KR101713115B1 - Cosmetic composition containing the fermented extract of allium monanthum, allium scorodorpasum, allium tuberosum, allium fistulosum and scilla scilloides - Google Patents
Cosmetic composition containing the fermented extract of allium monanthum, allium scorodorpasum, allium tuberosum, allium fistulosum and scilla scilloides Download PDFInfo
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- KR101713115B1 KR101713115B1 KR1020140158106A KR20140158106A KR101713115B1 KR 101713115 B1 KR101713115 B1 KR 101713115B1 KR 1020140158106 A KR1020140158106 A KR 1020140158106A KR 20140158106 A KR20140158106 A KR 20140158106A KR 101713115 B1 KR101713115 B1 KR 101713115B1
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- TUHBEKDERLKLEC-UHFFFAOYSA-N squalene Natural products CC(=CCCC(=CCCC(=CCCC=C(/C)CCC=C(/C)CC=C(C)C)C)C)C TUHBEKDERLKLEC-UHFFFAOYSA-N 0.000 description 1
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
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- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/59—Mixtures
- A61K2800/592—Mixtures of compounds complementing their respective functions
- A61K2800/5922—At least two compounds being classified in the same subclass of A61K8/18
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
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Abstract
본 발명은 달래, 마늘, 부추, 파 및 흥거로 구성된 발효 오신채 추출물을 함유하는 화장료 조성물에 관한 것이다. 본 발명의 화장료 조성물은 매우 우수한 손상된 피부세포 회복 및 면역증강 효과를 나타낸다.The present invention relates to a cosmetic composition containing an extract of fermented bean curd refuse comprising soya bean, garlic, leek, persimmon and persimmon. The cosmetic composition of the present invention exhibits a very excellent damaged skin cell recovery and immunostimulating effect.
Description
본 발명은 오신채인 달래, 마늘, 부추, 파 및 흥거로 구성된 오신채의 발효 추출물을 함유하는 화장료 조성물에 관한 것이다.The present invention relates to a cosmetic composition containing a fermented extract of Alaska pollack, consisting of garlic, garlic, leek, persimmon and horseradish.
피부(skin)는 약 1.6 m2이며 일반적으로 전신을 둘러싸고 있는 신체의 한 기관으로 총 면적은 성인의 경우 중량이 약 9 kg이다. 피부 두께는 나이, 성별, 부위에 따라 다르며 남성 피부가 여성 피부보다 두꺼운 편이고, 신체에서 손바닥과 발바닥 피부가 가장 두껍다. 이 부분은 체중을 지탱하고 물건을 잡고 그리고 피부에 기계적인 자극이 미치는 곳이기도 하다. 피부는 다른 기관에 비하여 새로운 세포의 생성과 소멸이 활발히 일어나는 기관으로, 다양한 기능을 가지고 있다. 물리적인 찰과상으로부터 보호하고 생활할 수 있도록 하며 사람이 몸안에 가지고 있는 물의 손실을 막아준다. 또한, 태양으로부터 나오는 해로운 자외선으로부터 보호해 주고, 몸의 체온을 조절해주는데 매우 중요한 역할을 담당하고 있다. 그리고, 피부는 외부환경과 전달이 가능한 다양한 기능을 가진 감각기관이며, 병원균의 침입으로부터 막아주고 비타민 합성을 한다. 끝으로, 피부의 상태와 외관은 건강의 상태를 쉽게 알 수 있는 지표로서도 중요한 역할을 하며 아름다움을 나타내기 위한 치장의 대상이 되고 있다.The skin is about 1.6 m 2 and is an organ of the body that is generally surrounded by the whole body. The total area is about 9 kg for adults. Skin thickness varies according to age, sex, and region. Male skin is thicker than female skin, and palms and soles are thickest in body. This part is also where you can support your weight, hold things, and have mechanical stimulation on your skin. Skin is an organ that actively produces and destroys new cells compared to other organs, and has various functions. It protects from physical abrasions and makes life possible and prevents the loss of water that a person has in the body. It also plays a very important role in protecting against harmful ultraviolet rays from the sun and controlling body temperature. And, the skin is a sensory organ with various functions that can communicate with the external environment and prevents the invasion of pathogens and synthesizes vitamins. Finally, the condition and appearance of the skin play an important role as an indicator of the health status, and have become objects of beauty for beauty.
그러나, 피부는 면역기능이 약하여 손상받기 쉽고, 이 손상을 회복하기 위해서는 활발한 세포 대사활성이 이루어져야 하는 한편, 민감한 피부는 일반인의 피부보다 훨씬 얇고 외부의 영향에도 더 민감하며 피부의 보호와 장벽 기능이 아직 완전하지 못한 상태로 직접 해로운 환경에 노출되어 있다. 또한, 민감한 피부는 연약하고 민감하기에 작은 외부의 자극에도 쉽게 손상을 입고, 면역 기능 또한 약화된다.However, the skin is susceptible to damage due to its weak immune function. In order to recover this damage, active cell metabolism activity must be performed, while sensitive skin is much thinner than ordinary skin and is more sensitive to external influences. It is still in an incomplete state and is exposed to direct harmful environment. In addition, sensitive skin is fragile and sensitive, so it is easily damaged by small external stimuli and weakens immune function.
특히, 아기 피부의 경우 땀샘 등의 주요 피부 부속 기관들이 아직 미성숙한 상태이고 땀을 내어 자신의 체온을 내리고 조절하는 체온 조절 능력이 부족하기 때문에 땀띠가 생기기도 하며, 수분을 잃어 피부 건조 및 저항력 저하로 인해 피부 손상이 쉽다. 즉, 아기 피부는 성인과 그 겉모습은 같지만 기능상으로 볼 때 미흡하며, 만3세가 되어야 비로소 어른 피부의 두께 정도가 되므로 아기에게 있어 피부에서의 수분 유지는 건강한 피부를 유지하는데 기본 요건이며 중요한 일이다. 또한, 아기 피부는 얇고, 연약하여 외부의 자극과 세균 감염에 민감하고 침투력이 성인보다 매우 빠르기 때문에 피부 위에 남아 있는 땀이나 세균 등이 다시 피부속으로 침투되어 이로인해 피부가 짓물러지게 되고 다른 피부 트러블이 발생할 수 있다. 다시 말하면, 아기 피부는 연약하고 섬세할 뿐만 아니라 민감하기 때문에 조그마한 외부 자극에도 피부 트러블이 생기기 쉬우므로 피부 면역을 강화시켜 줄 수 있는 원료를 선택하여 사용하는 것이 중요하다. 이렇듯 아기 피부는 건조한 피부가 되기 쉽고 이 건조한 피부는 면역기능이 약화되어 아토피 피부염뿐만 아니라 여러 피부 질환으로 발전할 수도 있음으로 피부면역 및 피부회복 기능의 강화가 절실히 요구된다.In particular, in the case of baby skin, major skin attachment organs such as sweat glands are still immature, and because of lack of body temperature control ability to lower and adjust their body temperature by sweating, a sweaty tendon may be formed, It is easy to damage the skin. In other words, the baby skin is the same as the adult skin, but it is insufficient in terms of function, and since it is only about the thickness of the adult skin when it is 3 years old, the moisture retention in the skin for the baby is a basic requirement for maintaining a healthy skin to be. In addition, baby skin is thin, soft, sensitive to external stimuli and bacterial infections, penetration is much faster than adults, sweat and germs remaining on the skin will penetrate back into the skin, A trouble may occur. In other words, it is important to select and use raw materials that can enhance skin immunity because baby skin is not only fragile, delicate, but also sensitive, and tends to cause skin troubles even with a small external stimulus. As such, baby skin tends to become dry skin, and this dry skin may weaken the immune function, and may develop not only atopic dermatitis but also various skin diseases, so that skin immunity and skin recovery function are urgently required.
이에, 본 발명자들은 상당한 기간의 연구 결과물로서 달래, 마늘, 부추, 파 및 흥거로 구성된 오신채 추출물이 세포를 활성화시키고 피부의 면역을 강화시켜 주어 손상 입은 피부를 신속하게 회복시켜 건강한 피부를 유지해 줄 수 있는 천연 원료임을 발견하여 상기한 바와 같이 오신채 추출물을 함유하는 화장료 조성물을 개발하고 이에 관한 특허를 받은 바 있다. 본 발명자들의 선행특허 제10-0877669호에 따르면, 화장료 조성물에 사용된 오신채 추출물은 당업계에 공지된 생약 추출용매로 추출된다. 예를 들어, 달래, 마늘, 부추, 파 및 흥거를 정제수로 깨끗이 씻은 후 끓는 정제수에 5 내지 10분 가량 침지시킨다. 그 후, 10℃ 이하의 찬물에 냉침시킨 후 물을 방출시키고, 물, 무수 또는 함수 에탄올, 글리세린, 부틸렌글리콜 및 프로필렌글리콜중에서 하나 또는 둘 이상의 추출용매를 그 원료의 부피에 대하여 5 내지 10배 부피량을 가한 후, 실온에서 침적시켜 유효성분을 추출함으로써 오신채 추출물을 수득한다.Accordingly, the inventors of the present invention have found that, as a result of research for a considerable period of time, the extract of Ochinacea comprising soya, garlic, leek, leek, and hunger activates cells and strengthens the skin's immunity so that damaged skin can be quickly restored to maintain healthy skin And discovered a cosmetic composition containing the extract of osmanthus as described above and received a patent related thereto. According to the prior art No. 10-0877669 of the present inventors, the extract of Chrysanthemums used in the cosmetic composition is extracted with a herbal medicine extracting solvent known in the art. For example, sole, garlic, leek, leek and hawthorn are thoroughly washed with purified water and then immersed in boiling purified water for about 5 to 10 minutes. Thereafter, water is released after cooling in cold water at 10 ° C or lower, and one or two or more extraction solvents selected from water, anhydrous or hydrolyzed ethanol, glycerin, butylene glycol and propylene glycol are added in an amount of 5 to 10 times After adding a volumetric amount, it is immersed at room temperature to extract an effective ingredient, thereby obtaining a crude extract.
원물에서 생리활성물질을 추출하는 가장 일반적인 방법은 열수추출 방법이다. 그러나, 이 방법은 고온처리에 의한 생리활성물질의 파괴와 변성이 수반되고, 수용성 성분 또는 지용성, 불용성 생리활성물질의 제한적인 추출로 다양한 생리활성성분의 완벽한 추출에 어려움이 있다. 게다가, 이 방법에 의해 추출되는 고분자 생리활성물질은 인체 흡수율이 낮다는 문제점이 있다. 본 발명자들의 선행특허 제10-0877669호의 경우에는 생성된 오신채 추출물이 면역강화와 손상된 피부의 회복을 빠르게 도와주는데 실질적인 효과가 있음은 분명하나 용매에 의해 추출된 것이므로 용매와 자극성 물질의 잔존 가능성으로 인하여 사용하는데 제한이 있을 수 있다. 본 발명자들은 이러한 선행 기술의 문제점을 극복함과 동시에 오신채 추출물의 안전성과 피부에 대한 효능을 높이기 위한 목적으로 끊임없이 계속 연구하였다.The most common method for extracting physiologically active substances from raw materials is hot water extraction. However, this method involves destruction and denaturation of physiologically active substances by high temperature treatment, and it is difficult to extract various physiologically active ingredients with limited extraction of water-soluble components or lipid-soluble and insoluble physiologically active substances. In addition, the polymer physiologically active substance extracted by this method has a problem that the rate of absorption of the human body is low. In the case of the prior art No. 10-0877669 of the present inventors, it is obvious that the resulting extract of osmanquat extract has a substantial effect for rapidly enhancing immunity and recovery of damaged skin. However, since it is extracted by a solvent, There may be restrictions on use. The present inventors have continuously studied to overcome the problems of the prior art and to enhance the safety and efficacy of the extract of Ochinacea.
본 발명자들은 달래, 마늘, 부추, 파 및 흥거로 구성된 오신채를 바실러스 서브틸러스 나토(Bacillus subtilis natto) 균주 배양액으로 접종하여 발효시킨 후, 정제, 숙성, 여과 과정을 거쳐 발효 오신채 추출물을 수득하고, 이와 같이 수득된 발효 오신채 추출물이 일반적인 열수추출로부터 수득된 오신채 추출물에 비하여 상당히 개선된 효능을 나타낸다는 것을 발견하였다.The inventors of the present invention have found that fermentation of a fermented oyster extract is obtained by inoculation with a culture solution of Bacillus subtilis natto strain, which is composed of soya, garlic, leek, leek and broccoli, followed by purification, aging and filtration, It has been found that the thus obtained fermented herbal extract has a significantly improved efficacy compared to the herbal extract obtained from ordinary hot water extraction.
본 발명에 따른 발효 오신채 추출물은 생리활성물질이 고농도로 함유되어 있고, 저온처리로 인해 생리활성물질의 파괴가 적으며, 발효과정에서 2차 생성물로 다양한 생리활성물질이 생성되고 또한 저분자 전환에 의한 인체 흡수율이 증가되는 특징을 갖는다. 실제로, 본 발명의 발효 오신채 추출물을 함유하는 화장료 조성물은 일반적인 열수추출로부터 수득된 오신채 추출물을 함유하는 화장료 조성물에 비하여 피부의 면역강화와 세포 대사 활성 등의 효능에서 현저히 개선된 것이다.The fermented ointment extract according to the present invention contains a high concentration of a physiologically active substance, a low degree of destruction of the physiologically active substance due to low temperature treatment, a variety of physiologically active substances are produced as a secondary product during the fermentation process, And the absorption rate of the human body is increased. In fact, the cosmetic composition containing the fermented ointment extract of the present invention is remarkably improved in the effects of skin immunity and cell metabolism activity, as compared with a cosmetic composition containing ointment extract obtained from general hot water extraction.
도 1은 본 발명의 발효 오신채 추출물에 의한 피부 면역 강화 효과로서 발효 오신채 추출물의 농도에 따른 면역세포로부터 분비된 IL-6의 양을 ELISA 키트로 측정한 결과이다.
도 2는 본 발명의 발효 오신채 추출물에 의한 피부 면역 강화 효과로서 발효 오신채 추출물의 농도에 따른 면역세포로부터 분비된 TNF-α의 양을 ELISA 키트로 측정한 결과이다.
도 3은 본 발명의 발효 오신채 추출물에 의한 세포 활성 증진 효과로서 발효 오신채 추출물의 농도에 따른 사람 피부 섬유아세포에 함입된 [3H]-티미딘의 양을 LSC(액체 방사능 계수기)로 측정한 결과이다.
도 4는 본 발명의 발효 오신채 추출물에 의한 세포 활성 증진 효과로서 발효 오신채 추출물의 농도에 따른 사람 피부 섬유아세포의 생존율을 MTT 검정법으로 측정한 결과이다.
도 5는 본 발명의 발효 오신채 추출물에 의한 세포 활성 증진 효과로서 발효 오신채 추출물의 농도에 따른 자외선-조사된 사람 피부 섬유아세포의 생존율을 MTT 검정법으로 측정한 결과이다.
도 6은 본 발명의 발효 오신채 추출물에 의한 세포 활성 증진 효과로서 발효 오신채 추출물의 농도에 따른 H2O2-처리된 사람 피부 섬유아세포의 생존율을 MTT 검정법으로 측정한 결과이다.
도 7은 본 발명에 따른 발효 오신채 추출물을 함유한 로션 제형을 염증에 의해 손상된 사람 표피 세포에 적용한 후 H&E 염색한 사진이다.
도 8은 본 발명에 따른 발효 오신채 추출물을 함유한 로션 제형을 정상 피부에 3일간 적용하고 자외선 조사 후 발현된 랑게르한스 세포의 사진이다.FIG. 1 shows the results of measurement of the amount of IL-6 secreted from immune cells by an ELISA kit according to the concentration of the extract of fermented ossein as a skin immunity enhancing effect of the fermented osuga extract of the present invention.
2 is a graph showing the results of measurement of the amount of TNF-? Secreted from immune cells by ELISA kit according to the concentration of the fermented oyster extract as a skin immunity enhancing effect of the fermented osuga extract of the present invention.
FIG. 3 is a graph showing the effect of the extract of fermented bean curd according to the present invention on the amount of [ 3 H] -thymidine incorporated into human skin fibroblasts by LSC to be.
FIG. 4 shows the results of measurement of the survival rate of human skin fibroblasts by the MTT assay according to the concentration of the fermented osteocalcin extract, which is an effect of promoting cell activity by the fermented osuga extract of the present invention.
FIG. 5 shows the results of measurement of the survival rate of ultraviolet-irradiated human dermal fibroblasts by MTT assay according to the concentration of fermented ossein extract as an effect of promoting cell activity by the fermented osculus extract of the present invention.
FIG. 6 shows the results of measurement of the survival rate of H 2 O 2 -treated human skin fibroblasts by MTT assay according to the concentration of the fermented osteocyte extract according to the present invention.
FIG. 7 is a photograph of H & E staining after applying a lotion formulation containing the fermented Orensech extract according to the present invention to human epidermal cells damaged by inflammation.
FIG. 8 is a photograph of Langerhans cells expressed after irradiation with ultraviolet rays, in which a lotion formulation containing a fermented osmanthus extract according to the present invention is applied to normal skin for 3 days.
본 발명의 화장료 조성물에 사용되는 달래, 마늘, 부추, 파 및 흥거는 시장에서 구입하거나 직접 그들의 씨앗으로부터 재배하여 사용할 수 있다.The soothing, garlic, leek, leek and flavoring agents used in the cosmetic composition of the present invention can be purchased from the market or directly grown from their seeds.
달래(Allium monanthum)는 강장식품으로 유명하며, 신경을 안정시키는 작용이 있어 불면증에도 효과가 있고 피부미용이나 빈혈 자궁출혈 등에도 이로우며, 특히 동맥경화에 좋은식품으로 알려져 있다. 달래는 피로를 풀어주는 비타민A, B1, B2, C 등이 풍부하고, 천연 비타민과 각종 미네랄을 골고루 함유하고 있으며, 달래에는 마늘의 매운 맛을 내는 알리인과 알리신이 들어 있어 작은 마늘 '산마늘'로도 불린다. 한방에서는 달래의 비늘줄기는 소산(小蒜)이라는 약재로 쓰는데 비장과 신장 기능을 돕고 기혈순환을 원활하게 하여 불면증, 장염, 위염에 효과가 있다고 하며 종기나 벌레물린곳, 협심통, 복통을 치료하는데 쓰인다. 본초습유라는 의서에는 달래는 적괴(암이나 종양 같은 것)를 다스리고 부인의 혈괴(부인과 계통의 종양이나 어혈 응어리)를 다스린다고 전한다. 비타민A, B1, B2, C 등의 함유량이 많은 달래는 세포와 세포를 잇는 결합 조직의 생성과 유지를 원활하게 하여 피부 결합조직의 활력을 증가시키고 피부의 영양흐름을 증진시켜 건강한 피부를 만드는데 도움을 준다. 풍부한 미네랄을 함유하고 특히 칼슘이 많아 표피 회복 및 장벽 항상성 유지에 영향을 주어 피부를 탄력있게 하고 면역기능 향상과 빈혈, 자궁출혈 및 동맥경화예방에 좋다. Allium monanthum is known for its tangible foods and it is effective for insomnia because it has a function to stabilize the nerves. It is also known to be good for atherosclerosis, especially for skin beauty and anemia bleeding. It is rich in vitamins A, B1, B2, and C that relieve soothing fat, contains natural vitamins and various minerals, and alliin and alicin, which give spicy taste of garlic, . In one room, the stem of the algae is used as a medicinal product called Xiaoshan, which helps the spleen and kidney function and smoothes the blood circulation, which is effective for insomnia, enteritis and gastritis. It treats boils, insect bites, . In the tradition of herbal medicine, it is said that it governs soothing masses (such as cancer and tumors) and governs the blood clots of the wife (gynecologic and systemic tumors and hemorrhoids). Vitamin A, B1, B2, C, etc., which contains a large amount of soothing cells and connect the cells to connect the formation and maintenance of smooth skin to increase the vitality of the connective tissue, skin nutrition to help create healthy skin give. Rich in minerals, especially calcium, it affects the skin's recovery and barrier homeostasis, and it makes skin elastic, and it is good for improving immune function and preventing anemia, uterine bleeding and arteriosclerosis.
마늘(Allium scorodorpasum var. viviparum Regel)은 질병을 치료하는 은빛탄환이라고 할 만큼 우리 몸에 좋은 성분들이 가득 들어있는 영양소의 보고라 할 수 있다. 서양 의학계에서는 마늘 속에서 항암물질을 뽑아 암환자의 치료에 이용하고, 국내에서도 마늘 속의 특정 성분을 추출하여 혈액순환 개선제로 개발하여 시판하고 있다. 마늘의 주성분은 단백질, 당질, 비타민 B1, B2, C, 칼슘, 인, 철분 등으로 매운맛을 내는 알리신을 함유한다. 생리 활성 물질로 스코르디닌과 크레아틴, 인체 내 해독장욕을 하는 시스테인, 메티오닌 성분이 들어있다. 그 밖에 항암기능을 가지고 있는 게르마늄과 셀레늄, 아연도 풍부하게 함유되어있다. 톡쏘는 맛과 향을 좌우하는 마늘의 알리신은 비타민 B1과 결합해 알리디아민이라는 성분으로 바뀌는데 알리디아민은 호르몬 활동을 조절하고 살균작용과 소화효소의 분비를 촉진한다. 세포 대사를 촉진시키고 암세포를 억제하는 효과가 있다. 마늘에는 비타민 C나 유지의 산화를 막으며 체내의 과산화지방생성을 방지하는 노화방지의 효능도 있음이 실험을 통하여 입증되었다. 또한, 게르마늄과 셀레늄, 아연을 다량 포함하여 아토피성 피부질환시 유리되는 베타헥기 소사미니데스 효소를 억제하여 아토피성 피부염을 억제하고 면역작용을 증가시키는 효과가 있다. 동의보감에서는 마늘이 종기나 옹종(癰腫)을 풀어지게 하고, 풍습(風濕)과 장기를 없애며, 복부에 생기는 적취(積聚)의 일종인 현벽을 삭히고, 냉증과 풍증을 없애며, 비장을 든든하게 하고, 위를 따뜻하게 한다고 쓰여있다. 혈관을 확장해서 혈행을 좋게 하므로 피부의 신진대사가 촉진되고 피부에 활력을 준다.Garlic ( Allium scorodorpasum var viviparum regel ) is a report of nutrients that are full of good ingredients in our body, which is a silver bullet to cure disease. In western medicine, it extracts anticancer substance from garlic and uses it for the treatment of cancer patients. In Korea, the specific ingredient of garlic is extracted and developed as a blood circulation improving agent. The main ingredient of garlic contains protein, carbohydrate, vitamins B1, B2, C, calcium, phosphorus, iron and so on. The physiologically active substances include scorodinin and creatine, and cysteine and methionine which cause detoxification in the human body. It is also rich in germanium, selenium and zinc, which have anti-cancer functions. Alicin of garlic, which affects the taste and flavor, is combined with vitamin B1 to become an ingredient called allydiamine. Allydiamine regulates hormonal activity and promotes the sterilization and secretion of digestive enzymes. It promotes cell metabolism and inhibits cancer cells. Garlic has been shown to prevent the oxidation of vitamin C and fat, and also has the anti-aging effect of preventing the production of peroxide in the body. In addition, it contains germanium, selenium and zinc in large amounts to inhibit beta-hexasusaminidase enzyme, which is released in atopic skin diseases, to suppress atopic dermatitis and increase immune function. In Dong-bo-gyung, garlic has the effect of releasing bruises and stomachs, eliminating customs and organs, cutting off the horn, a kind of abundance in the abdomen, eliminating cold and wind, It says that it warms the stomach. It enlarges the blood vessels and improves blood circulation, thus promoting the metabolism of the skin and vitality of the skin.
부추(Allium tuberosum)는 백합과에 속하는 다년생 풀로써 아시아가 원산지이다. 자생력이 강해 뿌리만 살아 있으면 4월부터 11월까지 계속해서 새순이 돋아 수확이 가능하고 겨울에도 잘 얼어 죽지 않는다. 지역에 따라 솔, 정구지라고도 부르며 한약재로는 구채, 난총이라고 하여 간장과 신장을 보해주는 중요한 약재로 쓰이고 있다. 봄에 비늘줄기에서 가는 선 모양의 육질의 잎이 여러가닥으로 나오는데, 이 부분을 식용으로 사용한다. 영양학적으로는 다른 식물들에 비해 단백질, 지질, 당분, 비타민A와 C가 월등히 많으며 철분, 인, 칼슘, 비타민 B군도 많이 함유하고 있다. 특히, 베타-카로틴의 함량은 호박중 늙은 호박의 4배 이상, 애호박의 19배 이상, 그리고 배추의 무려 83배 이상이며 세포손상을 방지하고 항산화작용을 하는 클로로필, 글루타티온도 함유하고 있다. 비타민B1의 흡수를 돕는 알리신을 함유하며 당질은 대부분이 포도당 또는 과당으로 구성되는 단당류이다. 일명 `기양초`라고 부르는 부추는 간장과 신장을 보해주어 자양강장효과가 뚜렷해 혈액을 맑게 하고 세포에 활력을 준다. 이뇨작용, 지사작용 등의 효과가 있으며 소화액분비를 증진시켜 소화불량, 위장염에도 효과가 좋다. 코피, 산후출혈 등에도 지혈효과가 있다. 부추에 든 베타-카로틴은 활성산소가 세포를 산화시키는 것을 강력하게 막아주는 항산화작용을 하는데, 이미 우리 몸에 생긴 활성산소를 꼭 붙잡아 활동을 못하게 하는 것뿐만 아니라 활성산소 발생 자체를 억제하는 뛰어난 물질이다. 다량 함유된 비타민 A와 C로 인해 피부나 점막의 저항력을 강화시켜 주고 세포분화와 신진대사를 촉진하는 효과가 있다. 풍부한 칼륨은 나트륨의 피해를 줄이고 피부의 영양 흐름을 증진시켜 준다. 동의보감에 따르면 부추는 더운 성질을 갖고 있어 인체의 열을 돋우는 보온효과가 있고 어혈을 풀고 혈액순환을 좋게 하여 어혈로 인한 근육통이나 생리통, 수족냉증 등에 진통효과가 있고, 얼굴이 검어지는 어혈성 기미가 있는 경우에도 부추를 쓴다.Leek ( Allium tuberosum ) is a perennial grass belonging to the lily family and originates in Asia. If the root is alive with strong self-sustenance, it can be harvested continuously from April to November, and freezing in the winter does not die well. Depending on the area, it is also called as a sole or a tongue. Herbal medicines are used as medicines to show the kidneys and kidneys. In spring, a line of fleshy leaves from the stem of the scales emerges in multiple strands, which are used for edible purposes. Nutritionally, proteins, lipids, sugars, vitamins A and C are significantly higher than other plants, and iron, phosphorus, calcium, and vitamin B groups are also abundant. In particular, the content of beta-carotene is more than four times that of old amber pumpkin, more than 19 times of zucchini, and more than 83 times of Chinese cabbage, and also contains chlorophyll and glutathione which prevent cell damage and antioxidative action. It contains allicin which helps absorption of vitamin B1, and the saccharides are monosaccharides mostly composed of glucose or fructose. The so - called "candy candy" is called "candy candy", which gives the liver and kidneys a strong nourishing tonic effect, which cleanses the blood and energizes the cells. Diuretic effect, the effect of the branch office, and digestive juice is promoted by the secretion of digestion is good for gastroenteritis. Nose bleeding and postpartum hemorrhage are also effective. Beta-carotene in leek has an antioxidant effect that strongly inhibits the oxidation of reactive oxygen species. It is an excellent substance that inhibits active oxygen generation itself to be. Vitamin A and C, which are contained in large amounts, strengthen the skin and mucous membrane resistance and promote cell differentiation and metabolism. Rich potassium reduces the damage of sodium and improves the nutritional flow of the skin. According to Donguibogam, leek has a hot temperament, it has a warming effect that arouses the heat of the body, loosens the ejaculation and improves the blood circulation. It has analgesic effect on myalgia, menstrual cramps and poor circulation due to ejaculation. Even if you have leek.
파(Allium fistulosum)는 외떡잎식물인 백합과(百合科 Liliaceae)에 속하는 다년생 풀로 음식을 조리하는 데에 있어 필수적인 향신료로 음식의 기호도를 높게 해주는 채소이다. 파는 가용성 탄수화물류가 많고 조섬유, 순단백질, 조지방, 조회분 펜토산 등을 함유한다. 비타민 A, B, C를 포함하며 비타민의 합성을 촉진시키고 면역기능을 향상시키는 피틴과 피톤치드를 함유한다. 황화아미노산인 알린을 함유해 알리신을 생성하며 비타민과 칼슘, 염류도 풍부하다. 파는 성인병 예방에 효과가 우수한 천연건강식품으로 몸을 따뜻하게 데워주고 위장 기능을 도아주어 복통, 소화장애 궤양 등에 사용한다. 기운을 통하게 하고 해독시켜 혈액을 맑게 하고 신진대사를 촉진하여 세포를 활성화시킨다. 이뇨, 건위, 거담, 구충 작용이 있으며 다량의 비타민과 피틴, 피톤치드를 함유하여 항산화, 항균작용이 뛰어나고 피부를 건강하게 한다. 암을 억제하는 효과가 입증된 바 있다. Allium fistulosum is a perennial plant belonging to the monocotyledonous Liliaceae and is an essential spice for cooking food. The digestible carbohydrates are abundant and contain crude fiber, pure protein, crude fat, and ascorbic pentanoic acid. Contains vitamins A, B and C, contains phytone and phytoncide to stimulate the synthesis of vitamins and improve immune function. It contains allysine, which is a sulfurized amino acid, to produce allysine. It is also rich in vitamins, calcium, and salts. It is a natural health food that is effective for prevention of adult diseases. It warms up the body and helps the gastrointestinal function. It is used for abdominal pain, digestive disorder ulcer and so on. It activates cells by activating and decrypting energy and clearing blood and promoting metabolism. It has diuretic, dryness, genomic and antiparasitic properties. It contains a lot of vitamins, phytin and phytoncide, so it has antioxidant, antimicrobial action and makes your skin healthy. The effect of suppressing cancer has been proven.
흥거(Scilla scilloides)는 그 꽃이 연한 홍자색이고 화피갈래조각과 수술은 각각 6개이며 암술은 1개이다. 씨방은 타원형이고 잔털이 3줄로 돋아 있다. 열매는 달걀을 거꾸로 세운 모양으로 길이 4 mm이다. 비늘줄기와 어린 잎을 엿처럼 오랫동안 조려서 먹으며, 구황식물(救荒植物)의 하나이다. 뿌리를 구충제로 사용한다. 한국, 아시아 동북부의 온대에서 아열대까지 널리 분포한다. 일부 지방에서는 물구지라고도 하며 한의학에서는 면조아(綿棗兒)라고 부른다. 백색 꽃이 피는 것을 흰무릇(for. alba)이라고 한다. 흥거의 비늘줄기와 잎, 꽃에 부바티에놀리드(강심 배당체)가 들어있으며 많은 양의 녹말과 자당, 실레인이 들어있다. 흥거는 흔히 약재로 알뿌리를 쓰며 진통효과가 있고 혈액순환을 왕성하게 해 피부의 영양 흐름을 증진시키고 세포에 활력을 준다. 구충제나 머리에 창진이 생겼을 때 찧어 붙이며 부어오른 것을 가시게 하는 효능이 있다. 허리와 팔다리가 쑤리고 아픈데, 타박상 등을 치료한다. 또한 종기나 유방염, 장염 등에도 효과가 있다. 비늘 줄기나 잎을 알코올에 담가 추출한 액체는 강심, 이뇨 작용을 한다. <동의학사전> 에서는 이렇게 기록하고 있다. "맛은 쓰고 성질은 서늘하다. 혈을 잘 돌게 하고 해독하며 부종을 내리고 통증을 멈춘다. 약리실험에서 강심작용, 이뇨작용, 자궁수축작용 등이 밝혀졌다. 유선염, 장옹, 타박상, 요통, 다리통증, 석림, 산후어혈, 부스럼 등에 쓴다. Scilla scilloides are light reddish purple flowers, with six scalloped pieces and one stamen and one pistil. The ovary is elliptical and has three lines of fine hairs. The fruit is egg shaped upside down and 4 mm long. It is one of the naturopathic plants that eat scallops and young leaves for a long time, like fish. Root is used as an insecticide. It is widely distributed in temperate and subtropical regions of Korea and northeastern Asia. In some provinces, it is also known as water bulb, and in oriental medicine, it is called cotton quat. White flowers are called white flowers (for. Alba). It contains a large amount of starch, sucrose, and silane. It contains scaly stem, leaf, and bovatinic nodule. Hengge is often used as a medicinal herb and has an analgesic effect. It promotes blood circulation and promotes nourishment of skin and revitalizes cells. It has an efficacy that makes it tingle and swollen when it comes to insect repellents or headaches. Waist and limbs are sore and sore, and bruises are cured. It also has effects on boils, mastitis and enteritis. The liquid extracted from the stalks or leaves by alcohol is a strong, diuretic effect. In <The Dictionary of the Donghak>, it is recorded as follows. "The taste is good, the nature is cool, the blood circulates well, it decays, the edema is stopped, the pain is stopped, and the pharmacological experiments revealed the mastication, the diuretic effect and the contraction of the uterus. , Stomach, postpartum hemorrhage, swelling.
한 가지 관점에서, 본 발명은 달래, 마늘, 부추, 파 및 흥거로 구성된 발효 오신채 추출물을 함유하는 화장료 조성물을 제공한다.In one aspect, the present invention provides a cosmetic composition comprising a fermented starch extract consisting of soya bean, garlic, leek, pars, and steamer.
본 발명의 화장료 조성물에 함유되는 발효 오신채 추출물의 제조는 정제수에 오신채 원물을 넣고 약 100℃로 약 8시간 정도 가열하여 추출하는 단계, 추출된 혼합액을 약 37℃로 냉각시킨 후 바실러스 서브틸리스 나토(Bacillus subtilis natto) 배양액을 첨가하고 약 37℃에서 약 72시간 발효시키는 단계, 수득된 발효액을 정제, 숙성 및 여과하는 단계를 포함한다. 따라서, 추가로, 본 발명은 상기 발효 오신채 추출물이 오신채 원물을 열수추출하는 단계, 추출된 혼합액을 냉각시킨 후 바실러스 서브틸리스 나토(Bacillus subtilis natto) 배양액을 첨가하여 발효시키는 단계, 수득된 발효액을 정제하는 단계, 정제된 발효액을 숙성하는 단계 및 숙성된 발효액을 여과하는 단계에 의해 제조된 것임을 특징으로 하는 화장료 조성물을 제공한다.In the preparation of the fermented ointment extract contained in the cosmetic composition of the present invention, the raw material is added to the purified water and heated at about 100 ° C for about 8 hours to extract the mixture. The extracted mixture is cooled to about 37 ° C and then extracted with Bacillus subtilis Adding a culture medium of Bacillus subtilis natto and fermenting at about 37 ° C for about 72 hours, and purifying, aging and filtering the obtained fermentation broth. Accordingly, the present invention also provides a method for producing fermented fruit of the present invention, comprising the steps of: extracting the fermented oyster extract with hot water, cooling the extracted mixed liquor, adding a culture medium of Bacillus subtilis natto to ferment, A step of purifying the purified fermented broth, a step of purifying the purified fermented broth, a step of aging the purified fermented broth, and a step of filtering the aged fermented broth.
상기 오신채 발효액의 정제 공정은 발효완결 후 잔존하는 원물슬러지 및 불용성 물질을 제거하는 단계이다. 발효가 완결된 후 이 혼합물에는 원물슬러지 및 불용성 물질이 잔존하는데 이를 제거하기위해 0.4 기압의 진공으로 감압여과를 진행하며 이때의 정제공정에서는 3 ㎛의 여과필터를 사용하여 불순물을 제거한다. 상기 숙성공정은 여과된 발효액을 4℃에서 24시간 정치하면 여과액 밑부분에 하얀 슬러지들이 생성되는데 이는 발효가 덜된 탄수화물 같은 고분자물질들과 잔존하는 미생물들이 밑부분에 침전물 형태로 존재하게 되어 이를 제거하기 위해 반드시 필요한 단계이다. 상기 여과공정은 정치후 침전된 침전물과 미생물을 제거하기위해 먼저 상등액을 회수하고 회수된 상등액을 0.2기압의 진공에서 0.25 ㎛의 여과필터를 사용하여 감압여과하여 미생물과 부유하고 있는 비활성 고분자 탄수화물들을 제거한다. 따라서, 추가로, 본 발명은 정제수에 오신채 원물을 넣고 약 100℃로 약 8시간 가열하여 추출하는 단계, 추출된 혼합액을 약 37℃로 냉각시킨 후 바실러스 서브틸리스 나토(Bacillus subtilis natto) 배양액을 첨가하고 약 37℃에서 약 72시간 발효시키는 단계, 수득된 발효액을 약 0.4 기압의 진공하에 약 3 ㎛ 여과필터로 정제하는 단계, 여과된 발효액을 약 4℃에서 약 24시간 정치하여 숙성하는 단계 및 숙성된 발효액을 약 0.2 기압의 진공하에 약 0.25 ㎛의 여과필터로 여과하는 단계에 의해 제조된 것임을 특징으로 하는 화장료 조성물을 제공한다.The process for purifying the fresh nutritional fermentation liquid is a step for removing the residual sludge and insoluble matter remaining after completion of fermentation. After the fermentation is completed, the crude sludge and insoluble matter remain in the mixture. To remove the sludge and the insoluble matter, the filtrate is vacuum filtered at a pressure of 0.4 atm. In this purification process, impurities are removed using a 3 ㎛ filter. When the filtered fermentation broth is left to stand at 4 ° C for 24 hours, white sludge is formed at the bottom of the filtrate. This is because polymer substances such as less fermented carbohydrates and residual microorganisms exist in the form of precipitate at the bottom, It is a necessary step to do. In the filtration step, the supernatant is recovered and the recovered supernatant is filtered under a vacuum of 0.2 atm and a filtration filter of 0.25 μm to remove microorganisms and inactive polymer carbohydrates suspended in the filtrate to remove sediment and microorganisms. do. Accordingly, the present invention further provides a method for producing a Bacillus subtilis natto culture, which comprises the steps of placing the raw material in purified water and heating the mixture at about 100 DEG C for about 8 hours, cooling the extracted mixture to about 37 DEG C, Fermenting the fermentation broth at about 37 캜 for about 72 hours, purifying the obtained fermentation broth with a filtration filter of about 3 탆 under a vacuum of about 0.4 atm, aging the filtered fermentation broth at about 4 캜 for about 24 hours and And filtering the aged fermentation broth with a filter of about 0.25 占 퐉 under a vacuum of about 0.2 atm.
본 발명의 발효추출 단계에서 사용된 서브틸러스 나토(Bacillus subtilis natto) 균주 배양액은 멸균수 2 L에 프로테오스펩톤 no. 3 310.0g, 비프추출물 10.0g, 효모추출물 5.0g, 덱스트로스 20.0g, 폴리소베이트80 1.0g, 암모니늄시트레이트 2.0g, 소듐아세테이트 5.0g, 마그네슘설페이트 0.1g, 망간설페이트 0.05g, 디포타슘포스페이트 20g을 가하고 바실러스 서브틸러스 나토(Bacillus subtilis natto)를 접종하여 37℃에서 하룻밤 동안 배양하여 수득한 것이다.The culture medium of the Bacillus subtilis natto strain used in the fermentation extraction step of the present invention was prepared by adding protease peptone no. 3, 10.0 g of beef extract, 5.0 g of yeast extract, 20.0 g of dextrose, 1.0 g of
본 발명의 발효 오신채 추출물은 달래, 마늘, 부추, 파, 흥거의 다섯가지 매운 맛을 지닌 식물을 사용하여 만든 추출물로서, 저항력을 강화시켜 주고 면역작용을 증가시켜 주는 데에 효과가 있어 연약한 피부에 좋고, 다량의 비타민과 풍부한 무기질을 함유, 신진대사를 촉진하고 피부 회복을 증진시켜 피부를 건강하게 해주며, 다섯가지 원료가 가지고 있는 세포 대사 활성 촉진 및 면역 강화에 대한 성분이 상승작용을 가져 더욱 더 우수한 효능을 가지고, 연약한 피부에 자극을 주지않는 안전성과 안정성을 갖춘 우수한 원료이다.The fermented omega extract of the present invention is an extract made by using plants having five pungent flavors of soya, garlic, leek, and pung, and is effective in enhancing resistance and enhancing immune function. Contains a large amount of vitamins and rich minerals, promotes metabolism, promotes skin recovery and promotes skin health. It promotes cellular metabolic activity and enhances immunity of the five raw materials. It has superior efficacy and is an excellent raw material with safety and stability that does not stimulate soft skin.
본 발명에서 원물로서 사용되는 오신채 달래, 마늘, 부추, 파, 흥거에는 알린(Alline)이라는 유기유황성분이 있는데 이 성분을 자르거나 빻게되면 이들 식물조직 세포의 파괴로 인해 알리나아제 효소가 산소에 접촉하면 그 작용으로 자기방어물질이며 항염, 항산화, 항균작용을 하는 알리신(Allicin)성분으로 변화된다. 그러나 이 알리신 성분은 지용성이고 자극성이 강하고 열에 쉽게 파괴가 되어 생물로 이용하는 경우가 많은데 근래에 와서는 보다 고농도의 알리신을 얻기위하여 다양한 추출농축방법이 개발되어져 왔다. 일반적인 열수추출로는 고온의 물을 사용하기 때문에 지용성이며 열에 쉽게 파괴되는 알리신을 극미량 추출할 수밖에 없고 자극성 특성을 제거하기 어렵다. 또한 용매를 이용한 추출방법으로는 잔류용매 및 자극성 물질의 잔존으로 사용에 제한이 있다. 그러나, 본 발명에 따른 발효에 의한 추출방법은 그 공정이 40℃ 이하의 저온에서 추출을 하며 지용성 성분들도 추출되는 특성이 있고 발효에 의한 저분자화로 빻거나 자르지 않아도 알린의 성분을 거의 전량 알리신으로 전환하여 고농도의 알리신을 얻을수 있다. 아래 표 1은 오신채 추출방법별 알리신 함량을 비교 분석한 결과를 보여준다.In the present invention, there is an organic sulfur component called Alline in the almonds, garlic, leeks, pork, and hunger which are used as the raw materials of the present invention. When these components are cut or crushed, It is a self-defense substance, and it is changed into an allicin component which has anti-inflammation, antioxidant and antibacterial action. However, this allysine component is fat-soluble, has strong irritation, is easily destroyed by heat, and is often used as an organism. Recently, various extractive concentration methods have been developed in order to obtain higher concentration of allysine. Since general hot water extraction uses high-temperature water, it is inevitable to extract trace amounts of allysine, which is fat-soluble and easily destroyed by heat, and it is difficult to remove the irritant property. In addition, the extraction method using a solvent has a limitation in its use as a residual solvent and a residual irritant. However, the fermentation-based extraction method according to the present invention is characterized in that the extraction is performed at a low temperature of 40 ° C or lower and lipophilic components are also extracted. Even if the fermentation does not grind or cut the fermentation by low-molecular-weight fermentation, To obtain a high concentration of allysine. Table 1 below shows the results of comparative analysis of allysine content by ocea extract method.
(100℃, 8시간)Hot water extraction
(100 占 폚, 8 hours)
(37℃, 72시간)The fermentation extraction of the present invention
(37 DEG C, 72 hours)
본 발명의 화장료 조성물에서 발효 오신채 추출물을 구성하는 달래, 마늘, 부추, 파 및 흥거의 중량비는 다양하게 변화될 수 있으며, 그 중 달래, 마늘, 부추, 파 및 흥거의 중량비가 3~4:5~6:3~4:2~3:2~3인 것이 바람직하며, 2:3:2:1.5:1.5일 때 보다 바람직하다.In the cosmetic composition of the present invention, the weight ratio of the soya extract, garlic, leek, leek, and pea constituting the fermentation extract can be variously changed, and the weight ratio of the soya, garlic, leek, 6: 3 to 4: 2 to 3: 2 to 3, and more preferably 2: 3: 2: 1.5: 1.5.
본 발명의 화장료 조성물에서 발효 오신채 추출물은 총 조성물의 중량에 대하여 건조 중량으로 0.1 내지 15 중량%, 바람직하게는 3 내지 10 중량%의 양으로 화장료에 첨가될 수 있다.In the cosmetic composition of the present invention, the fermented starch extract may be added to the cosmetic in an amount of 0.1 to 15% by weight, preferably 3 to 10% by weight, based on the weight of the total composition, on a dry weight basis.
본 발명의 화장료 조성물은 손상된 피부 세포 회복 및 면역증강 효과를 나타내는 것을 특징으로 한다.The cosmetic composition of the present invention is characterized by exhibiting damaged skin cell recovery and immunity enhancing effect.
본 발명의 화장료 조성물은 일반적인 화장료 제형에 적용시킬 수 있다. 예를 들면, 화장수(스킨로션), 영양로션, 영양크림, 맛사지크림, 영양에센스, 팩 등의 제형에 적용시킬 수 있다. 또한, 본 발명의 화장료 조성물은 피부의 면역력이 약한 유아용 화장수(스킨로션), 로션, 크림, 자외선차단 로션, 자외선차단 크림, 목욕 세정제, 샴푸, 비누일 수 있다. 따라서, 본 발명의 화장료 조성물은 제조하려는 제형에 따라, 화장료 조성물을 제조하는 업계에서 통상적으로 사용되는 성분을 임의로 포함할 수 있다.
The cosmetic composition of the present invention can be applied to general cosmetic formulations. For example, it can be applied to formulations such as lotion (skin lotion), nutrition lotion, nutritional cream, massage cream, nutritional essence, pack and the like. In addition, the cosmetic composition of the present invention may be an infant lotion (skin lotion), a lotion, a cream, an ultraviolet barrier lotion, an ultraviolet barrier cream, a bath cleaner, a shampoo or a soap having weak skin immunity. Accordingly, the cosmetic composition of the present invention may optionally contain ingredients conventionally used in the industry for producing cosmetic compositions, depending on the formulation to be prepared.
이하, 본 발명을 하기 실시예 및 실험예에 의해 상세히 설명하나, 이들은 본 발명의 이해를 돕기 위한 것일 뿐, 본 발명의 보호범위가 이들로 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to the following Examples and Experimental Examples. However, the present invention is only intended to facilitate understanding of the present invention, and the scope of protection of the present invention is not limited thereto.
실시예 1 내지 10. 다양한 비율의 발효 오신채 추출물 제조Examples 1 to 10. Preparation of various proportions of fermented starch extract
5가지 식물들을 아래 표 1에 따라 배합하여 하기의 방법을 사용하여 발효 오신채 추출물을 생성하였다.Five plants were blended according to Table 1 below to produce a fermented oyster extract using the following method.
멸균수 2 L에 프로테오스펩톤 no. 3 310.0 g, 비프추출물 10.0 g, 효모추출물 5.0 g, 덱스트로스 20.0 g, 폴리소베이트 80 1.0 g, 암모니늄시트레이트 2.0 g, 소듐아세테이트 5.0 g, 마그네슘설페이트 0.1 g, 망간설페이트 0.05 g, 디포타슘포스페이트 20 g에 바실러스 서브틸러스 나토(Bacillus subtilis natto)를 접종하여 37℃에서 하룻밤 동안 배양하여 바실러스 서브틸러스 나토(Bacillus subtilis natto) 균주 배양액을 생성한다.Sterile water 2 L protease peptone no. 3 310.0 g of beef extract, 5.0 g of yeast extract, 20.0 g of dextrose, 1.0 g of
아래 표 2와 같이 조성된 원료 500 g을 깨끗하게 세척하고 정제수 5 kg에 넣고 100℃로 승온하여 8시간 추출한다. 생성된 추출물을 37℃까지 냉각시킨 후 바실러스 서브틸리스 나토 균주 배양액 50 g을 첨가하고 37℃에서 72시간 발효시킨다. 생성된 발효액을 3 ㎛의 여과 필터를 사용하여 0.4 기압의 진공으로 감압 여과하여 원물슬러지와 불용성물질을 제거하고, 여과된 발효액을 4℃에서 24시간 정치한 후, 0.2 기압의 진공에서 0.25 ㎛ 필터를 이용하여 감압 여과하여 미생물과 부유하고 있는 탄수화물들을 제거하여 목적하는 발효 오신채 추출물을 수득한다.500 g of the raw material as shown in Table 2 below was cleanly washed, put into 5 kg of purified water, heated to 100 ° C and extracted for 8 hours. After the resulting extract is cooled to 37 ° C, 50 g of Bacillus subtilis strain culture is added and fermented at 37 ° C for 72 hours. The resultant fermentation broth was filtered under a reduced pressure of 0.4 atm using a filtration filter of 3 탆 to remove the raw sludge and the insoluble matter. The filtered fermentation broth was allowed to stand at 4 캜 for 24 hours, and then 0.25 탆 filter To remove the microorganisms and floating carbohydrates to obtain the desired fermented oyster extract.
실험예 1. 면역세포의 생육활성 측정(MTT Assay)EXPERIMENTAL EXAMPLE 1 Measurement of Growth Activity of Immune Cells (MTT Assay)
사람 면역세포인 Raji(Human B 세포)과 Jurkat(Human CD4+ T 세포)을 MTT(3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) 검정법을 이용하여 실시하였다. 세포의 생육은 RPMI1640(GIBCO, USA) 배지에서 10% FBS를 사용하여 37℃, 5% CO2 인큐베이터에서 배양하였다. 면역세포 생육활성 측정은 24 웰 평판에 세포 농도를 4x104 세포/㎖로 하여 배양 24시간 후 각 시료(실시예 1)를 농도별로 적용하였다. 48시간 배양 후 각각의 웰에 있는 배지를 버리고 MTT 용액을 웰당 1000 ㎕씩 가한 후 4시간 후에 MTT를 제거하고 DMSO를 웰당 1000 ㎕씩 가한 후 하룻밤 37℃에서 인큐베이션 후 570 nm에서 흡광도를 측정하였다. 이때 발효 오신채 추출물을 첨가하지 않고 같은 방법으로 실험을 진행한 것을 대조군으로 하여 상대적인 흡광도의 차이를 총 8일에 걸쳐 측정하여 세포의 생육활성을 관찰하였다. 그 결과는 아래 표 3과 같다.Human immunodeficient cells, Raji (Human B cells) and Jurkat (Human CD4 + T cells) were assayed by MTT (3- [4,5-dimethylthiazole-2-yl] -2,5-diphenyltetrazolium bromide) assay. The cells were cultured in RPMI1640 (GIBCO, USA) at 37 ° C in a 5% CO 2 incubator with 10% FBS. Immunocytochemical growth activity was determined on a 24-well plate at a cell concentration of 4
상기 표 3에서 보는 바와 같이 본 발명에 따른 발효 오신채 추출물의 피부 면역 강화 효과를 측정한 결과 상기 B세포 생존율과 T세포 생존율에서 효능이 뛰어났다. 이 결과로 볼 때 본 발명의 발효 오신채 추출물은 뛰어난 피부 면역 강화능을 가지고 있는 원료임을 알 수 있다.
As shown in the above Table 3, the skin fermentation enhancing effect of the fermented omega extract according to the present invention was excellent in the B cell survival rate and the T cell survival rate. From these results, it can be understood that the fermented omega extract of the present invention is a raw material having excellent skin immunity enhancing ability.
실험예 2. 면역세포로부터 사이토카인 분비량 측정Experimental Example 2. Measurement of cytokine secretion amount from immune cells
사이토카인은 여러 가지 면역세포들에 의하여 만들어지며, 여러 가지 면역세포의 활성화, 성장, 분화 등에 영향을 미칠 수 있다. 사이토카인 단백질들은 자연면역과 획득면역 반응에 관여할 뿐 아니라, 면역세포의 성숙과정에서도 중요한 작용을 나타낸다. 사이토카인 중에서 식균작용이 있는 단핵세포에 의해서 만들어지는 것을 모노카인이라고 부르기도 하며, 림프구가 만들어 다른 림프구의 작용을 조절하는 것을 림포카인 또는 인터루킨이라고 부르기도 한다. 또한, 골수의 전구세포로부터 각종 면역세포의 성숙을 조절하는 사이토카인들을 콜로니자극인자라고 부르기도 한다. 그러나, 하나의 사이토카인이 특정한 하나의 세포에 의해 만들어지는 것도 아니고, 심지어 하나의 사이토카인의 작용이 한 가지 세포에만 정해져 있는 것이 아니기 때문에, 이러한 구분이 아주 명확한 것은 아니다.Cytokines are produced by various immune cells and can affect the activation, growth, and differentiation of various immune cells. Cytokine proteins not only play a role in innate and acquired immune responses, but also play an important role in immune cell maturation. The cytokine that is produced by mononuclear cells with phagocytosis is also called monocaine. It is also called lymphocaine or interleukin that lymphocytes produce and regulate the action of other lymphocytes. Cytokines that regulate the maturation of various immune cells from bone marrow precursor cells are also referred to as colony stimulating factors. However, this distinction is not very clear, since a single cytokine is not made by a single cell, and even the action of one cytokine is not specific to one cell.
IL-6은 간세포에 작용하여 급성기 반응 단백질(acute phase response protein)을 만들게 하며, B 세포에 작용하여 B 세포의 성장을 촉진하고, T 세포나 흉선세포에 공동자극인자로 작용한다. TNF-α는 그람음성세균 감염에 의해서 만들어지는데, 세균의 세포막에 있는 내독소인 지당류(LPS)에 의해 활성화된 림프구에 의해서 만들어진다. LPS의 양이 적은 경우는 TNF-α가 적은 양으로 생성되어, 백혈구나 혈관세포에 작용하여 국소적인 염증반응이 나타나 항원이 제거된다. 또한, T 와 B 세포의 활성화에 공동자극인자로 작용하기도 한다. TNF-α는 또한 CSF의 합성을 유도할 수도 있으며, class I MHC의 발현을 증가시키기도 한다. TNF-α는 또한 종양세포에 작용하여 세포자살(apoptosis)을 유도하기도 한다.IL-6 acts on hepatocytes to produce acute phase response proteins, acts on B cells to promote the growth of B cells, and acts as a co-stimulatory factor in T cells and thymocytes. TNF-α is produced by gram-negative bacterial infections, which are produced by activated lymphocytes (LPS), an endotoxin in the bacterial cell membrane. When the amount of LPS is low, TNF-α is produced in a small amount and acts on leukocyte or blood vessel cells, resulting in a local inflammatory reaction and elimination of the antigen. It also acts as a co-stimulatory factor in the activation of T and B cells. TNF- [alpha] may also induce the synthesis of CSF and increase the expression of class I MHC. TNF-α also acts on tumor cells to induce apoptosis.
면역세포들이 배지 내에 분비하는 사이토카인인 종양괴사인자-α(TNF-α)와 인터루킨-6(IL-6)의 양을 ELISA 키트(Genzyme, USA)를 이용하여 측정하였다. 우선 사람 면역세포인 B 세포(Raji)를 이용하여 RPMI 1640(Gibco, USA) 배지에서 10% FBS를 사용하여 세포의 농도를 4x104 세포/㎖의 농도 정도를 배양시킨 후 이 배양 배지를 원심분리하여 상등액을 취한 다음 다양한 농도의 표준물질로서 TNF-α, IL-6들과 함께 37℃에서 30분간 배양 후 450 nm에서 흡광도를 측정하여 표준곡선을 작성하였다. ELISA 키트를 이용하여 인간 면역세포인 B 세포(Raji)에 발효 오신채 추출물 시료(실시예 1) 투여 후 얻어진 O.D.값을 비교하여 표준곡선에서 얻어진 값을 이용 사이토카인의 양을 측정하였다. 그 결과는 도 1 및 2와 같다. 도 1 및 2는 각각 IL-6 및 TNF-α의 분비량을 보여준다.
(TNF-α) and interleukin-6 (IL-6), which are cytokines secreted by immune cells in the medium, were measured using an ELISA kit (Genzyme, USA). First, B cells (Raji), which is a human immune cell, were cultured in RPMI 1640 (Gibco, USA) to a concentration of 4 × 10 4 cells / ml using 10% FBS, and the culture medium was centrifuged The supernatant was taken and cultured at 37 ° C for 30 minutes with TNF-α and IL-6 as standard substances at various concentrations. The absorbance was measured at 450 nm to prepare a standard curve. The amount of cytokine was measured using the value obtained from the standard curve by comparing the OD values obtained after the administration of the fermented osteocyte extract sample (Example 1) to B cells (Raji), which are human immune cells, using an ELISA kit. The results are shown in FIGS. 1 and 2. Figures 1 and 2 show the secretion levels of IL-6 and TNF-a, respectively.
실험예 3. Experimental Example 3. 33 H-티미딘에 의한 세포 활성 측정Measurement of cell activity by H-thymidine
건강한 피부를 갖기 위해서는 세포의 대사 활성 촉진 및 증식 능력은 필수적이다. 그래서 다음과 같이 발효 오신채 추출물의 농도에 따른 세포에 대한 영향을 조사하였다.In order to have healthy skin, the ability to promote and proliferate the metabolic activity of cells is essential. Therefore, we examined the effects on the cells depending on the concentration of the fermented oyster extract as follows.
사람의 피부세포로부터 섬유아세포를 분리하여 발효 오신채 추출물 시료(실시예 1)을 첨가 시킨 것과 무첨가시킨 것을 배양시킨다. 배양 3일 후 3H-티미딘을 넣고 5시간 배양하여 삽입된 양을 액체 방사능계수기(Liquid Scintillation Counter, LSC)를 사용하여 측정하였다. 그 결과는 도 3과 같다.Fibroblasts are isolated from human skin cells and cultured in the same manner as in Example 1 except that the fermented osteocyte extract sample (Example 1) is added. Three days after the incubation, 3 H-thymidine was added and incubated for 5 hours. The amount of the inserted product was measured using a liquid scintillation counter (LSC). The results are shown in FIG.
도 3의 그래프에서 보는 바와 같이 섬유아세포의 대조군에서는 증식이 지연되고, 3H-티미딘의 삽입량은 적었다. 그렇지만, 본 발명의 발효 오신채 추출물을 첨가한 세포 배양계에서는 농도 의존적인 증가를 나타냈으며, 이 결과로 볼때 세포 대사활성 촉진에 의해 증식이 촉진됨을 확인할 수 있었다.
As shown in the graph of FIG. 3, in the fibroblast control group, the proliferation was delayed, and the insertion amount of 3 H-thymidine was small. However, in the cell culture system containing the fermented osuga extract of the present invention, the concentration-dependent increase was observed. As a result, it was confirmed that the proliferation was promoted by promoting the cell metabolic activity.
실험예 4. 세포 대사 활성 촉진 측정EXPERIMENTAL EXAMPLE 4. Measuring Promotion of Cell Metabolism Activity
영양분이 부족한 배지에서 신생아 섬유아세포의 대사 활성 즉, 본 발명에 따른 발효 오신채 추출물의 세포 자극제로서의 능력을 측정하였다. 대사 활성은 서로 다른 조성의 배지에 신생아 섬유아세포를 48시간 배양 후 MTT assay 방법을 사용하여 측정하였다.The metabolic activity of neonatal fibroblasts in the nutrient-deficient medium, that is, the ability of the fermented nutritional extract according to the present invention as a cell stimulating agent, was measured. Metabolic activity was measured by MTT assay after culturing neonatal fibroblasts for 48 h in different media.
사람 섬유아세포주(ATCC, CRL-2076)를 5x104 세포/㎖의 농도로 하여 24웰 평판에 씨딩하였다. 배지는 FBS 10%를 함유한 IMDM(Iscove's Modified Dubelcco's Medium, BRL, USA)를 사용하였다. 씨딩 24시간 후 FBS 2%와 FBS 0%를 함유한 DMEM를 교체하고, FBS 0%를 함유한 웰 평판에 본 발명의 발효 오신채 추출물(실시예 1)을 농도별로 첨가 한 후 3일 동안 37℃, 5% CO2 인큐베이터에서 배양하였다. 배양이 끝난 후 상등액을 제거하고, MTT 용액을 1.0 ㎖/웰 씩 가한 후 4시간 후에 MTT를 제거하고 DMSO를 처리한 후 다음 날 570 nm에서 흡광도를 측정하였다. 그 결과는 도 4와 같다.Human fibroblast cell line (ATCC, CRL-2076) was seeded into a 24 well plate at a concentration of 5 x 10 4 cells / ml. The medium was Iscove's Modified Dubelco's Medium (BRL, USA) containing 10% FBS. After 24 hours of seeding, the DMEM containing 2% FBS and 0% FBS was replaced, and the fermented ointment extract of the present invention (Example 1) was added to the well plate containing 0% FBS for each concentration. , And incubated in a 5% CO 2 incubator. After the incubation, the supernatant was removed, MTT solution was added at 1.0 ml / well, MTT was removed after 4 hours, DMSO was treated, and the absorbance was measured at 570 nm the next day. The result is shown in Fig.
도 4의 그래프에서 보는 바와 같이 2% 소혈청 첨가 배지와 무첨가 배지에 발효 오신채 추출물을 첨가한 배지의 증식력이 농도가 높아질수룩 거의 비슷함을 보여주었다. 이 결과로부터 본 발명의 발효 오신채 추출물은 세포의 자극제로서 세포의 대사 활성을 촉진함을 알수 있다.
As shown in the graph of FIG. 4, the proliferation power of the medium supplemented with 2% bovine serum-supplemented medium and the non-supplemented medium was almost similar to that of the medium supplemented with 2% bovine serum-supplemented medium. From these results, it can be seen that the fermented osmotic extract of the present invention promotes the metabolism activity of cells as stimulants of cells.
실험예 5. 자외선에 대한 손상으로부터 세포 회복 활성 측정Experimental Example 5. Measurement of Cell Recovery Activity from Damage to Ultraviolet Light
배양된 사람 섬유아세포에 UV-A를 조사하여 손상을 준 후, 본 발명의 발효 오신채 추출물을 첨가한 세포와 첨가하지 않은 세포의 대사 활성에 의한 증식능을 5일간 배양 후 측정하였다.After the cultured human fibroblasts were irradiated with UV-A for damage, the proliferative capacity by the metabolism activity of the cells to which the fermented extract of the present invention was added and the cells not to be added were measured for 5 days.
사람 섬유아세포주(ATCC, CRL-2076)을 5x104 세포/㎖의 농도로 하여 24 웰 평판에 씨딩하였다. 배지는 FBS 10%를 함유한 IMDM (Iscove's Modified Dubelcco's Medium, BRL, USA)를 사용하였다. 씨딩 24시간 후 배지를 PBS로 교체하고, UV-A를 조사하여 손상을 준 후 배지를 넣고, 발효 오신채 추출물(실시예 1)을 농도별로 첨가 한 후 3일 동안 37℃, 5% CO2 인큐베이터에서 배양하였다. 배양이 끝난 후 상등액을 제거하고 MTT 용액을 1.0 ㎖/웰 씩 가한 후 4시간 후에 MTT를 제거하고 DMSO를 처리한 후 다음 날 570nm에서 흡광도를 측정하였다. 그 결과는 도 5와 같다.Human fibroblast cell line (ATCC, CRL-2076) was seeded into a 24 well plate at a concentration of 5 x 10 4 cells / ml. The medium was Iscove's Modified Dubelco's Medium (BRL, USA) containing 10% FBS. After 24 hours of seeding, the medium was replaced with PBS, and UV-A was irradiated for damage. Then, the medium was added to the medium, and the fermented ointment extract (Example 1) was added for each concentration, followed by incubation for 3 days at 37 ° C in a 5% CO 2 incubator Lt; / RTI > After the incubation, the supernatant was removed and MTT solution was added at a rate of 1.0 ml / well. After 4 hours, the MTT was removed and the absorbance was measured at 570 nm the next day after the treatment with DMSO. The results are shown in Fig.
도 5의 그래프에서 보는 바와 같이 자외선을 조사하여 섬유아 세포에 손상을 준 후 발효 오신채 추출물의 대사 활성에 의한 세포 증식능을 측정한 결과 자외선을 조사하지 않은 세포배양계와 비교해 볼 때 조사후 발효오신채 추출물을 첨가한 세포 배양계는 증식력이 약간 뒤떨어졌으나 첨가하지 않은 세포 배양계보다 세포 증식능이 훨씬 뛰어남을 알 수 있다. 이 결과로 볼 때 본 발명의 발효 오신채 추출물은 손상을 입은 세포의 대사 활성을 촉진시켜 세포 증식을 촉진시킨다.
As shown in the graph of FIG. 5, when the cell proliferative activity was measured by the metabolic activity of the fermented oyster extract after damaging the fibroblasts by irradiating ultraviolet rays, it was found that compared with the cell culture system not irradiated with ultraviolet light, The cell culture system to which the extract was added had a slightly lower proliferative capacity, but the cell proliferative capacity was much better than that of the cell culture system without the addition. As a result, the fermented oyster extract of the present invention promotes the metabolism of damaged cells and promotes cell proliferation.
실험예 6. 하이드록실 라디칼에 대한 손상으로부터 세포 회복 효과 측정Experimental Example 6. Measurement of Cell Recovery Effect from Damage to Hydroxyl Radicals
배양된 사람 섬유아세포에 30 mM의 H2O2를 가하여 손상을 준 후 24시간 후에 발효 오신채 추출물의 섬유아 세포 회복 효과를 측정하였다.30 mM H 2 O 2 was added to the cultured human fibroblasts. After 24 hours, the fibroblast recovery effect of the fermented oyster extract was measured.
사람 섬유아세포주(ATCC, CRL-2076)을 5x104 세포/㎖의 농도로 하여 24웰 평판에 씨딩하였다. 배지는 FBS 10%를 함유한 IMDM (Iscove's Modified Dubelcco's Medium, BRL, USA)를 사용하였다. 씨딩 24시간 후 배지를 교체하고, H2O2를 가하여 손상을 준 후 4시간 후에 발효 오신채 추출물(실시예 1)을 농도별로 첨가 한 후 24시간 동안 37℃, 5% CO2 인큐베이터에서 배양하였다. 배양이 끝난 후 상등액을 제거 하고 MTT 용액을 1.0 ㎖/웰 씩 가한 후 4시간 후에 MTT를 제거하고 DMSO로 처리한 후 다음 날 570nm에서 흡광도를 측정하였다. 그 결과는 도 6과 같다.Human fibroblast cell line (ATCC, CRL-2076) was seeded into a 24 well plate at a concentration of 5 x 10 4 cells / ml. The medium was Iscove's Modified Dubelco's Medium (BRL, USA) containing 10% FBS. 24 hours after seeding, the medium was replaced with H 2 O 2 , and after 4 hours from the addition of H 2 O 2 , the fermented oyster extract (Example 1) was added in concentration-dependent manner and cultured in a 5% CO 2 incubator at 37 ° C. for 24 hours . After the incubation, the supernatant was removed and MTT solution was added at 1.0 ml / well. After 4 hours, MTT was removed and treated with DMSO, and the absorbance was measured at 570 nm the next day. The result is shown in Fig.
도 6의 그래프에서 보는 바와 같이 발효 오신채 추출물을 첨가한 세포 배양계와 첨가하지 않은 세포 배양계에 H2O2를 가하여 섬유아 세포에 손상을 준 후 세포 회복능을 측정한 결과 발효 오신채 추출물을 첨가한 세포 배양계에서 보다 세포 회복능이 훨씬 뛰어남을 알 수 있었다. 이 결과로 볼 때 본 발명의 발효 오신채 추출물은 라디칼에 의한 손상으로부터 세포 회복능이 있음을 알 수 있다.
As shown in the graph of FIG. 6, H 2 O 2 was added to the cell culture system to which the fermented New Zealand extract was added and the cell culture system to which no fermentation was added. And the ability to recover the cells was much better than in the cell culture system added. From these results, it can be seen that the fermented oyster extract of the present invention is capable of recovering cells from damage by radicals.
실험예 7. 독성 측정(MTT Assay)Experimental Example 7. Toxicity Measurement (MTT Assay)
발효 오신채 추출물의 안전성 여부를 확인하기 위하여 독성의 정도와 알러지 유발 가능성에 대한 영향을 조사하였다.To investigate the safety of the fermented ointment extracts, the effects of the degree of toxicity and the possibility of allergy were investigated.
발효 오신채 추출물에 대하여 단층 세포 배양에서의 세포독성을 측정 하였다(MTT assay, J. Immunological Methods 65, 55(1983)). MTT 검정법은 살아있는 세포의 수를 측정함으로서 세포 증식이나 독성에 많이 사용되는 실험법으로 살아있는 세포는 미토콘드리아 내에서 수용성이고 노란색의 염인 MTT(3-[4,5-디메틸티아졸-2-일]-2,5-디페닐테트라졸륨 브로마이드)가 숙신산탈수소효소에 의해 수불용성인 파란색의 포르마잔(formazan) 유도체로 환원되는 원리를 이용하는 것이다. MTT는 NADH에 의해서 포르마잔으로 환원되어 570 nm의 파장에서 최대 흡광을 가진다. 따라서, 이를 이용하면 세포의 증식 및 독성에 관한 연구를 비교적 쉽게 수행할 수 있기 때문에 일반적으로 많이 이용되고 있다.The cytotoxicity of the fermented osseous extract was measured in a monolayer cell culture (MTT assay, J. Immunological Methods 65 , 55 (1983)). MTT assay is an experimental method widely used for cell proliferation and toxicity by measuring the number of living cells. The living cells are soluble in mitochondria and have a yellow salt, MTT (3- [4,5-dimethylthiazol- , 5-diphenyltetrazolium bromide) is reduced to a blue formazan derivative which is insoluble in water by succinic acid dehydrogenase. MTT is reduced to formazan by NADH and has maximum absorption at a wavelength of 570 nm. Therefore, it is generally used because it can relatively easily perform research on cell proliferation and toxicity.
사람 섬유아세포주(ATCC, CRL-2076)을 1x105 세포/의 농도로 하여 24웰 평판에 씨딩하였다. 배지는 FBS 10%를 함유한 IMDM (Iscove's Modified Dubelcco's Medium, BRL, USA)를 사용하였다. 씨딩 24시간 후 FBS 0%를 함유한 DMEM를 교체하고, 발효 오신채 추출물(실시예 1)을 농도별로 10 ㎕ 첨가한 후 3일 동안 37℃, 5% CO2 인큐베이터에서 배양하였다. 배양이 끝난 후 상등액을 제거 하고 MTT 용액을 1.0 ㎖/웰 씩 가한 후 4시간 후에 MTT를 제거하고 DMSO로 처리한 후 다음 날 570 nm에서 흡광도를 측정 하였다. 그 결과는 아래 표 4와 같다.Human fibroblast cell line (ATCC, CRL-2076) was seeded in a 24 well plate at a concentration of 1 x 10 cells / ml. The medium was Iscove's Modified Dubelco's Medium (BRL, USA) containing 10% FBS. After 24 hours of seeding, DMEM containing 0% FBS was replaced, and 10 μl of the fermented oyster extract (Example 1) was added for each concentration, followed by culturing in a 5% CO 2 incubator at 37 ° C. for 3 days. After the incubation, the supernatant was removed and MTT solution was added at 1.0 ml / well. After 4 hours, the MTT was removed and treated with DMSO, and the absorbance was measured at 570 nm the following day. The results are shown in Table 4 below.
상기 표 4에서 보는 바와 같이 본 발명에 따른 발효 오신채 추출물의 안전성을 확인하기 위하여 MTT 방법에 의해 피부 섬유아세포로 독성을 시험한 결과 독성이 없는 안전한 원료임을 알 수 있다.
As shown in Table 4, the toxicity of the extracts of fermented omnivores according to the present invention was tested by MTT to confirm the safety of the extracts.
실험예 8. 알러지 유발 여부 측정Experimental Example 8. Measurement of allergen induction
발효 오신채 추출물에 대한 알러지 유발여부를 확인하기 위하여 국부임파절평가(LLNA: local lymph node assay)를 실시하였다. 일주일간의 순화기간을 거친 후 한 개의 농도당 3마리의 마우스를 사용하였다. 3일 동안 하루 한 번씩 시료를 마우스 귀에 25 ㎕씩 도포하여 4일째 임파절을 떼어내어 임파구를 분리 후 5% 이산화탄소 배양기에서 24 - 48시간 배양한 후 증폭정도를 방사선 동위원소 [3H]-메틸 티미딘의 삽입량으로 측정한다. 국부임파절평가 결과는 대조군에 비해 시료군의 임파구 증폭정도로 나타내며 한 가지 농도에서라도 발효 오신채 추출물 시료의 자극지수(S.I.: stimulation index)가 3이상이고 농도별로 증가하는 경향을 보일 경우 알러젠으로 간주하였다. 그 결과는 아래 표 5와 같다.A local lymph node assay (LLNA) was performed to determine whether allergen was induced in the fermented oyster extract. Three mice were used per concentration after a week of purifying period. After 3 days, 25 μl of each sample was applied to the mouse ear. After 4 days, the lymph nodes were removed and the lymphocytes were separated and cultured in a 5% CO 2 incubator for 24 - 48 hours. The degree of amplification was measured by radioisotope [ 3 H] Is measured by the insertion amount of dean. The results of local lymph node evaluation were expressed as the degree of lymphocyte amplification in the sample group as compared with the control group. When the stimulation index (SI) of the fermented osteocyte extract sample was more than 3 at one concentration and the concentration was increased by concentration, it was regarded as allergen. The results are shown in Table 5 below.
* cpm(분당 계수): 림프구와 결합한 3H-티미딘의 방사능* cpm (coefficient per minute): radioactivity of 3 H-thymidine combined with lymphocytes
* S.I.(자극 지수): ≤3이어야 자극이 없는 것으로 인정한다.* S.I. (irritation index): It is recognized that there is no irritation with ≤3.
상기 표 3에서 보는 바와 같이 본 발명에 따른 발효 오신채 추출물의 알러지 유발 여부를 LLNA 법으로 측정한 결과 알러지 유발 가능성이 없음을 확인하였다.
As shown in Table 3, it was confirmed by the LLNA assay that allergen induction of the fermented oyster extract according to the present invention is unlikely to cause allergy.
제제예 1: 영양로션 제조Formulation Example 1: Production of nutrition lotion
하기 표 6의 조성 및 하기 제조 방법으로 실시예 1의 발효 오신채 추출물을 함유한 화장료중 영양로션 제제예 1-A 및 제제예 1-B를 제조하였다.Nutrition Lotion Formulation Example 1-A and Formulation Example 1-B were prepared from the cosmetic compositions containing the fermented ointment extract of Example 1 according to the composition shown in Table 6 below and the following manufacturing method.
하기 표의 10번, 11번, 13번 및 16번을 혼합 교반하면서 80 내지 85℃사이로 가열하고 제조부에 투입한 후 유화기를 작용시키고 2번, 3번, 4번, 5번, 6번, 7번, 8번, 9번 및 12번을 80 내지 85℃사이로 가열하여 투입한 뒤 유화한다. 유화가 끝나면 교반기를 이용하여 교반하면서 50℃까지 열냉각한 뒤 15번을 투입하고 45℃까지 냉각한 뒤 14번을 투입하고 35℃에 1번을 투입하여 25℃까지 냉각한 뒤 숙성시켜 표제의 화장품을 수득하였다.No. 10, No. 11, No. 13 and No. 16 in the following table were heated to between 80 and 85 ° C with mixing and stirring, No. 8, No. 9 and No. 12 are heated to 80 to 85 ° C and emulsified. After the emulsification was completed, the mixture was thermally cooled to 50 ° C. with stirring using an agitator, and then the mixture was cooled to 45 ° C. After the mixture was cooled to 45 ° C., the mixture was stirred at 35 ° C. and cooled to 25 ° C. A cosmetic product was obtained.
스테아레이트Glyceryl stearate / PEG-400
Stearate
제제예 2: 영양크림 제조Formulation Example 2: Preparation of nutritional cream
하기 표 7의 조성 및 하기 제조 방법으로 실시예 1의 발효 오신채 추출물을 함유한 화장료중 영양크림을 제조하였다.Nutritive creams were prepared from the cosmetic compositions containing the fermented starch extract of Example 1 by the composition shown in the following Table 7 and the following manufacturing method.
12번, 13번, 14번, 16번을 혼합 교반하면서 80 내지 85℃사이로 가열하여 제조부에 투입한 후 유화기를 작용시키고 2번, 3번, 4번, 5번, 6번, 7번, 8번, 9번, 10번, 11번을 80 내지 85℃사이로 가열하여 투입한 뒤 15번을 투입하여 유화하였다. 유화가 끝나면 교반기를 이용하여 교반하면서 35℃까지 냉각하고 1번을 투입하여 25℃까지 냉각한 뒤 숙성시켜 표제의 화장품을 수득하였다.12, 13, 14 and 16 were heated to between 80 and 85 ° C with mixing and stirring, and then put into a manufacturing part, 8, 9, 10 and 11 were heated to between 80 and 85 ° C, and the mixture was emulsified by introducing 15 times. After the emulsification was completed, the mixture was cooled to 35 DEG C while stirring with an agitator, and the mixture was cooled to 25 DEG C by the addition of 1 time, followed by aging to obtain the title cosmetic product.
/폴리옥시에틸렌스테아레이트Glyceryl monostearate / glyceryl stearate
/ Polyoxyethylene stearate
제제예 3: 워시폼 제조Formulation Example 3: Wash foam production
하기 표 8의 조성 및 하기 제조 방법으로 실시예 1의 발효 오신채 추출물을 함유한 화장료중 워시폼을 제조하였다.A wash foam was prepared from the cosmetic compositions containing the fermented omega-3 extract of Example 1 according to the composition shown in the following Table 8 and the following production method.
2번, 3번, 4번, 5번, 6번, 7번, 8번, 9번, 10번을 순차적으로 제조부에 투입하고 60 내지 65℃사이로 가열한 후 15분간 교반하였다. 교반이 끝난 후 12번의 일부를 투입하고 30분간 교반한 후 다시 12번의 일부를 천천히 투입하고 30분간 교반한 후에 35℃까지 냉각하고 1번과 11번을 투입하여 25℃까지 냉각한 뒤 숙성시켜 표제의 화장품을 수득하였다.No. 2, No. 3, No. 4, No. 5, No. 6, No. 7, No. 8, No. 9 and No. 10 were sequentially added to the production part and heated to 60 to 65 ° C. and then stirred for 15 minutes. After the stirring, a portion of 12 was added and stirred for 30 minutes. Then, a portion of 12 was added slowly and stirred for 30 minutes. After cooling to 35 ° C, 1 and 11 were added and cooled to 25 ° C, Of a cosmetic product.
제제예 4: 샴푸 제조Formulation Example 4: Shampoo preparation
하기 표 9의 조성 및 하기 제조 방법으로 실시예 1의 발효 오신채 추출물을 함유한 화장료중 샴푸를 제조하였다.A shampoo was prepared from the cosmetic compositions containing the fermented starch extract of Example 1 by the composition shown in the following Table 9 and the following manufacturing method.
2번, 3번, 4번, 5번, 6번, 7번, 8번, 9번, 10번, 11번, 12번, 13번, 14번, 17번, 18번을 순차적으로 제조부에 투입하고 60 내지 65℃사이로 가열한 후 30분간 교반하였다. 별도 용해부에서 15번, 16번, 20번의 일부를 투입하고 30분간 교반한 후 제조부에 투입하고 30분간 교반한다. 다시 20번의 일부를 천천히 투입하고 30분간 교반한 후에 35℃까지 냉각하고 1번과 19번을 투입하여 25℃까지 냉각시킨 뒤 숙성시켜 표제의 화장품을 수득하였다.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 17, The mixture was heated to between 60 and 65 ° C and stirred for 30 minutes. Add part of 15, 16 and 20 in the separate dissolution part, stir for 30 minutes, put into the manufacturing part and stir for 30 minutes. Then, a portion of 20 times was slowly added, stirred for 30 minutes, cooled to 35 DEG C, and 1 and 19 were added thereto, cooled to 25 DEG C, and aged to obtain the title cosmetic product.
비교제제예 1: 영양로션 제조Comparative formulation Example 1: Preparation of nutrition lotion
제제예 1-B에서 실시예 1의 발효 오신채 추출물 대신 물을 넣은 것을 제외하고는 제제예 1-B와 동일한 방법으로 영양로션을 제조하였다.
Nutrition lotion was prepared in the same manner as in Preparation Example 1-B, except that water was added to Formulation Example 1-B in place of the fermentation product of Example 1.
적용 실험예 1. 염증에 의한 피부 손상 억제 효과Application Example 1. Inhibition of skin damage by inflammation
발효 오신채 추출물을 함유한 로션제형(제제예 1-A)과 함유하지 않은 로션제형을 시험에 사용하였다. 재구성된 사람 표피세포에 7일째, 염증생성 물질로 LPS(lipopolysaccharide) (E. Coli, 20.000 endotoxin U/ml) 30 ㎕를 적용하였고 점점 양을 늘려 적용하였다. 적용한 후 24시간 동안 37℃, 5% CO2에서 배양하였다. 후에 조직은 10% 포르말린으로 고정시키고, 파라핀에 담가 두었다가 수직으로 잘라 H(Hematoxylin) & E(Eosin)로 염색하였다. 그 결과는 도 7과 같다.
The lotion formulation (formulation example 1-A) containing the fermented primate extract and the lotion formulation not containing it were used for the test. On the 7th day, 30 μl of lipopolysaccharide (LPS) (E. Coli, 20.000 endotoxin U / ml) was applied to the reconstructed human epidermal cells and the dose was gradually increased. The cells were cultured at 37 ° C and 5% CO 2 for 24 hours. The tissue was fixed with 10% formalin, immersed in paraffin, cut vertically and stained with H (Hematoxylin) & E (Eosin). The results are shown in FIG.
적용실험예 2. UV 조사 후 랑게르한스 세포 발현에 의한 피부 면역 증강Application Example 2. Skin immunity enhancement by Langerhans cell expression after UV irradiation
랑게르한스 세포(Langerhans cell)는 수지상세포라고도 하며 표피의 모든 부분에 위치하고 있으나 가시층(spinous layer)의 가장 위쪽에 제일 많이 있다. 이들 세포들은 급성 염증반응이나, 피부에서 림프구 쪽으로 이주하는데 관여하고 있다. 랑게르한스 세포는 단클론 항체들(monoclonal antibodies)을 사용할 수 있는 마커(marker)가 되며 이것의 수를 셀 수 있다. 이와 같은 원리를 이용하여, 랑게르한스 세포는 면역반응시 발현되므로, UV B 조사 후에 면역반응 활성을 알 수 있어 피부 면역 증강에의 발효 오신채 추출물의 능력을 결정할 수 있다.Langerhans cells, also called dendritic cells, are located in all parts of the epidermis, but most are at the top of the spinous layer. These cells are involved in acute inflammatory reactions or migration from the skin to the lymphocytes. Langerhans cells can be counted as a marker that can use monoclonal antibodies. Using this principle, since the Langerhans cells are expressed in the immune response, the immune reaction activity can be known after the irradiation with UVB, and thus the ability of the fermented extract to enhance the skin immunity can be determined.
아토피 피부염과 다른 습진의 현병력 및 과거력이 없는 현재 피부질환이 없다고 판단된 건강한 남, 여를 대상으로 하였다. 팔 안쪽에 발효 오신채 추출물을 함유한 로션제형(제제예 1-A)과 함유하지 않은 로션제형을 3일 동안 매일 두 번씩 발라주었다. 그 다음 1.5J/㎠ 의 UV B를 조사하였다. 그 결과는 도 8과 같다.This study was aimed at healthy men and women who have no history of atopic dermatitis and other eczema and no history of skin disease. The lotion formulation (Formulation Example 1-A) containing the fermented primate extract on the inside of the arm and the lotion formulation not containing the extract were applied twice daily for three days. Then, UV B of 1.5 J / cm < 2 > was irradiated. The results are shown in Fig.
자외선 조사 부위는 랑게르한스 세포 감소시켜 항원 제시 능력을 저해한다. 도 8의 사진에서도 볼 수 있듯이 함유하지 않은 로션제형은 세포가 많이 감소되었다. 반면 본 발명의 발효 오신채 추출물을 함유한 제형에서는 랑게르한스 세포의 수가 많이 발현되는 것으로 미루어 볼 때 본 발명의 발효 오신채 추출물은 UV로 조사 후 유발되는 피부 손상으로부터 면역세포의 활성이 증가하여 피부를 보호하는 면역기능이 강화되었음을 알 수 있다.Ultraviolet irradiation sites inhibit the antigen presentation ability by decreasing Langerhans cells. As can be seen in the photograph of FIG. 8, the lotion formulation not containing the cells showed much decreased cells. On the other hand, in the formulation containing the fermentation extract of the present invention, the number of Langerhans cells is increased. Therefore, the fermented extract of the present invention exhibits an increased activity of immune cells due to skin damage caused by UV irradiation, Immune function is enhanced.
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