KR20130122066A - A composition comprising extract of fermented ginseng using lactobacillus plantarum crnb-22 for treating or preventing atopic dermatitis - Google Patents
A composition comprising extract of fermented ginseng using lactobacillus plantarum crnb-22 for treating or preventing atopic dermatitis Download PDFInfo
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- KR20130122066A KR20130122066A KR1020120045101A KR20120045101A KR20130122066A KR 20130122066 A KR20130122066 A KR 20130122066A KR 1020120045101 A KR1020120045101 A KR 1020120045101A KR 20120045101 A KR20120045101 A KR 20120045101A KR 20130122066 A KR20130122066 A KR 20130122066A
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- KR
- South Korea
- Prior art keywords
- extract
- lactobacillus plantarum
- ginseng
- atopic dermatitis
- fermented ginseng
- Prior art date
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Abstract
Description
본 발명은 락토바실러스 플랜타럼 CRNB-22(Lactobacillus plantarum CRNB-22, 미생물 수탁번호 : KCTC11931BP)를 이용한 발효인삼의 추출물을 함유하는 아토피 피부염의 예방 또는 치료용 조성물에 관한 것이다.The invention Lactobacillus plan tareom CRNB-22 (Lactobacillus plantarum CRNB-22, microorganism accession no.: KCTC11931BP). The present invention also relates to a composition for preventing or treating atopic dermatitis.
더욱 상세하게는, 본 발명은 MCP-1(monocyte chemoattractant protein-1), IL-8(interleukin-8) 및 IL-6(interleukin-6)의 활성을 억제하는 효과가 뛰어난 락토바실러스 플랜타럼 CRNB-22(Lactobacillus plantarum CRNB-22, 미생물 수탁번호 : KCTC11931BP)를 이용한 발효인삼의 추출물을 함유하는 아토피 피부염의 예방 또는 치료용 조성물에 관한 것이다.More particularly, the present invention relates to a method for inhibiting the activity of MCP-1 (monocyte chemoattractant protein-1), IL-8 (interleukin-8) and IL-6 (interleukin-6), Lactobacillus plantarum CRNB- 22 ( Lactobacillus plantarum CRNB-22, microorganism accession no.: KCTC11931BP). The present invention also relates to a composition for preventing or treating atopic dermatitis.
아토피 피부염의 병인기전은 아직 완전하게 밝혀지지 않았지만 환경 내에서 흔한 물질(알레르겐)에 대한 과민한 면역반응(알레르기반응)으로 인하여 피부에 만성적인 염증반응을 유발하여 아토피 피부염이 발생한 것으로 알려져 왔다. 또한, 아토피 피부염(atopic dermatitis)은 천식과 알레르기성 질환의 발달 전에 발생하는 만성적인 염증질환으로, 케모카인의 농도, 양적변화나 활성 정도에 따라 그 증상에 차이가 있으며(Curr. Allergy Asthma Rep., 2005, 5, 284~290), Th2 타입의 림프구가 병변부(lesional skin)로 침입하는 현상을 말하기도 한다.Although the pathogenesis of atopic dermatitis has not yet been fully elucidated, it has been known that atopic dermatitis is caused by a chronic inflammatory reaction caused by an irritant immune reaction (allergic reaction) to a common substance (allergen) in the environment. In addition, atopic dermatitis is a chronic inflammatory disease that occurs before the development of asthma and allergic diseases, and the symptoms vary depending on the concentration of chemokine, the quantitative change, and the degree of activity (Curr. Allergy Asthma Rep., 2005, 5, 284-290), and also refers to a phenomenon in which a Th2-type lymphocyte invades a lesional skin.
현재 세계 인구의 0.5%~1%, 특히, 어린이의 경우 5~10%가 아토피 피부염으로 고생하는 것으로 알려져 있으며, 그 발증과 악화에는 환경인자가 크게 관여하고 있는 것으로 알려져 있다. 그 예로서, 아토피 피부염의 발병 빈도는 집먼지 진드기에 노출된 양과 상관성을 보이며 아토피 피부염의 중증도와도 유의한 상관관계를 보인다고 알려져 있다. 또한 환경 내에서 집먼지 진드기에 대한 노출을 줄임으로써 아토피 피부염의 중증도를 감소시킬 수 있다고 알려져 있다. 그 외에 아토피 피부염은 건조한 피부, 정상인에 비해 쉽게 피부 가려움증을 느끼는 특성, 세균·바이러스·곰팡이 등에 의한 감염, 정서적 요인, 환경적 요인 등이 서로 복합적으로 작용하여 일어나는 것으로 보인다. 최근 수십 년 동안 아토피 피부염의 발병률이 상승하고 있으며, 우리나라 또한 그 환자 수가 크게 늘고 있는 현실이다. It is known that 0.5% to 1% of the world's population, especially 5% to 10% of children, suffer from atopic dermatitis. Environmental factors are known to be involved in the onset and deterioration. As an example, the incidence of atopic dermatitis is correlated with the amount of exposure to house dust mites, and it is known that the severity of atopic dermatitis also has a significant correlation. It is also known to reduce the severity of atopic dermatitis by reducing exposure to house dust mites in the environment. In addition, atopic dermatitis appears to be caused by a combination of dry skin, easily itchy skin, infection by bacteria, viruses, fungi, emotional factors, and environmental factors. In recent decades, the incidence of atopic dermatitis has been rising, and the number of patients in Korea is also increasing.
아토피 피부염을 일으키는 사이토카인 중의 하나인 IL-8(interleukin-8)은 기도 상피세포에서 분비되는 중요한 염증성 케모카인(chemokine)으로서 염증 초기에 주로 발현되며, IL-8에 의해 기관지 과민성이 유발되고 알레르기 비염이나 기관지 천식에서도 증가되는 것으로 알려져 있다. 이에 대한 예로서, 아토피 피부염을 유발하는 집먼드기 추출물을 THP-1 세포나 EoL-1 세포에 처리하면 아토피 피부염을 유발하는 사이토카인(cytokine)인 IL-8의 양이 증가하게 된다. IL-8 (interleukin-8), one of the cytokines that cause atopic dermatitis, is an important inflammatory chemokine secreted from the airway epithelium. It is mainly expressed at the early stage of inflammation. IL-8 induces bronchial hyperresponsiveness. And bronchial asthma. As an example of this, treatment of THP-1 cells or EoL-1 cells with an extract of Phellinus linteus causing atopic dermatitis increases the amount of IL-8, a cytokine that causes atopic dermatitis.
한편, 아토피 피부염을 일으키는 다른 종류의 사이토카인으로는 MCP-1(monocyte chemoattractant protein-1)이 있는데, 상기 MCP-1은 CCR2(chemokine receptor)에 결합하며 MCP-1 유전자가 제거된 생쥐는 단핵구(monocytes)에 대한 화학주성이 손상을 받고, 특정 균의 감염에 저항성이 약화되는 것이 관찰되었으며, 이러한 현상은 CCR2 유전자가 제거된 동물에서 관찰되는 증상과 유사하다고 보고된 바 있다. 이 밖에도 MCP-1은 시험관에서 Th0 세포를 Th2 사이토카인을 분비하는 세포로 전환시키며, MCP-1을 정맥주사 하였을 때 IL-12의 생성이 감소되고 IL-4의 생성이 증가하는 것이 보고된 바 있다. The other cytokine that causes atopic dermatitis is MCP-1 (monocyte chemoattractant protein-1). MCP-1 binds to CCR2 (chemokine receptor) and MCP-1 gene- monocytes have been observed to be impaired in resistance to certain bacterial infections and have been reported to be similar to those observed in animals with the CCR2 gene removed. In addition, MCP-1 converts Th0 cells into cells that secrete Th2 cytokines in the test tube, and when intravenously injected with MCP-1, the production of IL-12 is decreased and the production of IL-4 is increased have.
한편, 기존에 아토피 피부염의 치료제로 가장 널리 이용되어 왔던 것은 주로 스테로이드제, 항히스타민제, 항생제 등과 같은 치료제이다. 이 중, 스테로이드제는 기관지천식 등의 염증성 질환에서 가장 널리 사용되는 치료제이기는 하지만, 아직까지도 스테로이드제가 체내에서 반응하는 기전이 다 알려져 있지는 않는 실정이다. 한편, 기관지 천식이나 류마티스 관절염 등의 환자들 중 일부는 스테로이드제에 반응하지 않는 스테로이드 저항성을 보인다. 또한, 아토피 피부염은 스테로이드 계통의 약제를 통해 완전히 치료가 되기보다 만성화되면서 조직의 변성 및 섬유화가 일어나는 부작용이 종종 발생한다. 한편, 스테로이드제에 대한 저항성이 커지면, 만성 염증성 반응에서 다양한 세포를 통해 섬유화에 관련된 TGF-β(tumor growth factor-β), IL-4, IL-13 등의 다양한 사이토카인이 분비되고, 이를 통해 섬유모세포(fibroblast)를 활성화시키는 IL-6의 분비가 증가되어 많은 섬유모세포의 분화증식이 초래되며, 세포외 기질(extra cellular matrix)이 과생산되어 세포 및 조직의 변형과 섬유화가 야기될 수도 있다. On the other hand, therapeutic agents such as steroids, antihistamines, and antibiotics have been widely used as treatments for atopic dermatitis. Among these, steroids are the most widely used therapeutic agents for inflammatory diseases such as bronchial asthma, but the mechanism by which steroids respond in the body is still unknown. On the other hand, some patients with bronchial asthma or rheumatoid arthritis show steroid resistance that does not respond to steroids. In addition, atopic dermatitis is often chronicized rather than completely treated with a steroid-based drug, resulting in side effects such as denaturation and fibrosis of the tissue. On the other hand, when resistance to steroids increases, various cytokines such as TGF-beta, IL-4 and IL-13 related to fibrosis are secreted through various cells in the chronic inflammatory reaction. The secretion of IL-6, which activates the fibroblast, is increased, resulting in the proliferation of many fibroblasts and the over-production of extra cellular matrix, resulting in cell and tissue transformation and fibrosis .
따라서, 스테로이드제와 같은 아토피 피부염의 치료제들은 상기와 같은 여러 가지 부작용으로 인해 그 사용을 점차 제한하고 있는 추세이며, 아토피 피부염에 효과가 있으면서도 부작용이 없는 새로운 개념의 아토피 피부염의 치료용 조성물이 요구되고 있다. Therefore, therapeutic agents for atopic dermatitis such as steroid agents are gradually restricted due to various side effects as described above, and a composition for treating a new concept of atopic dermatitis which is effective for atopic dermatitis but has no side effects is required have.
인삼(Panax ginseng)은 오갈피나무과(Aralaiaceae) 인삼 속에 속하는 다년생 초본류로 과거 수천년 전부터 우리나라를 비롯한 동양에서 약제 또는 건강식품으로 널리 사용되어 왔다. 또한 인삼은 수삼, 홍삼 등의 다양한 형태로 이용이 가능하며, 특히 수삼을 증기로 쪄서 가공한 홍삼이 인체에 유용한 다양한 진세노사이드를 함유하고 있는 것으로 알려져 있다. Panax ginseng ) is a perennial herbaceous plant belonging to the genus Ginseng ( Araliaceae ). It has been widely used as medicines or health foods in Korea and the Orient for thousands of years. In addition, ginseng can be used in various forms such as ginseng and red ginseng. Especially, it is known that red ginseng processed by steaming steam ginseng contains various ginsenosides useful for human body.
인삼의 주요 효능을 나타내는 사포닌(saponin)은 30종류 이상의 진세노사이드(ginsenoside)로 구성되는데, 진세노사이드는 면역력강화, 항염증작용, 항알러지작용, 항암효과, 발기부전에 대한 효과, 혈압강하작용, 항콜레스테롤작용, 항혈전작용, 성인병 및 노화에 대한 예방 및 치료효과가 있다고 알려져 있다. Saponin, which represents the major efficacy of ginseng, is composed of more than 30 kinds of ginsenosides. Ginsenosides are used to enhance immunity, antiinflammatory action, antiallergic action, anticancer effect, effect on erectile dysfunction, Action, anticholesterol action, antithrombotic action, adult disease and aging.
인삼의 사포닌은 트리터페노이드(triterpenoid) 계열의 다마란(dammarane) 골격에 글루코즈(glucose), 아라비노즈(arabinose), 자일로즈(xylose) 그리고 람노즈(rhamnose) 등이 결합되어 있는 중성배당체이다. 인삼을 물과 알코올로 추출할 때 인삼 사포닌의 진세노사이드 중에서 20-O-β-D-글루코피라노실-20(S)-프로토파낙사디올(20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol; Rb1), 20-O-[α-L-아라비노피라노실(1→6)-β-D-글루코피라노실]-20(S)-프로토파낙사디올(20-O-[α-L-arabinopyranosyl(1→6)-β-D-glucopyranosyl]-20(S)-protopanaxadiol; Rb2), 20-O-[α-L-아라비노푸라노실(1→6)-β-D-글루코피라노실]-20(S)-프로토파낙사디올(20-O-[α-L-arabinofuranosyl(1→6)-β-D-glucopyranosyl]-20(S)-protopanaxadiol; Rc), 20(S)-프로토파낙사트리올(20(S)-protopanaxatriol; Rg1) 등이 다량 존재하는 것이 확인된다. Saponin of ginseng is a neutral glycoside in which glucose, arabinose, xylose, and rhamnose are bonded to a triterpenoid-based dammarane skeleton. When extracting ginseng with water and alcohol, 20-O-? -D-glucopyranosyl-20 (S) -protonanaxadiol 20-O-? -D-glucopyranosyl-20 (S) -protopanaxadiol; Rb 1 ), 20-O- [? -L-arabinopyranosyl (1? 6) -? - D-glucopyranosyl] - [α-L-arabinopyranosyl (1 → 6) -β-D-glucopyranosyl] -20 (S) -protopanaxadiol Rb 2 , 20- β-D-glucopyranosyl] -20 (S) -protopanaxadiol; Rc (S) -protopanaxadiol (20-O-α-L-arabinofuranosyl (20 (S) -protopanaxatriol; Rg 1 ) and 20 (S) -protopanaxatriol (Rg 1 ).
한편, 사포닌은 체내에서 장내 미생물에 의해 분해되어 흡수되는데(Planta. Med., 1996, 62(5), 453~457), 진세노사이드 Rb1의 경우, 장내 미생물에 의해 진세노사이드 Rd, 화합물 K(compound K)와 프로토파낙사디올로 순차적으로 전환되어 흡수된다. 하지만 인삼 사포닌을 분해하는 장내 미생물은 사람의 체질과 식습관에 따라 그 존재의 유무와 보유하고 있는 정도가 다르며, 대사 산물로 전환하는데 차이가 있어서 인삼을 복용한 후의 효능의 차이가 나타날 수 있다. 즉, 장내 미생물 균총이 사람마다 다르기 때문에 진세노사이드의 전환율과 생체이용율이 달라지게 된다(J. Pharm. Pharmacol. 1998, 50(10), 1155~1160). On the other hand, saponin is degraded and absorbed by intestinal microorganisms in the body (Planta. Med., 1996, 62 (5), 453 ~ 457). In the case of ginsenoside Rb 1 , ginsenoside Rd, compound K (compound K) and protopanaxadiol. However, intestinal microorganisms that degrade ginseng saponin have different existence and existence degree depending on the constitution and eating habits of humans, and there is a difference in conversion to metabolites, so that there may be a difference in efficacy after taking ginseng. In other words, since intestinal microflora vary from person to person, the conversion and bioavailability of ginsenosides are different (J. Pharm. Pharmacol. 1998, 50 (10), 1155-1160).
따라서 장내 미생물의 차이로 인한 인삼 내의 진세노사이드의 흡수율의 차이를 없애기 위해, 다양한 미생물을 이용하여 인삼을 미리 발효함으로써 진세노사이드의 체내 흡수율을 제고하고, 또한 새로운 형태의 유용한 인삼 발효물을 제공하고자 하는 시도가 지속적으로 이루어지고 있다. Therefore, in order to eliminate the difference in the absorption rate of ginsenosides in ginseng due to differences in intestinal microorganisms, various kinds of microorganisms are used to ferment ginseng in advance, thereby increasing the absorption of ginsenoside into the body and providing a new type of useful ginseng fermentation There is an ongoing attempt to do so.
그러나, 상기 미생물들을 이용해 발효된 인삼 내의 진세노사이드의 성분 및 함량을 확인했을 때, 진세노사이드의 전환능이 기대했던 것만큼 아주 높지는 않은 것으로 확인되고 있어 새로운 진세노사이드 전환 활성을 갖는 미생물을 개발하는 것에 대한 필요성이 제기되고 있다. However, when the components and contents of ginsenosides in ginseng fermented using the above-mentioned microorganisms were confirmed, it was confirmed that the conversion of ginsenosides was not as high as expected. Therefore, microorganisms having a novel ginsenoside conversion activity There is a need for development.
한편, 본 발명자들은 락토바실러스 플랜타럼 CRNB-22(Lactobacillus plantarum CRNB-22, 미생물 수탁번호 : KCTC11931BP) 균주가 진세노사이드의 전환능이 우수함으로써 장내 흡수율이 높은 진세노사이드를 다량으로 포함하는 고기능성을 갖는 발효인삼을 제조할 수 있음을 확인한 바 있다(특허출원 제10-2010-0029649호). 이에, 본 발명자들은 상기 락토바실러스 플랜타럼 CRNB-22을 이용한 발효인삼의 활성을 연구하던 중, 상기 발효인삼의 추출물이 아토피 피부염의 예방 또는 치료 효과가 우수함을 확인함으로써 본 발명을 완성하였다. On the other hand, the inventors are Lactobacillus plantarum CRNB-22 ( Lactobacillus plantarum CRNB-22, microorganism accession number: KCTC11931BP) It was confirmed that the fermented ginseng having a high functionality containing a large amount of ginsenoside with high intestinal absorption rate because the strain has excellent ginsenoside conversion ability (patent application) 10-2010-0029649). Accordingly, the inventors of the present invention have completed the present invention by confirming that the extract of fermented ginseng has an excellent effect of preventing or treating atopic dermatitis, while studying the activity of fermented ginseng using Lactobacillus plantarum CRNB-22.
본 발명의 목적은 락토바실러스 플랜타럼 CRNB-22(Lactobacillus plantarum CRNB-22, 미생물 수탁번호 : KCTC11931BP)를 이용한 발효인삼의 추출물을 함유하는 아토피 피부염의 예방 또는 치료용 조성물을 제공하는 데에 있다.It is an object of the present invention to provide a pharmaceutical composition containing Lactobacillus plantarum CRNB-22 plantarum CRNB-22, microorganism accession no.: KCTC11931BP) as an effective ingredient for preventing or treating atopic dermatitis.
더욱 상세하게는, 본 발명의 목적은 MCP-1(monocyte chemoattractant protein-1), IL-8(interleukin-8) 및 IL-6(interleukin-6)의 활성을 억제하는 효과가 뛰어난 락토바실러스 플랜타럼 CRNB-22(Lactobacillus plantarum CRNB-22, 미생물 수탁번호 : KCTC11931BP)를 이용한 발효인삼의 추출물을 함유하는 아토피 피부염의 예방 또는 치료용 조성물을 제공하는 데에 있다.More specifically, it is an object of the present invention to provide a method of inhibiting the activity of MCP-1 (monocyte chemoattractant protein-1), IL-8 (interleukin-8) and IL- CRNB-22 ( Lactobacillus plantarum CRNB-22, microorganism accession no.: KCTC11931BP) as an effective ingredient for preventing or treating atopic dermatitis.
본 발명은 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물을 함유하는 아토피 피부염의 예방 또는 치료용 조성물에 관한 것이다. The present invention relates to a composition for preventing or treating atopic dermatitis containing an extract of fermented ginseng using Lactobacillus plantarum CRNB-22.
상기 추출물은, 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼을 물, C1 내지 C4의 저급 알코올, 또는 이들의 혼합용매로 추출한 추출물일 수 있다. The extract may be an extract obtained by extracting fermented ginseng using Lactobacillus plantarum CRNB-22 with water, C1 to C4 lower alcohols, or a mixed solvent thereof.
상기 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼은 인삼 100 중량부에 락토바실러스 플랜타럼 CRNB-22 균주 0.001~1.0 중량부를 접종하고 20~50℃에서 5~9일간 발효하여 제조할 수 있다.Fermented ginseng using Lactobacillus plantarum CRNB-22 was prepared by inoculating 100 parts by weight of ginseng with 0.001 to 1.0 part by weight of Lactobacillus plantarum CRNB-22 strain And fermenting at 20 to 50 ° C for 5 to 9 days.
또한, 본 발명은 상기 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물을 함유하는 아토피 피부염의 예방 또는 개선용 건강기능식품에 관한 것이다. The present invention also relates to a health functional food for preventing or ameliorating atopic dermatitis containing the extract of fermented ginseng using Lactobacillus plantarum CRNB-22.
이하, 본 발명을 더 자세하게 설명한다.Hereinafter, the present invention will be described in more detail.
상기 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물은 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼에, 상기 발효인삼 중량의 2~20배(w/w)의 물, 탄소수 1 내지 4개의 저급 알코올 또는 이들의 혼합용매를 가하여 제조할 수 있다. The extract of fermented ginseng using Lactobacillus plantarum CRNB-22 was prepared by adding 2 to 20 times (w / w) weight of the fermented ginseng to the fermented ginseng using Lactobacillus plantarum CRNB-22, 1 to 4 A lower alcohol or a mixed solvent thereof.
상기 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물은 필요에 따라, 유기용매(알코올, 에테르, 아세톤 등)에 의한 추출, 유기용매(n-헥산, 에틸아세테이트, n-부탄올)와 물의 분배, 컬럼크로마토그래피에 의한 방법, 식물체 성분의 분리 추출에 이용되는 공지의 방법 등을 단독 또는 적합하게 조합하여 더욱 정제할 수 있으며, 필요에 따라 상법에 따라서 더욱 정제할 수 있다.The extract of fermented ginseng using Lactobacillus plantarum CRNB-22 may be extracted with an organic solvent (alcohol, ether, acetone, etc.), an organic solvent (n-hexane, ethyl acetate, n-butanol) , A method by column chromatography, a known method used for separation and extraction of a plant component, etc., alone or in combination, can be further purified and, if necessary, further purification can be carried out according to a conventional method.
본 발명에서 사용하는 크로마토그래피에는 실리카겔 컬럼 크로마토그래피(silica gel column chromatography), 엘에이취-20 컬럼 크로마토그래피(LH-20 column chromatography), 이온교환수지 크로마토그래피(ion exchange resin chromatography), 박층크로마토그래피(TLC; thin layer chromatography), 중압크로마토그래피(medium pressure liquid chromatography) 및 고성능 액체 크로마토그래피(high performance liquid chromatography) 등이 이용될 수 있다.Chromatography used in the present invention includes silica gel column chromatography, L-20 column chromatography, ion exchange resin chromatography, thin layer chromatography ( Thin layer chromatography (TLC), medium pressure liquid chromatography, high performance liquid chromatography, and the like.
본 발명의 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼은 인삼 100 중량부에 락토바실러스 플랜타럼 CRNB-22 균주 0.001~1.0 중량부를 접종하고, 20~50℃, 바람직하게는 25~45℃에서 5~9일간 발효하여 제조할 수 있으며, 필요에 따라 쌀겨, 옥수수, 전분, 포도당, 서당, 과당, 유당 등의 탄소원을 추가하여 발효할 수도 있다. Fermented ginseng using Lactobacillus plan tareom CRNB-22 of the present invention is inoculated to 100 parts by weight of ginseng Lactobacillus plan tareom CRNB-22 strain 0.001 ~ 1.0 parts by weight, 20 ~ 50 ℃, preferably at 25 ~ 45
상기 발효인삼의 제조에 이용되는 인삼은 파낙스(Panax) 속에 속하는 다년생 식물로, 고려인삼(Panax ginseng), 화기삼(Panax quinquefolia), 전칠삼(삼칠, Panax notoginseng), 죽절삼(Panax japonicus), 히말라야삼(Panaxa pseudoginseng). 베트남삼(Panax vietnamensis), 파낙스 엘레가티오르(Panax elegatior), 파낙스 완지아누스(Panax wangianus) 및 파낙스 비핀라티피두스(Panax bipinratifidus), 파낙스 안구스티폴리움(Panax angustifolium)에서 선택되는 1종 이상의 인삼을 이용할 수 있으나 이에 한정되는 것은 아니다. 아울러 이들 인삼은 단독으로 또는 2종 이상 조합하여 사용할 수도 있다. Ginseng is a perennial plant belonging to the genus Panax (Panax) for use in the production of the fermented ginseng, ginseng (Panax ginseng), hwagisam (Panax quinquefolia), jeonchilsam (thirty-seven, Panax notoginseng , Panax japonicus , Panaxa pseudoginseng ). Vietnamese Panax vietnamensis), Panax elegans tee climb (Panax elegatior), Panax Wan Jia Augustine (Panax wangianus ) and Panax bifunlatipidus ( Panax bipinratifidus , and Panax angustifolium . However, the present invention is not limited thereto. These ginsengs may be used alone or in combination of two or more.
한편, 상기 인삼은 4~6년근의 인삼 뿌리를 이용하는 것이 바람직하다.Meanwhile, the ginseng preferably uses ginseng roots of 4 to 6 years old.
또한, 본 발명은 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물을 함유하는 아토피 피부염의 예방 또는 치료용 약학 조성물을 제공한다. 상기 약학 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 상기 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물을 함유하는 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. 제제화 할 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 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로스 또는 락토오스, 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 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 present invention also provides a pharmaceutical composition for preventing or treating atopic dermatitis containing extract of fermented ginseng using Lactobacillus plantarum CRNB-22. The pharmaceutical compositions may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method. Examples of carriers, excipients and diluents that can be contained in the composition containing the extract of fermented ginseng using Lactobacillus plantarum CRNB-22 include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch , Hydroxypropyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethylcellulose, . 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. The solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations can be prepared by adding to the extract of fermented ginseng using Lactobacillus plantarum CRNB-22 at least one excipient such as starch , Calcium carbonate, sucrose or lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.
상기 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물의 투여량은 치료받을 대상의 연령, 성별, 체중과, 치료할 특정 질환 또는 병리 상태, 질환 또는 병리 상태의 심각도, 투여경로 및 처방자의 판단에 따라 달라질 것이다. 이러한 인자에 기초한 투여량 결정은 당업자의 수준 내에 있으며, 일반적으로 투여량은 0.01㎎/㎏/일 내지 대략 2000㎎/㎏/일의 범위이다. 더 바람직한 투여량은 0.1㎎/㎏/일 내지 500㎎/㎏/일이다. 투여는 하루에 한 번 투여할 수도 있고, 수 회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. 본 발명의 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물은 쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁내 경막 또는 뇌혈관내 주사에 의해 투여될 수 있다. 또한 본 발명의 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물은 천연물 유래의 조성물이기 때문에, 독성 및 부작용은 거의 없으므로 예방 목적으로 장기간 복용시에도 안심하고 사용할 수 있는 약제이다. The dose of the extract of fermented ginseng using the above-mentioned Lactobacillus plantarum CRNB-22 is determined by the age, sex, and weight of the subject to be treated, the severity of the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, Will vary. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 0.1 mg / kg / day to 500 mg / kg / day. The administration may be carried out once a day or several times. The dose is not intended to limit the scope of the invention in any way. The extract of fermented ginseng using Lactobacillus plantarum CRNB-22 of the present invention can be administered to mammals such as rats, livestock, and humans in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. Also, since the extract of fermented ginseng using Lactobacillus plantarum CRNB-22 of the present invention is a composition derived from a natural product, it has little toxicity and side effects, and thus can be safely used for long-term use even for prophylactic purposes.
또한, 본 발명은 상기 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물을 함유하는 아토피 피부염의 예방 또는 개선용 건강기능식품을 제공한다. 상기 건강기능식품에는 식품학적으로 허용 가능한 식품보조 첨가제가 첨가될 수 있다. 또한, 상기 건강기능식품은 정제, 캡슐제, 환제 또는 액제 등의 형태를 포함하며, 상기 건강기능식품으로는 드링크제, 육류, 소세지, 빵, 캔디류, 스넥류, 면류, 아이스크림을 포함한 낙농제품, 스프, 이온음료를 포함한 음료수, 알코올 음료 및 비타민 복합제를 포함한 영양 공급용 제품이 포함될 수 있다. The present invention also provides a health functional food for preventing or ameliorating atopic dermatitis containing the extract of fermented ginseng using Lactobacillus plantarum CRNB-22. Food-acceptable food-aid additives may be added to the health functional food. The health functional food may be in the form of tablets, capsules, pills or liquids. The health functional foods include dairy products such as drinks, meat, sausage, bread, candy, snacks, noodles, It may include nutritional products, including beverages, alcoholic beverages and vitamin complexes, including ionic beverages.
또한 상기 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼 추출물은 그대로 건강기능식품으로 제품화할 수도 있고, 풍미나 향취를 높이기 위해 다양한 첨가물을 배합하여 제품화할 수 있다. 첨가물의 예로서는 각종 당질, 유화제, 감미료, 산미료, 과즙 등이 있다. 구체적으로는 포도당, 서당, 과당, 봉밀 등의 당질과 솔비톨, 키실리톨, 에레스리콜, 락티톨, 파라티니드 등의 당알코올, 글리세린지방산에스테르와 같은 지방산에스테르, 레시틴 등의 유화제 등이 사용될 수 있으며, 이외에도 비타민A, 비타민B, 비타민C, 비타민E 등의 각종 비타민류나 식물엑기스, 곡물성분, 야채성분 등도 첨가제로 사용가능하다.In addition, the fermented ginseng extract using Lactobacillus plantarum CRNB-22 can be directly commercialized as a health functional food, and various additives can be mixed and commercialized to enhance flavor and flavor. Examples of additives include various saccharides, emulsifiers, sweeteners, acidulants, juices, and the like. Specific examples thereof include saccharides such as glucose, sucrose, fructose, and acacia, sugar alcohols such as sorbitol, xylitol, erysuricol, lactitol, and paratinide, fatty acid esters such as glycerin fatty acid esters, and emulsifiers such as lecithin In addition, various vitamins such as vitamin A, vitamin B, vitamin C and vitamin E, plant extract, cereal ingredient and vegetable ingredient can be used as additives.
본 발명은 락토바실러스 플랜타럼 CRNB-22(Lactobacillus plantarum CRNB-22, 미생물 수탁번호 : KCTC11931BP)를 이용한 발효인삼의 추출물을 함유하는 아토피 피부염의 예방 또는 치료용 조성물에 관한 것이다. 상기 조성물은 MCP-1(monocyte chemoattractant protein-1), IL-8(interleukin-8) 및 IL-6(interleukin-6)의 활성을 억제하는 효과가 탁월하여 아토피 피부염과 같은 각종 피부질환의 증세를 치료 또는 경감시키는 치료제로 이용될 수 있다. The present invention relates to a composition for preventing or treating atopic dermatitis containing an extract of fermented ginseng using Lactobacillus plantarum CRNB-22 (Microorganism Accession Number: KCTC11931BP). The composition is excellent in inhibiting the activities of MCP-1 (monocyte chemoattractant protein-1), IL-8 (interleukin-8) and IL-6 (interleukin-6), and thus exhibits various symptoms of skin diseases such as atopic dermatitis Can be used as therapeutic agents for treatment or alleviation.
도 1은 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물의 MCP-1 활성 억제 효과를 확인한 결과이다.
도 2는 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물의 IL-6 활성 억제 효과를 확인한 결과이다.
도 3은 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물의 IL-8 활성 억제 효과를 확인한 결과이다.FIG. 1 shows the results of confirming the inhibitory effect of extracts of fermented ginseng on MCP-1 activity using Lactobacillus plantarum CRNB-22.
FIG. 2 shows the results of confirming the inhibitory effect of extract of fermented ginseng on IL-6 activity using Lactobacillus plantarum CRNB-22.
FIG. 3 shows the results of confirming the inhibitory effect of the extract of fermented ginseng on IL-8 activity using Lactobacillus plantarum CRNB-22.
이하 본 발명의 바람직한 실시예를 상세히 설명하기로 한다. 그러나, 본 발명은 여기서 설명되는 실시예에 한정되지 않고 다른 형태로 구체화될 수도 있다. 오히려, 여기서 소개되는 내용이 철저하고 완전해질 수 있도록 그리고 당업자에게 본 발명의 사상을 충분히 전달하기 위해 제공하는 것이다. Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art.
<실시예 1. 미생물의 분리 및 동정>≪ Example 1: Isolation and identification of microorganism &
실시예 1-1. 미생물의 분리 Example 1-1. Isolation of Microorganisms
본 발명의 미생물은 김치와 몽고의 마유로부터 분리하였다. 김치 시료 및 마유를 멸균수에 희석하고, 희석액 1㎖을 BCP 아가 플레이트(agar plate, 표 1의 조성 참조)에 분주하여 30℃와 37℃에서 3일간 배양하였다. 배양 후 BCP 아가 플레이트상에서 콜로니(colony) 주위에 노란색의 환을 형성하는 균주를 선발하여 BCP 아가 상에서 5회 계대 배양하여 순수 분리하였다. The microorganism of the present invention was isolated from Kimchi and Mung oil of Mongolia. Kimchi samples and horse oil were diluted in sterilized water and 1 ml of the diluted solution was divided into BCP agar plates (see composition of Table 1) and cultured at 30 ° C and 37 ° C for 3 days. After culturing, strains forming a yellow circle around a colony on a BCP agar plate were selected and cultured subcultured five times on BCP agar to be purely isolated.
사포닌 분해능력을 가진 균주를 분리하기 위하여 분리된 균주를 에스컬린-아가(Esculin-agar, 표 2의 조성 참조) 배지에 접종하여 배지 내의 색깔 변화를 관찰하였다. 사포닌 분해능력을 가진 균주는 β-글루코시다제(glucosidase)를 생성하고 에스컬린(esculin)에 β-글루코스(glucose)가 반응하여 생성된 에스컬레틴(esculetin)은 배지 내에서 페릭 암모늄 시트레이트(ferric ammonium citrate)와 반응하여 콜로니 주위에 검정색 환을 형성하게 된다. 따라서 콜로니 주위에 검정색 환이 형성된 균주들을 β-글루코시다제 활성을 가지는 균주라고 판단하고, 사포닌 분해능력이 있을 것으로 판단되어 사포닌 분리 균주로서 선발하였다. 이러한 과정을 거쳐 김치로부터 6개, 몽고 마유로부터 10개의 미생물을 분리하였다.In order to isolate strains having saponin-decomposing ability, the isolates were inoculated into Esculin-agar (see composition in Table 2) medium to observe the color change in the medium. The strain having saponin-decomposing ability produces β-glucosidase and esculetin produced by the reaction of β-glucose to esculin is produced by ferric ammonium citrate ferric ammonium citrate) to form a black circle around the colonies. Therefore, strains with black circles around the colonies were regarded as strains having? -Glucosidase activity, and as saponin-decomposing ability was determined, they were selected as isolates of saponin. Through these processes, six microorganisms were isolated from kimchi and ten microorganisms from Mongolian horse.
(Bromcresol purple) Bromocresol Purple
(Bromcresol purple)
(Pancreatic digest of casein) Casein extract
(Pancreatic digest of casein)
(Ferric ammonium citrate)Ferric ammonium citrate
(Ferric ammonium citrate)
실시예Example 1-2. 분리된 미생물의 동정 1-2. Identification of Isolated Microorganisms
상기 실시예 1-1에서 선발된 균주 중 β-글루코시다제 활성 및 사포닌 분해능력 우수한 균주를 선택하여 사포닌 분해 미생물로 선발된 균주 중 β-글루코시다제 활성이 가장 뛰어난 미생물에 대하여 16S rDNA 염기서열에 기초한 분자계통 분류학적 분석을 이용하여 동정을 실시하였다. 이 후, 상기 균주 DNA를 추출하고, 범용 프라이머(universal primer) (5'-GAGTTTGATCCTGGCTCAG-3', 5'-AGAAAGGAGGTGATCCAGCC-3')를 이용하여 PCR을 행하여 16S rDNA(16S rRNA 코딩 유전자)를 증폭하였다. 증폭조건은 96℃에서 2분간 반응한 다음 96℃에서 변성 10초, 50℃에서 annealing 5초, 60℃에서 extension을 4분간 25회 반복하고, 60℃에서 2분간 최종 extension을 실시하였다. 증폭된 16S rDNA는 PCR 산물 정제 키트(Product Purification Kit, Qiagen)를 사용하여 정제한 후, Genetic analyzer 377(Perkin-Elmer)을 사용하여 결정하였다.Glucosidase activity and saponin decomposition ability among the strains selected in Example 1-1 were selected and microorganisms having the highest activity of? -Glucosidase among the strains selected as saponin-degrading microorganisms were selected for the 16S rDNA sequence Based on molecular systematic taxonomic analysis. Thereafter, the strain DNA was extracted and subjected to PCR using a universal primer (5'-GAGTTTGATCCTGGCTCAG-3 ', 5'-AGAAAGGAGGTGATCCAGCC-3') to amplify 16S rDNA (16S rRNA coding gene) . Amplification conditions were 96 ℃ for 2 min, denaturation at 96 ℃ for 10 sec, annealing at 50 ℃ for 5 sec, extension at 60 ℃ for 25 min, and final extension at 60 ℃ for 2 min. Amplified 16S rDNA was purified using a PCR product purification kit (Qiagen) and then determined using a genetic analyzer 377 (Perkin-Elmer).
그 결과 β-글루코시다제 활성 및 사포닌 분해능력이 가장 우수한 균주를 락토바실러스 플랜타럼 CRNB-22(Lactobacillus plantarum CRNB-22)로 명명하였으며, 한국미생물자원센터(수탁번호 KCTC11931BP)에 기탁하였다(특허출원 제10-2010-0029649호). As a result, Lactobacillus plantarum CRNB-22 ( Lactobacillus) strains with the highest β-glucosidase activity and saponin degrading ability were identified. plantarum CRNB-22) and deposited in the Korea Microbial Resources Center (Accession No. KCTC11931BP) (Patent Application No. 10-2010-0029649).
<< 실시예Example 2. 발효인삼의 제조> 2. Preparation of fermented ginseng>
6년근 수삼 100g에 상기 실시예 1에서 분리한 락토바실러스 플랜타럼 CRNB-22 균주 배양물 0.1g을 넣고 37℃에서 7일간 발효하여 발효인삼을 제조하였다. 0.1 g of the Lactobacillus plantarum CRNB-22 culture obtained in Example 1 was added to 100 g of 6-year old ginseng and fermented at 37 ° C for 7 days to prepare fermented ginseng.
<< 실시예Example 3. 발효인삼 추출물의 제조> 3. Preparation of Fermented Ginseng Extract>
실시예Example 3-1. 발효인삼 물 추출물 3-1. Fermented ginseng water extract
상기 실시예 2의 발효인삼을 수분 14%가 되도록 건조하고, 물 10kg에 상기 발효인삼 건조물 1kg을 넣고 90℃에서 8시간 동안 추출한 후, 고형분을 제거한 여액을 동결건조하여 발효인삼 물 추출물을 제조하였다. The fermented ginseng of Example 2 was dried to a moisture content of 14%, 1 kg of the dried fermented ginseng was added to 10 kg of water, and the mixture was extracted at 90 ° C for 8 hours. Then, the filtrate from which the solid content was removed was lyophilized to prepare a fermented ginseng water extract .
실시예Example
3-2. 발효인삼 70% 에탄올 수용액 추출물 3-2.
상기 실시예 2의 발효인삼을 수분 14%가 되도록 건조하고, 70% 에탄올 수용액 4kg에 상기 발효인삼 건조물 1kg을 넣고 50℃에서 8시간 동안 추출한 후, 고형분을 제거한 여액을 감압증류 및 동결건조하여 발효인삼 70% 에탄올 수용액 추출물을 제조하였다. The fermented ginseng of Example 2 was dried to a moisture content of 14%, 1 kg of the fermented ginseng dried product was added to 4 kg of 70% ethanol aqueous solution, and the mixture was extracted at 50 ° C for 8 hours. Then, the filtrate was subjected to vacuum distillation and freeze- Extract of 70% ethanol aqueous solution of ginseng was prepared.
실시예Example 3-3. 발효인삼 100% 에탄올 추출물 3-3. 100% ethanol extract of fermented ginseng
상기 실시예 2의 발효인삼을 수분 14%가 되도록 건조하고, 100% 에탄올 4kg에 상기 발효인삼 건조물 1kg을 넣고 50℃에서 8시간 동안 추출한 후, 고형분을 제거한 여액을 감압증류하여 발효인삼 100% 에탄올 추출물을 제조하였다. The fermented ginseng of Example 2 was dried to a moisture content of 14%, 1 kg of the dried fermented ginseng was added to 4 kg of 100% ethanol, and the mixture was extracted at 50 ° C for 8 hours. Then, the filtrate with the solid content removed was distilled under reduced pressure, The extract was prepared.
<< 비교예Comparative Example 1. 인삼 추출물의 제조> 1. Preparation of ginseng extract>
비교예Comparative Example 1-1. 인삼 물 추출물의 제조 1-1. Preparation of ginseng water extract
실시예 3-1과 동일하게 추출물을 제조하되, 발효인삼 건조물 대신에 6년근 백삼(수분 14% 이하)을 사용하였다.The extract was prepared in the same manner as in Example 3-1 except that 6-year-old white ginseng (moisture content of 14% or less) was used instead of the fermented ginseng dried product.
비교예Comparative Example 1-2. 인삼 70% 에탄올 추출물의 제조 1-2. Preparation of 70% Ethanol Extract of Ginseng
실시예 3-2와 동일하게 추출물을 제조하되, 발효인삼 건조물 대신에 6년근 백삼(수분 14% 이하)을 사용하였다.The extract was prepared in the same manner as in Example 3-2 except that 6-year-old white ginseng (moisture content of 14% or less) was used instead of the fermented ginseng dried product.
<< 비교예Comparative Example 2. 홍삼 추출물의 제조> 2. Preparation of red ginseng extract>
비교예Comparative Example 2-1. 홍삼 물 추출물의 제조 2-1. Production of red ginseng water extract
실시예 3-1과 동일하게 추출물을 제조하되, 발효인삼 건조물 대신에 수분이 14% 이하로 건조된 홍삼(6년근 인삼으로 제조한 것)을 사용하였다.The extract was prepared in the same manner as in Example 3-1, except that red ginseng (manufactured from 6-year-old ginseng) having a moisture content of 14% or less was used instead of the fermented ginseng dried ginseng.
비교예Comparative Example 2-2. 홍삼 70% 에탄올 추출물의 제조 2-2. Preparation of 70% Ethanol Extract of Red Ginseng
실시예 3-2와 동일하게 추출물을 제조하되, 발효인삼 건조물 대신에 수분이 14% 이하로 건조된 홍삼(6년근 인삼으로 제조한 것)을 사용하였다.The extract was prepared in the same manner as in Example 3-2 except that red ginseng (manufactured from 6-year-old root ginseng) having a moisture content of 14% or less was used instead of fermented ginseng dried ginseng.
<< 실험예Experimental Example 1. 세포배양 및 발효인삼 추출물의 처리> 1. Cell culture and treatment of fermented ginseng extract>
사람의 단핵구(monocyte)인 THP-1 세포(human acute monocytic leukemia cell)를 RPMI 1640 배지, 항생제(penicillinm 104U/㎖, streptomycin 10㎎/㎖, amphotericin B 25㎍/㎖)와 10% FBS(fetal bovine serum)를 넣고, 37℃, 5% CO2조건에서 3일간 배양하였다. 이 후, MCP-1, IL-6, IL-8의 활성을 측정하기 위해 상기 THP-1 세포를 트립신-EDTA를 이용하여 2.0×106/㎖의 농도로 24웰 플레이트에 분주한 후 0.5% FBS가 포함된 RPMI에서 37℃, 5% CO2조건에서 16시간 동안 배양하였다. 배양 후 실시예 3의 발효인삼 추출물(200㎍/㎖), 비교예 1의 인삼 추출물(200㎍/㎖) 및 비교예 2의 홍삼 추출물(200㎍/㎖), 덱사메타손(dexamethasone, 50㎍/㎖), 진드기 추출물(연세대학교 의과대학에서 구입, 1㎍/㎖)을 14시간 동안 처리한 후 ELISA(enzyme-linked immunospecific assay ; Cytokine. 2008, 42(3), 365~371)를 이용하여 상층액에서 MCP-1, IL-6, IL-8의 양을 측정하여 도 1~3에 나타내었다. 또한 정상군에는 물 추출물의 용매인 생리식염수를 처리하였다(에탄올 처리군의 용매인 DMSO를 처리해도 결과는 동일하게 나옴). Human monocyte THP-1 cells (human acute monocytic leukemia cells) were cultured in RPMI 1640 medium, antibiotics (penicillin 10 4 U / ml,
도 1~3에서와 같이 THP-1 세포에 아토피를 유발한다고 알려진 진드기 추출물을 처리하면 MCP-1, IL-6 및 IL-8의 분비가 증가됨을 알 수 있었다. 양성대조군으로 사용한 덱사메타손은 MCP-1의 분비를 거의 정상수준으로 떨어뜨리는 것으로 확인되었으며, 본 발명의 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼 추출물을 처리한 세포에서는 MCP-1, IL-6 및 IL-8의 분비가 현저하게 감소되는 것으로 확인되어, 상기 발효인삼 추출물이 아토피 피부염의 치료 효과가 우수함을 알 수 있었다. 반면, 발효과정을 거치지 않은 인삼이나 홍삼의 추출물은 본 발명의 추출물보다는 MCP-1, IL-6 및 IL-8의 분비를 억제하는 효과가 높지 않은 것으로 나타났다. As shown in FIGS. 1 to 3, when mite extracts known to induce atopy in THP-1 cells were treated, secretion of MCP-1, IL-6 and IL-8 was increased. Dexamethasone used as a positive control was found to lower the secretion of MCP-1 to almost normal level. In the cells treated with fermented ginseng extract using Lactobacillus plantarum CRNB-22 of the present invention, MCP-1, IL-6 and It was confirmed that the secretion of IL-8 was remarkably reduced, and thus the fermented ginseng extract showed excellent therapeutic effect of atopic dermatitis. On the other hand, the extracts of ginseng and red ginseng which had not undergone fermentation showed less inhibitory effect on the secretion of MCP-1, IL-6 and IL-8 than the extract of the present invention.
<< 실험예Experimental Example 2. 독성실험> 2. Toxicity test>
실험예Experimental Example 2-1. 2-1. 급성독성Acute toxicity
본 발명의 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼 추출물을 단기간에 과량을 섭취하였을 시 급성적(24시간 이내)으로 동물체내에 미치는 독성을 조사하고, 치사율을 결정하기 위하여 본 실험을 수행하였다. 일반적인 마우스인 ICR 마우스 계통 20마리를 대조군에 10마리, 실험군에 30마리씩 배정하였다. 대조군에는 아무것도 투여하지 않았으며, 실험군은 각 10마리 당 실시예 3-1 내지 3-3에서 제조한 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물 2.0g/㎏(일반적인 동물실험에서 사용되는 양의 50배 정도)을 경구 투여하였다. 투여 24시간 후에 각각의 치사율을 조사한 결과, 대조군과 2.0g/㎏ 농도의 실시예 3-1 내지 3-3에서 제조한 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물을 투여한 실험군은 모두 생존하였다. This experiment was conducted to investigate the toxicity of the fermented ginseng extract using Lactobacillus plantarum CRNB-22 of the present invention to an animal body in an acute (within 24 hours) when an excessive amount of the fermented ginseng extract was consumed in a short period of time and to determine the mortality rate . Twenty ICR mice were injected into the control group and 30 mice, respectively. In the control group, nothing was administered. In the experimental group, 2.0 g / kg of the extract of fermented ginseng using Lactobacillus plantarum CRNB-22 prepared in Examples 3-1 to 3-3 per 10 animals (used in general animal experiments About 50 times the dose) was orally administered. After 24 hours of administration, the mortality rate was examined. As a result, the control group and the experimental group administered with the extract of fermented ginseng using Lactobacillus plantarum CRNB-22 prepared in Examples 3-1 to 3-3 at a concentration of 2.0 g / kg Survived.
실시예Example 5-2. 5-2. 실험군Experimental group 및 대조군의 장기 및 조직 독성 실험 And control organ organs and tissue toxicity experiments
C57BL/6J 생쥐를 대상으로 동물의 각 장기(조직)에 미치는 영향을 조사하기 위하여 실시예 3에서 제조한 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물을 투여한 실험군과 용매만을 투여한 대조군의 동물들로부터 8주 후 혈액을 채취하여 GPT(glutamate-pyruvate transferase) 및 BUN(Blood Urea Nitrogen)의 혈액 내 농도를 Select E(Vital Scientific NV, Netherland) 기기를 이용하여 측정하였다. 그 결과, 간독성과 관계있는 것으로 알려진 GPT와 신장독성과 관계있는 것으로 알려진 BUN의 경우, 대조군과 비교하여 실험군은 별다른 차이를 보이지 않았다. 또한, 각 동물로부터 간과 신장을 절취하여 통상적인 조직절편 제작과정을 거쳐 광학현미경으로 조직학적 관찰을 시행하였으며 특이한 이상이 관찰되지 않았다. In order to investigate the effect of C57BL / 6J mice on the organs (tissues) of animals, the experimental group administered with the extract of fermented ginseng using Lactobacillus plantarum CRNB-22 prepared in Example 3 and the control group The blood was collected from the animals for 8 weeks and the blood levels of glutamate-pyruvate transferase (GPT) and blood urine nitrogen (BUN) were measured using Select E (Vital Scientific NV, Netherland). As a result, GPT, which is known to be related to hepatotoxicity, and BUN, which is known to be related to renal toxicity, showed no significant difference compared to the control group. In addition, liver and kidney were cut from each animal and histological observation was carried out with an optical microscope through a conventional tissue section production process. No abnormal abnormalities were observed.
<< 사용예Examples 1. 약학적 1. Pharmaceutical 제제예Formulation example >>
사용예Examples 1-1. 정제의 제조 1-1. Manufacture of tablets
실시예 3-1에서 제조한 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물 20g을 락토오스 175.9g, 감자전분 180g 및 콜로이드성 규산 32g과 혼합하였다. 이 혼합물에 10% 젤라틴 용액을 첨가시킨 후, 분쇄해서 14 메쉬체를 통과시켰다. 이것을 건조시키고 여기에 감자전분 160g, 활석 50g 및 스테아린산 마그네슘 5g을 첨가해서 얻은 혼합물을 정제로 만들었다. 20 g of fermented ginseng extract using Lactobacillus plantarum CRNB-22 prepared in Example 3-1 was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. To this mixture was added a 10% gelatin solution, which was pulverized and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.
사용예Examples 1-2. 주사액제의 제조 1-2. Injection preparation
실시예 3-1에서 제조한 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물 2g을 증류수에 용해시켜서 100㎖를 만들었다. 이 용액을 병에 넣고 20℃에서 30분간 가열하여 멸균시켰다.2 g of fermented ginseng extract using Lactobacillus plantarum CRNB-22 prepared in Example 3-1 was dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.
<< 사용예Examples 2. 식품 2. Food 제조예Manufacturing example >>
사용예Examples 2-1. 조리용 양념의 제조 2-1. Manufacture of cooking seasonings
실시예 3-1에서 제조한 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물을 0.2~10 중량%로 하여 건강 증진용 조리용 양념을 제조하였다.Healthy cooking sauce was prepared using 0.2 to 10% by weight of fermented ginseng extract using Lactobacillus plantarum CRNB-22 prepared in Example 3-1.
사용예Examples 2-2. 밀가루 식품의 제조 2-2. Manufacture of flour food products
실시예 3-1에서 제조한 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물을 0.1~5.0 중량%로 하여 밀가루에 첨가하고, 이 혼합물을 이용하여 빵, 케이크, 쿠키, 크래커 및 면류를 제조하여 건강 증진용 식품을 제조하였다.The extract of fermented ginseng using Lactobacillus plantarum CRNB-22 prepared in Example 3-1 was added to flour at a concentration of 0.1-5.0 wt%, and the mixture was used to prepare bread, cake, cookies, crackers and noodles To produce health promotion foods.
사용예Examples 2-3. 2-3. 스프soup 및 육즙( And juicy ( graviesgravies )의 제조Manufacturing
실시예 3-1에서 제조한 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물을 0.1~1.0 중량%로 하여 스프 및 육즙에 첨가하여 건강 증진용 육가공 제품, 면류의 수프 및 육즙을 제조하였다.The health-enhancing meat product, soup soup and juice were prepared by adding the fermented ginseng extract of Lactobacillus plantarum CRNB-22 prepared in Example 3-1 to the soup and the juice at 0.1 to 1.0 wt%.
사용예Examples 2-4. 유제품( 2-4. dairy product( dairydairy productsproducts )의 제조Manufacturing
실시예 3-1에서 제조한 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물을 0.1~1.0 중량%로 하여 우유에 첨가하고, 상기 우유를 이용하여 버터 및 아이스크림과 같은 다양한 유제품을 제조하였다.The extract of fermented ginseng using Lactobacillus plantarum CRNB-22 prepared in Example 3-1 was added to milk in an amount of 0.1-1.0 wt%, and various dairy products such as butter and ice cream were prepared using the milk.
<< 사용예Examples 3. 음료 3. Drink 제조예Manufacturing example >>
사용예Examples 3-1. 3-1. 야채쥬스Vegetable juice 제조 Produce
실시예 3-1에서 제조한 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물 0.5g을 토마토 또는 당근 쥬스 1,000㎖에 가하여 건강 증진용 야채쥬스를 제조하였다.Healthy vegetable juice was prepared by adding 0.5 g of fermented ginseng extract to 1,000 ml of tomato or carrot juice using Lactobacillus plantarum CRNB-22 prepared in Example 3-1.
사용예Examples 3-2. 3-2. 과일쥬스Fruit juice 제조 Produce
실시예 3-1에서 제조한 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼의 추출물 0.1g을 사과 또는 포도 쥬스 1,000㎖에 가하여 건강 증진용 과일쥬스를 제조하였다.Health enhancing fruit juice was prepared by adding 0.1 g of fermented ginseng extract to 1,000 ml of apple or grape juice using Lactobacillus plantarum CRNB-22 prepared in Example 3-1.
Claims (6)
상기 추출물은 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼을 물, C1 내지 C4의 저급 알코올, 또는 이들의 혼합용매로 추출한 추출물인 것을 특징으로 하는 아토피 피부염의 예방 또는 치료용 조성물.The method of claim 1,
The extract is a composition for the prevention or treatment of atopic dermatitis, characterized in that the extract extracted with fermented ginseng using Lactobacillus plantarum CRNB-22 with water, C1 to C4 lower alcohol, or a mixed solvent thereof.
상기 락토바실러스 플랜타럼 CRNB-22(Lactobacillus plantarum CRNB-22, 미생물 수탁번호 : KCTC11931BP)를 이용한 발효인삼은 인삼 100 중량부에 락토바실러스 플랜타럼 CRNB-22 균주 0.001~1.0 중량부를 접종하고, 20~50℃에서 5~9일간 발효하여 제조하는 것을 특징으로 하는 아토피 피부염의 예방 또는 치료용 조성물.The method of claim 1,
In the fermented ginseng using the above-mentioned Lactobacillus plantarum CRNB-22 ( Lactobacillus plantarum CRNB-22, microorganism accession number: KCTC11931BP), 0.001 to 1.0 part by weight of Lactobacillus plantarum CRNB-22 strain was inoculated into 100 parts by weight of ginseng , ≪ / RTI > for 5 to 9 days. ≪ RTI ID = 0.0 > 5. < / RTI >
상기 추출물은 락토바실러스 플랜타럼 CRNB-22를 이용한 발효인삼을 물, C1 내지 C4의 저급 알코올, 또는 이들의 혼합용매로 추출한 추출물인 것을 특징으로 하는 아토피 피부염의 예방 또는 개선용 건강기능식품.5. The method of claim 4,
The extract is a functional food for the prevention or improvement of atopic dermatitis, characterized in that the extract extracted with fermented ginseng using Lactobacillus plantarum CRNB-22 with water, lower alcohols of C1 to C4, or a mixed solvent thereof.
상기 락토바실러스 플랜타럼 CRNB-22(Lactobacillus plantarum CRNB-22, 미생물 수탁번호 : KCTC11931BP)를 이용한 발효인삼은 인삼 100 중량부에 락토바실러스 플랜타럼 CRNB-22 균주 0.001~1.0 중량부를 접종하고, 20~50℃에서 5~9일간 발효하여 제조하는 것을 특징으로 하는 아토피 피부염의 예방 또는 개선용 건강기능식품.5. The method of claim 4,
In the fermented ginseng using the above-mentioned Lactobacillus plantarum CRNB-22 ( Lactobacillus plantarum CRNB-22, microorganism accession number: KCTC11931BP), 0.001 to 1.0 part by weight of Lactobacillus plantarum CRNB-22 strain was inoculated into 100 parts by weight of ginseng , ≪ / RTI > for 5 to 9 days. ≪ RTI ID = 0.0 >
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KR102032110B1 (en) * | 2018-07-17 | 2019-10-15 | 건국대학교 글로컬산학협력단 | Composition for prevention or treatment of allergic skin diseases comprising fermented extract of ginseng |
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KR20160018118A (en) * | 2014-08-08 | 2016-02-17 | 유향자 | Enhancing method for pharmaceutical effect of herb, preparation method for fermented chitosan for immune stimulating and its use |
KR102032110B1 (en) * | 2018-07-17 | 2019-10-15 | 건국대학교 글로컬산학협력단 | Composition for prevention or treatment of allergic skin diseases comprising fermented extract of ginseng |
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