KR20140107031A - Composition for immune-enhancing comprising aged black garlic polysaccharides and white ginseng - Google Patents
Composition for immune-enhancing comprising aged black garlic polysaccharides and white ginseng Download PDFInfo
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- KR20140107031A KR20140107031A KR1020130021519A KR20130021519A KR20140107031A KR 20140107031 A KR20140107031 A KR 20140107031A KR 1020130021519 A KR1020130021519 A KR 1020130021519A KR 20130021519 A KR20130021519 A KR 20130021519A KR 20140107031 A KR20140107031 A KR 20140107031A
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- South Korea
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- ginseng
- composition
- white ginseng
- black garlic
- polysaccharide
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Abstract
Description
본 발명은 흑마늘 유래 다당류 성분 및 백삼을 유효성분으로 함유하는 면역증강용 조성물에 관한 것으로, 더욱 상세하게는 백삼의 면역활성 작용을 상승시키는 흑마늘 유래 다당류 성분 및 백삼을 유효성분으로 함유하는 면역증강용 조성물에 관한 것이다.The present invention relates to a polysaccharide component derived from black garlic and an immunostimulating composition containing white ginseng as an active ingredient. More particularly, the present invention relates to a polysaccharide component derived from black garlic, which enhances the immunological activity of white ginseng, ≪ / RTI >
인삼은 오래 전부터 사용되고 있는 대표적인 약용식물이며, 건강기능식품으로 현재 세계 곳곳에서 각광을 받고 있다. 이러한 인삼은 가열 처리 여부와 건조 방법에 따라 백삼, 홍삼, 태극삼과 흑삼 등으로 분류된다(이영상 외 7인, Korean J. Food & Nutr. 24(1): 138-143, 2011). 백삼(白蔘)은 수삼(水蔘)을 익히지 아니하고 햇볕, 열풍 또는 기타의 방법으로 건조한 것이며, 홍삼(紅蔘)은 수삼을 수증기 또는 기타 방법으로 찐 후 건조한 것이고, 태극삼은 수삼을 물로 삶은 후 건조한 것을 말한다. 또한, 흑삼은 수삼을 아홉번 찌고 아홉번 말리는 구증구포 처리한 것이다(이영상 외 7인, Korean J. Food & Nutr. 24(1): 138-143, 2011).Ginseng is a typical medicinal plant that has been in use for a long time, and it is a health functional food, and it is currently attracting attention all over the world. These ginsengs are classified into white ginseng, red ginseng, taegeuk ginseng and black ginseng according to the heat treatment and drying method (Lee, Young-Sang et al., 7, Korean J. Food & Nutr. 24 (1): 138-143, 2011). White ginseng is not dried ginseng but is dried by sunlight, hot air or other methods. Red ginseng is steamed or otherwise dried and then dried. After fermenting ginseng with water, Dry. In addition, black ginseng is made by nourishing ginseng nine times and drying it nine times (Lee Young Sang et al., 7, Korean J. Food & Nutr. 24 (1): 138-143, 2011).
홍삼 중 여러 특이성분은 인삼을 수증기로 찌는 과정(steam process)에서 생성되는 것으로 알려져 있다(LiXG, Korean J Ginseng Sci 16:64-67, 1992). 홍삼의 찌는 과정 중 맥아당과 아미노산이 반응해서 불안정한 4-O-α-D-글루코실-1-디옥시-2,3-디케토사카라이드(4-0-α-D-glucosyl-1-deoxy-2,3-diketosaccharide)를 생성하고, 다시 2-케톤기(2-ketone group)와 C-6-하이드록실기(C-6-hydroxyl group)가 고리화(cyclization)되어 글리코시드 B(glycoside B)로 되며, 이 물질은 다시 자리옮김(rearrangement) 및 당의 이탈 반응으로 백삼에는 함유되어 있지 않은 비배당체 성분인 말톨(maltol)이 생성된다(Li XG, Korean J Ginseng Sci 16:64-67,1992).Several specific components of red ginseng are known to be produced in the steam process of ginseng (LiXG, Korean J Ginseng Sci 16: 64-67, 1992). D-glucosyl-1-deoxy-2,3-diketosaccharide (4-O-α-D-glucosyl- 2-ketone group and C-6-hydroxyl group are cyclized to form glycoside B (2,3-diketosaccharide) ), And this substance is rearrangement and elimination reaction of sugar to produce maltol, an unglycosylated component not contained in white ginseng (Li XG, Korean J Ginseng Sci 16: 64-67, 1992 ).
상기 반응은 전체적으로 메일라이드 반응(Maillard reaction)을 따르는 것으로 알려져 있으며(SK Grandheeand VM Monnier, J. Biol. Chem. 266(18):11649-11653. 1991), 일반적으로 말톨은 항산화 효과가 있어 생체노화와 관련된 지질의 과산화를 억제한다고 보고되었다(Choi et al., Korean J Food Sci Technol. 34:493-497, 2002). 하지만 말톨은 홍삼 제조뿐만 아니라 백삼 제조 시 열풍 건조와 같은 열처리에 의해서도 미량생성될 수도 있다(Nam KY, J Ginseng Res. 29:1-18, 2005).The reaction is generally known to follow the Maillard reaction (SK Grandhee and VM Monnier, J. Biol. Chem. 266 (18): 11649-11653, 1991). In general, maltol has antioxidative effects, (Choi et al., Korean J Food Sci Technol. 34: 493-497, 2002). However, maltol may be produced in a small amount not only by red ginseng production but also by heat treatment such as hot air drying in the production of white ginseng (Nam KY, J Ginseng Res. 29: 1-18, 2005).
말톨 외에도 사포닌 성분이 인삼의 주요 약리활성 성분으로 알려져 있다. 또한, 사포닌 성분은 수치법에 의해 함량과 조성이 변화하는 것으로 보고되었는데(Shoji KY., Natural Medicines 53:55-59, 1999), 지금까지 백삼과 홍삼으로부터 분리된 진세노사이드(ginsenoside, GS)는 각각 24종과 32종이고, 이 중 18종이 백삼과 홍삼 모두에서, 6종이 백삼에서, 14종이 홍삼에서 발견되었다(Lee et al., Korean J. Food & Nutr. 24(1):138-143,2011).In addition to maltose, saponin is known as the main pharmacologically active ingredient of ginseng. Ginsenoside (GS), isolated from white ginseng and red ginseng, has been reported to change the content and composition (Shoji KY., Natural Medicines 53: 55-59, 1999) 24 (1): 138-136. Among them, 18 were found in white ginseng and red ginseng, 6 were found in white ginseng, and 14 were found in red ginseng (Lee et al., Korean J. Food & Nutr. 143,2011).
홍삼 특유의 진세노사이드는 찌는 과정 중 열처리(heating)에 의한 가수분해 반응에 의해 생성되고, 홍삼의 높은 약리적 효능이 이 성분에서 기인된다(Park et al., Food Industry and Nutrition 8:10-23, 2003).The ginsenosides unique to red ginseng are produced by hydrolysis reaction by heating during the steaming process and the high pharmacological efficacy of red ginseng originates from this component (Park et al., Food Industry and Nutrition 8: 10-23 , 2003).
그러나, 인삼 중의 비사포닌계 화합물에서도 여러 약리활성이 있음이 보고되었으며(Kong et al., J Ginseng Res 33:111-114, 2009), 홍삼 제조과정 중 여러 비사포닌계 화합물은 구조가 변화하여 새로운 성분이 되기도 하고, 존재하던 약리활성 성분의 함량 증가도 일어난다고 보고된바 있다(Ryu GH., Food Industry and Nutrition 8:8-42, 2003).However, several non-saponin compounds in ginseng have been reported to have various pharmacological activities (Kong et al., J Ginseng Res 33: 111-114, 2009) (Ryu GH., Food Industry and Nutrition 8: 8-42, 2003).
지금까지 국내에서, 림프구 증식과 대식세포 활성에 있어서 홍삼의 효능이 있음을 발표되었지만(김기환 등, Korean J. Ginseng Sci., 21(2):78-84, 1997; 이혜연 등, Korean J. Ginseng Sci., 22(1):60-65, 1998), 항체생산에 있어 홍삼의 효능은 미미하다(Park et al., Natural Product Sciences, 61(1):31-35, 2000). 게다가, 근래에 홍삼과 백삼의 면역효능 비교실험에 있어서 홍삼이 백삼보다 조금 더 높은 효능을 가지지만 큰 차이를 보이지 않는다는 것이 발표되기도 하였다(최재호 등, J. Ginseng Res., 33(1):33-39, 2009).In Korea, it has been reported that the efficacy of red ginseng in lymphocyte proliferation and macrophage activity (Korean J. Ginseng Sci., 21 (2): 78-84, 1997, Korean J. Ginseng Sci., 22 (1): 60-65, 1998), the efficacy of red ginseng in antibody production is minimal (Park et al., Natural Product Sciences, 61 (1): 31-35, 2000). In addition, it has recently been reported that red ginseng has slightly higher efficacy than white ginseng in comparison with the immunological efficacy tests of red ginseng and white ginseng, but does not show a large difference (Choi, JH, et al., J. Ginseng Res., 33 -39, 2009).
또한, 인삼 사포닌 성분 중의 하나인 진세노사이드(ginsenoside)가 B 림프구를 활성화하여 항체생산에 도움을 주는 아주반트(adjuvant) 역할을 한다고 알려져 있지만, 그 효능이 크지 않고(이혜영 등, Korean J. Ginseng Sci., 22(1):60-65, 1998; Nam et al., Natural Product Sciences, 6(1):31-35, 2000; Sun Y et al., Vaccine, 5;26(47):5911-5917, 2008), 복잡한 제조공정으로 인해 경제성이 떨어지는 문제가 있다.In addition, although it is known that ginsenoside, which is one of ginseng saponin components, acts as an adjuvant to activate B lymphocytes to help the production of antibodies, but its effect is not great (Lee, HJ et al., Korean J. Ginseng Natural Product Sciences, 6 (1): 31-35, 2000; Sun Y et al., Vaccine, 5; 26 (47): 5911 -5917, 2008), there is a problem that economical efficiency is deteriorated due to a complicated manufacturing process.
게다가, 일반적으로 백삼 추출물에는 20%의 관세를 부과하나, 홍삼 추출물에는 농림축산물양허관세 754.3%를 부과하여 관세율의 차이가 약 38배에 이른다(한국관세무역개발원 2011; 이영상 외 7인, Korean J. Food & Nutr. 24(1): 138-143, 2011). 따라서 홍삼이 백삼보다 효능이 특별히 크지 않는데 반해 복잡한 제조공정이나 높은 수입관세로 인해 제품가격이 효능에 비해 턱없이 높아 소비자에게 많은 부담을 주고 있는 것이 현실정이다.In addition, 20% tariffs are generally imposed on white ginseng extracts, but the difference in tariff rates is about 38 times that of 754.3% of tariffs for agriculture, forestry and livestock products in the red ginseng extract (Korea Customs Trade Development 2011; Food & Nutr 24 (1): 138-143, 2011). Therefore, while red ginseng is not particularly large in efficacy than white ginseng, it is a reality that the price of the product is very high compared to the efficacy due to complicated manufacturing process and high import tariff, which puts a burden on consumers.
한편, 면역반응은 초기 면역반응인 선천성 면역반응과 후기 면역반응인 후천성 면역반응으로 구분될 수 있다. 초기 면역반응에서는 대식세포와 자연살해세포(natural killer cell; NK 세포)의 활성에 의해 병원체를 억제하여 숙주를 보호하는데, 이때 대식세포는 병원체를 탐식하면서 활성표지인 TNF-α를 많이 생산해 분비하고 NK 세포는 활성표지인 퍼포린(perforin)을 많이 생산하여 분비함으로써 병원체 감염세포를 살해한다. 이어서 후천성 면역에 관여하는 세포독성(cytotoxic) T 림프구, 헬퍼(helper) T 림프구 그리고 B 림프구가 활성화해 감염세포를 살해하거나 항체를 만들어 숙주를 보호한다. 세포독성 T 림프구는 NK 세포처럼 퍼포린을 많이 생산분비해 병원체 감염세포를 죽이고 B 림프구는 헬퍼 T 림프구의 의존 또는 비의존적으로 항체를 만들어 숙주를 보호한다(최인성, 생화학 총설집, 4:129-133, 1992; 최재호 등, J. Ginseng Res., 33(1):33-39, 2009).On the other hand, the immune response can be classified into an innate immune response, which is an initial immune response, and an acquired immune response, which is a later immune response. In the early immune response, macrophages and natural killer cells (NK cells) inhibit the pathogen by inhibiting the activity of the host. In this case, macrophages digest pathogens and produce and secrete TNF- NK cells produce and secrete the active label perforin, killing pathogen-infected cells. It then activates cytotoxic T lymphocytes, helper T lymphocytes, and B lymphocytes that are involved in acquired immunity to kill infected cells or protect the host by making antibodies. Cytotoxic T lymphocytes produce perforin as much as the NK cells, killing the pathogen-infected cells and B lymphocytes protect the host by making antibodies dependent on or dependent on helper T lymphocytes (Choi et al., Biochemistry Review, 4: 129-133 , 1992; Choi, Jaeho, et al., J. Ginseng Res., 33 (1): 33-39, 2009).
이에 본 발명자들은 홍삼 못지않은 약효를 지니면서 가격이 저렴한 백삼의 면역효능을 증진시킬 수 있는 면역 상승제를 찾기 위하여 노력을 계속한 결과, 백삼과 흑마늘 유래 다당류 성분을 함께 사용하면 부작용이 없이 단독으로 사용할 때보다 적은 백삼의 양, 즉 홍삼의 1/10의 양에서도 홍삼의 면역활성보다 수배 이상 크게 상승되는 것을 확인하고, 본 발명을 성공적으로 완성하였다.Therefore, the present inventors continued their efforts to find an immune enhancer that can enhance the immunity efficacy of white ginseng having low drug efficacy as much as red ginseng. As a result, when white ginseng and black ginseng derived polysaccharide components are used together, It was confirmed that the amount of white ginseng, that is, 1/10 of the amount of red ginseng, is much higher than that of red ginseng several times more than that of using red ginseng. Thus, the present invention has been successfully completed.
본 발명자들은 이전 연구에서 마늘을 고온, 고습의 조건에서 장기간동안 숙성시키면 흑색 또는 붉은색이 진한 발효 흑마늘이 제조되는 방법을 고안하였으며, 이러한 방법에 의해 제조된 흑마늘은 원료 성분인 생마늘에 비해 마늘의 유효성분이 수배이상이 증가한다는 사실을 발견하였다(한국특허등록 제10-857270호 및 한국특허등록 제10-900988). 이에 흑마늘의 유효성분들을 비롯한 흑마늘 성분들은 열에 약한 단백질이 아닌 열에 강한 단백질 이외의 물질들로부터 유래됨을 강하게 시사하고 있다.The inventors of the present invention have devised a method in which garlic is fermented for a long period of time under high temperature and high humidity conditions to produce a dark fermented black garlic having a black or red color. The black garlic produced by this method has a higher content of garlic It has been found that the efficacy increases more than several times (Korean Patent No. 10-857270 and Korean Patent No. 10-900988). Thus, the black garlic ingredients, including the active ingredients of black garlic, strongly suggest that they are derived from substances other than heat-sensitive proteins rather than heat-sensitive proteins.
흑마늘에 관한 이용한 종래기술로 본 발명자들의 발명인 한국특허등록 제10-857270호 및 한국특허등록 제10-900988 이외에도 흑마늘 제조방법에 대한 다수의 특허문헌 등이 있으나, 흑마늘 유래 다당류 성분이 백삼의 효능(특히, 면역관련 효능)을 증진시키는 효과에 대해서는 지금까지 어떠한 개시나 교시된 바 없다.In addition to Korean Patent Registration No. 10-857270 and Korean Patent Registration No. 10-900988, which are inventions of the present inventors, there have been numerous patent documents on the method of manufacturing black garlic. However, the effect of polysaccharide derived from black garlic on the efficacy of white ginseng In particular, the effect of enhancing the immune-related efficacy has not been disclosed or taught until now.
본 발명은 복잡한 제조공정으로 제품가격이 비싼 홍삼 또는 진세노사이드를 대신하여 부작용이 거의 없고 홍삼과 유사한 약효를 가지면서 가격이 저렴한 백삼을 이용하여 탁월한 면역활성을 갖는 면역증강제를 제공하고자 안출된 것으로, 본 발명은 흑마늘 유래 다당류 성분 및 백삼을 유효성분으로 함유하는 면역증강용 조성물을 제공하는데 그 목적이 있다.The present invention has been made to provide an immune enhancer having excellent immunity activity by using white ginseng, which has little side effects in place of red ginseng or ginsenoside, which is expensive in price, The present invention provides a polysaccharide component derived from black garlic and an immunostimulating composition containing white ginseng as an effective ingredient.
상기 목적을 달성하기 위하여, 본 발명은 백삼의 면역활성 작용을 상승시키는 흑마늘 유래 다당류 성분 및 백삼을 유효성분으로 함유하는 면역증강용 조성물을 제공한다.In order to achieve the above object, the present invention provides a polysaccharide component derived from black gum, which enhances the immunological activity of white ginseng, and a composition for enhancing immunity comprising white ginseng as an active ingredient.
바람직하게는, 상기 흑마늘 유래 다당류 성분은 10kD 이상의 분자량을 갖는다.Preferably, the black-germ-origin polysaccharide component has a molecular weight of 10 kD or more.
또한, 상기 흑마늘 유래 다당류 성분 및 백삼은 0.5:99.5 내지 99.5:0.5의 중량비로 혼합된 것을 특징으로 한다.The black garnet-derived polysaccharide component and white ginseng are mixed at a weight ratio of 0.5: 99.5 to 99.5: 0.5.
또한, 상기 흑마늘 유래 다당류 성분은 조성물 총 중량에 대하여 0.5 내지 20중량%로 함유되며, 상기 백삼은 조성물 총 중량에 대하여 5 내지 30중량%로 함유되는 것을 특징으로 한다.The black germ-derived polysaccharide component is contained in an amount of 0.5 to 20 wt% based on the total weight of the composition, and the white ginseng is contained in an amount of 5 to 30 wt% based on the total weight of the composition.
또한 본 발명의 목적은 흑마늘 유래 다당류 성분 및 백삼(白蔘)을 유효성분으로 함유하는 면역증강용 건강기능식품에 의해 달성된다.The object of the present invention is also achieved by a health functional food for immunity enhancement comprising a component of polysaccharide derived from black garlic and white ginseng as an active ingredient.
상기와 같은 본 발명에 따르면, 흑마늘 유래 다당류 성분은 백삼의 면역활성 작용을 상승시키므로, 흑마늘 유래 다당류 성분과 백삼을 유효성분으로 함유하는 본 발명의 면역증강용 조성물은 값비산 홍삼 또는 진세노사이드 대신 저렴한 백삼을 이용할 수 있어서 경제적이다.According to the present invention, the polysaccharide derived from black garlic raises the immunological activity of white ginseng. Therefore, the composition for improving immunity of the present invention, which contains polysaccharide derived from black garlic and white ginseng as an active ingredient, can be used instead of red ginseng or ginsenoside It is economical to use cheap white ginseng.
또한, 본 발명의 면역증강용 조성물은 적은 투여량으로도 우수한 면역증강 효과를 나타내므로 면역결핍질환, 세균성 및 비루스성 감염 질환, 종양 등의 예방 및 치료를 위한 의약품 또는 건강기능식품으로 유용하게 이용될 수 있다.In addition, since the immunoconjugate composition of the present invention exhibits excellent immunity enhancing effect even at a small dose, it is useful as a medicament or a health functional food for the prevention and treatment of immunodeficiency diseases, bacterial and viral infectious diseases, tumors and the like .
이하, 본 발명을 상세히 설명하면 다음과 같다.
Hereinafter, the present invention will be described in detail.
본 발명은 흑마늘 유래 다당류 성분 및 백삼을 유효성분으로 함유하는 면역증강용 조성물을 제공한다.The present invention provides a composition for enhancing immunity comprising a component of polysaccharide derived from black garlic and white ginseng as an active ingredient.
본 발명의 상기 흑마늘 유래 다당류 성분은, 세절한 흑마늘에 대하여, 시료 중량의 약 1 내지 20배 부피의 물, 메탄올, 에탄올, 부탄올 등과 같은 C1 내지 C4의 저급 알코올의 극성 용매 또는 이들의 약 1 : 0.1 내지 1 : 10의 혼합비를 갖는 혼합용매로, 바람직하게는 약 5 내지 10배에 달하는 부피의 물로, 약 80℃ 내지 100℃에서 1 내지 24시간 동안 초음파추출, 환류냉각추출, 교반추출, 열수추출 등의 추출방법을 사용하여 추출한 후, 믹서기로 분쇄하여 원심분리한 다음 얻은 상등액을 한외여과기(멤브레인 필터(membrane filter): 10K cut, PLCGC, Bedford, MA, USA)를 이용하여 분자량 10kD 이상의 고분자량 화합물을 수득하고, 3배 부피의 95%(v/v) 에탄올을 가하여 4℃에서 하루 동안(24시간) 방치하고, 다시 원심분리하여 얻은 침전물을 증류수에 용해시킨 것을 감압농축 또는 건조하여 수득할 수 있다.The black garlic-derived polysaccharide component of the present invention is prepared by dissolving a polarized solvent of a C1 to C4 lower alcohol such as water, methanol, ethanol, butanol, etc., in a volume of about 1 to 20 times the weight of the sample, Refluxing cooling extraction, stirring extraction, hydrothermal extraction, hydrothermal condensation, hydrothermal condensation, or the like in a mixed solvent having a mixing ratio of 0.1 to 1: 10, preferably about 5 to 10 times the volume of water at about 80 DEG C to 100 DEG C for 1 to 24 hours The resulting supernatant was pulverized with a blender and centrifuged. The resulting supernatant was extracted with an ultrafilter (membrane filter: 10K cut, PLCGC, Bedford, Mass., USA) The resulting precipitate was dissolved in distilled water to obtain a molecular weight compound, and the solution was concentrated by evaporation under reduced pressure or in the same manner as in Example 1, except that a 3-fold volume of 95% (v / v) ethanol was added and left at 4 캜 for one day (24 hours) And it can be obtained.
또는, 흑마늘을 열수추출 등의 방법으로 추출한 후 믹서기로 분쇄하고, 교반기를 사용하여 1 내지 12시간 동안 저속으로 교반하여 원심분리하고, 이때 얻은 상등액을 헥산 등의 유기용매로 처리하여 탈지한 다음 한외여과기(멤브레인 필터(membrane filter): 10K cut, PLCGC, Bedford, MA, USA)를 이용하여 수득한 분자량 10kD 이상의 고분자량 화합물을, 3배 부피의 95%(v/v) 에탄올을 가하여 4℃에서 하루 동안(24시간) 방치하고, polymymix-B 컬럼(Biorad, Hercules, CA, USA)를 사용하여 오염된 지질당지질(lipopolysaccharide, LPS)를 제거한 후 동결건조를 통해 최종적으로 본 발명의 흑마늘 유래 다당류 성분을 수득하는 것이 가능하다.Alternatively, the black garlic is extracted by a method such as hot water extraction, followed by pulverization with a blender, and the mixture is centrifuged at low speed for 1 to 12 hours using a stirrer. The resulting supernatant is treated with an organic solvent such as hexane and then degreased The high molecular weight compound having a molecular weight of 10 kD or more obtained by using a filter (membrane filter: 10K cut, PLCGC, Bedford, Mass., USA) was added at 3 ° C with 95% (v / v) (24 hours), and the contaminated lipopolysaccharide (LPS) was removed using a polymymix-B column (Biorad, Hercules, Calif., USA), followed by lyophilization to finally obtain the black- Can be obtained.
본 발명에서 상기 흑마늘 유래 다당류 성분은 조성물 총 중량에 대하여 0.5 내지 20 중량%로 함유되는 것이 바람직한데, 0.5중량% 미만이면 그 효과가 미비하고, 20중량%를 초과하면 백삼과의 동반상승효과(시너지 효과)가 반감하기 때문이다.In the present invention, the content of the polysaccharide derived from the black garlic is preferably 0.5 to 20% by weight based on the total weight of the composition. When the content is less than 0.5% by weight, the effect is insufficient. When the content is more than 20% by weight, Synergy effect) is halved.
또한, 상기 백삼은 당업계의 통상적인 방법에 따라 백삼 추출물로 수득할 수 있으며, 그 방법은 특별히 한정되지 않는다. 또한, 본 발명의 상기 백삼을 조성물 총 중량에 대하여 5 내지 30중량%로 함유되는 것이 바람직한데, 5중량% 미만이면 그 효과가 미비하고, 30중량%를 초과하면 흑마늘 유래 다당류 성분과의 동반상승효과(시너지 효과)가 반감될 수 있다.In addition, the white ginseng can be obtained as white ginseng extract according to a conventional method in the art, and the method is not particularly limited. It is preferable that the white ginseng according to the present invention is contained in an amount of 5 to 30% by weight based on the total weight of the composition. If it is less than 5% by weight, the effect is insufficient. If it exceeds 30% by weight, The effect (synergy effect) can be halved.
또한, 본 발명의 면역증강용 조성물은 흑마늘 유래 다당류 성분과 백삼이 각각 0.5 :99.5 ~ 99.5 : 0.5의 중량비로 혼합되는 것이 바람직하다.Further, in the composition for immune enhancement of the present invention, the polysaccharide derived from black garlic and white ginseng are preferably mixed at a weight ratio of 0.5: 99.5 to 99.5: 0.5, respectively.
본 발명의 면역증강용 조성물은 면역증강 효과가 뛰어나기 때문에 면역증강제, 항종양제, 항암제 등으로 유용하게 사용될 수 있으며, 상기 유효성분 외에 필요에 따라 부형제, 당류, 향류, 색소, 유지류, 단백질 등을 적의 함유할 수 있다.The immunoconjugation composition of the present invention is useful as an immunostimulating agent, an antitumor agent, an anticancer agent or the like because of its excellent immunity enhancing effect. In addition to the above active ingredients, excipients, saccharides, May contain an enemy.
또한, 본 발명의 면역증강용 조성물은 경구 투여할 수 있는 정제, 캡슐제, 또는 수액제 등의 제제로서 제공될 수 있다.In addition, the composition for enhancing immunity of the present invention can be provided as a preparation for oral administration such as tablets, capsules, or liquids.
또한, 본 발명의 면역증강용 조성물은 해당 용도에 따라 약학적으로 허용 가능한 담체와 함께 독립적으로 또는 약품 첨가제로 사용되거나, 사람에게 투여하기 적합한 기타 모든 형태로 사용될 수 있다. 본 발명의 면역증강제에 함유될 수 있는 담체로는 증량제, 고섬유 첨가제, 캡슐화제 및 지질 등이 포함될 수 있으며, 이러한 담체들의 예는 당업계에 충분히 공지되어 있다.In addition, the immunoconjugate composition of the present invention may be used independently or in combination with a pharmaceutically acceptable carrier according to the intended use, or in any other form suitable for administration to humans. The carrier which may be contained in the immunostimulant of the present invention may include an extender, a high fiber additive, an encapsulating agent and a lipid, and examples of such carriers are well known in the art.
본 발명의 면역증강제는 질환의 진행 정도, 나이, 성별, 신체 상태, 투여 기간, 투여 방법, 환자의 체중, 식사, 배출 속도 등에 따라 용량을 달리하여 투여될 수 있다. 그러나, 바람직한 효과를 위해서는 상기 면역증강용 조성물 1일 0.01 내지 1,000 ㎎/㎏으로, 더욱 바람직하게는 0.1 내지 500 ㎎/㎏의 범위로 비경구 또는 경구 투여하는 것이 좋다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면이로든 본 발명의 범위를 한정하는 것은 아니다.The immunomodulating agent of the present invention can be administered in different doses depending on the progression of the disease, age, sex, physical condition, administration period, administration method, body weight of the patient, diet, However, for the desired effect, parenteral or oral administration is preferably carried out in the range of 0.01 to 1,000 mg / kg, more preferably 0.1 to 500 mg / kg, per day of the above immunoconjugate composition. The administration may be carried out once a day or divided into several times. The dose does not in any way limit the scope of the invention.
또한, 본 발명의 면역증강용 조성물은 건강기능식품으로 사용될 수도 있는데, 이때 건강기능식품의 제형은 특별히 제한되지 않으며, 구체적인 예를 들면 분말, 과립, 캡슐, 페이스트 또는 음료 등으로 제형화될 수 있다.In addition, the composition for immuno-enhancement of the present invention may be used as a health functional food. The formulation of the health functional food is not particularly limited and may be formulated into powder, granule, capsule, paste, .
또한, 본 발명은 백삼과 흑마늘 유래 다당류 성분을 유효성분으로 함유하는 면역증강용 조성물이 대식세포에서 면역 활성 작용을 나타내는데 있어서 동반상승효과(synergic effect)가 있음을 보여주기 위하여, 다음과 같은 효능 검사들을 수행한다:(1) 마우스 복강으로부터 대식세포의 분리; (2) 종양괴사인자-알파(tumor necrosis factor-α, TNF-α) mRNA 분석을 위한 대식세포 자극 및 전체 RNA의 분리; (3) 역전사-중합효소 연쇄반응(RT-PCR)을 이용한 TNF-α mRNA 분석; (4) TNF-α 단백질의 정량적 분석을 위한 대식세포 자극 및 배양; 및 (5) 면역 블럿팅을 이용한 TNF-α 단백질의 정량적 분석.In order to show that the composition for enhancing immunity comprising an ingredient of polysaccharide derived from white ginseng and black garlic as an active ingredient has a synergic effect on immunocompetent activity in macrophages, (1) separation of macrophages from the mouse abdominal cavity; (2) stimulation of macrophages and isolation of total RNA for tumor necrosis factor-alpha (TNF-alpha) mRNA analysis; (3) analysis of TNF-α mRNA using reverse transcription-polymerase chain reaction (RT-PCR); (4) macrophage stimulation and culture for quantitative analysis of TNF-a protein; And (5) quantitative analysis of TNF-α protein using immunoblotting.
또한, 본 발명은 본 발명의 면역증강용 조성물이 T 림프구와 자연살해세포 (natural killer cell; NK 세포)활성화에 있어 동반상승효과(synergic effect)가 있음을 보여주기 위하여, 다음과 같은 효능 검사들을 수행한다:(1) 페포린 mRNA 분석을 위한 마우스에 시료 투여 및 마우스의 비장으로부터 전체 RNA의 분리; (2) 역전사-중합효소 연쇄반응(RT-PCR)을 이용한 마우스 비장내의 페포린 mRNA 분석; (3) 페포린 단백질의 정량적 분석을 위한 마우스에 시료 투여 및 마우스의 비장으로부터 단백질의 분리; 및 (4) 면역 블럿팅(immunoblotting)을 이용한 마우스 비장내의 perforin 단백질의 정량적 분석.The present invention also provides the following effects tests to show that the immunoenhancing composition of the present invention has a synergic effect on the activation of T lymphocytes and natural killer cells (NK cells) (1) administration of samples to mice for pheophorin mRNA analysis and isolation of total RNA from the spleen of mice; (2) analysis of pheophorin mRNA in mouse spleen using reverse transcription-polymerase chain reaction (RT-PCR); (3) administration of a sample to the mouse for quantitative analysis of the pheophorin protein and separation of the protein from the spleen of the mouse; And (4) quantitative analysis of perforin protein in mouse spleen using immunoblotting.
또한, 본 발명은 본 발명의 면역증강용 조성물이 B 림프구 항체생산에 있어 동반상승효과(synergic effect)가 있음을 보여주기 위하여, 다음과 같은 효능 검사들을 수행한다:(1) 마우스에 시료 투여; 및 (2) 직접 용혈 항체 플라크 형성세포(Direct Hemolytic Antibody Plaque Forming Cell, AFC)를 이용한 항체 생산의 정량적 분석.In addition, the present invention provides the following efficacy tests to show that the immunoconjugation composition of the present invention has a synergic effect in the production of B lymphocyte antibodies: (1) administration of a sample to a mouse; And (2) quantitative analysis of antibody production using direct Hemolytic Antibody Plaque Forming Cell (AFC).
또한, 본 발명은 본 발명의 면역증강용 조성물이 세포독성이 없음을 보여주기 위하여, 다음과 같은 효능 검사들을 수행한다:(1) 비장세포 추출 및 배양; 및 (2) 화학적 세포독성 검사를 위한 MTT 분석.In addition, the present invention performs the following efficacy tests to show that the immunoenhancing composition of the present invention is free from cytotoxicity: (1) splenocyte extraction and culture; And (2) MTT assay for chemical cytotoxicity.
또한, 본 발명은 본 발명의 면역증강용 조성물이 장기독성이 없음을 보여주기 위하여, 간과 신장 독성 검사(동물실험)를 수행한다.In addition, the present invention performs liver and kidney toxicity tests (animal experiments) to show that the composition for immune enhancement of the present invention has no long-term toxicity.
또한, 본 발명은 본 발명의 면역증강용 조성물이 성장에 미치는 독성이 없음을 보여주기 위하여, 미성숙 마우스 및 성숙 마우스의 성장 독성 검사를 수행한다.
In addition, the present invention tests the growth toxicity of immature and adult mice to show that the immunoconjugation composition of the present invention has no toxicity to growth.
이하, 실시 예에 의하여 본 발명을 더욱 상세히 설명하고자 한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시 예에 한정되는 것은 아니다.
Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
실시예 1: 흑마늘 유래 다당류 성분의 제조 Example 1 : Preparation of black sugar-derived polysaccharide components
실험에 사용한 흑마늘은 본 발명자가 고안한 한국특허등록 제10-857270호의 제조방법으로 제조된 것을 사용하였다.The black garlic used in the experiment was prepared by the manufacturing method of Korean Patent No. 10-857270, which was invented by the present inventor.
잘게 세절한 흑마늘을 증류수에 넣고 80~100℃의 불로 2~3 시간 동안 끓인 후 믹서기(솔잎 82A, 한국)로 분쇄하고, 4시간 동안 교반기(제일과학, 한국)를 사용해 저속으로 교반한 다음, 15~30분 동안 원심분리(6000 x g)하여 얻은 상등액을 헥산으로 처리하여 탈지한 후 탈지한 용액을 한외여과기(Ultrafilteration kit, Millipore, Pellicon 2, Bedford, MA, USA)의 멤브레인 필터(10K cut, PLCGC, Bedford, MA, USA)를 이용하여 분자량 10kD 이상의 고분자량 화합물을 얻었다.
The finely divided black garlic was placed in distilled water and boiled for 2 to 3 hours at 80 to 100 ° C. The mixture was pulverized with a blender (pine needle 82A, Korea) and stirred for 4 hours at a low speed using a stirrer (Jeil Scientific, Korea) The supernatant obtained by centrifugation (6000 xg) for 15 to 30 minutes was degreased by treating with hexane, and the degreased solution was filtered through a membrane filter (10K cut, manufactured by Ultrafilteration Kit, Millipore, Pellicon 2, Bedford, Mass., USA) PLCGC, Bedford, Mass., USA) to obtain a high molecular weight compound having a molecular weight of 10 kD or more.
상기 분자량 10kD 이상의 고분자량 화합물에 3배 부피(v/v)의 95%(v/v) 에탄올 가하여 4℃에서 24시간 방치한후 다시 원심분리(6000 x g)하여 얻은 침전물을 증류수에 용해시켜 polymyxin-B 컬럼(Biorad, Hercules, CA, USA)를 사용하여 오염된 지질당지질(LPS)을 제거한 다음, 동결건조하여 최종적으로 연갈색 분말의 흑마늘 유래 다당류 성분을 얻었으며, 다음 실험 때까지 -20℃에서 보관하였다.
95% (v / v) ethanol of 3 times volume (v / v) was added to the above high molecular weight compound having a molecular weight of 10 kD or more and allowed to stand at 4 ° C for 24 hours. The precipitate obtained by centrifugation (6000 xg) was dissolved in distilled water to obtain polymyxin (LPS) was removed using a B-column (Biorad, Hercules, Calif., USA), and then lyophilized to finally obtain a component of polysaccharide derived from dark brown powder of dark brown powder. Respectively.
상기에서 수득한 흑마늘 유래 다당류 성분 내의 LPS 잔류량 검사는 Limulus ES Ⅱ 키트(Wako, Osaka, Japan)로 검사하였으며, 잔류량은 10 내지 60 EU(endotoxin unit)/㎖이었다.
The residual amount of LPS in the polysaccharide-derived polysaccharide thus obtained was examined with a Limulus ES II kit (Wako, Osaka, Japan) and the residual amount was 10 to 60 EU (endotoxin unit) / ml.
유럽국가에서 실험 시약의 LPS 잔류량 허용범위는 350 EU/㎖인데(Scheer R., Arzneimittelforschung.43(7):795-800. 1993), 본 발명에 따른 흑마늘 유래 다당류 성분은 LPS 잔류량 허용범위에 상당히 못 미치는 것으로 확인되었다.
In European countries, the LPS residual amount tolerance range of the test reagent is 350 EU / ml (Scheer R., Arzneimittelforschung. 43 (7): 795-800. 1993) Respectively.
또한, 흑마늘 유래 다당류 성분 내의 렉틴성분 잔류량 유무검사인 혈액응집반응시험에서 응집반응을 보이지 않아 본 발명의 흑마늘 유래 다당류 성분에는 렉틴성분 잔류량이 없음을 확인하였으며, 당분해 효소인 비스코자임 L(viscozyme L)에 의해 인터루킨-1β 생산효능을 완전히 제거하는 것으로 나타나 상기 흑마늘 유래 다당류 성분의 유효성분이 당류임을 확인하였다.
In addition, coagulation reaction was not observed in the blood coagulation reaction test for the presence or absence of the residual amount of lectin component in the component of the polysaccharide derived from black garlic. Thus, it was confirmed that the residue of the lectin component was not present in the component of the polysaccharide derived from the black garlic of the present invention, and viscoszyme L ), Indicating that the active ingredient of the polysaccharide derived from the black garlic was the saccharide.
더욱이, 상기 흑마늘 유래 다당류 성분은 추출분리과정에서 멤브레인 필터(10K cut, PLCGC, Bedford, MA, USA)를 이용하여 얻은 당류이기 때문에, 당류 중에서도 분자량 10kD 이상의 다당류임을 알 수 있다.
Furthermore, since the polysaccharide derived from the black garlic is a saccharide obtained by using a membrane filter (10K cut, PLCGC, Bedford, Mass., USA) during the extraction and separation, it can be seen that the polysaccharide is a polysaccharide having a molecular weight of 10 kD or more among the saccharides.
실시예 2. 백삼에 의한 대식세포 활성화에 있어 흑마늘 유래 다당류 성분에 의한 동반상승효과 확인 Example 2 . Identification of synergistic effects of white-ginseng-derived polysaccharide components in the activation of macrophages
본 발명에서는 다당류인 흑마늘 유래 다당류 성분이 백삼의 대식세포 활성화에 있어 동반상승효과를 나타내는지 확인하기 위하여, 대식세포 활성화 지표가 되는 종양괴사인자-알파(TNF-α)의 유전자 TNF-α mRNA 발현 유도농도와 TNF-α 단백질 생산분비 유도능에 있어 동반상승효과 여부와 이들 유도능 동반상승효과가 홍삼보다 탁월한지를 확인하였다.
In the present invention, in order to confirm whether polysaccharide derived from polysaccharide, which is a polysaccharide derived from black garlic, has synergistic effect on the macrophage activation of white ginseng, gene TNF-α mRNA expression of tumor necrosis factor-alpha (TNF- Induced concentration and induction of TNF-α protein production secretion, we confirmed whether the synergistic effect and the synergistic effect of induction were better than that of red ginseng.
2-(1). 마우스 복강으로부터의 대식세포 분리2- (1). Macrophage separation from mouse abdominal cavity
대식세포(macrophage)를 추출하기 위하여, 2개월 이상 배양된 티오글리콜레이트 배지(thioglycolate medium;Gibco, USA)를 화학연구소(대전, 한국)에서 제공한 6~10주령 Balb/c 마우스에 투여한 다음 4일이 경과한 후 마우스의 경추를 탈골시켰다.To extract macrophages, thioglycolate medium (Gibco, USA), which had been cultured for 2 months or more, was administered to Balb / c mice at 6 to 10 weeks of age, which was provided by Chemical Laboratories (Daejeon, Korea) After 4 days, the cervical spine of the mouse was disassembled.
희생시킨 마우스의 복강에 100 U/㎖ 농도로 페니실린-스트렙토마이신이 첨가된 불완전 RPMI-1640 배지(Gibco, USA)를 주입하여 복강 내에 있던 대식세포를 추출하고, 다시 원심분리한 다음 24-웰 플레이트에 1×106세포/웰의 농도로 분주하였다.Incomplete RPMI-1640 medium (Gibco, USA) supplemented with penicillin-streptomycin at a concentration of 100 U / ml was added to abdominal cavity of sacrificed mice to extract macrophages in the abdominal cavity, centrifuged again, to 1 × 10 6 it was dispensed at a concentration of cells / well.
분주된 세포 플레이트는 약 1시간 정도 CO2 배양기에서 배양하여, 상기 플레이트에 부착하지 않은 세포와 기타 부유물들을 불완전 RPMI-1640 배지를 사용하여 2번 씻은 다음, 100 U/㎖ 농도의 페니실린-스트렙토마이신과 10% 우태아 혈청이 첨가된 완전 RPMI-1640 배지를 1.5 ㎖/웰의 양으로 주입하고 다시 3일 동안 CO2 배양기(습도 100%, 37℃, 5% CO2 조건)에서 배양하였다.The dispensed cell plate was incubated in a CO 2 incubator for about 1 hour. Cells and other suspensions not attached to the plate were washed twice with incomplete RPMI-1640 medium, and then diluted with 100 U / ml of penicillin-streptomycin And 10% fetal bovine serum were added at a dose of 1.5 ml / well and cultured for 3 days in a CO 2 incubator (humidity 100%, 37 ° C, 5% CO 2 ).
이때, 대식세포의 순수도는 부착된 세포들을 분리하고 사이토스핀-Ⅲ(Cytospin-Ⅲ; 500 rpm, 5분)을 이용하여 슬라이드 글라스에 부착시켜 Wright and Geimsa 염색법으로 세포를 염색하여 측정하였으며, 그 결과 순수도가 95% 이상임을 확인하였다.
At this time, the purity of macrophages was measured by staining the cells with Wright and Geimsa staining method by attaching the cells to a slide glass using Cytospin-III (Cytospin-Ⅲ; 500 rpm, 5 minutes) It was confirmed that the result was more than 95% pure.
2-(2). TNF-α mRNA 분석을 위한 대식세포 자극 및 전체 RNA 분리2- (2). Macrophage stimulation and total RNA isolation for TNF-α mRNA analysis
상기와 같이 분리한 마우스의 대식세포는 6-웰 플레이트(Falcon, USA)에 3×106세포/웰 농도로 도말하고 100U/㎖의 페니실린-스트렙토마이신과 10% 우태아 혈청이 첨가된 완전 RPMI-1640 배지(Gibco, USA)를 사용하여 3일 동안 배양하였다. 세포 배양이 끝난 다음 배양 배지를 씻어내고 새로운 100 U/㎖의 페니실린-스트렙토마이신만이 첨가된 불완전 RPMI-1640 배지(Gibco, USA)를 주입하여 적당한 배지 조건 하에서 상기 대식세포들을 백삼(KT&G, 한국; 10~100 ㎍/㎖), 홍삼(KT&G, 한국; 10~100 ㎍/㎖), 흑마늘 유래 다당류 성분(10 ㎍/㎖), 및 복합제제([백삼(10 ㎍) + 흑마늘 유래 다당류 성분(10 ㎍)]/㎖)를 사용하여 4시간 동안 자극시켰다.The macrophages of the mice thus isolated were plated on a 6-well plate (Falcon, USA) at a concentration of 3 × 10 6 cells / well and washed with 100 U / ml of penicillin-streptomycin and 10% fetal bovine serum -1640 medium (Gibco, USA) for 3 days. After the cell culture was completed, the culture medium was rinsed, and the macrophages were infected with white ginseng (KT & G, Korea) under appropriate medium conditions by injecting incomplete RPMI-1640 medium (Gibco, USA) supplemented with 100 U / ml penicillin- (10 ㎍ / ㎖), red ginseng (KT & G, Korea; 10 ~ 100 ㎍ / ㎖), black garlic-derived polysaccharide component (10 ㎍ / 10 [mu] g / ml) for 4 hours.
어떠한 시료도 처리하지 않은 군을 음성 대조구(negative control)로, 홍삼을 처리한 군을 양성 대조구(positive control)로 사용하였다.Negative control was used for the group not treated with any sample, and positive control was used for the group treated with red ginseng.
세포 자극이 끝나면, 차가운 인산 완충용액(PBS)으로 배양 세포를 2회 씻어내고 PBS를 제거한 각 웰에 1 ㎖의 트리졸(Trisol)을 투여하여 5분 동안 항온에서 배양하였다. 배양이 끝난 다음, 트리졸에 녹인 세포 용액을 3회 피펫팅하여 E-튜브에 담고 0.2 ㎖의 클로로포름을 첨가 후 10초 정도 볼텍스(vortex)하여 세포를 잘 혼합하고 다시 원심분리(속도 12,000 rpm, 60분, 4℃)하였다.After cell stimulation, the cultured cells were washed twice with cold phosphate buffered saline (PBS), and 1 ml of Trisol was added to each well from which the PBS had been removed, followed by incubation at constant temperature for 5 minutes. After the incubation, the cell solution dissolved in the trizol was pipetted 3 times into the E-tube, 0.2 ml of chloroform was added, vortexed for about 10 seconds, the cells were well mixed and centrifuged again (speed 12,000 rpm, 60 min, 4 < 0 > C).
그 다음 전체 RNA 층인 상층액만을 E-튜브에 옮기고, 차가운 100% 이소프로판올을 넣어 적어도 하루 동안 침전시켰다. 다시 동일한 조건에서 20분 정도 원심분리하여 상층액을 따라 버리고 이 과정을 반복하여 순수한 전체 RNA를 분리하였다.Only the supernatant, the entire RNA layer, was then transferred to the E-tube and precipitated with cold 100% isopropanol for at least one day. After centrifuging for 20 minutes under the same conditions, the supernatant was discarded, and this procedure was repeated to isolate pure total RNA.
이어서, 75%(v/v) 에탄올을 첨가하여 손으로 잘 저어 혼합하고 원심분리 하였으며, 원심분리가 끝나면 상층액을 버리고 0.1% DEPC(diethyl pyrocarbonate)-H2O를 넣어 65℃에서 10분 동안 가열한 후 -20℃에서 사용할 때까지 저장하였다.
Subsequently, 75% (v / v) ethanol was added, and the mixture was thoroughly mixed by hand and centrifuged. After centrifugation, the supernatant was discarded and 0.1% DEPC (diethyl pyrocarbonate) -H2O was added and heated at 65 ° C for 10 minutes And stored at -20 < 0 > C until use.
2-(3). 역전사-중합효소 연쇄반응(RT-PCR)을 이용한 TNF-α mRNA 분석2- (3). Analysis of TNF-α mRNA Using Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
역전사-중합효소 연쇄반응을 수행하기 위하여 하기에 기술하는 모든 프라이머는 주식회사 바이오니아(청원, 충북)에 의뢰하여 주문 합성하였다.In order to perform the reverse transcription-polymerase chain reaction, all the primers described below were ordered and synthesized by Biona Co., Ltd. (Cheongwon, Chungbuk).
구체적으로, TNF-α의 정방향 프라이머(forward primer; 5'-GGCAGGTCTTTGGAGTCATTGC-3') 및 역방향 프라이머(reverse primer; 5'-CATTCGAGGCTCCAGTGAATTCCAG-3'), 그리고 베타-액틴의 정방향 프라이머(β-actin forward primer; 5'-CAAAGAAAATGGACGCCGCCGAACTTGG-3') 및 역방향 프라이머(β-actin reverse primer; 5'-CCTGCTTGCTTGCTGATCCACATCTGCTGG-3')를 사용하였다.Specifically, a forward primer (5'-GGCAGGTCTTTGGAGTCATTGC-3 ') and a reverse primer (5'-CATTCGAGGCTCCAGTGAATTCCAG-3') of TNF-α and a β-actin forward primer ; 5'-CAAAGAAAATGGACGCCGCCGAACTTGG-3 ') and a β-actin reverse primer (5'-CCTGCTTGCTTGCTGATCCACATCTGCTGG-3').
중합효소 연쇄반응으로 cDNA 를 합성하기 위하여, 5× 역전사효소 완충용액, 2.5 mM dNTP, 500 ng 프라이머/㎕, 200 U/㎕ MMLV 역전사효소(reverse trancriptase; Takara, Japan), 전체 RNA, 0.1% DEPC-처리된 이차 증류수(DDW)를 첨가하여 반응 혼합액을 준비하였다. 이때 사용한 프라이머는 역방향 프라이머였으며, 42℃에서 30분 동안 배양, 75℃에서 30분 동안 배양을 실시하여 cDNA를 얻었다.To prepare cDNA by polymerase chain reaction, 5 × reverse transcriptase buffer, 2.5 mM dNTP, 500 ng primer / μl, 200 U / μl MMLV reverse transcriptase (Takara, Japan), total RNA, 0.1% DEPC - treated secondary distilled water (DDW) was added to prepare a reaction mixture. The primer used here was a reverse primer, which was cultured at 42 ° C for 30 minutes and then cultured at 75 ° C for 30 minutes to obtain cDNA.
그 다음 상기 역전사 반응 과정에서 얻은 cDNA가 포함된 RT 혼합액, 10× Taq 폴리머라제 완충용액, 2.5 mM dNTP, 정방향 프라이머(25 pmol/㎕), 역방향 프라이머(25 pmol/㎕), Taq 폴리머라제(2.5 units/㎕; Takara,Japan), 이차 증류수를 잘 혼합하여 반응 조건 95℃에서 30초, 55℃에서 1분, 72℃에서 30초를 30회로 반복하여 cDNA를 증폭하였다. 그리고 나서 상기 cDNA를 전기영동하여 TNF-α 유전자의 mRNA 발현 상의 차이를 확인하였다.
Then, the PCR reaction was performed using the RT mixed solution containing cDNA, 10 × Taq polymerase buffer, 2.5 mM dNTP, forward primer (25 pmol / μl), reverse primer (25 pmol / μl), Taq polymerase cDNA was amplified by repeating 30 cycles of 95 ° C for 30 seconds, 55 ° C for 1 minute, and 72 ° C for 30 seconds. Then, the cDNA was electrophoresed to confirm the difference in mRNA expression of the TNF-α gene.
2-(4). TNF-α 단백질의 정량적 분석을 위한 대식세포 자극 및 배양2- (4). Macrophage stimulation and culture for quantitative analysis of TNF-α protein
상기와 같이 분리한 마우스의 대식세포를 6-웰 플레이트(Falcon, USA)에 3×106세포/웰 농도로 접종하고 100U/㎖ 페니실린-스트렙토마이신과 10% 우태아 혈청이 첨가된 완전 RPMI-1640배지(Gibco, USA)를 사용하여 3일 동안 배양하였다.The macrophages of the thus-isolated mice were inoculated into 6-well plates (Falcon, USA) at a concentration of 3 × 10 6 cells / well and cultured in RPMI-1640 medium supplemented with 100 U / ml penicillin-streptomycin and 10% fetal bovine serum. 1640 medium (Gibco, USA) for 3 days.
세포 배양이 끝나면, 배양 배지를 씻어내고 새로운 불완전 RPMI-1640 배지로 적당한 배지 조건 하에서 백삼(KT&G, 한국; 10～100 ㎍/㎖), 홍삼(KT&G, 한국; 10～100 ㎍/㎖), 흑마늘 유래 다당류 성분(10 ㎍/㎖), 및 복합제제([백삼(10㎍) + 흑마늘 유래 다당류 성분(10 ㎍)]/㎖)를 사용하여 6시간 동안 자극시켰다.After the cell culture was completed, the culture medium was rinsed, and red ginseng (KT & G, Korea; 10-100 ㎍ / ml), white ginseng (KT & G, Derived polysaccharide component (10 占 퐂 / ml) and the combination preparation ([white ginseng (10 占 퐂 + black garlic-derived polysaccharide component (10 占 퐂)] / ml) for 6 hours.
상기 실시예 2-(2)와 마찬가지로 어떠한 시료도 처리하지 않은 군을 음성 대조구(negative control)로, 홍삼을 처리한 군을 양성 대조구(positive control)로 사용하였다.As in the case of Example 2- (2), the groups not treated with any sample were used as a negative control and the group treated with red ginseng was used as a positive control.
세포 자극을 위한 배양이 끝난 다음 즉시 각 배양액을 채취하여 원심분리(1000 x g, 4℃, 5분)하고 배양 상층액을 수거하여 하기에 기술한 바와 같은 면역 블럿팅을 사용하여 배양 상등액에 존재하는 TNF-α단백질의 양을 측정하였다.
After the culture for cell stimulation is completed, each culture is immediately collected by centrifugation (1000 xg, 4 캜, 5 minutes), and the culture supernatant is collected, and the culture supernatant is recovered from the culture supernatant using immunoblotting The amount of TNF-a protein was measured.
2-(5) 면역 블럿팅을 이용한 TNF-α 단백질의 정량적 분석2- (5) Quantitative analysis of TNF-α protein by immunoblotting
0.1% SDS(Sodium Dodecyl Sulfate) 폴리아크릴아미드 전기영동 상의 분리 겔과 상단 겔은 각각 16% 및 3%를 사용하였으며, 전기영동시 전류는 55 mA(Powersupply; Pharmacia, Sweden)로 공급하고, 온도는 쿨링 시스템(cooling system; LKB, Sweden)을 사용하여 4℃를 유지하며 약 4~5시간 동안 러닝하였다. 또한, 전기영동 시스템은 Hoefer SE 600(Hoefer-Pharmacia, USA)을 사용하였다.Separation gel and top gel of 0.1% SDS (Sodium Dodecyl Sulfate) polyacrylamide electrophoresis were used at 16% and 3%, respectively. The electrophoresis current was supplied to 55 mA (Powersupply; Pharmacia, Sweden) And maintained for 4 to 5 hours at 4 ° C using a cooling system (LKB, Sweden). The electrophoresis system was a Hoefer SE 600 (Hoefer-Pharmacia, USA).
상기 전기영동이 끝난 다음 얻은 겔은 트랜스퍼 완충용액(transfer buffer; 39 mM 글리신 2.9 g, 48 mM 트리스 베이스 5.8 g, 0.037% SDS, 20% 메탄올, pH 8.3)에 미리 적신 니트로셀룰로스 멤브레인(nitrocellulose membrane; NC)을 위에 올려놓고 섬유 패드(fiber pad)에 넣어 조립하였으며, 면역 블럿팅 트랜스퍼 카세트(immunoblotting transfer cassette)를 니트로셀룰로스 멤브레인이 양극 (positive charge) 방향으로 오고, 겔이 음극(negative charge) 방향으로 오도록 맞추어 트랜스퍼 챔버에 넣어두었다. 전류는 1 A로 공급하고, 온도는 쿨링 시스템을 사용하여 4℃를 유지하면서 1시간 30분 동안 트랜스퍼시켰다.After completion of the electrophoresis, the obtained gel was immersed in a nitrocellulose membrane pre-wetted with transfer buffer (39 mM glycine, 2.9 g, 48 mM tris base, 5.8 g, 0.037% SDS, 20% methanol, pH 8.3) NC) was placed on a fiber pad and assembled. An immunoblotting transfer cassette was prepared by placing the nitrocellulose membrane in the direction of a positive charge and placing the gel in the direction of the negative charge And placed in the transfer chamber. The current was supplied at 1 A, and the temperature was transferred for 1 hour and 30 minutes while maintaining the temperature at 4 ° C using a cooling system.
상기 니트로셀룰로스 멤브레인은 블로킹 완충용액(blocking buffer; 5% 무지방 분유(nonfat dry milk)를 녹인 TBS-T 완충용액(20 mM 트리스-HCl pH 7.5, 150 mM NaCl, 0.05% 트윈-20) 약 200 ㎖에 1시간 동안 Red-Rocker(Hoefer; CA, USA)를 사용하여 블로킹하고 항체(antibody)의 비특이적 결합(non-specific binding)을 블로킹한 다음, 다시 TBS-T 완충용액으로 15분 동안 1회, 10분 동안 2회 씻어내었다.The nitrocellulose membrane was washed with blocking buffer (TBS-T buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20) dissolved in 5% nonfat dry milk at about 200 Ml of the antibody was blocked with Red-Rocker (Hoefer; CA, USA) for 1 hour to block the non-specific binding of the antibody, and then once again with TBS-T buffer for 15 minutes , And washed twice for 10 minutes.
그 다음 TBS-T 완충용액 3 ㎖를 실리콘 용기(siliconized bottle)에 분주하고 여기에 토끼 항-생쥐 TNF-α 다클론 일차 항체(rabbit anti-murine TNF-α polyclonal primary antibody; PBS에 500 ㎍/1.5 ㎖ 농도) 30 ㎕(1 : 300 희석 비율)를 각각 첨가하여 니트로셀룰로스 멤브레인을 실리콘 용기에 넣은 후 4℃로 맞춘 하이브리디제이션 반응기(hybridization incubator; Robbins, USA)에서 10 rpm 속도로 12시간 동안 반응시켰다.Next, 3 ml of TBS-T buffer was dispensed into a siliconized bottle, and a rabbit anti-murine TNF-alpha polyclonal primary antibody (500 / / 1.5 (1: 300 dilution ratio) was added to the nitrocellulose membrane, and the nitrocellulose membrane was placed in a silicone container. The mixture was incubated at 4 rpm in a hybridization incubator (Robbins, USA) at 10 rpm for 12 hours .
반응완료 후, TBS-T 완충용액 70 ㎖를 사용하여 20분 동안 각각 3번 하이브리디제이션 반응기에서 12 rpm 속도로 씻어내고 이차 항체로서 염소 항-토끼 IgG 퍼옥시다제 결합 항체(goat anti-rabbit IgG horseradish peroxidase conjugated affinity purified antibody; 스톡 용액은 PBS에 0.02 ㎎/4 ㎖ 농도)를 친화성 분리하여 3 ㎖ TBS-T 완충용액에 30 ㎕(1 : 20,000 희석 비율)을 첨가한 다음 4℃로 맞춘 하이브리디제이션 반응기에서 3시간 동안 배양하고 다시 70 ㎖ TBS-T 완충용액을 사용하여 20분 동안 각각 4번 씻어내었다.After the completion of the reaction, 70 ml of TBS-T buffer solution was used to wash the cells in a hybridization reactor for 3 minutes at 12 rpm for 20 minutes, and a goat anti-rabbit IgG peroxidase-conjugated antibody (goat anti-rabbit IgG 30 μl (1: 20,000 dilution ratio) was added to 3 ml of TBS-T buffer solution, and the mixture was incubated at 4 ° C for 4 h. The hives were then incubated with a horseradish peroxidase conjugated affinity purified antibody (0.02 mg / The cells were incubated in a rehydration reactor for 3 hours and then washed four times for 20 minutes each time with 70 ml of TBS-T buffer.
그 다음 니트로셀룰로스 멤브레인을 얇고 투명한 비닐에 넣고 퍼옥시다제 기질(ECL; peroxidase substrate)반응용액 1 및 반응용액 2를 50%씩 혼합한 반응용액 3 ㎖를 멤브레인 위에 분주하고 5분 동안 배양하였다. 반응이 끝난 다음 암실(10 W 안전 램프; Kodak, USA)에서 NC가 들어있는 카세트에 X-선 필름을 끼우고 5분 또는 10분 이상 감광시킨 후 X-선 필름 현상용 박스 안의 현상 용액(develop solution)에 약 3분 동안 넣어 반응시켰다.Next, the nitrocellulose membrane was placed in a thin, transparent vinyl, and 3 ml of the reaction solution in which 50% of peroxidase substrate (ECL) reaction solution 1 and reaction solution 2 were mixed was placed on the membrane and cultured for 5 minutes. After the reaction was completed, the X-ray film was inserted into a cassette containing NC in a dark room (10 W safety lamp; Kodak, USA) and exposed for 5 minutes or 10 minutes or more. solution for about 3 minutes.
마지막으로, 상기 X-선 필름을 다시 2분 동안 물로 씻은 다음 고정 용액(fixing solution)에서 3분 동안 반응시키고, 다시 2분 동안 물로 씻어 나타나는 밴드를 분석하였다.
Finally, the X-ray film was washed again with water for 2 minutes, then reacted in a fixing solution for 3 minutes, and again washed with water for 2 minutes to analyze the band appearing.
실시예 3. 백삼에 의한 T 림프구와 자연살해세포(NK 세포) 활성화에 있어 흑마늘 유래 다당류 성분에 의한 동반상승효과 확인 Example 3 . Identification of synergistic effects of white-ginseng-derived polysaccharide components in the activation of T lymphocytes and natural killer cells (NK cells)
본 발명의 면역증강용 조성물의 면역활성 검사의 일환으로, T 림프구와 자연살해세포(NK 세포)가 많이 존재하는 비장을 이용하여 해당 살해인자인 퍼포린(perforin)의 mRNA 발현 상승능과 단백질 생산분비 상승능 효과를 확인하였다.
As a part of the immunoactivity test of the immunoadjuvant composition of the present invention, using the spleen in which T lymphocytes and natural killer cells (NK cells) are abundant, the ability of perforin, which is a killer, Secretion enhancing effect was confirmed.
3-(1). 퍼포린 mRNA 분석을 위한 마우스에 시료 투여 및 마우스의 비장으로부터 전체 RNA의 분리3- (1). Administration of samples to mice for perforin mRNA analysis and isolation of total RNA from spleen of mice
백삼에 의한 비장내의 T 림프구와 NK 세포 활성화에 있어 흑마늘 다당류인 흑마늘 유래 다당류 성분에 의한 상승효과여부를 조사하기 위하여 7주령의 Balb/c 마우스(대전 화학연구소, 한국)에 백삼(KT&G, 한국; 30 ~ 300 ㎎/㎏of body weight), 홍삼(KT&G, 한국; 30 ~ 300 ㎎/㎏ of body weight), 흑마늘 유래 다당류 성분(30 ㎎/㎏ of body weight), 복합제제([백삼 30 ㎎ + 흑마늘 유래 다당류 성분 30 ㎎]/㎏ of body weight)을 4일 동안 하루 2회 응용시켰다. 어떠한 시료도 음용시키지 않은 군을 음성 대조구(negative control)로, 홍삼을 음용시킨 군을 양성 대조구(positive control)로 사용하였다.To investigate the synergistic effect of white ginseng on the T lymphocyte and NK cell activation by white ginseng, black ginseng polysaccharide, white ginseng (KT & G, Korea, Korea) was administered to 7 week old Balb / c mice (Daejeon Chemical Research Institute, Korea). (30 mg / kg body weight), 30 mg / kg body weight of red ginseng, 30 mg / kg body weight of Korean red ginseng (30 mg / kg body weight) The component of polysaccharide derived from black garlic was 30 ㎎] / ㎏ of body weight) was applied twice a day for 4 days. Negative controls were used for the non-consumed groups and positive control groups were used for the red ginsengs.
음용시킨 후 경추 탈골해서 희생시키고 일반적으로 T 림프구와 NK 세포가 많이 존재하는 비장을 무균 상태에서 회수한 다음 100 U/㎖의 페니실린-스트렙토마이신만이 첨가된 차가운 불완전 RPMI-1640 배지(Gibco, USA)로 2회 씻고 다시 차가운 불완전 RPMI-1640 배지를 주입하여 Daunce 세포균질기(Wheaton, USA)를 사용하여 각각 하나의 세포를 만든 후 비장 내의 적혈구를 모두 제거하였다. 제거한 후 1× 106 비장의 세포들을 5 ㎖튜브에 옮긴 후 1.5 ㎖의 트리졸(Trisol)을 투여하여 5분 동안 항온에서 배양하였다.The spleen, which generally contains T lymphocytes and NK cells, is recovered in an aseptic state, and then cultured in cold incomplete RPMI-1640 medium (Gibco, USA) supplemented with 100 U / ml of penicillin-streptomycin alone ), And then cold incomplete RPMI-1640 medium was injected. One cell was made using a Daunce cell homogenizer (Wheaton, USA), and all of the red blood cells in the spleen were removed. After removing the cells, 1 × 10 6 spleen cells were transferred to 5 ml tubes and 1.5 ml of Trisol was added thereto, followed by incubation at a constant temperature for 5 minutes.
트리졸 배양이 끝난 다음의 모든 과정은 실시예 2-(2)와 동일한 방법으로, 순수한 전체 RNA를 분리한 후 75%(v/v) 에탄올을 첨가하여 손으로 잘 저어 혼합하고 원심분리 하였으며, 원심분리가 끝나면 상층액을 버리고 0.1% DEPC-H2O 를 넣어 65℃에서 10분 동안 가열한 후 -20℃에서 사용할 때까지 저장하였다.
After the completion of the trizol culture, the pure total RNA was separated, and 75% (v / v) ethanol was added thereto by the same procedure as in Example 2- (2) After centrifugation, the supernatant was discarded and 0.1% DEPC-H2O was added, and the mixture was heated at 65 ° C for 10 minutes and stored at -20 ° C until use.
3-(2). 역전사-중합효소 연쇄반응(RT-PCR)을 이용한 마우스 비장내의 퍼포린 mRNA 분석3- (2). Analysis of perforin mRNA in mouse spleen using reverse transcription-polymerase chain reaction (RT-PCR)
역전사-중합효소 연쇄반응을 이용한 마우스 비장내의 퍼포린(perforin) mRNA 분석을 위해 퍼포린의 정방향 프라이머(forward primer)는 5'-TGGAGTGCCGCTTCTACAGTT-3'과 역방향 프라이머(reverse primer)는 5'-GTGGGTGCCGTAGTTGGAGAT-3'을 이용해 cDNA 합성, cDNA 증폭, cDNA 전기영동 실험과정을 상기 실시예 2-(3)과 동일하게 수행하여 비장내 T 림프구와 NK 세포로부터 퍼포린 mRNA 유전자 발현능을 분석하였다.
5'-TGGAGTGCCGCTTCTACAGTT-3 'for the forward primer and 5'-GTGGGTGCCGTAGTTGGAGAT-3' for the reverse primer for perforin mRNA analysis in mouse spleen using the reverse transcription-polymerase chain reaction 3 ', cDNA amplification, cDNA amplification, and cDNA electrophoresis were performed in the same manner as in Example 2- (3) above, and the ability of perforin mRNA gene expression from spleen T lymphocytes and NK cells was analyzed.
3-(3). 퍼포린 단백질의 정량적 분석을 위한 마우스에 시료 투여 및 마우스의 비장으로부터 단백질의 분리3- (3). Administration of samples to mice for quantitative analysis of perforin proteins and separation of proteins from the spleen of mice
상기 실시예 3-(1)과 동일하게 7주령의 Balb/c 마우스(대전 화학연구소, 한국)에 백삼(KT&G, 한국; 30 ～ 300㎎/㎏ of body weight), 홍삼(KT&G, 한국; 30 ～ 300 ㎎/㎏ of body weight), 흑마늘 유래 다당류 성분(30 ㎎/㎏ of body weight), 복합제제([백삼 30 ㎎ + 흑마늘 유래 다당류 성분 30 ㎎]/㎏ of body weight)을 4일 동안 하루 2회 응용시켰다. 어떠한 시료도 음용시키지 않은 군을 음성 대조구(negative control)로, 홍삼을 음용시킨 군을 양성 대조구(positive control)로 사용하였다.(KT & G, Korea; 30-300 mg / kg of body weight), red ginseng (KT & G, Korea; Korea) in 7-week-old Balb / c mice (30 mg of white ginseng + 30 mg of polysaccharide derived from black garlic) / kg of body weight) was added for 4 days to the body weight of 4 mg / kg body weight, 2 times. Negative controls were used for the non-consumed groups and positive control groups were used for the red ginsengs.
음용시킨 후 경추 탈골해서 희생시키고 일반적으로 T 림프구와 NK 세포가 많이 존재하는 비장을 무균 상태에서 회수한 다음 100 U/㎖의 페니실린-스트렙토마이신만이 첨가된 차가운 불완전 RPMI-1640 배지(Gibco, USA)로 2회 씻고 다시 차가운 불완전 RPMI-1640 배지를 주입하여 Daunce 세포균질기(Wheaton, USA)를 사용하여 각각 하나의 세포를 만든 후 비장 내의 적혈구를 모두 제거하였다. 제거한 후 원심분리하여 상층액을 버리고 단백질 분리를 위한 라이시스 완충용액(lysis buffer)을 첨가한 후 100℃에서 5분 동안 가열한 후 퍼포린량 측정을 위한 면역 블럿팅 전까지 -20℃에서 저장하였다.
The spleen, which generally contains T lymphocytes and NK cells, is recovered in an aseptic state, and then cultured in cold incomplete RPMI-1640 medium (Gibco, USA) supplemented with 100 U / ml of penicillin-streptomycin alone ), And then cold incomplete RPMI-1640 medium was injected. One cell was made using a Daunce cell homogenizer (Wheaton, USA), and all of the red blood cells in the spleen were removed. After removing the supernatant, the supernatant was discarded. The lysis buffer for protein isolation was added, and the mixture was heated at 100 ° C for 5 minutes and stored at -20 ° C until the immunofluorescence for the measurement of perforin.
3-(4). 면역 블럿팅(immunoblotting)을 이용한 마우스 비장내의 퍼포린 단백질의 정량적 분석3- (4). Quantitative analysis of perforin protein in mouse spleen using immunoblotting
마우스 비장내의 퍼포린 단백질 정량 분석을 위한 면역 블럿팅의 과정을 상기 실시예 2-(5)와 같은 방법과 과정으로 전기영동, 겔 트랜스퍼, 및 니트로셀룰로스 멤브레인 블로킹과정을 실시하였다.Immunoblotting for the quantitative analysis of perforin protein in mouse spleen was performed by electrophoresis, gel transfer, and nitrocellulose membrane blocking process by the same method and procedure as in Example 2- (5).
블로킹 과정 후 항체와의 하이브리디제이션 반응(hybridization reaction)에는 토끼 항-생쥐 퍼포린 다클론일차 항체(rabbit anti-murine TNF-α polyclonal primary antibody)를 사용하였고, 이차 항체로는 염소 항-토끼 IgG 퍼옥시다제 결합 항체(goat anti-rabbit IgG horseradish peroxidase conjugated affinity purified antibody)를 사용하였다.Rabbit anti-murine TNF-α polyclonal primary antibody was used for the hybridization reaction with the antibody after blocking, and secondary anti-rabbit anti-rabbit IgG A goat anti-rabbit IgG horseradish peroxidase conjugated affinity purified antibody was used.
항체와의 하이브리디제이션 반응 후 X-선 필름 현상까지 모든 실험진행 과정은 상기 실시예 2-(5)와 동일하게 수행하고, 상기 X-선 필름을 다시 2분 동안 물로 씻어 고정 용액(fixing solution)에서 3분 동안 반응시키고 다시 2분 동안 물로 씻어 나타나는 밴드를 분석하였다.
The hybridization reaction with the antibody and the X-ray film development were carried out in the same manner as in Example 2- (5), and the X-ray film was again washed with water for 2 minutes to remove the fixing solution ) For 3 minutes and washed again with water for 2 minutes to analyze the bands that appeared.
실시예 4. 백삼에 의한 B 림프구 항체생산에 있어 흑마늘 유래 다당류 성분에 의한 동반상승효과 확인 Example 4 . Identification of synergistic effects of Bacillus subtilis polysaccharide components in the production of B lymphocyte antibodies by white ginseng
본 발명에서는 항체를 생산하는 세포를 검출하기 위한 실험인 항체플라크 형성 세포(Antibody Plaque FormingCell; AFC) assay 기법(장성옥 외 2인, 천식 및 알레르기, 23(4):818-825, 2003년)을 이용하여, 백삼에 의한 B 림프구 항체생산에 있어 흑마늘 다당류인 흑마늘 유래 다당류 성분에 의한 동반상승효과(synergic effect)를 확인하였다.
In the present invention, an antibody-plaque forming cell (AFC) assay technique (Jiang et al., Et al., Asthma and allergy, 23 (4): 818-825, 2003), which is an experiment for detecting an antibody- In the production of B lymphocyte antibodies by white ginseng, the synergic effect of the black gum polysaccharide-derived polysaccharide component was confirmed.
4-(1). 마우스에 시료 투여4- (1). Samples are administered to mice
7주령의 BALB/c 마우스(대전 화학연구소, 한국)에 1×109 개의 sheep red blood cell(sRBC)를 정맥주사하여 감작시켰다. 감작 당일 날부터 시작하여 4일 동안 백삼(KT&G, 한국; 30 ~ 300 ㎎/㎏ of body weight), 홍삼(KT&G, 한국; 30 ~ 300 ㎎/㎏ of body weight), 흑마늘 유래 다당류 성분(30 ㎎/㎏ of body weight), 복합제제([백삼 30 ㎎ +흑마늘 유래 다당류 성분 30 ㎎]/㎏ of body weight)를 하루 2회 응용시켰다. 어떠한 시료도 음용시키지 않은 군을 음성 대조구(negative control)로, 홍삼을 음용시킨 군을 양성 대조구(positive control)로 사용하였다.
Seven week old BALB / c mice (Daejeon Chemical Research Institute, Korea) were sensitized by intravenous injection of 1 x 10 9 sheep red blood cells (sRBC). (30 ~ 300 ㎎ / ㎏ of body weight), red ginseng (KT & G, Korea, 30 ~ 300 ㎎ / ㎏ of body weight) and black garlic - derived polysaccharide component (30 ㎎ / Kg body weight) and a combination preparation ([white ginseng 30 mg + polysaccharide derived from black garlic 30 mg] / kg of body weight) twice a day. Negative controls were used for the non-consumed groups and positive control groups were used for the red ginsengs.
4-(2). Direct Hemolytic Antibody Plaque Forming Cell(AFC)를 이용한 항체 생산의 정량적 분석 4일 뒤에 마우스를 희생시키고 비장을 적출하여 용혈(hemolytic) PFC assay를 실시하였다(장성옥 외 2인, 천식 및 알레르기 23(4):818-825, 2003).
4- (2). Quantitative Analysis of Antibody Production Using Direct Hemolytic Antibody Plaque Forming Cell (AFC) Four days later, mice were sacrificed and spleens were extracted to perform a hemolytic PFC assay (Jung et al., 2, Asthma and allergy 23 (4) 818-825, 2003).
구체적으로, 비장을 단일세포 부유액(single cell suspension)으로 만든 뒤 비장 내의 적혈구를 모두 제거하고, 비장세포와 sRBC를 아가로오스(agarose)와 섞은 뒤 0.3% 아가로오스로 코팅한 슬라이드 글래스 상에 도포하였다. 실온에서 굳힌 뒤 습상에 넣어 5% CO2, 37℃ 배양기에서 30분간 배양하였다. 이후 1:30으로 희석한 보체(complement; Sigma, Missouri, USA)를 분주하고 역시 습상에 넣어 5% CO2, 37℃ 배양기에서 1시간 이상 배양하여 항체 플라크(antibody plaque)의 수를 육안으로 관찰하여 항체의 생산 정도를 분석하였다.
Specifically, the spleen was made into a single cell suspension, all of the red blood cells in the spleen were removed, and the splenocytes and sRBC were mixed with agarose and then immersed in a 0.3% agarose-coated slide glass Respectively. After curing at room temperature, it was put in a humidified condition and incubated in a 5% CO 2 incubator at 37 ° C for 30 minutes. After that, 1: 30 diluted complement (Sigma, Missouri, USA) was added and incubated in a humidified 5% CO 2 incubator at 37 ° C for 1 hour or more to observe the number of antibody plaques with naked eyes And the level of antibody production was analyzed.
실시예 5. 복합제제의 세포독성 확인Example 5. Cytotoxicity of Compound Preparation
본 발명에 따른 백삼과 흑마늘 유래 다당류 성분의 복합제제가 정상 세포에 미치는 세포독성을 하기와 같이 확인하였다.
The cytotoxicity of the complex agent of white ginseng and black garlic-derived polysaccharide components to normal cells according to the present invention was confirmed as follows.
5-(1). 비장세포 추출 및 배양5- (1). Splenocyte Extraction and Culture
7주령의 Balb/c 마우스(대전 화학연구소, 한국)를 경추 탈골해서 희생시키고, 비장을 무균 상태에서 회수한 다음 100 U/㎖의 페니실린-스트렙토마이신만이 첨가된 차가운 불완전 RPMI-1640 배지(Gibco, USA)로 2회 씻고 다시 차가운 불완전 RPMI-1640 배지를 주입하여 Daunce 세포균질기(Wheaton, USA)를 사용하여 각각 하나의 세포를 만들고 얼음물에 10분 동안 배양하였다.Seven weeks old Balb / c mice (Daejeon Chemical Research Institute, Korea) were sacrificed by cervical dislocation, the spleen was recovered in aseptic state, and then cold incomplete RPMI-1640 medium supplemented with 100 U / ml penicillin-streptomycin , USA), cold incomplete RPMI-1640 medium was added, and one cell was prepared using Daunce cell homogenizer (Wheaton, USA) and incubated in ice water for 10 minutes.
배양이 끝나면 세포 상층액만을 수집하여 원심분리(1000 x g, 5분, 4℃)하고 다시 상층액을 따라버리고 세포를 얻었다. 비장세포는 96-웰 플레이트에 1.5×105세포/웰 농도로 분주하여 100 U/㎖의 페니실린-스트렙토마이신과 10% 우태아 혈청이 첨가된 완전 RPMI-1640 배지(Gibco, USA)로 배양하였다. 그 다음 각 웰에 면역증강복합제제(백삼 + 흑마늘 유래 다당류 성분)를 농도별로 첨가하고 2일 동안 배양하여 MTT 분석을 수행하였다.
After culturing, only the supernatant was harvested, centrifuged (1000 xg, 5 min, 4 ° C) and the supernatant was discarded to obtain cells. Splenocytes were plated in 96-well plates at a concentration of 1.5 × 10 5 cells / well and cultured in complete RPMI-1640 medium (Gibco, USA) supplemented with 100 U / ml penicillin-streptomycin and 10% fetal bovine serum . Then, MTT assay was performed by adding the immunopotentiating combination (white ginseng + black garlic-derived polysaccharide component) to each well at a concentration and culturing for 2 days.
5-(2). 화학적 세포독성 검사를 위한 MTT 분석5- (2). MTT assay for chemical cytotoxicity
본 발명에서는 비장세포에 대한 면역증강 복합제제(백삼 + 흑마늘 유래 다당류 성분)의 세포 독성 유무를 MTT 방법을 사용하여 검증하였다.In the present invention, the cytotoxicity of the combined immunosuppressant preparation (white ginseng + black garlic-derived polysaccharide component) against spleen cells was verified by MTT method.
MTT 분석은 프랑소아와 리타(Francois D and Lita L., J. Immunological methods, 89, 271-277, 1986)의 방법에 준하여 다음과 같이 실시하였다.MTT assays were performed according to the method of Francois D and Lita L., J. Immunological methods, 89, 271-277, 1986 as follows.
비장세포의 배양이 완료되면 MTT 반응액(HBSS 완충용액에서 농도 1 ㎎/㎖)을 각 웰 당 50 ㎕씩 첨가하고 37℃에서 4시간 동안 다시 배양하였다. 이후 배양 상등액은 모두 제거하고 침전된 불용성 포르마잔(formazan)을 녹이기 위하여 디메틸설포옥사이드(dimethylsulfoxide; DMSO)를 각 웰 당 100 ㎕씩 첨가하여 15분 동안 저어주고(stirring), 엘리자 판독기(ELISA reader; Pharmacia, USA)를 사용하여 540 nm 파장에서의 흡광도(O.D.)를 측정하여 % 세포 독성(cytotoxicity)을 측정하였다.
When the spleen cells were cultured, 50 μl of MTT reaction solution (concentration 1 mg / ml in HBSS buffer solution) was added to each well, followed by incubation at 37 ° C for 4 hours. The culture supernatant was then removed and 100 μl of dimethylsulfoxide (DMSO) was added to dissolve the precipitated insoluble formazan, stirred for 15 minutes, and resuspended in an ELISA reader. (OD) at a wavelength of 540 nm was measured using a fluorescence microscope (Sigma, Pharmacia, USA) to determine% cytotoxicity.
실시예 6. 복합제제의 실험동물 장기독성 확인 Example 6 . Identification of long-term toxicity of compound preparation in experimental animals
본 발명에서는 복합제제가 생체의 신진대사와 독성에 민감한 주요 장기인 간과 신장에 미치는 독성을 확인하였다.In the present invention, the combination agent was confirmed to be toxic to liver and kidney, major organs sensitive to the metabolism and toxicity of living organisms.
이때, 신장 기능은 혈액 크레아틴(blood creatin, CRE)와 혈액 우레아 질소 (blood urea nitrogen, BUN)의 혈중 농도를 측정하여 검사하고, 간 기능은 간세포가 함유하는 GOT(glutamic-oxalacetate transaminase)와 GPT(glutamic pyruvic transaminase)의 두 효소의 혈중 농도를 측정하여 검사하였다.The kidney function is measured by measuring the blood levels of blood creatin (CRE) and blood urea nitrogen (BUN), and hepatic function is measured by GOT (glutamic-oxalacetate transaminase) and GPT glutamic pyruvic transaminase) were measured and examined.
7주령의 2e마우스(대전 화학연구소, 한국)를 사용하여 실험군에는 복합제제(백삼 + 흑마늘 유래 다당류 성분; 10 ㎎/㎏ of body weight 또는 300 ㎎/㎏of body weight) 매일 2회 음용시켰고, 비교구인 정상군에는 어떠한 시료도 투여하지 않고 일반 음용수만 공급하였다. 음용시킨 후 1, 2, 5 및 10일이 경과하면 마우스의 눈으로부터 채혈하여 얻은 혈청 10 ㎕를 다층 필름(multilayer film) 상에 놓고 후지 드라이 켐 시스템(Fugi Dry Chem System;Fugi Photo Film, Japan)을 이용하여 각각 CRE, BUN, GOT 및 GPT값을 측정하였다.
(10 mg / kg body weight or 300 mg / kg body weight) was used twice daily in the experimental group using 7 week old 2e mouse (Daejeon Chemical Research Institute, Korea) In the normal group, only normal drinking water was supplied without any sample. Fugi Dry Chem System (Fugi Photo Film, Japan) was used to place 10 μl of serum obtained from the eyes of a mouse on a multilayer film after 1, 2, 5 and 10 days after drinking, Were used to measure CRE, BUN, GOT, and GPT values, respectively.
실시예 7. 복합제제의 실험동물 성장독성 확인 Example 7 . Toxicity of experimental animal growth
본 발명의 복합제제(백삼 + 흑마늘 유래 다당류 성분)의 안전성을 확인하기 위하여, 미성숙 또는 성숙한 쥐의 신체 발육에 미치는 영향을 체중 증감 변화로 조사하였다.In order to confirm the safety of the combination preparation (white ginseng + black garlic-derived polysaccharide component) of the present invention, the effect on the body development of immature or adult rats was examined by weight change.
복합제제(백삼 + 흑마늘 유래 다당류 성분; 10 ~ 300 ㎎/㎏ of body weight)를 생후 1일째의 미성숙 마우스에는 매일 1회 피하주사하고, 생후 7주령된 성숙 마우스에는 매일 2회 음용시켰으며, 대조군(0 ㎎/㎏ of body weight)에는 0.01M 인산 완충용액(PBS)을 피하주사(미성숙 마우스)하거나 음용(성숙 마우스)시켰다.
The immature mice at day 1 were subcutaneously injected once daily, and the mice at 7 weeks of age were consecutively administered twice daily, while the control mice (control group) (0 mg / kg body weight) was subcutaneously injected (immature mouse) or drinking (maturation mouse) with 0.01 M phosphate buffered saline (PBS).
이상, 본 발명의 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific descriptions are only for the preferred embodiment and that the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
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| KR20220169089A (en) | 2021-06-18 | 2022-12-27 | 엠케이바이오파낙스 주식회사 | Health supplement composition using Hwangchil wood and black garlic, and its manufacturing method and product |
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| CN112812196A (en) * | 2019-11-18 | 2021-05-18 | 晨光生物科技集团股份有限公司 | Industrial method for producing garlic polysaccharide by using garlic processing waste |
| KR20220169089A (en) | 2021-06-18 | 2022-12-27 | 엠케이바이오파낙스 주식회사 | Health supplement composition using Hwangchil wood and black garlic, and its manufacturing method and product |
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