KR20110098122A - Composition containing ginseng berry fermentation extract using bacteria - Google Patents
Composition containing ginseng berry fermentation extract using bacteria Download PDFInfo
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- KR20110098122A KR20110098122A KR1020100017580A KR20100017580A KR20110098122A KR 20110098122 A KR20110098122 A KR 20110098122A KR 1020100017580 A KR1020100017580 A KR 1020100017580A KR 20100017580 A KR20100017580 A KR 20100017580A KR 20110098122 A KR20110098122 A KR 20110098122A
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- composition
- bacillus
- ginseng fruit
- bacteria
- skin
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- A61K36/18—Magnoliophyta (angiosperms)
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- A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
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Abstract
본 발명은 인삼 열매 발효 추출물을 함유하는 조성물에 관한 것으로서, 보다 상세하게는 인삼의 지상부 중에서 독특한 성분과 조성을 갖는 인삼 열매를 균을 이용하여 발효시킨 추출물을 함유함으로써 미백, 항노화, 보습 등의 효능을 제공할 수 있는 조성물에 관한 것이다.The present invention relates to a composition containing the ginseng fruit fermented extract, and more particularly, by containing the extract fermented ginseng fruit having a unique ingredient and composition in the ground portion of ginseng using a bacterium, such as whitening, anti-aging, moisturizing It relates to a composition capable of providing.
Description
본 발명은 인삼 열매 발효 추출물을 함유하는 조성물에 관한 것으로서, 보다 상세하게는 인삼의 지상부 중에서 독특한 성분과 조성을 갖는 인삼 열매를 균을 이용하여 발효시킨 추출물을 함유함으로써 미백, 항노화, 보습 등의 효능을 제공할 수 있는 조성물에 관한 것이다.
The present invention relates to a composition containing the ginseng fruit fermented extract, and more particularly, by containing the extract fermented ginseng fruit having a unique ingredient and composition in the ground portion of ginseng using a bacterium, such as whitening, anti-aging, moisturizing It relates to a composition capable of providing.
인삼(Panax ginseng C.A. Meyer)은 오가피과 인삼 속에 속하는 식물로서 한국, 중국 및 일본 등지에서 2,000여년 전부터 사용되어 온 생약이며, 경험적으로 질병을 예방하고 수명을 연장시킬 목적으로 사용되어 왔다. 지금까지 알려진 인삼의 효능 및 효과는 중추신경계에 대한 작용, 항발암 작용, 항암활성, 면역기능 조절작용, 항당뇨 작용, 간기능 항진효능, 심혈관 장해개선, 항동맥경화 작용, 혈압조절 작용, 갱년기 장애 개선, 골다공증에 미치는 효과, 항스트레스 및 항피로 작용, 항산화 활성 및 노화억제 효능 등이 있다(최신고려인삼 ‘성분 및 효능편’, 한국인삼연초연구원, 56-112, 1996).Ginseng (Panax ginseng C.A. Meyer) is a plant belonging to the genus Ogapi ginseng and has been used in Korea, China, and Japan for more than 2,000 years, and has been used to prevent disease and extend life. Known efficacy and effects of ginseng to the central nervous system, anticarcinogenic effect, anticancer activity, immune function regulation, antidiabetic effect, liver function anti-inflammatory effect, cardiovascular disorders improvement, anti-arteriosclerosis, blood pressure control action, menopausal Disability improvement, effects on osteoporosis, antistress and anti-fatigue, antioxidant activity and anti-aging effects (Korea Ginseng 'Ingredients and Efficacy', Korea Ginseng and Tobacco Research Institute, 56-112, 1996).
인삼의 대표적 생리활성 성분인 진세노사이드(Ginsenoside)는 인삼의 지상 및 지하부에 고르게 분포되어 있으며, 특히 인삼 근(뿌리), 인삼엽 및 인삼 열매 등 부위에 따라 진세노사이드 함량뿐만 아니라 조성도 다른 것으로 알려져 있다(Attele AS et al, Biochem Pharmacol, 58; 1685-1693, 1999). Ginsenoside, a representative physiologically active ingredient of ginseng, is distributed evenly in the ground and underground parts of ginseng. Especially, the composition and composition of ginsenosides are different depending on the parts such as ginseng root (root), ginseng leaf and ginseng fruit. (Attele AS et al, Biochem Pharmacol, 58; 1685-1693, 1999).
한편, 피부는 신체 표면을 완전히 덮고 있는 가장 큰 기관으로, 외부로부터 신체를 보호하는 최외곽 방어막을 형성하고 있다. 나이가 증가함에 따라 다른 신체 기관과 마찬가지로 노화가 진행되는데, 피부의 경우 미적 판단의 기준이 되는 경우가 많아 최근 피부 노화의 예방 및 억제에 관심이 모아지고 있다. On the other hand, the skin is the largest organ that completely covers the body surface, forming the outermost shield to protect the body from the outside. As age increases, aging proceeds like other body organs, and in the case of skin, it is often used as a criterion for aesthetic judgment.
진피의 구조를 살펴보면, 교원섬유(콜라겐, collagen)가 입체구조를 이루고 그 사이에 신축성이 강한 탄력섬유(elastin)가 스프링처럼 작용하여 피부의 탄력을 유지해 준다. 나이가 들어감에 따라 진피 중의 교원섬유가 파괴되고 탄력섬유도 체내에서 생성되는 말론 디알데히드(MDA)에 의해 분자 사슬 사이의 가교 결합이 형성되어 신축성이 떨어지게 된다. 노화가 진행되면 피부 구조를 유지하는 섬유 외에 세포외 공간을 채우고 있는 물질들의 체내 생성량도 줄어들게 된다. 건강한 피부의 각질층은 15~20%의 수분을 함유하고 있으며 수분이 10% 이하로 떨어지면 피부가 건조해지고 윤기와 탄력이 없어져 주름이 증가하게 된다. 이 모든 결과로 인해 노화된 피부는 탄력을 잃고 주름이 발생하게 된다. Looking at the structure of the dermis, collagen fibers (collagen, collagen) forms a three-dimensional structure between the elastic elastic fibers (elastin) between the spring to maintain the elasticity of the skin. As the body ages, collagen fibers in the dermis are destroyed, and elastic fibers also lose their elasticity by forming crosslinks between molecular chains by malon dialdehyde (MDA). As aging progresses, the body's production of substances that fill the extracellular space in addition to the fibers that maintain the skin's structure also decrease. The stratum corneum of healthy skin contains 15-20% of moisture, and when the moisture drops below 10%, the skin becomes dry, no gloss and elasticity, and wrinkles increase. All of this results in aging skin losing elasticity and wrinkles.
또한, 피부에 대하여 관심이 집중되는 다른 부분은 피부색과 관련된 것으로서, 피부색 결정 요인 중에서 가장 큰 부분을 차지하는 것이 피부 중 멜라닌의 분포상태 및 양이다. 멜라닌(melanin)은 멜라노사이트(melanocyte)에서 생성이 되는데 이 멜라노사이트에는 티로시나제 등의 효소가 존재하며, 이들이 함께 작용하여, 생체내에 항상 존재하는 티로신(tyrosine)이라는 아미노산을 기질로 중합화 산화반응을 함으로 흑갈색의 색소인 멜라닌을 형성 하게 된다. 이렇게 형성된 멜라닌은 멜라노사이트의 수지상 돌기를 통하여 케라티노사이트(keratinocyte)라는 표피 세포로 이동한다. 여기서 멜라닌은 핵주변에 모자와 같은 구조를 형성하여 자외선으로부터 유전자를 보호하고 자유 라디칼(free radical)을 제거하여 세포 내 단백질을 보호하는 등 중요한 역할을 하게 된다. 생체 내에는 멜라닌을 분해하는 효소가 없고 다만 케라티노사이트가 표피에서 떨어져나갈 때 같이 피부에서 떨어져나가는 것으로 제거된다. 하지만 멜라닌이 필요 이상으로 많이 생기게 되면 기미나 주근깨, 점 등과 같은 과색소침착증을 유발하여 미용상으로 좋지 않은 결과를 가져오게 된다. 멜라닌 생성에 영향을 미치는 인자는 여러 가지가 알려져 있는데, 자외선에 의하여 일어나는 멜라닌 생산의 항진 그리고 이에 따른 색소 침착이 화장품 분야에서 아주 중요하다. 색소 침착 예방목적으로 미백화장품에 배합되는 약제의 기본적인 메커니즘은 티로시나아제(tyrosinase) 작용 억제, 티로시나아제 생성 억제, 멜라닌 생성 미디에이터의 억제, 기존 멜라닌의 환원 및 광산화 억제, 멜라닌 배설의 촉진, 및 자외선 커트 등이다. In addition, another area of concern for skin is related to skin color, and the distribution and amount of melanin in the skin is the largest part of skin color determinants. Melanin is produced in melanocytes, which contain enzymes such as tyrosinase, which work together to polymerize and oxidize tyrosine, an amino acid that is always present in vivo. As a result, melanin, a dark brown pigment, is formed. The melanin thus formed migrates to epidermal cells called keratinocytes through dendritic processes of melanocytes. Here, melanin plays an important role such as forming a hat-like structure around the nucleus to protect genes from ultraviolet rays and to remove free radicals to protect intracellular proteins. There are no enzymes that break down melanin in vivo, but they are removed by falling off the skin as keratinocytes break off the epidermis. However, if more melanin is produced than necessary, it causes hyperpigmentation such as blemishes, freckles, and spots, resulting in cosmetically bad results. There are many known factors that affect melanin production, and the increase of melanin production caused by ultraviolet light and the subsequent pigmentation are very important in the cosmetic field. The basic mechanisms of drugs formulated in whitening cosmetics for the purpose of preventing pigmentation include: inhibiting tyrosinase action, inhibiting tyrosinase production, inhibiting melanogenesis mediators, inhibiting reduction and photooxidation of existing melanin, promoting melanin excretion, and UV cut and the like.
자외선 노출 환경속에서도 하얀 피부를 갖고자 하는 여성들의 욕구에 따라서 피부 색소 이상 증상과 과색소 침착 등의 예방과 개선에 대한 수요가 더욱 늘어 나고 있으며 이에 과도한 멜라닌 생성을 막는 미백제품 개발이 필요하게 되었고 그 동안 많은 노력들이 이루어졌다. 그 구체적인 예를 들면, 코지산(kojic acid), 알부틴(arbutin) 등과 같은 티로시나제 활성을 저해하는 억제제들, 하이드로퀴논(hydroquinone), 비타민 A, 비타민 C 및 이들의 유도체 등이 있다. 그러나, 이들은 피부에 대한 안전성, 제형 내에서의 안정성 및 미백효과의 불충분으로 인해 그 사용이 제한되고 있다. In response to women's desire to have white skin even under UV exposure, there is an increasing demand for the prevention and improvement of abnormal skin pigmentation and hyperpigmentation, and it is necessary to develop a whitening product that prevents excessive melanin production. Many efforts have been made. Specific examples thereof include inhibitors that inhibit tyrosinase activity such as kojic acid, arbutin, hydroquinone, vitamin A, vitamin C, and derivatives thereof. However, they are limited in their use due to insufficient skin safety, stability in formulations and whitening effects.
최근 화장품 분야에서는 천연물의 사용이 두드러지고 있으며, 보습력과 노화 개선 기능, 항산화 효과가 우수하고, 특히 피부 노화 개선 효과 면에서 우수하다고 보고된 천연물 추출물을 이용한 화장료 조성물에 대해서도 많은 연구가 이루어지고 있다. 그러나, 통상의 방법으로 추출한 천연물 추출물을 사용하는 조성물들은 원하는 미백, 주름 개선, 항산화 등의 효과가 미미할 뿐 아니라, 이러한 추출물의 활성을 지속적으로 유지, 제어하기가 어려워 실용성이 높지 않았다.
Recently, the use of natural products in the cosmetic field has been prominent, and many researches have been made on cosmetic compositions using natural extracts that have been reported to be excellent in moisturizing and aging improving functions and antioxidant effects, and particularly in terms of improving skin aging effects. However, the compositions using natural extracts extracted by conventional methods have not only the desired effects such as whitening, anti-wrinkle improvement, antioxidant, etc., but also have difficulty in maintaining and controlling the activity of such extracts.
이에, 본 발명자들은 피부 상태 개선 효과를 제공할 수 있는 물질을 찾고자 연구한 결과, 균을 이용한 인삼 열매 발효 추출물을 함유하는 조성물이 항노화, 보습, 미백 등의 피부 상태 개선 효과가 있음을 확인하고 본 발명을 완성하였다.Thus, the present inventors conducted a study to find a substance that can provide a skin condition improvement effect, confirming that the composition containing the ginseng fruit fermentation extract using the bacterium has a skin condition improvement effect such as anti-aging, moisturizing, whitening and The present invention has been completed.
따라서, 본 발명의 목적은 항노화, 보습, 미백 등의 피부 상태 개선 효과가 우수하면서 피부에 안전한 천연물을 함유한 조성물을 제공하는 것이다.
Accordingly, it is an object of the present invention to provide a composition containing natural products which are excellent in anti-aging, moisturizing, whitening and the like and are safe for skin.
상기한 목적을 달성하기 위하여, 본 발명은 균을 이용한 인삼 열매 발효 추출물을 함유하는 조성물을 제공한다.
In order to achieve the above object, the present invention provides a composition containing the ginseng fruit fermented extract using the bacteria.
본 발명의 조성물은 균을 이용한 인삼 열매 발효 추출물을 함유함으로써 피부 노화를 억제하고 피부 수분 보유력을 증가시키며, 또한 피부 미백에 우수한 효능이 있어 피부에 안전하면서도 전반적인 피부 상태를 개선시키는 효과를 제공할 수 있다.
The composition of the present invention contains the ginseng fruit fermentation extract using the bacteria to inhibit skin aging and increase skin moisture retention, and also has an excellent effect on skin whitening can provide an effect that is safe for the skin and improves the overall skin condition have.
본 발명의 조성물은 균을 이용한 인삼 열매 발효 추출물을 함유하며, 화장품용 조성물 또는 식품용 조성물로서 제공될 수 있다.The composition of the present invention contains the ginseng fruit fermented extract using bacteria, and may be provided as a cosmetic composition or a food composition.
또한, 본 발명의 조성물은 항노화, 보습, 미백 등의 효과를 제공한다.In addition, the composition of the present invention provides the effect of anti-aging, moisturizing, whitening and the like.
이하 본 발명에 대하여 보다 구체적으로 살펴본다.Hereinafter, the present invention will be described in more detail.
본 발명에서 사용되는 인삼 열매는 분쇄한 분말 상태나, 물, 또는 유기용매를 사용하여 추출할 수 있다. 구체적으로 물이나 탄소수 1 내지 4의 무수 또는 함수 저급 알코올(예를 들어, 메탄올, 에탄올, 프로판올 및 부탄올 등), 상기 저급 알코올과 물의 혼합 용매, 아세톤, 에틸아세테이트, 클로로포름, 1,3-부틸렌글리콜, 헥산 및 디에틸에테르로 구성된 군으로부터 선택되는 어느 하나의 용매, 바람직하게는 물 또는 탄소수 1 내지 4의 무수 또는 함수 저급 알코올로 환류냉각기가 달린 추출기에 50 내지 100℃, 1 내지 5시간 동안 가열하여 감압 농축하는 방법이다. 상기 추출 용매의 양은 이용되는 추출 용매의 종류에 따라 다양할 수 있으나, 전형적으로 발효물의 건조 중량에 대하여 1~20배이며, 바람직하게는 5~20배이다. 또한 상기 추출은 1회 이상 실시될 수 있으며, 또한 1회 이상 추출한 추출액을 혼합하고, 이를 감압 농축할 수도 있다.Ginseng fruit used in the present invention can be extracted using a pulverized powder state, water, or an organic solvent. Specifically, water or anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (for example, methanol, ethanol, propanol and butanol), a mixed solvent of the lower alcohol and water, acetone, ethyl acetate, chloroform, 1,3-butylene Any one solvent selected from the group consisting of glycol, hexane and diethyl ether, preferably water or an anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms in an extractor with reflux cooler for 50 to 100 ° C. for 1 to 5 hours It is a method of concentrating under reduced pressure by heating. The amount of the extraction solvent may vary depending on the type of extraction solvent used, but is typically 1 to 20 times with respect to the dry weight of the fermentation product, preferably 5 to 20 times. In addition, the extraction may be carried out one or more times, it is also possible to mix the extract extracted one or more times, it may be concentrated under reduced pressure.
본 발명의 조성물은 인삼 열매에 균을 첨가, 배양하여 발효시킴으로써 수득한 추출물을 사용한다. 본 발명에서 이용가능한 균의 종류로는 특별히 한정되지는 않지만, 효모균, 버섯균, 유산균, 누룩균, 바실러스속균(고초균) 등을 1종 이상 사용할 수 있다.The composition of the present invention uses the extract obtained by adding the bacteria to the ginseng fruit, cultured and fermented. Although it does not specifically limit as a kind of bacterium which can be used by this invention, One or more types of yeasts, mushrooms, lactic acid bacteria, yeast bacteria, Bacillus genus bacterium, etc. can be used.
구체적으로 효모균으로는 사카로마이세스(Saccharomyces sp .), 스키조사카로마이세스(Schizosaccharomyces sp .), 토룰로프시스(Torulopsis sp .), 로도토룰라(Rhodotorula sp .), 또는 칸디다(Candida sp .) 등의 균을 사용할 수 있다. Specifically, Saccharomyces as a yeast sp . , Ski investigation caromyces ( Schizosaccharomyces sp . ), Torulopsis sp . Rhodotorula sp . ), Or Candida sp . Bacteria such as) can be used.
또한, 버섯균으로는 식용으로 가능한 버섯균이라면 어느 것이나 가능하나, 바람직하게는 차가버섯(Inonnotus - obliquus), 동충하초균류(Cordyceps sinensis , Paecilomyces japonica), 송이버섯(Tricholomamatsutake), 상황버섯(Phellinus linteus) 또는 영지버섯(Ganoderma lucidum) 등의 균을 사용할 수 있다.Further, if the fungus is a fungus can be an edible one which would, preferably, Chaga (Inonnotus - obliquus), the fungus Cordyceps sinensis (Cordyceps sinensis , Paecilomyces japonica ), Matsutake mushroom ( Tricholomamatsutake ), Situation mushroom ( Phellinus linteus ) or Ganoderma lucidum ( Ganoderma) bacteria such as lucidum ) can be used.
또한, 유산균으로는 락토바실러스(Lactobacillus sp .), 스트렙토코쿠스(Streptococcus sp .), 류코노스톡(Leuconostoc sp .), 또는 비피도박테리아(Bifidobacteria sp .) 등의 균을 사용할 수 있다.In addition, Lactobacillus ( Lactobacillus) sp . ), Streptococcus sp . ), Leuconostoc sp . ), Or Bifidobacteria sp . Bacteria such as) can be used.
또한, 누룩균으로는 라이조푸스(Rhyzopus), 우사미(Usami) 및 오리재(Oryzae), 아스퍼질러스 오리재(Aspergillus oryzae), 아스퍼질러스 소재(Aspergillus sojae) 등의 균을 사용할 수 있다.In addition, yeast bacteria include Rhyzopus , Usami , and Oryzae , and Aspergillus. oryzae ), Aspergillus bacteria such as sojae ) can be used.
또한, 바실러스속균(고초균)으로는 바실러스 리체니포르미스(Bacillus licheniformis) F1017, 바실러스 리체니포르미스(Bacillus licheniformis) F1232, 바실러스 리체니포르미스(Bacillus licheniformis) F1267, 바실러스 리체니포르미스(Bacillus licheniformis) F1268, 바실러스 리체니포르미스(Bacillus licheniformis) F1200, 바실러스 푸미루스(Bacillus pumilus) F1337, 바실러스 소노렌시스(Bacillus sonorensis) F1005, 바실러스 서브틸리스(Bacillus subtilis) F1236 및 바실러스 서브틸리스(Bacillus subtilis) F1279 등의 균을 사용할 수 있다.Moreover, Bacillus spp. (Bacillus subtilis) with the Bacillus piece you miss formate (Bacillus licheniformis) F1017, Bacillus piece you miss formate (Bacillus licheniformis) F1232, Bacillus piece you miss PORTO (Bacillus licheniformis) F1267, Bacillus piece you miss PORTO (Bacillus licheniformis) F1268, Bacillus piece you miss PORTO (Bacillus licheniformis) F1200, Bacillus pumi Ruth (Bacillus pumilus) F1337, Bacillus Sono alkylene sheath (Bacillus sonorensis ) F1005, Bacillus subtilis subtilis) F1236 and Bacillus subtilis (Bacillus subtilis ) bacteria such as F1279 can be used.
본 발명의 조성물에 사용되는 인삼 열매의 발효 추출물의 제조방법은 특별히 한정되지는 않고, 당업계에 공지된 일반적인 방법을 이용할 수 있고, 구체적으로는 인삼 열매에 상기 균을 첨가, 배양하고 발효시켜 수득할 수 있다.The production method of the fermented extract of ginseng fruit used in the composition of the present invention is not particularly limited, and can be used a general method known in the art, specifically, by adding the above bacteria to the ginseng fruit, incubated and fermented can do.
보다 구체적으로, 발효시 효모를 사용할 경우에는 인삼 열매 분말 또는 추출물이 함유된 기본 배지에 일정한 농도(약 105~107 cell/mL)로 효모를 접종하여, pH 5.5~6.5, 온도 25~50℃, 통기량 0.05~0.5VVM의 조건을 유지하면서 배양한 후, 이 발효액을 원심분리하여 고분자 침전물과 효모균제가 제거된 최종 인삼 열매 발효 추출물을 수득할 수 있다. 배양 시간은 24 내지 120 시간이 바람직하다.More specifically, when using the fermentation when yeast was inoculated yeast at a constant concentration (about 10 5 ~ 10 7 cell / mL ) in a basic medium containing a ginseng fruit powder or extract, pH 5.5 ~ 6.5, a temperature between 25 and 50 After culturing while maintaining the conditions of aeration, 0.05 to 0.5VVM, the fermentation broth can be centrifuged to obtain a final ginseng fruit fermentation extract from which polymer precipitates and yeasts are removed. Incubation time is preferably 24 to 120 hours.
또한, 발효시 버섯균을 사용할 경우에는 인삼 열매 분말에 버섯균을 접종하여, 온도 18~25℃, 습도 60~80%의 조건을 유지하면서 배양한 후, 물이나 탄소수 1~4의 무수 또는 함수 저급 알코올(예를 들어, 메탄올, 에탄올, 프로판올 및 부탄올 등), 상기 저급 알코올과 물의 혼합 용매, 아세톤, 에틸아세테이트, 클로로포름, 1,30부틸렌글리콜, 헥산 또는 디에틸에테르 중 1종 이상을 이용하고, 50~100℃, 1~5시간 동안 가열하여 감압 농축하여 최종 인삼 열매 발효 추출물을 수득할 수 있다. 배양 시간은 1일 내지 100일이 바람직하다. In addition, in the case of using mushrooms during fermentation, inoculate mushrooms to ginseng fruit powder, incubating them under conditions of temperature of 18 to 25 ° C and humidity of 60 to 80%, followed by anhydrous or water-containing Using at least one of lower alcohols (e.g., methanol, ethanol, propanol and butanol), a mixed solvent of the lower alcohols and water, acetone, ethyl acetate, chloroform, 1,30 butylene glycol, hexane or diethyl ether And, the final ginseng fruit fermentation extract can be obtained by heating under 50 ~ 100 ℃, 1-5 hours and concentrated under reduced pressure. Incubation time is preferably 1 day to 100 days.
또한, 발효시 유산균을 사용할 경우에는 인삼 열매 분말 또는 추출물이 함유된 기본 배지에 일정한 농도(약 105~107 cell/mL)로 유산균을 접종하여, pH 6.5~7.5, 온도 25~50℃, 통기량 1VVM의 조건을 유지하면서 배양한 후, 이 발효액을 원심분리하여 고분자 침전물과 유산균제가 제거된 최종 인삼 열매 발효 추출물을 수득할 수 있다. 배양 시간은 24 내지 120 시간이 바람직하다.In addition, the use of fermentation when lactic acid bacteria is inoculated with lactic acid bacteria at a constant concentration (about 10 5 ~ 10 7 cell / mL ) in basal medium containing a ginseng fruit powder or extract, pH 6.5 ~ 7.5, temperature 25 ~ 50 ℃, After culturing while maintaining conditions of aeration of 1 VVM, the fermentation broth can be centrifuged to obtain a final ginseng fruit fermentation extract from which polymer precipitates and lactic acid bacteria are removed. Incubation time is preferably 24 to 120 hours.
또한, 발효시 누룩균을 사용할 경우에는 인삼 열매 분말 또는 추출물에 누룩균을 접종하여, 온도 15~45℃의 조건을 유지하면서 배양한 후, 이 발효액을 원심분리하여 고분자 침전물과 누룩균제가 제거된 최종 인삼 열매 발효 추출물을 수득할 수 있다. 배양 시간은 18 내지 120 시간이 바람직하다.In the case of using yeast bacteria during fermentation, inoculate yeast bacteria to ginseng fruit powder or extract, and incubate while maintaining the temperature of 15-45 ° C., and then centrifuge the fermentation broth to remove the polymer precipitate and yeast fungus. Fermented extracts can be obtained. Incubation time is preferably 18 to 120 hours.
또한, 발효시 바실러스속균을 사용할 경우에는 인삼 열매 분말 또는 추출물이 함유된 기본 배지에 일정한 농도(약 105~107 cell/mL)로 바실러스속균을 접종하여, 온도 10~60℃의 조건을 유지하면서 배양한 후, 이 발효액을 원심분리하여 pH 5.5~8.5, 고분자 침전물과 바실러스균제가 제거된 최종 인삼 열매 발효 추출물을 수득할 수 있다. 배양 시간은 24 내지 96 시간이 바람직하다.In addition, in case of using Bacillus genus during fermentation, Bacillus genus is inoculated at a constant concentration (about 10 5 to 10 7 cell / mL) in the basic medium containing ginseng fruit powder or extract, and maintained at a temperature of 10 to 60 ° C. After culturing, the fermentation broth may be centrifuged to obtain a final ginseng fruit fermentation extract having a pH of 5.5 to 8.5 and a polymer precipitate and bacillus bacteria removed. Incubation time is preferably 24 to 96 hours.
상기의 방법으로 제조한 인삼 열매 발효 추출물은 조성물 총 중량에 대하여 0.001~90중량%의 양으로 함유하는 것이 바람직하다. 이는 상기 유효성분의 함량이 0.001중량% 미만일 경우에는 효능, 효과가 미약하고, 90중량%를 초과할 경우에는 제형 안정화에 문제가 있으며, 함량의 증가에 따른 효능의 증가가 미약하기 때문이다.The ginseng fruit fermented extract prepared by the above method is preferably contained in an amount of 0.001 to 90% by weight based on the total weight of the composition. This is because when the content of the active ingredient is less than 0.001% by weight, the efficacy and effect is weak, and when it exceeds 90% by weight, there is a problem in stabilization of the formulation, and the increase in efficacy due to the increase of the content is weak.
본 발명에 의한 조성물은 화장품용 조성물 또는 식품용 조성물로서 제조될 수 있으며, 본 발명에 의한 조성물은 경피 조성물 또는 경구 조성물로서 제조될 수 있다.The composition according to the invention can be prepared as a cosmetic or food composition, and the composition according to the invention can be prepared as a transdermal or oral composition.
구체적으로 본 발명의 화장료 조성물은 그 제형에 있어서 특별히 한정되는 바가 없으며, 예를 들면 유연화장수, 영양화장수, 유액, 마사지크림, 영양크림, 팩, 패치, 젤, 스틱, 고 타입 또는 피부 점착 타입, 분무제 화장료의 제형을 갖는 화장료 조성물일 수 있다. 예를 들면 유연화장수, 수렴화장수, 영양화장수, 영양크림, 마사지크림, 에센스, 아이크림, 아이에센스, 클렌징크림, 클렌징폼, 클렌징워터, 팩, 파우더, 보디로션, 보디크림, 보디오일, 보디에센스 등의 화장료 제형을 가질 수 있으며 또한 로션, 연고, 겔, 크림, 패취 또는 분무제와 같은 경피 투과형 제형을 갖는 화장료 조성물 일 수 있다Specifically, the cosmetic composition of the present invention is not particularly limited in the formulation, for example, soft cosmetics, nourishing cosmetics, milky lotion, massage cream, nutrition cream, pack, patch, gel, stick, high type or skin adhesive type, It may be a cosmetic composition having a formulation of a spray cosmetic. For example, supple cosmetics, astringent cosmetics, nourishing cosmetics, nourishing cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body oil, body essence It may have a cosmetic formulation such as and may also be a cosmetic composition having a transdermal penetration formulation such as lotion, ointment, gel, cream, patch or spray.
또한 본 발명의 경구 투여용 제형으로는 예를 들면, 정제, 환제, 경질 및 연질 캅셀제, 액제, 현탁제, 유화제, 시럽제, 과립제 등이 있는데, 이들 제형은 유효성분 이외에 희석제(예: 락토즈, 덱스트로즈, 수크로즈, 만니톨, 솔비톨, 셀룰로즈 및 글리신), 활택제(예: 실리카, 탈크, 스테아르산 및 그의 마그네슘 또는 칼슘염 및 폴리에틸렌 글리콜)를 함유하고 있다. 정제는 또한 마그네슘 알루미늄 실리케이트, 전분 페이스트, 젤라틴, 트라가칸스, 메틸셀룰로즈, 나트륨 카복시메틸셀룰로즈 및 폴리비닐피롤리딘과 같은 결합제를 함유할 수 있으며, 경우에 따라 전분, 한천, 알긴산 또는 그의 나트륨 염과 같은 붕해제, 흡수제, 착색제, 향미제, 및 감미제 등의 약제학적 첨가제를 함유할 수 있다. 정제는 통상적인 혼합, 과립화 또는 코팅 방법에 의해 제조될 수 있다. 또한, 비경구 투여용 제형으로는, 피부 외용제, 주사제 등을 포함하고, 피부 외용제로는 연고제가 바람직하며, 주사제 제형으로 등장성 수용액 또는 현탁액일 수 있다.In addition, the oral dosage form of the present invention includes, for example, tablets, pills, hard and soft capsules, liquids, suspensions, emulsifiers, syrups, granules, etc. These formulations may contain diluents (e.g., lactose, Dextrose, sucrose, mannitol, sorbitol, cellulose and glycine), lubricants such as silica, talc, stearic acid and its magnesium or calcium salts and polyethylene glycols. Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, optionally starch, agar, alginic acid or its sodium salt Pharmaceutical additives such as disintegrants, absorbents, colorants, flavors, and sweeteners. Tablets may be prepared by conventional mixing, granulating or coating methods. In addition, formulations for parenteral administration include external skin preparations, injections and the like, ointment is preferred as an external preparation for skin, may be an isotonic aqueous solution or suspension in the injection formulation.
또한 본 발명을 식품 조성물로 제형화할 경우 육류, 소시지, 빵, 초코렛, 캔디류, 스넥류, 과자류, 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 식품을 모두 포함하고 이에 한정되지는 않는다.In addition, when the present invention is formulated as a food composition, dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, noodles, gum, ice cream, various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes, etc. This includes, but is not limited to, all foods in the ordinary sense.
또한, 각각의 제형에 있어서 상기한 필수성분 이외에 다른 성분들은 기타 제형의 종류 또는 사용목적 등에 따라 당업자가 어려움 없이 적합하게 선정하여 배합할 수 있다.In addition, in each formulation, other components in addition to the above-mentioned essential components may be appropriately selected and blended by those skilled in the art without difficulty depending on the type or purpose of use of other formulations.
본 발명에 의한 조성물은 항노화 효능이 우수하여 주름 개선 및 예방의 효과를 제공하며, 피부 보습력을 증진시킨다. 또한 피부 미백 효과도 제공하여 본 발명에 의한 조성물은 피부 상태를 전반적으로 개선시킬 수 있다.
The composition according to the present invention is excellent in anti-aging efficacy, provides an effect of wrinkle improvement and prevention, and enhances skin moisturizing power. It also provides a skin lightening effect so that the composition according to the present invention can improve the overall skin condition.
이하, 하기 실시예에 의하여 본 발명을 더욱 상세하게 설명하고자 한다. 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐 본 발명의 범위가 이들만으로 한정되는 것은 아니다.
Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.
[비교예 1] 인삼 열매 추출물 제조Comparative Example 1 Preparation of Ginseng Fruit Extract
1. 인삼 열매 전처리1. Ginseng Fruit Pretreatment
생(生)인삼 열매를 수확하여 종자를 분리하여 제거한 후 인삼 열매의 과육과 과피를 일광건조 또는 열풍건조를 통하여 인삼 열매 건조원료를 제조하였다.Raw ginseng fruit was harvested, seed was separated and removed, and then the ginseng fruit dried raw material was prepared by sun drying or hot air drying.
2. 인삼 열매 추출물 제조2. Ginseng Fruit Extract Manufacturer
인삼 열매 건조물 1kg에 물 3L를 가하여 환류 추출한 다음 여과한 후 40~45?에서 감압농축하여 인삼 열매 추출물 300g을 얻었다.
3L of water was added to 1 kg of dried ginseng fruit, and the mixture was extracted under reflux, filtered and concentrated under reduced pressure at 40 to 45 ° to obtain 300g of ginseng fruit extract.
[실시예 1] 효모를 이용한 인삼 열매 발효 추출물Example 1 Ginseng Fruit Fermented Extract Using Yeast
상기 비교예 1의 인삼 열매 건조원료를 분쇄한 분말을 1.5L의 정제수에 5중량%가 되도록 첨가하여 탄소원, 질소원, 인삼염, 무기염류, 미량영양성분 등을 기본 조성으로 하고, 수산화나트륨을 이용하여 적절한 pH 5.5~6.5으로 맞추었다. 상기 인삼 열매 분말이 함유된 배양 배지에 사카로마이세스(Saccharomyces sp .)를 106 cell/mL로 접종하고, pH 5.5~6.5, 온도 25~35℃, 통기량 0.5VVM의 조건을 유지하면서 72시간 동안 배양하였다.Add the ground powder of the ginseng fruit dry material of Comparative Example 1 to 5% by weight in 1.5L purified water to make a carbon composition, nitrogen source, ginseng salt, inorganic salts, micronutrient components, etc., using sodium hydroxide Was adjusted to an appropriate pH 5.5-6.5. Saccharomyces in the culture medium containing the ginseng fruit powder sp . ) Was inoculated at 10 6 cell / mL, and cultured for 72 hours while maintaining the conditions of pH 5.5 ~ 6.5, temperature 25 ~ 35 ℃, aeration 0.5VVM.
상기 발효액을 1.000 X g에서 30분간 원심분리하여 고분자 침전물과 효모균체가 제거된 최종 인삼 열매 발효 추출물을 얻었다.
The fermentation broth was centrifuged at 1.000 X g for 30 minutes to obtain a final ginseng fruit fermentation extract from which polymer precipitates and yeast cells were removed.
[실시예 2] 버섯균을 이용한 인삼 열매 발효 추출물Example 2 Ginseng Fruit Fermented Extract Using Mushroom Bacteria
상기 비교예 1의 인삼 열매 건조원료를 분쇄한 분말에 동충하초균류(Paecilomyces japonica)를 5중량%로 첨가하고, 18~25℃, 습도 60~70%에서 60일간 발효시킨 후 건조시킨 다음, 이를 분쇄하여 얻어진 분쇄물 100g씩을 80% 메탄올 3L에 각각 넣고 냉각콘덴서가 달린 환류추출기에서 3시간 끓여 추출하였다. 그 다음 100 메쉬 여과포로 여과한 후 잔사를 같은 방법으로 1회 더 추출하였다. 각각의 추출액을 합하여 상온에서 와트만 2번 여과지로 여과하여 불용성 물질을 제거한 후, 냉각 콘덴서가 달린 증류장치에서 60℃로 감압농축한 후 동결건조하여 최종 인삼 열매 발효 추출물을 얻었다.
Cordyceps sinensis fungi ( Paecilomyces) in the powder of ground ginseng fruit dry material of Comparative Example 1 japonica ) was added at 5% by weight, fermented at 18-25 ° C. and 60-70% humidity for 60 days and dried. Then, 100 g of the pulverized product was put in 3 L of 80% methanol and refluxed with a cooling capacitor. Boil for 3 hours in the extractor and extracted. Then filtered through 100 mesh filter cloth and the residue was extracted once more in the same way. Each extract was combined, filtered at room temperature with Whatman No. 2 filter paper to remove insoluble materials, concentrated under reduced pressure at 60 ° C. in a distillation apparatus equipped with a cooling condenser, and lyophilized to obtain a final ginseng fruit fermentation extract.
[실시예 3] 유산균을 이용한 인삼 열매 발효 추출물Example 3 Ginseng Fruit Fermented Extract Using Lactic Acid Bacteria
상기 비교예 1의 인삼 열매 건조원료를 분쇄한 분말을 1.5L의 정제수에 5중량%가 되도록 첨가하여 탄소원, 질소원, 인삼염, 무기염류, 미량영양성분 등을 기본 조성으로 하고, 수산화나트륨을 이용하여 적절한 pH 5.5~6.5으로 맞추었다. 상기 인삼 열매 분말이 함유된 배양 배지에 락토바실러스(Lactobacillus sp .)를 106 cell/mL로 접종하고, pH 6.5~7.5, 온도 35~38℃, 통기량 1VVM의 조건을 유지하면서 72시간 동안 배양하였다.Add the ground powder of the ginseng fruit dry material of Comparative Example 1 to 5% by weight in 1.5L purified water to make a carbon composition, nitrogen source, ginseng salt, inorganic salts, micronutrient components, etc., using sodium hydroxide Was adjusted to an appropriate pH 5.5-6.5. Lactobacillus in the culture medium containing the ginseng fruit powder sp . ) Was inoculated at 10 6 cells / mL, and cultured for 72 hours while maintaining the conditions of pH 6.5-7.5, temperature 35-38 ° C, and aeration rate 1VVM.
상기 발효액을 1.000 X g에서 30분간 원심분리하여 고분자 침전물과 효모균체가 제거된 최종 인삼 열매 발효 추출물을 얻었다.
The fermentation broth was centrifuged at 1.000 X g for 30 minutes to obtain a final ginseng fruit fermentation extract from which polymer precipitates and yeast cells were removed.
[실시예 4] 누룩균을 이용한 인삼 열매 발효 추출물Example 4 Ginseng Fruit Fermented Extract Using Nuruk
상기 비교예 1의 인삼 열매 건조원료를 분쇄한 분말에 아스퍼질러스 오리재(Aspergillus oryzae)를 5중량%로 첨가하고, 25℃에서 48시간 동안 발효시킨 후 건조시킨 다음, 이를 분쇄하여 얻어진 분쇄물 100g씩을 80% 메탄올 3L에 각각 넣고 냉각콘덴서가 달린 환류추출기에서 3시간 끓여 추출하였다. 그 다음 100 메쉬 여과포로 여과한 후 잔사를 같은 방법으로 1회 더 추출하였다. 각각의 추출액을 합하여 상온에서 와트만 2번 여과지로 여과하여 불용성 물질을 제거한 후, 냉각 콘덴서가 달린 증류장치에서 60℃로 감압농축한 후 동결건조하여 최종 인삼 열매 발효 추출물을 얻었다.
Aspergillus in a pulverized ginseng fruits dry ingredients of the comparative example 1 powder material's duck (Aspergillus oryzae ) was added at 5% by weight, fermented at 25 ° C. for 48 hours, and then dried. Then, 100 g of the pulverized product obtained by grinding was put into 3 L of 80% methanol and boiled for 3 hours in a reflux extractor equipped with a cooling capacitor. . Then filtered through 100 mesh filter cloth and the residue was extracted once more in the same way. Each extract was combined, filtered at room temperature with Whatman No. 2 filter paper to remove insoluble materials, concentrated under reduced pressure at 60 ° C. in a distillation apparatus equipped with a cooling condenser, and lyophilized to obtain a final ginseng fruit fermentation extract.
[실시예 5] 바실러스균을 이용한 인삼 열매 발효 추출물Example 5 Ginseng Fruit Fermented Extract Using Bacillus
상기 비교예 1의 인삼 열매 건조원료를 분쇄한 분말을 1.5L의 정제수에 5중량%가 되도록 첨가하여 탄소원, 질소원, 인삼염, 무기염류, 미량영양성분 등을 기본 조성으로 하고, 수산화나트륨을 이용하여 적절한 pH 5.5~6.5으로 맞추었다. 상기 인삼 열매 분말이 함유된 배양 배지에 바실러스 서브틸리스(Bacillus subtilis) F1279를 접종하고, 45℃에서 72시간 동안 배양하였다.Add the ground powder of the ginseng fruit dry material of Comparative Example 1 to 5% by weight in 1.5L purified water to make a carbon composition, nitrogen source, ginseng salt, inorganic salts, micronutrient components, etc., using sodium hydroxide Was adjusted to an appropriate pH 5.5-6.5. Bacillus subtilis ( Bacillus) in the culture medium containing the ginseng fruit powder subtilis ) F1279 was inoculated and incubated at 45 ° C. for 72 hours.
상기 발효액을 1.000 X g에서 30분간 원심분리하여 고분자 침전물과 효모균체가 제거된 최종 인삼 열매 발효 추출물을 얻었다.
The fermentation broth was centrifuged at 1.000 X g for 30 minutes to obtain a final ginseng fruit fermentation extract from which polymer precipitates and yeast cells were removed.
[시험예 1] 엘라스타아제 발현 억제 효능 측정Test Example 1 Elastase Expression Inhibition Effect Measurement
상기 비교예 1과 실시예 1~5로부터 얻은 인삼 열매 및 인삼 열매 발효 추출물의 엘라스타아제 생성 저해능을 토코페롤 및 EGCG와 비교하여 측정하였다.The inhibition of elastase production of ginseng fruit and ginseng fruit fermented extracts obtained from Comparative Examples 1 and 1 to 5 was measured by comparing with tocopherol and EGCG.
2.5%의 우태아 혈청이 함유된 DMEM(Dulbecco's Modified Eagle's Media) 배지가 들어있는 96공 평판배양기(96-well microtiter plate)에 인간의 섬유아세포를 5,000 세포/공(well)이 되도록 넣고, 90% 정도 자랄 때까지 배양하였다. 그 후 무혈청 배지로 24시간 배양하고, 무혈청 배지에 녹여진 상기 비교예 1과 실시예 1~5의 인삼 열매 및 인삼 열매 발효 추출물, 토코페롤 및 EGCG를 10-4 몰농도로 24시간 동안 처리한 다음, 세포배양액을 채취하였다. Place human fibroblasts at 5,000 cells / well in a 96-well microtiter plate containing Dulbecco's Modified Eagle's Media (DMEM) medium containing 2.5% fetal calf serum, 90% Incubate until growth. After 24 hours of incubation in serum-free medium, the ginseng fruit and ginseng fruit fermented extracts, tocopherol and EGCG of Comparative Example 1 and Examples 1 to 5 dissolved in serum-free medium were treated for 10 hours at 10 -4 molarity. After that, the cell culture was collected.
상업적으로 이용가능한 엘라스타아제 측정기구(미국 아머샴파마샤 사)를 이용하여 채취한 세포배양액의 엘라스타아제 생성 정도를 측정하였다. 먼저 1차 엘라스타아제 항체가 균일하게 도포된 96-공 평판(96-well plate)에 채취된 세포 배양액을 넣고 3시간 동안 항원-항체 반응을 항온조에서 실시하였다. 3시간 후 발색단이 결합된 2차 엘라스틴 항체를 96-공 평판(96-well plate)에 넣고 다시 15분간 반응시켰다. 15분 후 발색유발물질을 넣어 실온에서 15분간 발색을 유발시키고, 다시 1M 황산을 넣어 발색반응을 중지시키면 반응액의 색깔은 노란색을 띠게 되며 반응 진행의 정도에 따라 노란색의 정도가 다르게 나타났다.The degree of elastase production of cell culture fluids collected using a commercially available elastase measuring instrument (Amersham Pharmacia, USA) was measured. First, the cell culture solution collected in a 96-well plate uniformly coated with the primary elastase antibody was subjected to antigen-antibody reaction in a thermostat for 3 hours. After 3 hours, the chromophore-bound secondary elastin antibody was placed in a 96-well plate and reacted again for 15 minutes. After 15 minutes, the color-producing substance was added, causing color development at room temperature for 15 minutes. Then, 1M sulfuric acid was added to stop the color reaction.
노란색을 띠는 96-공 평판(96-well plate)의 흡광도를 흡광계를 이용하여 405nm에서 측정하고, 하기 수학식 1에 의해 엘라스타아제의 발현정도를 계산하였으며, 그 결과는 하기 표 1에 나타내었다. 이때 조성물을 처리하지 않은 군에서 채취된 세포배양액의 반응 흡광도를 대조군으로 하였다. The absorbance of the yellow 96-well plate (96-well plate) was measured at 405 nm using an absorbance meter, and the expression level of elastase was calculated by Equation 1 below, and the results are shown in Table 1 below. Indicated. At this time, the reaction absorbance of the cell culture liquid collected from the group not treated with the composition was used as a control.
상기 표 1에 나타낸 바와 같이, 시험관내(in vitro)에서 실시예 1~5의 엘라스타아제 발현 정도가 인삼 열매 추출물 비교예 1과 비교하여 낮은 것을 통해 본 발명에 의한 인삼 열매 발효 추출물의 엘라스타아제 발현 저해 효과가 더 좋음을 확인할 수 있다. 또한, 엘라스타아제 발현 저해제로 알려져 있는 토코페롤 및 EGCG와 비교하여서도 본 발명에 의한 인삼 열매 발효 추출물의 엘라스타아제 발현 저해 효과가 토코페롤보다는 우수하고, EGCG와는 비슷한 수준임을 알 수 있다.
As shown in Table 1 above, the elastase expression level of Examples 1 to 5 in vitro in the elastase of the ginseng fruit fermented extract according to the present invention through the lower than the ginseng fruit extract Comparative Example 1 It can be seen that the effect of inhibiting the expression of the enzyme is better. In addition, compared to tocopherol and EGCG known as an elastase expression inhibitor, the effect of inhibiting the elastase expression of the ginseng fruit fermentation extract according to the present invention is superior to tocopherol and is similar to EGCG.
[시험예 2] 콜라게나아제(MMP-1) 저해능Test Example 2 Collagenase (MMP-1) Inhibitory Activity
상기 비교예 1과 실시예 1~5로부터 얻은 인삼 열매 및 인삼 열매 발효 추출물의 콜라게나아제 생성 저해능을 레티노익산과 비교하여 측정하였다.Inhibition of collagenase production of ginseng fruit and ginseng fruit fermentation extracts obtained from Comparative Example 1 and Examples 1 to 5 was measured in comparison with retinoic acid.
2.5%의 우태아 혈청이 함유된 DMEM(Dulbecco's Modified Eagle's Media) 배지가 들어있는 96공 평판배양기(96-well microtiter plate)에 인간의 섬유아세포를 5,000 세포/공(well)이 되도록 넣고, 70~80% 정도 자랄 때까지 CO2 5%, 37℃ 배양기(incubator)에서 배양하였다. 그 후 비교예 1과 실시예 1~5의 인삼 열매 및 인삼 열매 발효 추출물을 10-4 몰농도로 24시간 동안 처리한 다음, 세포배양액을 채취하였다. Place human fibroblasts at 5,000 cells / well in 96-well microtiter plates containing DMEM (Dulbecco's Modified Eagle's Media) containing 2.5% fetal calf serum. Incubated in a CO 2 5%, 37 ℃ incubator until the growth of about 80%. Thereafter, the ginseng fruit and the ginseng fruit fermented extracts of Comparative Example 1 and Examples 1 to 5 were treated with 10 −4 molarity for 24 hours, and cell culture liquids were collected.
상업적으로 이용가능한 콜라게나아제 측정기구(미국 아머샴파마샤 사, Catalog #: RPN 2610)를 이용하여 채취한 세포배양액의 콜라게나아제 생성 정도를 측정하였다. 먼저 1차 콜라게나아제 항체가 균일하게 도포된 96-공 평판(96-well plate)에 채취한 세포 배양액을 넣고 3시간 동안 항원-항체 반응을 항온조에서 실시하였다. 3시간 후 발색단이 결합된 2차 콜라겐 항체를 96-공 평판(96-well plate)에 넣고 다시 15분간 반응시켰다. 15분 후 발색유발물질(3,3',5,5'-tetramethylbenzidine, sigma)을 넣어 실온에서 15분간 발색을 유발시키고, 다시 1M 황산을 넣어 발색반응을 중지시키면 반응액의 색깔은 노란색을 띠게 되며 반응 진행의 정도에 따라 노란색의 정도가 다르게 나타났다. The degree of collagenase production of cell cultures was measured using a commercially available collagenase measuring instrument (Amersham Pharmacia, USA, Catalog #: RPN 2610). First, the cell culture solution collected in a 96-well plate uniformly coated with primary collagenase antibody was placed in an incubator for 3 hours. After 3 hours, the chromophore-bound secondary collagen antibody was placed in a 96-well plate and reacted for another 15 minutes. After 15 minutes, color-causing substances (3,3 ', 5,5'-tetramethylbenzidine, sigma) were added to induce color development at room temperature for 15 minutes, and 1M sulfuric acid was added to stop the color reaction. The degree of yellow was different according to the degree of reaction progress.
노란색을 띠는 96-공 평판(96-well plate)의 흡광도를 흡광계를 이용하여 405nm에서 측정하고, 하기 수학식 2에 의해 콜라게나아제의 합성 정도를 계산하였으며, 그 결과는 하기 표 2에 나타내었다. 이때 조성물을 처리하지 않은 군에서 채취한 세포 배양액의 반응 흡광도를 대조군으로 하였다. The absorbance of the yellow 96-well plate was measured at 405 nm using an absorbance meter, and the degree of synthesis of collagenase was calculated by Equation 2 below, and the results are shown in Table 2 below. Indicated. At this time, the reaction absorbance of the cell culture liquid collected from the group not treated with the composition was used as a control.
상기 표 2에 나타낸 바와 같이, 시험관내(in vitro)에서 실시예 1~5의 콜라게나아제 발현 정도가 인삼 열매 추출물 비교예 1과 비교하여 낮은 것을 통해 본 발명에 의한 인삼 열매 발효 추출물의 콜라게나아제 발현 저해 효과가 더 좋음을 확인할 수 있다. 또한, 콜라게나아제 발현 저해제로 알려져 있는 레티노익산과 비교하여서도 본 발명에 의한 인삼 열매 발효 추출물의 콜라게나아제 발현 저해 효과가 레티노익산과 비슷한 수준임을 알 수 있다.As shown in Table 2, the collagenase of the ginseng fruit fermented extract according to the present invention through the collagenase expression level of Examples 1 to 5 in vitro in low compared with the ginseng fruit extract Comparative Example 1 It can be seen that the effect of inhibiting the expression of the enzyme is better. In addition, it can be seen that the collagenase expression inhibitory effect of the ginseng fruit fermentation extract according to the present invention is comparable to that of retinoic acid as compared to retinoic acid known as a collagenase expression inhibitor.
이와 같은 결과를 통하여, 본 발명에 의한 인삼 열매 발효 추출물은 기질 메탈로 프로테아제(MMP-1)를 저해시키는 효과를 가짐을 확인할 수 있다.
Through these results, it can be seen that the ginseng fruit fermentation extract according to the present invention has the effect of inhibiting the substrate metalloprotease (MMP-1).
[시험예 3] 피부 탄력 향상 효능 확인Test Example 3 Confirmation of Skin Elasticity Improvement Efficacy
피부 탄력 향상 효능 확인을 위하여 상기 비교예 1 및 실시예 1~5에서 얻은 인삼 열매 및 인삼 열매 발효 추출물을 활용하여 하기 표 3의 조성으로 비교제형예 1 및 제형예 1~5의 화장수를 제조하고 인삼 열매 또는 인삼 열매 발효 추출물을 함유하지 않는 화장수를 대조군으로 제조하였다.In order to confirm the skin elasticity improvement effect to prepare the skin preparation of Comparative Formulation Example 1 and Formulation Examples 1 to 5 by using the ginseng fruit and ginseng fruit fermented extract obtained in Comparative Example 1 and Examples 1 to 5 A lotion containing no ginseng fruit or ginseng fruit fermented extract was prepared as a control.
상기 대조군, 비교제형예 1 및 제형예 1~5 화장수의 피부 탄력 향상 효과를 비교하기 위하여, 30~40대 여성 140명을 대상으로 20명을 1개군으로 하여 7개군으로 나누어 측정하였다. 실험 방법은 다음과 같다.In order to compare the skin elasticity improvement effect of the control group, Comparative Formulation Example 1 and Formulation Examples 1 to 5, the skin was measured by dividing the group into seven groups of 20 people in a group of 140 women in their 30s to 40s. The experimental method is as follows.
각 조에 대조군, 비교제형예 1 또는 제형예 1~5의 화장수를 각각 나누어 주고 매일 1회 12주간 눈가에 일정량을 도포하게 하였다. 이때, 피검자의 눈가의 왼쪽에는 상기 대조군의 화장수를, 오른쪽에는 비교제형예 1 또는 제형예 1~5의 화장수를 도포하게 하였다. 12주 뒤 피부탄력을 측정할 수 있는 큐토미터(cutometer; SEM474, Courage+Khazaka electronic GmbH. Germany)를 이용하여 각 그룹의 피부 탄력을 측정하고 그 평균을 구하였으며, 그 결과는 하기 표 4에 나타내었다.In each group, the lotion of the control group, Comparative Formulation Example 1 or Formulation Examples 1 to 5 was divided and applied to an eye area for 12 weeks once daily. At this time, the lotion of the control group was applied to the left side of the eyes of the subject, and the lotion of Comparative Formulation Example 1 or Formulation Examples 1 to 5 was applied to the right side. After 12 weeks, the skin elasticity of each group was measured using a cutometer (SEM474, Courage + Khazaka electronic GmbH. Germany), which can measure skin elasticity, and the average of the results was shown in Table 4 below. It was.
상기 표 4의 결과를 보면, 본 발명에 의한 인삼 열매 발효 추출물을 함유한 제형예 1~5의 화장수를 사용한 경우에 인삼 열매 추출물을 함유하고 있지 않은 대조군과 비교하여 피부 탄력 향상도가 3배 정도 높게 나타남을 확인할 수 있으며, 인삼 열매의 단순 추출물을 함유한 비교제형예 1의 화장수를 사용한 경우와 비교하여서도 피부 탄력 향상도가 월등히 우수함을 알 수 있다.In the results of Table 4, when the lotion of Formulation Examples 1 to 5 containing the ginseng fruit fermentation extract according to the present invention is used, the skin elasticity improvement degree is three times higher than that of the control group which does not contain the ginseng fruit extract. It can be seen that it appears high, and compared with the case of using the lotion of Comparative Formulation Example 1 containing a simple extract of ginseng fruit, it can be seen that the skin elasticity improvement is excellent.
따라서, 본 발명의 인삼 열매 발효 추출물을 함유하는 화장료 조성물은 피부 탄력 향상에 매우 효과적임을 확인할 수 있다.
Therefore, it can be confirmed that the cosmetic composition containing the ginseng fruit fermentation extract of the present invention is very effective for improving skin elasticity.
[시험예 4] 쥐의 색소세포를 이용한 멜라닌 생성 억제효과 측정Test Example 4 Measurement of Melanin Inhibitory Effect Using Pigment Cells
C57BL/6 마우스 유래의 쥐의 색소세포(Mel-Ab cell)(Dooley, T.P. et al, Skin pharmacol, 7, pp 188-200)를 DMEM에 10% 우태반 혈청, 100nM 12-O-테트라데카노일포르볼(tetradecanoylphorbol)-13-아세테이트, 1nM 콜레라 독소(cholera toxin)를 첨가한 배지에서 37℃, 5% CO2의 조건에서 배양하였다. 배양된 Mel-Ab 세포를 0.25% 트립신-EDTA로 떼어내고, 24-웰 플레이트에 105 세포/웰(cells/well)의 농도로 세포를 배양한 다음, 이틀째부터 3일 연속으로 각 시험물질을 가하여 배양하였다. 시험물질로는 하이드로퀴논과 상기 실시예 1~5의 인삼 열매 발효 추출물을 10ppm의 농도로 하여 사용하였다. 이때, 상기 하이드로퀴논은 양성대조군으로 사용하였다. 그 다음 배양액을 제거하고, PBS로 세척한 후, 1N 수산화나트륨으로 세포를 녹여 400nm에서 흡광도를 측정한 다음, 하기 수학식 3에 따라 멜라닌 생성 억제율을 계산하여 그 결과를 표 5에 나타내었다(Dooley의 방법).Mel-Ab cells derived from C57BL / 6 mice (Dooley, TP et al, Skin pharmacol, 7, pp 188-200) in DMEM with 10% fetal placental serum, 100nM 12-O-tetradecanoyl Incubated at 37 ° C. and 5% CO 2 in a medium to which phorbol (tetradecanoylphorbol) -13-acetate and 1 nM cholera toxin were added. The cultured Mel-Ab cells were detached with 0.25% trypsin-EDTA, the cells were incubated at a concentration of 10 5 cells / well in 24-well plates, and then each test material was added for 3 consecutive days. And incubated. As a test substance, hydroquinone and the ginseng fruit fermentation extracts of Examples 1 to 5 were used at a concentration of 10 ppm. At this time, the hydroquinone was used as a positive control group. Then, the culture medium was removed, washed with PBS, dissolved in 1N sodium hydroxide, the absorbance was measured at 400 nm, and the melanin production inhibition rate was calculated according to Equation 3 below and the results are shown in Table 5 (Dooley). Method).
상기 표 5에 나타낸 바와 같이, 본 발명의 인삼 열매 발효 추출물이 공지된 미백 물질인 하이드로 퀴논과 유사한 정도의 멜라닌 생성 억제율을 보이는 것을 확인할 수 있다.
As shown in Table 5, it can be confirmed that the ginseng fruit fermentation extract of the present invention shows a melanin production inhibition rate similar to hydroquinone which is a known whitening substance.
[시험예 5] PPARs 활성화 확인[Test Example 5] PPARs activation confirmation
인체 각질형성 세포주(human keratinocyte)인 HaCaT을 10% 우태아 혈청을 포함하는 DMEM 배지에 계대 배양하고 페놀 레드의 에스트로겐에 의한 효과를 제거하기 위해 페놀 레드-프리(phenol red-free) 배지를 사용하였다. 플라스미드는 일반적인 배양 조건에서도 발현되는 유니버설(universal) 프로모터 뒤에 PPARα, PPARβ/δ, PPARγ 유전자를 지닌 플라스미드와 리간드 결합형의 PPARs가 결합하여 활성화되는 PPARs 반응 요소(PPARs Response Element, “PPRE”)를 프로모터로 가지고 뒤에 리포터로써 역할을 하는 반디불(firefly) 루시퍼라제(luciferase) 유전자를 지닌 것, 그리고 참조(reference)로 사용될 유니버설 프로모터에 β-갈락토시다제(β-galactosidase) 유전자가 결합된 플라스미드가 사용되었다(Proc. Natl. Acad. Sci. USA, 91 (1994) 7355-7359).HaCaT, a human keratinocyte, was passaged in DMEM medium containing 10% fetal bovine serum and phenol red-free medium was used to eliminate the effects of phenol red by estrogen. . The plasmid is a promoter of the PPARs Response Element (“PPRE”), which is activated by the combination of PPARα, PPARβ / δ, and PPARγ genes with ligand-binding PPARs followed by a universal promoter that is expressed under normal culture conditions. Plasmids with a firefly luciferase gene that acts as a reporter later, and a β-galactosidase gene bound to a universal promoter to be used as a reference. (Proc. Natl. Acad. Sci. USA, 91 (1994) 7355-7359).
HaCaT 세포를 5x104의 농도로 24 웰(well)형 플레이트에 분주하고 24 시간 배양한 후, 상기 플라스미드 유전자를 일시적 트랜스펙션(transient transfection)하였다. 24 시간 배양 후 인산완충용약(PBS)으로 세척한 후 7,3',4'-트리하이드록시이소플라본과 양성대조군으로 기존에 알려진 PPARs 리간드(PPARα 리간드인 Wy-14,643, PPARγ 리간드인 트로글리타존(troglitazone, “TGZ”))를 명시된 농도로 처리하였다. 음성대조군으로는 시료들을 녹일 때 사용한 에탄올을 처리한 군을 이용하였다. 24 시간 배양 후 PBS로 세척하고 세포를 수확하여 루시퍼라제 활성을 측정하여 표 6 및 표 7에 나타내었다.HaCaT cells were aliquoted into 24 well plate at a concentration of 5 × 10 4 and incubated for 24 hours, followed by transient transfection of the plasmid gene. After 24 hours of incubation, the cells were washed with phosphate buffer (PBS), and then PPARs ligand (PPARα ligand, Wy-14,643, PPARγ ligand, troglitazone (PPARα ligand) was previously known as a positive control. , “TGZ”)) was treated at the indicated concentrations. As a negative control group was used a group treated with ethanol used to dissolve the samples. After incubation for 24 hours, washed with PBS and harvested cells to measure luciferase activity is shown in Table 6 and Table 7.
표 6은 연구에 사용되는 HaCaT 세포주에 PPRE 프로모터 리포터 플라즈미드와 PPARα 발현 플라즈미드를 트랜스펙션 시킨 후, 약물을 처리하고 루시퍼라제 활성을 측정한 결과이다. 표 7은 PPARγ 발현 플라즈미드와 PPRE 프로모터 리포터 플라즈미드를 동시에 트랜스펙션시킨 후 약물을 처리하고 루시퍼라제 활성을 측정한 결과이다. Table 6 shows the results of transfecting the PPRE promoter reporter plasmid and PPARα expressing plasmid into the HaCaT cell line used in the study, followed by treatment with the drug and measuring luciferase activity. Table 7 shows the results of transfecting PPARγ expressing plasmid and PPRE promoter reporter plasmid at the same time, treating the drug and measuring luciferase activity.
표 6 및 표 7을 보면, 본 발명에 의한 실시예 1~5를 사용한 경우 유의한 정도의 PPARα 및 PPARγ의 활성을 유도하여 높은 루시퍼라제 활성을 나타냄을 확인할 수 있다.
Looking at Tables 6 and 7, it can be seen that when using Examples 1 to 5 according to the present invention, a high degree of luciferase activity is induced by inducing a significant degree of PPARα and PPARγ activity.
[시험예 6] 각질형성세포 분화 촉진 효과 [Test Example 6] keratinocyte differentiation promoting effect
상기 샘플들의 각질형성세포의 분화 촉진 효과를 알아보기 위해, 하기와 같이 각질형성세포의 분화시 생성되는 CE(Cornified Envelop)양을 흡광도를 이용하여 측정하였다. To determine the differentiation promoting effect of keratinocytes of the samples, the amount of CE (Cornified Envelop) generated during differentiation of keratinocytes was measured using absorbance.
먼저, 신생아의 표피로부터 분리해 일차 배양한 사람의 각질형성세포를 배양용 플라스크에 넣어 바닥에 부착시킨 뒤 하기 표 8의 시험물질을 배양액에 1ppm 농도로 처리한 후 세포가 바닥 면적의 70~80 % 정도 자랄 때까지 5일간 배양하였다. 이때, 저칼슘(0.03mM) 처리군과 고칼슘(1.2mM) 처리군을 각각 음성 대조군, 양성 대조군으로 하였다. 그 다음 상기 배양한 세포를 수확하여 PBS(Phophate buffered saline)로 세척한 뒤 2% SDS(sodium dodecyl sulfate)와 20mM 농도의 DTT(Dithiothreitol)를 함유한 10mM 농도의 트리스-염산 완충액(Tris-HCl, pH 7.4) 1ml를 가하여 소니케이션(sonication), 보일링(boiling), 원심분리한 후 침전물을 다시 PBS 1ml에 현탁시켜 340 nm 에서의 흡광도를 측정하였다. 이와 별도로 상기 소니케이션 후의 용액을 일부 취하여 단백질 함량을 측정하고, 세포 분화정도 평가시 기준으로 삼았다. 그 결과를 하기 표 8에 나타내었다. First, the keratinocytes of the primary cultured humans isolated from the epidermis of the newborn were put in a culture flask and adhered to the bottom, and then the test substance of Table 8 was treated at 1 ppm in the culture medium. It was incubated for 5 days until it grew about%. At this time, the low calcium (0.03mM) treated group and the high calcium (1.2mM) treated group were used as negative and positive controls, respectively. Then, the cultured cells were harvested and washed with PBS (Phophate buffered saline), followed by 10 mM Tris-HCl buffer (Tris-HCl, containing 2% SDS (sodium dodecyl sulfate) and 20 mM Dithiothreitol (DTT). pH 7.4) After adding 1 ml of sonication, boiling, and centrifugation, the precipitate was again suspended in 1 ml of PBS to measure absorbance at 340 nm. Separately, a portion of the solution after the sonication was taken to measure the protein content and used as a reference when evaluating the degree of cell differentiation. The results are shown in Table 8 below.
상기 표 8에 나타낸 바와 같이, 본 발명에 의한 실시예 1~5가 유의한 정도의 각질형성세포의 분화를 촉진하는 것을 알 수 있다.
As shown in Table 8, it can be seen that Examples 1 to 5 according to the present invention promote the differentiation of keratinocytes of a significant degree.
[시험예 7] 무모생쥐 피부에서의 피부 장벽기능 회복 및 피부 보습력 증가 효과 측정Test Example 7 Measurement of Skin Barrier Function Recovery and Skin Moisturizing Effect in Hairless Mouse Skin
상기 샘플들이 피부 손상으로 인해 손상된 피부 장벽기능의 회복에 미치는 효과 및 피부 보습력 증가에 미치는 효과를 측정하기 위하여, 하기와 같은 실험을 수행하였다. In order to measure the effects of the samples on the recovery of damaged skin barrier function due to skin damage and the increase in skin moisturizing power, the following experiments were performed.
먼저, 출생 후 8∼10 주 경과한 무모생쥐(Charles River, Japan)의 등에 아세톤을 1일 2회씩 5일 동안 주기적으로 도포하여 실험동물 등 피부의 장벽 기능 손실을 유도한 다음 증발계(Evaporimeter)로 TEWL(Transepidermal water loss, 경피수분손실)을 측정하여 40g/m2/hr 이상의 경피수분손실을 나타내는 피부를 가진 실험동물만을 대상으로 하였다. 선택한 실험동물에 대하여 시험물질로 프로필렌 글리콜과 에탄올을 7:3의 비율로 혼합한 비히클(Vehicle)(비히클 처리군)과 실시예 1~6을 각각 1% 함유한 샘플을 피부면적 5cm2 당 200μl의 용량으로 1일 2회씩 3일 동안 연속 도포하면서 일정 시간마다 TEWL을 측정하였으며, 그 결과를 표 9에 나타내었다. First, acetone is applied periodically to the back of hairless mice (Charles River, Japan) 8 to 10 weeks after birth for 5 days twice a day to induce the loss of barrier function of skin such as experimental animals, and then with an evaporimeter. Transepidermal water loss (TEWL) was measured and only experimental animals with skin showing percutaneous moisture loss above 40 g / m 2 / hr were included. For the selected experimental animals, 200 μl per 5 cm 2 of skin area containing a vehicle (vehicle treated group) containing 1% of propylene glycol and ethanol at a ratio of 7: 3 and 1% to Examples 1 to 6, respectively. TEWL was measured at regular intervals while continuously applying twice a day for 3 days at a dose of, and the results are shown in Table 9.
또한, 코니오미터(Corneometer, 독일 Courage Khazaka 사)를 사용하여 피부 수분량을 동시에 측정하였으며, 그 결과를 표 10에 나타내었다. 이때 무처리군의 피부 수분량도 함께 측정하였다.In addition, the skin moisture content was simultaneously measured using a coronometer (Corneometer, German Courage Khazaka), the results are shown in Table 10. At this time, the skin moisture content of the untreated group was also measured.
표 9에 나타낸 바와 같이 상기 실시예 1~5의 처리군이 빠르게 장벽 손상이 회복되었음을 알 수 있다. 또한 표 10에 나타낸 바와 같이 피부 수분량에 있어서도 실시예 1~5의 처리군이 높게 나타남을 확인할 수 있다. As shown in Table 9, it can be seen that the treatment groups of Examples 1 to 5 quickly recovered barrier damage. In addition, as shown in Table 10, it can be seen that the treatment groups of Examples 1 to 5 were also high in the skin moisture content.
따라서, 이를 통해 상기 인삼 열매 발효 추출물을 처리한 경우 장벽 손상 회복과 더불어 피부 수분량도 증가함을 알 수 있으며, 또한 인삼 열매 발효 추출물을 사용할 경우 인삼 열매 생품의 추출물을 사용할 경우보다 그 효능이 더 우수함을 확인할 수 있다.
Therefore, when the ginseng fruit fermented extract is treated, it can be seen that the moisture content of the skin increases as well as the barrier damage recovery. Also, when the ginseng fruit fermented extract is used, its efficacy is superior to that of the extract of the ginseng fruit product. can confirm.
Claims (15)
The composition of claim 1, wherein the composition is for moisturizing.
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|---|---|---|---|---|
| KR101314762B1 (en) * | 2011-09-27 | 2013-10-04 | 주식회사 한국지네틱팜 | Manufacturing method of Fermented material extracted from ginseng seed and fermented food thereof |
| KR101345735B1 (en) * | 2013-10-11 | 2013-12-30 | 주식회사 아미코스메틱 | Cosmetic composition with the extract of ginseng berry fermented with pleurotus ferulae |
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| KR101485896B1 (en) * | 2014-10-27 | 2015-01-26 | 주식회사 더트루메틱 | Cosmetic composition for improving anti-oxidation, anti-inflammatory and atopic skin and method of preparing the same |
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| WO2018080139A1 (en) * | 2016-10-25 | 2018-05-03 | 코스맥스 주식회사 | Cosmetic composition containing ginseng fruit fermented material obtained by fermentation using aureobasidium pullulans and lactic acid bacteria |
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2010
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| WO2014038822A1 (en) * | 2012-09-06 | 2014-03-13 | Kim Jong-Seog | Paecilomyces variotii var. brunneolus gpp1101b, and preparation using same |
| KR101434741B1 (en) * | 2012-09-06 | 2014-08-29 | 김종석 | Paecilomyces variotii var. brunneolus gpp1101b strains and agents using the same |
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| KR101485896B1 (en) * | 2014-10-27 | 2015-01-26 | 주식회사 더트루메틱 | Cosmetic composition for improving anti-oxidation, anti-inflammatory and atopic skin and method of preparing the same |
| KR20160065245A (en) * | 2014-11-28 | 2016-06-09 | (주)지에프씨 | Composition of culture medium for cultivation of Lactobacillus, ginseng berry-fermented extracts comprising thereof and cosmetic composition for improving skin conditions |
| WO2018080139A1 (en) * | 2016-10-25 | 2018-05-03 | 코스맥스 주식회사 | Cosmetic composition containing ginseng fruit fermented material obtained by fermentation using aureobasidium pullulans and lactic acid bacteria |
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| KR20210038019A (en) * | 2019-09-30 | 2021-04-07 | 에이앤펩주식회사 | Preparing method of natural fermentation extracts and use having improved contents of high functional meterials |
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