at -150 ° C for 2 to 4 days, preferably 12 hours to 2 days To 0 占 폚, preferably -50 to -100 占 폚 and lyophilization at 20 to -100 占 폚, preferably -20 to -60 占 폚 to obtain a powder; The red ginseng, purple sweet potato and mulberry leaf extract powder are preferably used in a relative weight ratio of 1-10: 0.1-20: 0.1-20 (w / w), preferably 1-10: 1-10: 1-10 (w / w), and a fifth step of blending the extract of step
따라서, 본 발명은 상기 제조방법 및 상기 제조방법으로 울금포함 복합물 추출물을 유효성분으로 함유하는 면역증강용 약학조성물을 제공한다.Accordingly, the present invention provides a pharmaceutical composition for enhancing immunity, comprising the extract of Composition containing Ulvae as an active ingredient by the above-mentioned method of manufacture and the above-mentioned method of manufacture.
본 발명의 면역저하증의 예방 및 치료용 조성물은, 조성물 총 중량에 대하여 상기 추출물을 0.1 내지 99% 중량으로 포함된다.The composition for the prevention and treatment of hypomagnesa of the present invention contains 0.1 to 99% by weight of the above extract relative to the total weight of the composition.
그러나 상기와 같은 조성은 반드시 이에 한정되는 것은 아니고, 환자의 상태 및 질환의 종류 및 진행 정도에 따라 변할 수 있다.However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.
본 발명의 추출물을 포함하는 조성물은 약학적 조성물의 제조에 통상적으로 사용되는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다.The composition comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.
본 발명에 따른 추출물을 포함하는 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제의 형태로 제형화하여 사용될 수 있으며, 또한 본 발명의 추출물을 포함하는 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 외용제제에는 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜 (propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈 (tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.The composition containing the extract according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like in the form of oral preparations or external preparations, Examples of carriers, excipients and diluents that can be included in the composition containing the extract of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . The external preparation for parenteral administration includes sterilized aqueous solution, non-aqueous solution, suspension, emulsion, freeze-dried preparation, and suppository. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.
본 발명의 추출물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나 바람직한 효과를 위해서, 본 발명의 추출물은 1일 0.01 mg/kg 내지 10 g/kg으로, 바람직하게는 1 g/kg 내지 5 g/kg으로 투여하는 것이 좋다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수 있다. 따라서 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The preferred dosage of the extract of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at a dose of 0.01 mg / kg to 10 g / kg per day, preferably 1 g / kg to 5 g / kg per day. The administration may be carried out once a day or divided into several doses. Accordingly, the dosage is not limited in any way to the scope of the present invention.
본 발명의 조성물은 쥐, 생쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁 내 경막 또는 뇌혈관 내 (intracerebroventricular) 주사에 의해 투여될 수 있다.The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.
또한, 본 발명은 상기 울금, 자색고구마 및 뽕잎으로 구성된 복합물 추출물을 유효성분으로 함유하는 면역저하증의 예방 및 개선용 건강기능식품을 제공한다.The present invention also provides a health functional food for prevention and improvement of hypoimmunities, which comprises a complex extract composed of the above-mentioned Uleum, purple sweet potato and mulberry leaf as an active ingredient.
따라서, 또한, 본 발명은 면역증강 효과를 갖는 상기 울금, 자색고구마 및 뽕잎으로 구성된 복합물 추출물을 유효성분으로 함유하는 식품 및 식품첨가제를 제공한다.Accordingly, the present invention also provides a food and food additive containing, as an active ingredient, a complex extract composed of the above-mentioned koi, purple sweet potato and mulberry leaf having an immunostimulating effect.
본 발명의 추출물을 포함하는 조성물은 면역저하증의 예방 및 개선을 위한 약제, 식품 및 음료 등에 다양하게 이용될 수 있다. 본 발명의 추출물을 첨가할 수 있는 식품으로는, 예를 들어, 캔디류의 각종 식품류, 음료, 껌, 차, 비타민 복합제, 건강보조 식품류 등이 있고, 분말, 과립, 정제, 캡슐 또는 음료인 형태로 사용할 수 있다.The composition containing the extract of the present invention can be used variously for medicines, foods and beverages for the prevention and improvement of hypoimmunities. Examples of foods to which the extract of the present invention can be added include various kinds of foods such as candy, beverages, gums, tea, vitamin complex, health supplement foods and the like, and they are in the form of powder, granule, tablet, capsule or beverage Can be used.
본 발명의 추출물은 독성 및 부작용은 거의 없으므로 예방 목적으로 장기간 복용 시에도 안심하고 사용할 수 있는 약제이다. Since the extract of the present invention has little toxicity and side effects, it can be safely used even for long-term administration for preventive purpose.
본 발명의 상기 추출물은 면역저하증의 예방 및 개선을 목적으로 식품 또는 음료에 첨가될 수 있다. 이때, 식품 또는 음료 중의 상기 추출물의 양은 일반적으로 본 발명의 건강식품 조성물은 전체 식품 중량의 0.01 내지 15 중량%로 가할 수 있으며, 건강 음료 조성물은 100 ㎖를 기준으로 0.02 내지 10 g, 바람직하게는 0.3 내지 1 g의 비율로 가할 수 있다. The extract of the present invention can be added to food or beverage for the purpose of prevention and improvement of hypoimmunities. At this time, the amount of the extract in the food or beverage is generally 0.01 to 15% by weight of the total food weight of the health food composition of the present invention, and 0.02 to 10 g, Can be added in a proportion of 0.3 to 1 g.
본 발명의 건강 음료 조성물은 지시된 비율로 필수 성분으로서 상기 추출물을 함유하는 것 외에 액체성분에는 특별한 제한점은 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등의 디사카라이드, 예를 들어 말토스, 슈크로스 등의 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 ㎖당 일반적으로 약 1 내지 20g, 바람직하게는 약 5 내지 12g이다.The health beverage composition of the present invention may contain various flavors or natural carbohydrates such as ordinary beverages as an additional ingredient, as long as it contains the extract as an essential ingredient at the indicated ratio, and there is no particular limitation to the liquid ingredient. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.
상기 외에 본 발명의 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 조성물들은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 조성물 100 중량부 당 0 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다.
In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.
이상에서 살펴본 바와 같이, 본 발명의 울금포함 복합물 추출물은 LP-BM5 leukemia retrovirus로 면역파괴를 유발시킨 동물모델에서 복합물 식이 제공이 사이토카인, T/B 세포의 증식, 항산화 효소 활성 등을 측정해 본 결과, 면역증진 효과가 탁월함을 확인하고 면역 저하증의 치료 및 예방용 식품, 의약품, 의약외품 등의 조성물로 유용하게 이용될 수 있음을 확인하였다.
As described above, the complex extract of Ulgum of the present invention was obtained by measuring the proliferation of cytokine, T / B cell, antioxidant enzyme activity and the like in an animal model in which immunodeficiency was induced by LP-BM5 leukemia retrovirus As a result, it was confirmed that the immunity enhancing effect is excellent, and it can be effectively used as a composition for foods and medicines and quasi-drugs for the treatment and prevention of hypoimmunities.
도 1은 면역 체계가 붕괴 된 마우스를 대상으로 한 해부학적 변화 소견을 나타낸 도이며;
도 2은 면역 체계가 붕괴 된 마우스를 대상으로 한 장기무게 변화를 나타낸 도이며;
도 3는 LP-BM5 retrovirus의 감염 시 primary splenocyte에서의 IL-2 발현에 미치는 영향을 나타낸 도이며;
도 4는 LP-BM5 retrovirus의 감염 시 primary splenocyte에서의 IFN-γ 발현에 미치는 영향을 나타낸 도이며;
도 5는 LP-BM5 retrovirus의 감염 시 primary splenocyte에서의 TNF-α 발현에 미치는 영향을 나타낸 도이며;
도 6는 LP-BM5 retrovirus의 감염 시 primary splenocyte에서의 IL-4 발현에 미치는 영향을 나타낸 도이며;
도 7는 LP-BM5 retrovirus의 감염 시 primary splenocyte에서의 IL-10 발현에 미치는 영향을 나타낸 도이며;
도 8는 LP-BM5 retrovirus의 감염 시 primary splenocyte에 존재하는 T세포의 ConA에 대한 증식능에 미치는 영향을 나타낸 도이며;
도 9는 LP-BM5 retrovirus의 감염 시 primary splenocyte에 존재하는 B 세포의 LPS에 대한 증식능에 미치는 영향을 나타낸 도이며;
도 10는 LP-BM5 retrovirus의 감염 시 혈액에서의 SOD 활성에 미치는 영향을 나타낸 도이며;
도 11는 LP-BM5 retrovirus의 감염 시 혈액에서의 glutathione peroxidase 활성에 미치는 영향을 나타낸 도이다.FIG. 1 is an illustration of anatomic change in a mouse whose immune system has collapsed;
FIG. 2 is a diagram showing organ weight changes for a mouse whose immune system has collapsed; FIG.
Figure 3 shows the effect of IL-2 on primary splenocytes when infected with LP-BM5 retrovirus;
Figure 4 shows the effect of IFN-y on primary splenocytes when infected with LP-BM5 retrovirus;
Figure 5 is a graph showing the effect of LP-BM5 retrovirus infection on TNF-α expression in primary splenocytes;
Figure 6 shows the effect of IL-4 on primary splenocytes during LP-BM5 retrovirus infection;
Figure 7 shows the effect of IL-10 on primary splenocytes in LP-BM5 retrovirus infection;
Fig. 8 shows the effect of T cells on primary splenocyte proliferating ability to ConA upon infection with LP-BM5 retrovirus; Fig.
FIG. 9 is a graph showing the effect of B cells present in the primary splenocyte on LPS proliferation in LP-BM5 retrovirus infection;
FIG. 10 shows the effect of SOD on blood in infecting the LP-BM5 retrovirus;
11 is a graph showing the effect of glutathione peroxidase activity in blood upon infection of LP-BM5 retrovirus.
이하, 본 발명을 실시예 및 실험예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.
단, 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 한정되는 것은 아니다.
However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.
실시예 1. 시료의 제조Example 1. Preparation of sample
1.1. 1.1. 울금추출물Ulgum extract 제조 Produce
건조된 울금 (전남 진도군, 진도울금농장) 4 kg에 10배수(v/w)의 물을 넣고 100℃에서 4시간 환류 추출하고 여과 및 감압농축하고 동결건조하여 울금 추출물 820 g을 수득하였다.
Water (v / w) was added to 4 kg of dried Ulgum (Jindo-gun, Jeollanam-do) and refluxed at 100 ° C for 4 hours. The mixture was filtered and concentrated under reduced pressure and lyophilized to obtain 820 g of Ulmus extract.
1.2. 자색고구마 추출물 제조1.2. Purple Sweet Potato Extract Manufacturing
건조된 자색고구마(전남 무안군 유기농플러스) 2kg에 10배수의 물을 넣고 100℃에서 4시간 환류 추출하고 여과 및 감압농축하고 동결건조하여 자색고구마 추출물 300 g을 수득하였다.
To 2 kg of dried purple sweet potato (Jeonnam Muangan Organic Plus), 10 times water was added, and the mixture was refluxed at 100 ° C for 4 hours, filtered and concentrated under reduced pressure, and lyophilized to obtain 300 g of purple sweet potato extract.
1.3. 뽕잎추출물 제조1.3. Mulberry leaf extract preparation
건조된 뽕잎(전남 함평군 동의나라) 3.5 kg에 10배수의 10%주정을 넣고 100℃에서 4시간 환류 추출하고 여과 및 감압농축하고 동결건조하여 뽕잎추출물 580 g을 수득하였다.
10 kg of 10% alcohol was added to 3.5 kg of dried mulberry leaves (Chonnam Hampyeong-gun, Korea), refluxed at 100 ° C for 4 hours, filtered, concentrated under reduced pressure and lyophilized to obtain 580 g of mulberry leaf extract.
1.4. 1.4. 울금추출물Ulgum extract 포함 복합물의 제조 Preparation of inclusion complexes
상기에서 수득한 추출물들을 울금추출물:자색고구마추출물:뽕잎추출물의 비율(중량비)을 1 : 7 : 1 으로 혼합하여 울금추출물 포함 복합물 300 g을 수득하였다(이하, “복합물”이라 함).
The extracts obtained above were mixed at a weight ratio of 1: 7: 1 to the extract of Purple ginseng extract: purple sweet potato extract: mulberry leaf extract to obtain 300 g of a complex containing the ginseng extract (hereinafter referred to as "complex").
실험예 1. 동물 실험Experimental Example 1. Animal experiment
상기 실시예 시료의 면역증진 효과를 확인하기 위하여 문헌에 개시된 방법을 응용하여 면역 체계가 붕괴 된 마우스를 대상으로 한 몸무게 변화량 및 식이효율 변화를 확인하였다
The immunoenhancing To confirm the effect, the method disclosed in the literature was applied to confirm the change in the body weight and the dietary efficiency of the mice with the collapsed immune system
1.1. 1.1. 식이제작Dietary production
울금 추출물등 복합물을 93G AIN식이에 첨가하여 결과적으로 소재의 농도는 150 mg/kg와 300 mg/kg가 되도록 조절하여 제작하였다.
The extract was added to the 93G AIN diet. As a result, the concentration of the material was adjusted to be 150 mg / kg and 300 mg / kg.
1.2. 실험동물1.2. Experimental animal
대한바이오링크 실험동물 사육장으로부터 공급받은 20 g 내외의 4주령 암컷 C57BL/6 마우스를 일주일 동안 설치류 사육실에서 적응시킨 후 적응기간 중 일반 상태를 관찰하여 건강한 개체만 실험에 사용하였다. 사육환경은 온도 23±3℃ 습도 50±5%에서 Light cycle이 12 시간으로 유지하였다. 순화기간 중 건강하다고 판정된 동물에 대하여 체중을 측정하고 평균체중에 가까운 개체를 선택하여 무작위법을 이용하여 군 분리를 실시하였다.A 4-week-old female C57BL / 6 mouse (20 g) supplied from Korea BioLink Experimental Livestock Breeding Center was adapted from rodent breeding room for one week, and normal state was observed during the adaptation period. The incubation environment was maintained at a temperature of 23 ± 3 ° C and a humidity of 50 ± 5%, with a light cycle of 12 hours. Body weight was measured for the animals that were judged to be healthy during the refinement period, and individuals close to the average body weight were selected and subjected to group separation using the random method.
면역파괴 유발 동물 모델을 만들기 위하여 마우스에서 면역체계를 무너뜨리는 바이러스인 LP-BM5(Immunopharmacol. Immunotoxicol. 23, pp 307-317, 2001)를 SC-1 세포의 배양액으로부터 얻은 뒤, 병원균에 민감한 종류인 C57BL/6 마우스에 복강 내 주사로 2회 주입, 바이러스에 감염시켜 백혈병을 유발해 면역을 파괴시켰다. 바이러스에 감염되어 면역 체계가 붕괴 된 마우스를 12주 동안 복합물이 함유된 식이를 제공하면서 식이량, 체중을 매주 2회씩 측정하고 12주 후 희생시켰다. (표 1 참조)(Immunopharmacol. Immunotoxicol. 23, pp 307-317, 2001), a virus that breaks down the immune system in mice to produce an immunocompromising animal model, is obtained from the culture medium of SC-1 cells, C57BL / 6 mice were injected twice by intraperitoneal injection to infect the virus, causing leukemia and destroying the immune system. Diets, body weights were measured twice a week, and mice were sacrificed 12 weeks after the mice were infected with the virus and the immune system collapsed, providing a diet containing the complex for 12 weeks. (See Table 1)
1.3. 1.3. 결 론conclusion
(1) 몸무게 변화량 및 식이효율(1) Weight change and diet efficiency
실험을 진행하는 동안의 식이 섭취량와 몸무게 변화량을 살펴보았다. 그 결과 모든 시험군들간의 식이 섭취량에 따른 유의적인 차이를 보이지 않았으며 이는 실험의 모든 데이터가 식이 섭취량의 차이에 의한 것이 아니라고 확인된다. 12주 동안의 체중의 증가는 LP-BM5 retrovirus 감염시키지 않은 군만 유의적으로 적게 나타났음을 볼 수 있다. 이는 LP-BM5 retrovirus 감염이 체내 및 조직의 부종과 염증반응을 일으켜 나타난 결과라고 본다. 양성대조군이나 복합물을 섭취한 군에서도 LP-BM5 retrovirus 감염군과의 유의적인 차이를 보이지 않고 체중이 증가 되었다. 식이효율(Food efficiency rate, FER=식이 섭취량 (g)/체중 증가량 (g))의 경우 체중 증가량에 따라 변화되었음을 알 수 있다. 정상군에 비하여 LP-BM5 retrovirus 감염군에서 유의적으로 증가하였으며, 양성대조군과 복합물 섭취군에서도 LP-BM5 retrovirus 감염군과 유의적인 차이없이 증가되었다. (표 2 참조)We examined the dietary intake and body weight change during the experiment. As a result, there was no significant difference in dietary intake between all test groups, which confirms that not all of the data from the experiment is due to differences in dietary intake. The increase in body weight over the 12-week period was significantly less in the group not infected with LP-BM5 retrovirus. This suggests that the LP-BM5 retrovirus infection is caused by edema and inflammation of the body and tissues. There was no significant difference between the LP-BM5 retrovirus-infected group and the positive control group or the composite group. The food efficiency rate (FER = dietary intake (g) / weight gain (g)) was found to change with weight gain. BM5 retrovirus infected group compared with the normal group, and the positive control group and the composite intake group were significantly increased compared to the LP-BM5 retrovirus infection group. (See Table 2)
(2) 해부학적 변화 소견 및 장기무게 변화 (도 1 및 2, 표 3 참조)(2) anatomical changes and organ weight changes (see Figures 1 and 2, Table 3)
LP-BM5 바이러스 감염을 시킨 군은 정상인 군에 비해 탈모가 진행되고 목과 양 앞다리와 몸통 사이, 양 뒷다리와 몸통 사이에 존재하는 림프절의 크기가 그렇지 않은 군에 비해 현저히 증가한 것을 알 수 있다. 이는 바이러스의 감염에 의해 체내 면역 반응이 활발해짐에 따른 변화인 것으로 보이며 C57BL/6 마우스는 탈모 유도가 쉬운 동물이며, 바이러스의 감염 이후 탈모 반응은 심해졌음을 확인하였다. 위에서 확인하였던 체중증가량의 변화는 장기의 크기와 무게가 크게 증가함에 따른 결과라는 것을 해부학적인 관찰을 통해 알 수 있었다. In the LP-BM5 virus-infected group, the size of the lymph nodes between the neck and the forelimb and the trunk, between the hind legs and the trunk, was significantly higher than that of the normal group. These results suggest that C57BL / 6 mice are more susceptible to dehydration than viruses, and that they are more susceptible to alopecia. Anatomical observations suggest that the change in weight gain observed above is the result of a significant increase in size and weight of organs.
가) 비장(spleen)의 무게 변화A) weight change of spleen
비장(spleen)의 무게 변화는 retrovirus의 감염에 의해 가장 큰 변화를 나타내는 기관으로 알려져 있다. spleen은 면역기능을 담당하는 제일 중요한 기관으로서 면역세포가 밀집되어 있고 memory 세포 또한 존재하기 때문에 항원이 제시되었을 때 이에 대해 반응하여 면역세포의 mitogenesis를 동반하여 급격하게 spleen의 크기가 증가하는 megasplenocyte를 유발하게 되며 이것은 LP-BM5의 감염에 따른 가장 일반적인 현상으로 인식되어져 왔다 (J Korean Soc Food Sci Nutr. 35, pp 1329-1335, 2006).The weight change of spleen is known to be the most significant organ of retrovirus infection. Spleen is the most important organ responsible for immune function. Since it has dense immune cells and memory cells, it responds to the antigen when it is presented and causes megasplenocyte which is accompanied by mitogenesis of immune cells and rapidly increases the size of spleen (J Korean Soc Food Sci Nutr. 35, pp. 1329-1335, 2006). It has been recognized that LP-BM5 infection is the most common phenomenon.
본 실험에서 감염되지 않은 정상군의 경우 0.063 ± 0.010 g으로 나타난 반면, LP-BM5 retrovirus 감염군의 경우 0.896 ± 0.308 g으로 유의적으로 비장의 무게가 증가하였음을 확인하였다. 이 결과는 LP-BM5 retrovirus의 감염이 정상적으로 진행이 되었음을 나타내는 결과이며 LP-BM5 외의 다른 병원체에 의한 영향은 없었음을 나타내는 것이다. In the present study, it was confirmed that 0.063 ± 0.010 g was observed in the non-infected normal group whereas 0.896 ± 0.308 g in the LP-BM5 retrovirus-infected group was significantly increased. These results indicate that the infection of LP-BM5 retrovirus has progressed normally and that there is no effect by other pathogens other than LP-BM5.
양성대조군인 홍삼 식이 섭취군과 복합물 50% , 100%군에는 각각 0.582 ± 0.099 g, 0.615 ± 0.057 g, 0.571 ± 0.135 g으로 세 군의 유의적인 차이는 보이지 않았으나 LP-BM5 retrovirus 감염군과 비교하였을 때 유의적으로 감소되었음을 볼 수 있다. 전 연구의 결과 세 가지 소재의 비장 크기 증가 억제 효능을 확인한 결과를 미루어 보아, 이러한 소재를 함유한 복합물의 섭취는 LP-BM5 retrovirus 감염에 의하여 증가된 비장의 크기를 감소시킨 효능을 보였음을 알 수 있다. There was no significant difference between the groups in the 50% and 100% groups of the red ginseng diets fed with the positive control group, 0.582 ± 0.099 g, 0.615 ± 0.057 g, and 0.571 ± 0.135 g, respectively, compared with the LP-BM5 retrovirus infection group , Respectively. As a result of the previous studies, it was found that the ingestion of the complex containing this material reduced the size of the spleen increased by the LP-BM5 retrovirus infection have.
나) 림프절(lymph node)의 무게 변화B) weight change of the lymph node
LP-BM5 retrovirus의 감염이 활성화 단계에 이르게 되면 면역세포들의 활성화에 따른 지속적인 mitogenesis로 인해 림프절이 급격하게 커지는 현상이 발생하게 되며 이것은 LP-BM5 retrovirus 감염이 정상적으로 발현되고 있음을 나타내는 지표가 되기도 한다. 따라서 림프절의 크기는 감염의 정도뿐 만 아니라 특정 샘플을 섭취시켰을 때 감염의 발현 억제 정도를 간접적으로 확인할 수 있는 수단이 되기도 한다. When the LP-BM5 retrovirus infection reaches the activation stage, the lymph node becomes rapidly enlarged due to the continuous mitogenesis due to the activation of the immune cells, which is an indicator that the LP-BM5 retrovirus infection is normally expressed. Thus, the size of the lymph nodes may be a means of indirectly confirming the degree of infection as well as the degree of inhibition of infection by ingesting a particular sample.
본 실험에서 감염시키지 않은 정상군에서는 림프절을 육안으로 관찰하기 어려웠으나 LP-BM5 retrovirus 감염군에서는 크기가 커졌으며 무게도 1.763 ± 0.668 g으로 나타났다. 반면 양성대조군인 홍삼 섭취군에서는 림프절의 크기가 감소되었음을 볼 수 있었으며, 무게도 1.365 ± 0.288 g으로 감소하여 LP-BM5 retrovirus 감염군과 통계적으로 유의적인 차이를 보였다. 복합물 섭취군에서도 양성대조군과 유의적인 차이를 보이지 않을 정도로 증가된 림프절의 무게가 감소되었으며, 50%에서는 1.360 ± 0.520 g, 100%에서는 1.343 ± 0.274 g으로 감소되었으며 이는 복합물의 농도의 차이에는 유의적인 차이를 보이지 않았다. 이러한 결과는 지난 연구를 통하여 소재들의 150 mg/kg와 300 mg/kg와의 농도에 따른 림프절의 변화가 차이가 없었던 결과와 같은 결과가 나타났음을 확인할 수 있었다. In the present study, it was difficult to observe the lymph nodes visually in the normal infected group, but the size was increased in the LP-BM5 retrovirus infection group and the weight was 1.763 ± 0.668 g. In contrast, the positive control group, the red ginseng group, showed a decrease in the size of the lymph nodes and the weight decreased to 1.365 ± 0.288 g, which was statistically significantly different from the LP-BM5 retrovirus group. In the composite intake group, the weight of the lymph node increased to the extent that it did not show a significant difference from the positive control, and decreased to 1.360 ± 0.520 g at 50% and to 1.343 ± 0.274 g at 100% There was no difference. These results show that the results of the previous study showed that the changes of the lymph nodes were not different according to the concentrations of 150 mg / kg and 300 mg / kg of the materials.
다) 간의 무게 변화C) weight change between
LP-BM5의 retrovirus의 감염 시 활성산소에 의한 산화적 스트레스가 급격히 증가하는 것으로 알려져 있으며 이렇게 생성된 산화적 스트레스는 간세포에 존재하는 비타민 C와 비타민 E를 포함한 항산화 비타민과 항산화성 물질들에 의해 일부 제거되지만 바이러스 감염에 의한 지속적인 산화적 스트레스의 생성은 간세포 내의 항산화 비타민과 항산화성 물질의 고갈을 초래하게 되고 염증반응을 유발하여 간의 크기가 증가하게 되는 것으로 일반적으로 알려져 있다(Immunopharmacology. 29, pp 225-233, 1995).It is known that oxidative stress induced by reactive oxygen species (LP-BM5) is rapidly increased during infection with retrovirus. The resulting oxidative stress is caused by antioxidant vitamins and antioxidative substances including vitamin C and vitamin E in hepatocytes However, it is generally known that the production of persistent oxidative stress due to viral infection leads to depletion of antioxidant vitamins and antioxidant substances in hepatocytes, and induction of inflammatory reaction, resulting in an increase in liver size (Immunopharmacology. 29, pp 225 -233, 1995).
본 실험 결과에서 정상군에서 간의 무게가 0.730 ± 0.071 g으로 나타난 반면, LP-BM5 retrovirus 감염군에서는 1.411 ± 0.102 g으로 정상군과 비교하여 유의적으로 간의 무게가 증가하였음을 확인할 수 있다. 양성대조군, 복합물 50%, 복합물 100% 군에서는 각각 1.196 ± 0.082 g, 1.256 ± 0.058 g, 1.240 ± 0.170 g으로 나타나 LP-BM5 retrovirus 감염군에 비하여 유의적으로 간의 무게가 감소되었음을 관찰하였다. 이는 LP-BM5 retrovirus에 의하여 발생한 간의 부종을 홍삼과 복합물에 포함된 소재들의 면역조절능에 의하여 부종이 억제되었음을 알 수 있다. In the present study, the liver weight was 0.730 ± 0.071 g in the normal group, while 1.411 ± 0.102 g in the LP-BM5 retrovirus-infected group was significantly increased compared with the normal group. In the positive control group, 50% of the composite and 100% of the composite 100% groups, the weight of the liver was significantly decreased compared with that of the LP-BM5 retrovirus group, which was 1.196 ± 0.082 g, 1.256 ± 0.058 g and 1.240 ± 0.170 g, respectively. This suggests that edema of the liver caused by the LP-BM5 retrovirus is suppressed by the immunoregulatory ability of the red ginseng and its components.
라) 심장의 무게 변화D) Weight change of heart
LP-BM5 retrovirus 감염 시 심장의 무게 또는 크기가 미세하지만 증가한다는 보고가 일부 있었지만 아직 결정적인 확인은 이루어지지 않았다. 본 연구에서도 정상군과 LP-BM5 retrovirus 감염군 뿐만 아니라 모든 실험군에서 심장의 무게 변화에 대한 유의적인 차이가 없었음을 확인하였다. There have been some reports that LP-BM5 retrovirus infection is associated with a slight increase in heart weight or size, but no definitive confirmation has been made. In this study, we also observed that there was no significant difference in heart weight changes between normal and LP-BM5 retrovirus infected groups as well as all experimental groups.
마) 신장의 무게변화E) Weight change of the kidney
LP-BM5 retrovirus 감염 시 신장의 무게에 있어서 큰 변화를 보인다는 연구결과는 아직 알려지지 않았다. 이에 근거하여 정상군과 LP-BM5 retrovirus 감염군의 신장무게가 통계적으로 유의적인 차이가 없었으며, 양성대조군과 복합물 50% 및 100% 섭취 모든 군에서 유의적인 차이를 보이지 않았다.The results of a study showing that LP-BM5 retrovirus infections show a large change in kidney weight are not yet known. On the basis of this, there was no statistically significant difference in kidney weight between the normal group and the LP-BM5 retrovirus infected group, and there was no significant difference between the positive control group and the 50% and 100%
실험예 2. 면역증진 효과 측정Experimental Example 2 Measurement of immunity enhancement effect
상기 실시예 시료의 면역증진 효과를 확인하기 위하여 문헌에 개시된 방법을 응용하여 LPS로 비장의 손상을 유도한 렛트에서 시료의 IL-2, IL-10, IL-4, TNF-α, IFN-γ 등의 면역조절 인자에 대한 활성을 확인하였다(Biosci. Biotechnol. Biochem. 75, pp 1234-1239, 2011).
The
2.1. 비장세포 사이토카인 생성 능력 측정2.1. Measurement of spleen cell cytokine production ability
마취시켜 혈액을 채취한 마우스를 해부하고 비장을 적출하여 PBS로 washing한 후 0.45 cell strainer(1343776, BD Falcon)에 놓고 조직을 갈아낸 뒤 10% FBS를 포함하는 RPMI 1640 배지(Hyclone Laboratories, Logan, Utah, USA)에 세포를 모아 원심분리 시킨 뒤 적혈구 분해 용액을 넣어 1분간 반응 시킨 뒤 2회 더 원심분리 하였다. 여기에 Trypan blue 염색약(1129155, Gibco)으로 세포를 염색시켜 숫자를 센 뒤, 96-well plate에 1×106 cells/200μL의 농도로 세포를 채운다. IL-2, IL-10 측정을 위하여 Con A (C5275, Sigma) 5㎍/mL 처리 후 24시간 동안 incubation 시켰으며, IFN-γ은 72시간 동안 incubation 시켰고, TNF-α 측정을 위하여 LPS 5㎍/mL 처리 후 24시간동안 incubation 시킨 후 상층의 배양액을 사용하여 사이토카인들의 양을 측정하였다. The mice were harvested by anesthesia and dissected. The spleens were harvested, washed with PBS, and placed on a 0.45 cell strainer (1343776, BD Falcon). The tissues were ground and cultured in RPMI 1640 medium (Hyclone Laboratories, Logan, Utah, USA), centrifuged and incubated for 1 minute with red cell dissolving solution and then centrifuged two more times. Cells are stained with trypan blue dye (1129155, Gibco) and counted. Fill cells in a 96-well plate at a concentration of 1 × 10 6 cells / 200 μL. IL-2 and IL-10 were incubated for 24 h after 5 ㎍ / mL of Con A (C5275, Sigma). IFN-γ was incubated for 72 h, mL and incubated for 24 hours. The amount of cytokines was measured using the culture medium of the upper layer.
사이토카인 측정은 Duoset sandwich ELISA Mouse kit(R&D system)을 이용하여 측정하였다. 코팅된 96-well plate에 IL-2, IL-10, IL-4, TNF-α, IFN-γ 측정에 특성화된 1차 항체를 PBS에 희석해 100 μl씩 분주해 하루 동안 처리하여 다음날, washing buffer (PBST, 0.05% Tween 20 in PBS)로 1차 항체를 씻어낸 뒤, 항체가 붙지 않은 plate의 다른 공간을 메워주기 위해 assay buffer (1% BSA in PBS)를 넣어 2시간 동안 처리한 뒤 washing buffer로 씻어냈다. Standard curve를 위한 용액과 위에서 LPS(032M4082V, Sigma)와 Con A를 처리한 비장세포 배양액을 100 μl씩 각 well에 넣어 2시간 동안 반응시켰다. 반응이 끝난 뒤 washing buffer로 씻어내고 assay buffer에 2차 항체를 희석시켜 준비한 뒤 각 well에 100 μL씩 분주하고 2시간 동안 처리하였다. 이 과정이 끝나면 washing buffer를 이용해 plate를 씻어내고 발색을 도와주는 시약을 넣어 반응 시킨 뒤 570 nm에서 흡광도를 측정하였고 standard curve를 이용해 세포에서 생성된 사이토카인 양을 계산하였다.
The cytokine measurement was performed using a DuoSet sandwich ELISA mouse kit (R & D system). In the coated 96-well plate, the primary antibody specific for IL-2, IL-10, IL-4, TNF-α and IFN-γ was diluted in PBS and dispensed in a 100 μl aliquot. After washing the primary antibody with PBS (0.05
2.2. 결 과(2.2. result( Th1Th1 typetype cytokinecytokine 의 발현 조절)Lt; / RTI >
2.2.1 2.2.1 ILIL -2 -2 cytokinecytokine 발현 Expression
IL-2는 면역조절에 관여하는 단백질로서, T-cell growth factor (TCGF)라고 알려져 있다. T-cell을 증식시키고 T-cell에 작용하여 Interferon-γ을 분비시키는 작용을 하며, B-cell을 자극시키는 활성이 있다. 또한 Natural killer (NK) cells, lymphokine-activated killer cells과 같은 면역세포를 활성화시킨다. 일반적으로 LP-BM5 retrovirus에 감염이 되면 Th1 type cytokine의 발현은 급격히 감소하는 경향을 나타내며 IL-2의 발현 또한 감소하는 경향을 나타낸다. LP-BM5 retrovirus의 감염 시 비장세포의 면역세포의 활성은 상당부분 억제를 받게 되는데 이에 대한 반응성을 확인하고자 mitogen으로서 Con A를 처리하여 Th1 type cytokine의 하나인 IL-2의 발현을 확인하였다. (도 3)
IL-2 is a protein involved in immunomodulation, known as T-cell growth factor (TCGF). It acts to proliferate T-cell, acts on T-cell, secretes interferon-γ, and has activity to stimulate B-cell. It also activates immune cells such as natural killer (NK) cells and lymphokine-activated killer cells. In general, when LP-BM5 retrovirus is infected, the expression of Th1 type cytokine tends to decrease rapidly and the expression of IL-2 tends to decrease. In the case of LP-BM5 retrovirus infection, the immunoreactivity of splenocytes was significantly inhibited. Con A was treated as a mitogen to confirm the reactivity of IL-2, which is one of the Th1 type cytokines. (Fig. 3)
그 결과 정상군에서는 168.32 ± 20.61 pg/mL으로 나타난 반면, LP- BM5 retrovirus의 감염군에서는 34.95 ± 4.82 pg/mL으로 유의적으로 크게 감소되었음을 확인하였다. 이는 앞서 말한 LP-BM5 retrovirus가 IL-2 발현을 억제시킨다는 이론과 같은 결과를 보였음을 알 수 있다. 양성대조군은 홍삼을 섭취한 군에서는 62.19 ± 3.99 pg/mL으로 LP-BM5 retrovirus감염군과 비교하여 유의적으로 증가되었음을 확인하였다. 또한 복합물 50%와 복합물 100% 각각 67.73 ± 8.75 pg/mL, 72.74 ± 7.25 pg/mL으로 양성대조군과 유의적인 차이를 보이지 않을 정도로 IL-2 발현이 증가되었음을 확인하였다. 따라서 복합물의 두 농도는 통계적으로 유의적인 차이를 보이지 않게 유사한 결과를 나타내었다.
The results were as follows: 168.32 ± 20.61 pg / mL in normal group, 34.95 ± 4.82 pg / mL in LP-BM5 retrovirus infection group. This suggests that the above-mentioned LP-BM5 retrovirus inhibits IL-2 expression. The positive control group was 62.19 ± 3.99 pg / mL in the red ginseng group, which was significantly increased compared with the LP-BM5 retrovirus-infected group. In addition, the expression of IL-2 was increased to a level of 67.73 ± 8.75 pg / mL and 72.74 ± 7.25 pg / mL, respectively, which were not significantly different from the positive control, 50% for the complex and 100% for the composite. Therefore, the two concentrations of the composite showed similar results with no statistically significant difference.
2.2.2. IFN-γ의 발현2.2.2. Expression of IFN-y
IFN-γ의 발현은 Th1 type cytokine과 Th2 type cytokine을 상대적으로 보완조절하는 중요한 역할을 IL-10과 함께 담당하고 있어서 전체적인 면역조절과 항상성 유지에 매우 중요한 cytokine으로 인식되고 있다. 이러한 IFN-γ의 발현은 LP-BM5 retrovirus 감염 시 급격히 감소하여 Th1 type cytokine은 감소하게 되고 Th2 type cytokine은 증가하게 되는 경향을 나타내는 조절 인자이기도 하다. The expression of IFN-γ plays an important role in regulating Th1-type and Th2-type cytokines relative to IL-10, and is recognized as an important cytokine for overall immune regulation and homeostasis. This expression of IFN-γ is abruptly decreased upon LP-BM5 retrovirus infection, and Th1 type cytokine is decreased and Th2 type cytokine is also increasingly regulated.
본 연구 결과에서 감염되지 않은 정상군에서는 1385.29 ± 170.48 pg/mL으로 유의적으로 가장 높은 발현을 나타냈으며, LP-BM5 retrovirus애 감염된 군에서는 251.39 ± 197.71 pg/mL로 크게 감소되었음을 확인하였다. 따라서 앞서 말한 이론과 맞는 결과가 나타나 LP-BM5 retrovirus 유발이 정상적으로 이루어졌음을 알 수 있다. 반면 홍삼을 섭취한 양성대조군에서는 934.42 ± 134.79 pg/mL으로 LP-BM5 retrovirus감염으로 감소된 IFN-γ의 발현을 억제시켰음을 확인하였다. 또한 복합물 섭취한 군에서는 50%와 100% 각각 992.62 ± 214.05 pg/mL, 1047.66 ± 187.60 pg/mL으로 LP-BM5 retrovirus감염군과 유의적인 차이가 나도록 발현이 증가되었다. (도 4)
In the present study, the highest expression level was 1385.29 ± 170.48 pg / mL in the uninfected normal group and 251.39 ± 197.71 pg / mL in the LP-BM5 retrovirus-infected group. Therefore, the LP-BM5 retrovirus induction was normalized in accordance with the above-mentioned theory. In contrast, the positive control group of red ginseng inhibited the expression of IFN-γ, which was reduced by LP-BM5 retrovirus infection to 934.42 ± 134.79 pg / mL. In addition, 50% and 100% of the complex-fed group showed 992.62 ± 214.05 pg / mL and 1047.66 ± 187.60 pg / mL, respectively, which were significantly increased compared with the LP-BM5 retrovirus infection group. (Figure 4)
2.3. 결 과 (Th2 type cytokine의 발현 조절)2.3. Results (Th2 type cytokine expression control)
2.3.1. 2.3.1. TNFTNF -α의 발현-α expression
TNF-α는 암세포를 파괴하는 작용뿐만 아니라 염증작용, 면역세포를 활성화하는 작용을 한다. 체내에 감염원이 유입되었을 때, 면역에 관련된 모든 세포들은 염증을 발생시키는 사이토카인을 분비하게 되며 이 염증 매개물질이 병적으로 급격히 과발현되면 체내 세포의 DNA, 지질, 항산화 영양소는 손상을 입게 되고 이로 인해 발암 가능성이 높아질 수 있다. 따라서 염증성 사이토카인의 발현을 억제하면 항염증 효과가 있고, 면역 조절에 도움을 준다고 말할 수 있다. 특히 LP-BM5 retrovirus 감염의 경우 TNF-α의 상승과 더불어 염증반응 및 산화적 스트레스와 관련된 인자로도 알려져 있다.TNF-a acts not only to destroy cancer cells, but also to activate inflammatory and immune cells. When an infectious agent is introduced into the body, all cells involved in the immune system secrete cytokines that cause inflammation. When the inflammatory mediator is rapidly over-expressed, DNA, lipids, and antioxidant nutrients in the body cells are damaged. The possibility of carcinogenesis can be increased. Thus, inhibiting the expression of inflammatory cytokines has anti-inflammatory effects and can be said to help control immune responses. In particular, LP-BM5 retrovirus infection is known to be associated with inflammatory response and oxidative stress as well as TNF-α uptake.
본 실험에서 LP-BM5 retrovirus 감염군의 TNF-α 발현은 422.0 ± 19.66 pg/mL으로 정상군(54.13 ± 12.49 pg/mL)의 발현과 비교하여 크게 증가되었다. 반면 양성대조군은 홍삼 식이 섭취군은 321.0 ± 3.00 pg/mL으로 LP-BM5 retrovirus 감염군과 비교하여 유의적으로 감소되었으며, 복합물 50%와 100%군에서는 각각 319.49 ± 61.46 pg/mL, 328.34 ± 25.08 pg/mL으로 양성대조군과 유의적인 차이를 보이지 않을 정도로 감소되었다. (도 5)
TNF-α expression in the LP-BM5 retrovirus-infected group was 422.0 ± 19.66 pg / mL, which was significantly higher than that in the normal group (54.13 ± 12.49 pg / mL). In contrast, the positive control group was significantly lower than the LP-BM5 retrovirus infected group with 321.0 ± 3.00 pg / mL in the red ginseng diet group, and 319.49 ± 61.46 pg / mL and 328.34 ± 25.08 in the 50% and 100% pg / mL, which was not significantly different from the positive control group. (Fig. 5)
2.3.2. 2.3.2. ILIL -- 4 의4 of 발현 Expression
IL-4는 CD-4 T-cell과 activated mast cell에 의해 만들어지며, B-cell growth factor의 작용을 한다. 또한 B-cell의 immunogloblin의 class switch에 관여하는 differentiation factor로 작용할 수 있으며, CD4 T-cell과 mast cell, macrophage등을 활성하기도 한다. LP-BM5 retrovirus에 감염되면 이러한 IL-4 발현은 증가하여 염증반응을 일으킨다고 알려져 있다. IL-4 is produced by CD-4 T-cells and activated mast cells and acts as a B-cell growth factor. In addition, it can act as a differentiation factor involved in the class switch of immunoglobulin of B-cell, and it also activates CD4 T-cell, mast cell, and macrophage. It is known that when infected with LP-BM5 retrovirus, IL-4 expression increases and inflammation reaction occurs.
본 연구 결과에서도, LP-BM5 retrovirus 감염(45.14 ± 17.01 pg/mL)으로 정상군(5.88 ± 3.64 pg/mL)에 비하여 IL-4 발현이 유의적으로 증가하였음을 확인하였다. 하지만 이러한 IL-4의 발현 증가를 홍삼 식이 섭취(24.78 ± 11.61 pg/mL)가 유의적으로 감소시켰다. 또한 복합물 50% 식이섭취군과 복합물 100% 식이섭취군은 각각 20.01 ± 6.93 pg/mL, 19.47 ± 5.59 pg/mL으로 발현이 LP-BM5 retrovirus 감염군에 비하여 감소되었다. 복합물의 농도에 따른 유의적인 차이를 보이지 않았으며 이 또한 위의 결과들과 마찬가지로 소재들의 농도별 유의적인 차이가 없었던 영향으로 보인다. (도 6)
In the present study, it was also confirmed that IL-4 expression was significantly increased in the LP-BM5 retrovirus infection (45.14 ± 17.01 pg / mL) compared to the normal group (5.88 ± 3.64 pg / mL) However, the increased expression of IL-4 was significantly reduced by the ingestion of red ginseng (24.78 ± 11.61 pg / mL). In addition, the 50% dietary intake of the compound and the 100% dietary intake of the complex were 20.01 ± 6.93 pg / mL and 19.47 ± 5.59 pg / mL, respectively, compared to the LP-BM5 retrovirus infection group. There was no significant difference according to the concentration of the composite, and this also seems to be the effect that there was no significant difference in the concentration of the materials like the above results. (Fig. 6)
2.3.3. 2.3.3. ILIL -- 10 의Ten 발현 Expression
IL-10의 발현은 Th1 type cytokine을 상대 보완적으로 억제하여 비감염시 Th1 type 과 Th2 type cytokine의 항상성을 유지하는 역할을 담당하고 있다. 하지만 LP-BM5 retrovirus감염 시 과도하게 증가하여 Th2 type cytokine은 증가시키고 상대적으로 Th1 type cytokine을 억제시켜 항상성을 파괴하는 원인이 되기도 한다. The expression of IL-10 plays a role in maintaining Th1 type and Th2 type cytokine homeostasis by inhibiting Th1 type cytokine. However, when LP-BM5 retrovirus is infected, it is excessively increased and Th2 type cytokine is increased and relatively Th1 type cytokine is inhibited, which causes destruction of homeostasis.
본 실험 결과 정상군에서는 IL-10의 발현양이 6.60 ± 5.14 pg/mL인 반면, LP-BM5 retrovirus 감염군에서는 165.12 ± 37.43 pg/mL으로 25배 이상 IL-10의 발현이 증가하였다. 양성대조군인 홍삼 섭취군에서는 72.23 ± 23.64 pg/mL으로 LP-BM5 retrovirus 감염군과 비교하여 유의적으로 발현이 감소되었음을 확인하였다. 또한 복합물 50%는 108.72 ± 30.76 pg/mL으로, 복합물 100%는 101.60 ± 21.84 pg/mL으로 발현양이 LP-BM5 retrovirus 감염군과 비교하여 유의적으로 감소하였으나, 이는 양성대조군보다는 유의적으로 높았다. 또한 다른 결과 값과 마찬가지로 복합물 50%와 100%의 유의적인 차이는 보이지 않았다.(도 7)
The expression of IL-10 in normal group was 6.60 ± 5.14 pg / mL, whereas that of LP-BM5 retrovirus group was 165.12 ± 37.43 pg / mL. In the positive control group, 72.23 ± 23.64 pg / mL in the red ginseng group was significantly decreased compared to the LP-BM5 retrovirus group. In addition, the expression level of the complex 50% was 108.72 ± 30.76 pg / mL and that of the complex 100% was 101.60 ± 21.84 pg / mL, which was significantly lower than that of the LP-BM5 retrovirus infection group, but was significantly higher than that of the positive control group . As with the other results, no significant difference was observed between the 50% and 100% complexes (Figure 7).
실험예Experimental Example 3. T 세포의 3. T cells mitogenmitogen 에 대한 For 증식능Proliferative ability 반응성 측정 Reactivity measurement
상기 실시예 시료의 T 세포의 mitogen에 대한 증식능에 미치는 영향을 확인하기 위하여 문헌에 게재된 방법을 응용하여 확인하였다 (Biosci. Biotechnol. Biochem. 75, pp 1234-1239, 2011).Biosci. Biotechnol. Biochem. 75, pp 1234-1239, 2011) was used to confirm the effect of the sample of the above example on the ability of the T cell to proliferate against the mitogen.
3.1. 실험 과정3.1. Experimental course
마취시켜 혈액을 채취한 마우스를 해부하고 비장을 적출하여 PBS로 washing한 후 0.45 cell strainer에 놓고 조직을 갈아낸 뒤 10% FBS를 포함하는 RPMI 1640 배지에 세포를 모아 원심분리 시킨 뒤 적혈구 분해 용액을 넣어 1분간 반응 시킨 뒤 2회 더 원심분리 한다. 여기에 Trypan blue 염색약(1129155, Gibco)으로 세포를 염색시켜 숫자를 센 뒤, 96-well plate에 1×107 cells/ml의 농도로 세포를 채운 뒤, Con A (5 μg/mL)를 각각 처리하여 48시간 동안 37℃, 5% CO2에서 incubation시킨다. 그 후 EZ-CyTox를각 well에 10 μL씩 분주한 후 4시간 동안 incubation 시킨 뒤 450 nm에서 흡광도를 측정하였다.
The cells were collected by centrifugation in RPMI 1640 medium containing 10% FBS, and then the red blood cell lysate solution was centrifuged. After incubation for 1 min, centrifuge two more times. Cells were stained with Trypan blue dye (1129155, Gibco), counted, filled in 96-well plates at a density of 1 × 10 7 cells / ml, and then Con A (5 μg / mL) And incubated at 37 ° C, 5% CO 2 for 48 hours. Then, 10 μL of EZ-CyTox was dispensed into each well, incubated for 4 hours, and absorbance was measured at 450 nm.
3.2. 결과 (T 세포의 3.2. Results (T cell mitogenmitogen 에 대한 For 증식능Proliferative ability 반응성 측정) Reactivity measurement)
외부로부터 유래한 항원이 체내에 침투 시, 면역체계에서는 antigen presenting cell이 이를 인식하여 MHC (Major Histocompatibility complex) molecule에 결합시켜, 작은 peptide 형태로 T cell에 전달되게 된다. 면역반응에서 중요한 역할을 담당하는 T cell은 다양한 항원에 대하여 특이적인 T cell receptor들을 가지고 있어, antigen presenting cell에 의해 전달되는 항원 유래 peptide에 특이적인 T cell만 활성화 되고, 이후 전개되는 면역 반응에 필요한 cytokine들의 분비와 활성화된 T cell clone의 증식이 일어나게 된다. LP-BM5 retrovirus의 감염으로 인한 T세포의 활성감소는 CD4 수용체를 경유하는 LP-BM5 retrovirus의 감염경로와 관련이 있는데 주도적으로 T helper 세포가 영향을 받아 사멸하거나 정상적인 작용을 하기 힘든 anergy 상태로 되기 때문이다. 따라서 본 실험에서는 세포를 활성화 시킬 수 있도록 mitogen을 처리한 후 각각의 식이를 투여한 군에서 어떠한 반응이 나타나는 지 확인하였다.When an antigen derived from the outside penetrates into the body, the antigen presenting cell recognizes it in the immune system and binds to MHC (Major Histocompatibility complex) molecule, and is transferred to T cell in small peptide form. T cells, which play an important role in the immune response, have specific T cell receptors for various antigens, so only T cells specific for antigen-derived peptides delivered by antigen presenting cells are activated, and are required for the subsequent immune response secretion of cytokines and proliferation of activated T cell clones. The reduction of T cell activity due to LP-BM5 retrovirus infection is related to the pathway of LP-BM5 retrovirus infection via the CD4 receptor, primarily due to the influence of T helper cells, resulting in anergy state Because. Therefore, in this experiment, mitogens were treated to activate the cells, and then the reaction was observed in each diet.
정상군의 T 세포 증식능을 100%로 하였을 때 LP-BM5 retrovirus의 감염군은 59.64 ± 15.92%의 증식능을 보여 정상군에 비하여 유의적으로 크게 감소하였음을 확인하였다. 반면 홍삼 식이 섭취군에서는 82.55 ± 6.27%로 LP-BM5 retrovirus의 감염군에 비하여 T 세포 증식능을 유의적으로 증가시켰음을 알 수 있다. 복합물 50%에서는 83.38 ± 12.43%, 100%에서는 93.41 ± 8.43%로 100%에서 높은 경향을 보였으나 두 군간에 유의적인 차이는 보이지 않았으며, 두 군 모두에서 양성대조군와 유의적인 차이를 보이지 않을 정도로 LP-BM5 retrovirus의 감염군에 비하여 T 세포 증식능이 증가하였다.(도 8)
When the T-cell proliferative capacity of the normal group was taken as 100%, the LP-BM5 retrovirus infected group showed a significant decrease of 59.64 ± 15.92% compared to the normal group. On the other hand, 82.55 ± 6.27% of the red ginseng group showed significant increase in T cell proliferative capacity compared to the LP-BM5 retrovirus group. In the 50% of the composite, 83.38 ± 12.43% and 93.41 ± 8.43% in 100%, respectively. However, there was no significant difference between the two groups, and the LP -BM5 retrovirus (Fig. 8). ≪ tb >< TABLE >
실험예Experimental Example 4. B 세포의 4. B cells mitogenmitogen 에 대한 For 증식능Proliferative ability 반응성 측정 Reactivity measurement
상기 실시예 시료의 B 세포의 mitogen에 대한 증식능에 미치는 영향을 확인하기 위하여 문헌에 게재된 방법을 응용하여 확인하였다(Biosci. Biotechnol. Biochem. 75, pp 1234-1239, 2011).Biosci. Biotechnol. Biochem. 75, pp 1234-1239, 2011). In order to confirm the effect of the sample of the example on the mitogen-promoting ability of B cells, the method described in the literature was applied.
4.1. 실험 과정4.1. Experimental course
마취시켜 혈액을 채취한 마우스를 해부하고 비장을 적출하여 PBS로 washing한 후 0.45 cell strainer에 놓고 조직을 갈아낸 뒤 10% FBS를 포함하는 RPMI 1640 배지에 세포를 모아 원심분리 시킨 뒤 적혈구 분해 용액을 넣어 1분간 반응 시킨 뒤 2회 더 원심분리 한다. 여기에 Trypan blue 염색약(1129155, Gibco)으로 세포를 염색시켜 숫자를 센 뒤, 96-well plate에 1×107 cells/ml의 농도로 세포를 채운 뒤, LPS (5 μg/mL)를 각각 처리하여 48시간 동안 37℃, 5% CO2에서 incubation시킨다. 그 후 EZ-CyTox를각 well에 10 μL씩 분주한 후 4시간 동안 incubation 시킨 뒤 450 nm에서 흡광도를 측정하였다.
The cells were collected by centrifugation in RPMI 1640 medium containing 10% FBS, and then the red blood cell lysate solution was centrifuged. After incubation for 1 min, centrifuge two more times. Cells were stained with trypan blue dye (1129155, Gibco) and counted. Cells were filled in a 96-well plate at a concentration of 1 × 10 7 cells / ml and treated with LPS (5 μg / mL) Incubate at 37 ° C, 5% CO 2 for 48 hours. Then, 10 μL of EZ-CyTox was dispensed into each well, incubated for 4 hours, and absorbance was measured at 450 nm.
4.2. 결과4.2. result
본 연구에서 정상군의 B 세포 증식능이 100%였을 때 LP-BM5 retrovirus의 감염군은 45.68 ± 7.24%으로 크게 감소되었다. 반면 홍삼 식이 섭취군에서는 63.59 ± 15.00%로 LP-BM5 retrovirus의 감염으로 감소되었던 B 세포 증식능이 유의적으로 증가되었다. 복합물 50% 식이 섭취군에서는 58.7 1 ± 11.18%으로 LP-BM5 retrovirus의 감염군과 유의적인 차이를 보이지 않았지만 복합물 100% 군에서는 70.43 ± 12.50%로 양성대조군과 유의적인 차이를 보이지 않을 정도로 증가되었다.(도 9)
In this study, LP-BM5 retrovirus infection was significantly reduced to 45.68 ± 7.24% when normal B-cell proliferative capacity was 100%. On the other hand, the B cell proliferative capacity, which was reduced by LP-BM5 retrovirus infection, was significantly increased to 63.59 ± 15.00% in the red ginseng diet group. In the 50% dietary supplement group, 58.7 1 ± 11.18% was not significantly different from the LP-BM5 retrovirus group, but in the 100% composite group, the difference was 70.43 ± 12.50%, which was not significantly different from the positive control group. (Fig. 9)
실험예Experimental Example 5. 5. SuperoxideSuperoxide dismutasedismutase (( SODSOD ) 효소 활성 측정) Enzyme activity measurement
상기 실시예 시료의 Superoxide dismutase(SOD) 효소 활성에 미치는 영향을 확인하기 위하여 문헌에 게재된 방법을 응용하여 확인하였다(J Korean Soc Food Sic Nutr. 40, pp 525-535, 2011).
(J Korean Soc Food Sic Nutr. 40, pp 525-535, 2011). In order to confirm the effect of SOD on the activity of superoxide dismutase (SOD) enzyme of the above example,
5.1. 실험 과정5.1. Experimental course
동물을 희생시켜 얻은 혈액을 16000 rpm에서 20분 동안 원심분리하여 혈청을 얻은 후 SOD 효소 활성을 측정하였다. Animal sacrifice was centrifuged at 16000 rpm for 20 minutes to obtain serum, and SOD enzyme activity was measured.
SOD 효소 활성은 SOD assay Kit-WST (Dojindo Molecular Technologies, Rockville, MD)를 이용하여 지시대로 측정하였다. 96-well plate에 세포 파쇄 상층액을 넣고 WST-1(2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl) -2H-tetrazolium, mono-sodium salt과 xanthine oxidase working solution을 첨가한 후 37℃에서 20분간 배양한 후 microplate reader(VERSAMAXSL-20, Molecular Devices)를 이용하여 450 nm 파장에서 흡광도를 측정하였다. SOD 활성도는 하기 수학식 1로 계산하였다. SOD enzyme activity was measured as directed using SOD assay Kit-WST (Dojindo Molecular Technologies, Rockville, Md.). The cell lysate was added to a 96-well plate and the cell supernatant was added with WST-1 (2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium, mono-sodium salt xanthine oxidase working solution was added and incubated at 37 ° C for 20 minutes and absorbance was measured at 450 nm wavelength using a microplate reader (VERSAMAXSL-20, Molecular Devices).
여기에서 A1,A2, A3, AS는 각각 uninhibited test, blank sample, blank reagent, 그리고 sample의 측정값이다.
Where A1, A2, A3, and AS are measured values of uninhibited test, blank sample, blank reagent, and sample, respectively.
5.2. 항산화효소 활성 변화5.2. Antioxidant enzyme activity change
LP-BM5 retrovirus의 감염에 의한 면역결핍 증상이 진행될 때 세포내의 산화적 스트레스가 급격히 증가하는 것으로 알려져 있다. 특히 iNOS의 발현증가, NF-kB의 활성화, 그리고 COX-2와 proinflammatory cytokine 유전자의 발현 또한 산화적 스트레스와 관련이 있어 면역결핍으로 인한 세포의 노화 및 손상에 영향을 미치는 것으로 알려져 있다. 따라서 본 연구에서는 이와 관련하여 간세포에서의 SOD, glutathione peroxidase의 항산화 효소의 활성을 측정하여 각 추출물의 투여가 항산화작용에 어떠한 영향을 미쳤는지를 확인하였다.It is known that oxidative stress in the cell rapidly increases when immunodeficiency symptoms due to infection of LP-BM5 retrovirus are progressed. In particular, increased expression of iNOS, activation of NF-kB, and the expression of COX-2 and the proinflammatory cytokine gene are also associated with oxidative stress and are known to affect cell senescence and damage due to immunodeficiency. Therefore, in this study, we measured the antioxidant enzyme activity of SOD and glutathione peroxidase in hepatocytes, and confirmed the effect of each extract on antioxidant activity.
SOD는 산화적 스트레스에 대한 세포의 방어에 일차적으로 관여하는 효소로써 과산화물을 과산화수소로 전환하는 과정을 촉매한다. 이 때 합성된 과산화수소는 GSH-Px 에 의해 물과 산소로 분해되고, 과산화수소의 농도가 증가하게 되면 CAT 도 작용하게 되고 GSH-Px는 세포질과 미토콘드리아에 존재하며 과산화수소의 축적을 막아 조직이 손상되는 것을 방어하는 기능을 하며, SOD에 의해 생성된 과산화수소를 GSH -Px와 함께 물과 산소로 분해시킴으로서 활성산소종을 분해시킨다. SOD is an enzyme primarily involved in the defense of cells against oxidative stress and catalyzes the conversion of peroxides to hydrogen peroxide. At this time, the synthesized hydrogen peroxide is decomposed into water and oxygen by GSH-Px. When the concentration of hydrogen peroxide increases, CAT also acts. GSH-Px exists in the cytoplasm and mitochondria and prevents accumulation of hydrogen peroxide And decomposes active oxygen species by decomposing hydrogen peroxide produced by SOD with water and oxygen together with GSH-Px.
본 연구에서는 정상군에서 SOD 활성이 57.17 ± 5.93%으로 나타난 반면 LP-BM5 retrovirus 감염군에서는 30.52 ± 2.74%으로 유의적으로 활성이 감소하였음을 확인하였다. 이는 LP-BM5 retrovirus 감염에 의하여 산화적 스트레스가 발생하였으며 이러한 산화적 스트레스가 항산화 효소의 정상적인 활성을 방해하였다고 할 수 있다. 이러한 SOD 활성 감소는 홍삼, 복합물 식이 섭취군에서 유의적으로 활성을 증가시켰다. 홍삼 식이 섭취군은 36.78 ± 4.21%, 복합물 50%는 40.37 ± 4.68%, 복합물 100%는 38.85 ± 5.77%로 나타났으며 세 군간의 유의적인 차이는 보이지 않았다. (도 10)
In this study, SOD activity in normal group was 57.17 ± 5.93%, whereas in LP-BM5 retrovirus group, activity was 30.52 ± 2.74%. This suggests that oxidative stress was induced by LP-BM5 retrovirus infection and that oxidative stress interfered with the normal activity of antioxidant enzymes. The decrease of SOD activity was significantly increased in red ginseng and mixed dietary group. 36.78 ± 4.21%, 40.37 ± 4.68%, and 38.85 ± 5.77%, respectively, of the red ginseng diet and 38% and 5.77% of the mixed ginseng were not different. (Fig. 10)
실험예Experimental Example 6. 6. GlutathioneGlutathione peroxidaseperoxidase (( GSHGSH -- PxPx ) 효소 활성 측정) Enzyme activity measurement
상기 실시예 시료의 Glutathione peroxidase(GSH-Px) 효소 활성에 미치는 영향을 확인하기 위하여 문헌에 게재된 방법을 응용하여 확인하였다(J Korean Soc Food Sic Nutr. 38, pp 423-429, 2009).(J Korean Soc Food Sic Nutr. 38, pp 423-429, 2009). In order to confirm the effect of Glutathione peroxidase (GSH-Px) on the enzyme activity of the above example.
6.1. 실험과정6.1. Experimental course
동물을 희생시켜 얻은 혈액을 16000 rpm에서 20분 동안 원심분리하여 혈청을 얻은 후 GSH-Px 효소 활성을 측정하였다. GSH-Px 효소 활성은 Tappel의 방법(Korean J Plant Res. 24, pp 105-112, 2011).을 사용하여 Glutathione peroxidase activity assay kit (BioVision Inc., CA, USA)를 이용하여 측정하였다. 96well plate에 NADH, Glutathione, Glutathione reductase용액을 첨가한 후 340nm에서 흡광도의 변화값을 측정하였다.
Animal sacrifice was centrifuged at 16000 rpm for 20 minutes to obtain serum, and GSH-Px enzyme activity was measured. GSH-Px enzyme activity was measured using the Glutathione peroxidase activity assay kit (BioVision Inc., CA, USA) using the method of Tappel (Korean J Plant Res. 24, pp 105-112, 2011). After adding NADH, Glutathione, and Glutathione reductase solution to 96 well plate, the change of absorbance at 340 nm was measured.
6.2. 결과6.2. result
Glutathione peroxidase(GPx) 활성을 측정한 결과, 정상군에서 63.13 ± 4.06 mU/mL으로 활성이 나타난 반면, LP-BM5 retrovirus의 감염에 의하여 39.21 ± 3.83 mU/mL으로 크게 감소하였다. 양성대조군인 홍삼 식이 섭취군에서는 52.52 ± 7.96 mU/mL으로 LP-BM5 retrovirus의 감염에 의하여 감소된 활성을 증가시켰다. 또한 복합물 50%에서는 51.25 ± 9.29 mU/mL으로, 100%에서는 54.16 ± 6.87 mU/mL으로 두 군간의 유의적인 차이가 나지 않게 두 군 모두 LP-BM5 retrovirus의 감염군과 비교하여 유의적으로 증가하였다.(도 11)
Activity of Glutathione peroxidase (GPx) activity was 63.13 ± 4.06 mU / mL in normal group, but decreased to 39.21 ± 3.83 mU / mL by LP-BM5 retrovirus infection. And 52.52 ± 7.96 mU / mL in the red ginseng diet group, which is a positive control group, by the LP-BM5 retrovirus infection. In addition, 51.25 ± 9.29 mU / mL in 50% of the complex and 54.16 ± 6.87 mU / mL in 100% were not significantly different between the two groups, which was significantly increased in both groups compared with the LP-BM5 retrovirus infection group . (Fig. 11)
본 발명의 추출물들을 포함하는 조성물의 제제 예를 설명하나, 본 발명은 이를 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다.
The formulation examples of the compositions containing the extracts of the present invention are illustrated, but the present invention is not intended to be limited thereto but is only specifically described.
제제예 1. 산제의 제조Preparation Example 1. Preparation of powder
복합물 ------------------------------------------- 20 ㎎Composition ------------------------------------------- 20 mg
유당 -------------------------------------------- 100 ㎎Lactose -------------------------------------------- 100 mg
탈크 --------------------------------------------- 10 ㎎Talc --------------------------------------------- 10 mg
상기의 성분들을 혼합하고 기밀포에 충진하여 산제를 제조한다.
The above components are mixed and filled in airtight bags to prepare powders.
제제예 2. 정제의 제조Formulation Example 2. Preparation of tablets
복합물 ------------------------------------------- 10 ㎎Composition ------------------------------------------- 10 mg
옥수수전분 -------------------------------------- 100 ㎎Corn starch -------------------------------------- 100 mg
유당 -------------------------------------------- 100 ㎎Lactose -------------------------------------------- 100 mg
스테아린산 마그네슘 ------------------------------- 2 ㎎Magnesium stearate ------------------------------- 2 mg
상기의 성분들을 혼합한 후 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조한다.
After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.
제제예 3. 캅셀제의 제조Formulation Example 3. Preparation of capsules
복합물 ------------------------------------------- 10 ㎎Composition ------------------------------------------- 10 mg
결정성 셀룰로오스 --------------------------------- 3 ㎎Crystalline cellulose - 3 mg
락토오스 --------------------------------------- 14.8 ㎎Lactose --------------------------------------- 14.8 mg
마그네슘 스테아레이트 --------------------------- 0.2 ㎎Magnesium stearate - 0.2 mg
통상의 캡슐제 제조방법에 따라 상기의 성분을 혼합하고 젤라틴 캡슐에 충전하여 캡슐제를 제조한다.
The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.
제제예 4. 주사제의 제조Formulation Example 4. Preparation of injection
복합물 ------------------------------------------- 10 ㎎Composition ------------------------------------------- 10 mg
만니톨 ------------------------------------------ 180 ㎎Mannitol ------------------------------------------ 180 mg
주사용 멸균 증류수 ----------------------------- 2974 ㎎Sterile sterilized distilled water for injection - 297 mg
Na2HPO4·12H2O ------------------------------------ 26 ㎎Na 2 HPO 4 .12H 2 O ------------------------------------ 26 mg
통상의 주사제의 제조방법에 따라 1 앰플당(2 ㎖) 상기의 성분 함량으로 제조한다.
(2 ml) per ampoule in accordance with the usual injection method.
제제예 5. 액제의 제조Formulation Example 5. Preparation of a liquid preparation
복합물 ------------------------------------------- 20 ㎎Composition ------------------------------------------- 20 mg
이성화당 ------------------------------------------ 10 gIsomerization Party ------------------------------------------ 10 g
만니톨 --------------------------------------------- 5 gMannitol --------------------------------------------- 5 g
정제수 -------------------------------------------- 적량Purified water --------------------------------------------
통상의 액제의 제조방법에 따라 정제수에 각각의 성분을 가하여 용해시키고 레몬향을 적량 가한 다음 상기의 성분을 혼합한 다음 정제수를 가하여 전체를 정제수를 가하여 전체 100㎖로 조절한 후 갈색병에 충진하여 멸균시켜 액제를 제조한다.
Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.
제제예 6. 건강 식품의 제조 Formulation Example 6. Preparation of Healthy Foods
복합물 ----------------------------------------- 1000 ㎎Compound ----------------------------------------- 1000 mg
비타민 혼합물 ------------------------------------- 적량Vitamin mixture -------------------------------------
비타민 A 아세테이트 ------------------------------ 70 ㎍Vitamin A Acetate ------------------------------ 70 g
비타민 E ---------------------------------------- 1.0 ㎎Vitamin E ---------------------------------------- 1.0 mg
비타민 B1 --------------------------------------- 0.13 ㎎Vitamin B 1 --------------------------------------- 0.13 mg
비타민 B2 --------------------------------------- 0.15 ㎎Vitamin B 2 --------------------------------------- 0.15 mg
비타민 B6 ---------------------------------------- 0.5 ㎎Vitamin B 6 ---------------------------------------- 0.5 mg
비타민 B12 --------------------------------------- 0.2 ㎍Vitamin B 12 --------------------------------------- 0.2 g
비타민 C ----------------------------------------- 10 ㎎Vitamin C ----------------------------------------- 10 mg
비오틴 ------------------------------------------- 10 ㎍Biotin ------------------------------------------- 10 [mu] g
니코틴산아미드 ---------------------------------- 1.7 ㎎Nicotinic acid amide 1.7 mg
엽산 --------------------------------------------- 50 ㎍Folic acid --------------------------------------------- 50 μg
판토텐산 칼슘 ----------------------------------- 0.5 ㎎Calcium pantothenate ----------------------------------- 0.5 mg
무기질 혼합물 ------------------------------------- 적량Inorganic mixture -------------------------------------
황산제1철 -------------------------------------- 1.75 ㎎Ferrous sulfate -------------------------------------- 1.75 mg
산화아연 --------------------------------------- 0.82 ㎎Zinc oxide --------------------------------------- 0.82 mg
탄산마그네슘 ----------------------------------- 25.3 ㎎Magnesium carbonate - 25.3 mg
제1인산칼륨 -------------------------------------- 15 ㎎Potassium phosphate monohydrate 15 mg
제2인산칼슘 -------------------------------------- 55 ㎎Secondary calcium phosphate -------------------------------------- 55 mg
구연산칼륨 --------------------------------------- 90 ㎎Potassium citrate --------------------------------------- 90 mg
탄산칼슘 ---------------------------------------- 100 ㎎Calcium carbonate ---------------------------------------- 100 mg
염화마그네슘 ----------------------------------- 24.8 ㎎Magnesium chloride ----------------------------------- 24.8 mg
상기의 비타민 및 미네랄 혼합물의 조성비는 비교적 건강식품에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 그 배합비를 임의로 변형 실시하여도 무방하며, 통상의 건강식품 제조방법에 따라 상기의 성분을 혼합한 다음, 과립을 제조하고, 통상의 방법에 따라 건강식품 조성물 제조에 사용할 수 있다.
Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.
제제예 7. 건강 음료의 제조Formulation Example 7. Preparation of health drink
복합물 ------------------------------------------- 100 ㎎Composition ------------------------------------------- 100 mg
비타민 C ------------------------------------------- 15 gVitamin C ------------------------------------------- 15 g
비타민 E(분말) ------------------------------------ 100 gVitamin E (powder) ------------------------------------ 100 g
젖산철 ------------------------------------------ 19.75 gLactic Acid Iron ------------------------------------------ 19.75 g
산화아연 ------------------------------------------ 3.5 gZinc oxide ------------------------------------------ 3.5 g
니코틴산아미드 ------------------------------------ 3.5 gNicotinic acid amide 3.5 g
비타민 A ------------------------------------------ 0.2 gVitamin A ------------------------------------------ 0.2 g
비타민 B1 ----------------------------------------- 0.25 gVitamin B 1 ----------------------------------------- 0.25 g
비타민 B2 ------------------------------------------ 0.3 gVitamin B 2 ------------------------------------------ 0.3 g
물 -------------------------------------------------- 정량Water ------------------------------------------------- - Quantity
통상의 건강음료 제조방법에 따라 상기의 성분을 혼합한 다음, 약 1시간동안 85 ℃에서 교반 가열한 후, 만들어진 용액을 여과하여 멸균된 2 ℓ 용기에 취득하여 밀봉 멸균한 뒤 냉장 보관한 다음 본 발명의 건강음료 조성물 제조에 사용한다. The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 ° C for about 1 hour. The resulting solution was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, It is used in the production of the health beverage composition of the invention.
상기 조성비는 비교적 기호음료에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만 수요계층이나, 수요국가, 사용용도 등 지역적, 민족적 기호도에 따라서 그 배합비를 임의로 변형 실시하여도 무방하다.
Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.
제제예 8. 씨리얼의 제조Preparation Example 8. Preparation of cereal
복합물 ------------------------------------------- 7.51 gComplex ------------------------------------------- 7.51 g
쌀 ---------------------------------------------- 40.49 gRice ---------------------------------------------- 40.49 g
현미 -------------------------------------------- 24.29 gBrown rice -------------------------------------------- 24.29 g
수수 --------------------------------------------- 2.02 gSorghum --------------------------------------------- 2.02 g
보리 --------------------------------------------- 2.02 gBarley --------------------------------------------- 2.02 g
찹쌀 --------------------------------------------- 2.02 gGlutinous rice --------------------------------------------- 2.02 g
소금 --------------------------------------------- 0.49 gSalt --------------------------------------------- 0.49 g
깨 ----------------------------------------------- 0.16 gSes ----------------------------------------------- 0.16 g
설탕 -------------------------------------------- 17.75 gSugar -------------------------------------------- 17.75 g
해바라기유 --------------------------------------- 1.21 gSunflower oil --------------------------------------- 1.21 g
물 ------------------------------------------------- 정량Water ------------------------------------------------- dose
통상의 씨리얼 제조방법에 따라 상기의 성분을 혼합한 다음, 압출성형, 1차건조, 숙성, 파칭, 시럽코팅, 2차건조 과정, 포장한 다음 본 발명의 씨리얼 조성물 제조에 사용한다. The ingredients are mixed according to a conventional cereal manufacturing method and then used in the production of the cereal composition of the present invention after extrusion molding, primary drying, aging, ripening, syrup coating, secondary drying, and packaging.
상기 조성비는 비교적 기호에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만 수요계층이나, 수요국가, 사용용도 등 지역적, 민족적 기호도에 따라서 그 배합비를 임의로 변형 실시하여도 무방하다. Although the compositional ratio is a mixture of the components suitable for the preference, it is also possible to arbitrarily modify the blending ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20160129199A (en) | 2015-04-29 | 2016-11-09 | 주식회사 불스원 | Compositions for immune enhancement comprising oil mixture |
| KR20200082426A (en) | 2018-12-28 | 2020-07-08 | 전남대학교산학협력단 | Vegetable soup composition comprising beta-glucan for enhancing function of immunity and method thereof |
| KR20210030797A (en) | 2019-09-10 | 2021-03-18 | 전남대학교산학협력단 | Vegetable Soup Composition Comprising Branched Beta-glucan for Enhancing Function of Immunity and Method thereof |
| KR20220105355A (en) | 2021-01-20 | 2022-07-27 | 전남대학교산학협력단 | Food composition comprising beta-glucan hydrolysate for enhancing of immune function and manufacturing method thereof |
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| CN104721759A (en) * | 2015-04-16 | 2015-06-24 | 王磊 | Chinese herbal compound for treating lung cancer |
| KR101722448B1 (en) | 2016-09-26 | 2017-04-03 | 주식회사 프롬바이오 | Food composition with the fruit extract of Phyllanthus emblica Linn. and the leaf extract of Psidium guajava for the improvement of immunity |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20160129199A (en) | 2015-04-29 | 2016-11-09 | 주식회사 불스원 | Compositions for immune enhancement comprising oil mixture |
| KR20200082426A (en) | 2018-12-28 | 2020-07-08 | 전남대학교산학협력단 | Vegetable soup composition comprising beta-glucan for enhancing function of immunity and method thereof |
| KR20210030797A (en) | 2019-09-10 | 2021-03-18 | 전남대학교산학협력단 | Vegetable Soup Composition Comprising Branched Beta-glucan for Enhancing Function of Immunity and Method thereof |
| KR20220105355A (en) | 2021-01-20 | 2022-07-27 | 전남대학교산학협력단 | Food composition comprising beta-glucan hydrolysate for enhancing of immune function and manufacturing method thereof |
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