KR101682101B1 - Composition for skin protection against uv comprising exopolysaccharide produced by ceriporia lacerata as an active ingredient - Google Patents
Composition for skin protection against uv comprising exopolysaccharide produced by ceriporia lacerata as an active ingredient Download PDFInfo
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- KR101682101B1 KR101682101B1 KR1020150166466A KR20150166466A KR101682101B1 KR 101682101 B1 KR101682101 B1 KR 101682101B1 KR 1020150166466 A KR1020150166466 A KR 1020150166466A KR 20150166466 A KR20150166466 A KR 20150166466A KR 101682101 B1 KR101682101 B1 KR 101682101B1
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- composition
- culture
- mycelium
- ultraviolet rays
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Abstract
Description
본 발명은 세리포리아 락세라타(Ceriporia lacerate)에 의해 생산되는 세포외다당체; 상기 세포외다당체를 포함하는 세리포리아 락세라타의 균사체 배양액; 상기 균사체 배양액의 건조분말; 또는 상기 균사체 배양액의 추출물을 유효성분으로 함유하는 자외선에 대한 피부 보호용 조성물에 관한 것이다.
The present invention relates to an extracellular polysaccharide produced by Ceriporia lacerate ; A mycelial culture solution of ceriplora lacerata containing the extracellular polysaccharide; A dry powder of the mycelial culture liquid; Or a composition for skin protection against ultraviolet rays containing an extract of the mycelial body as an active ingredient.
일반적으로 태양광에 포함된 자외선이 피부에 과도하게 조사되는 경우 피부에 홍반이 형성될 수 있고, 멜라닌 생성 촉진을 통한 기미 및 잡티 발생의 원인이 되며, 장기적으로는 주름의 증가, 피부의 처짐, 이완 등과 같은 피부 노화를 일으킬뿐만 아니라, 심할 경우 피부암 발생의 원인이 되기도 한다.Generally, when ultraviolet rays contained in sunlight are excessively irradiated to skin, erythema can be formed on the skin, and it can cause stains and dullness through promotion of melanin production. In the long term, Not only causes skin aging such as relaxation, but also causes skin cancer in severe cases.
자외선은 태양에서 발산되는 400 ㎚이하의 파장을 가지는 광선으로 피부를 손상시키며 가시광선보다 파장이 짧아 강렬한 작용을 한다. 자외선은 장파 자외선(UV-A; 320-400 ㎚), 중파 자외선(UV-B; 280-320 ㎚), 단파 자외선(UV-C; 200-250 ㎚)으로 나누어지며 이들은 피부에 광생물학적 반응을 일으켜 일광화상, 색소침착, 광노화, 피부암 등을 유발하며 파장에 따라 다양한 피부손상을 유발한다.Ultraviolet rays are rays that have a wavelength of 400 ㎚ or less emitted from the sun, and damage the skin. The ultraviolet rays have a shorter wavelength than the visible light and act intensely. Ultraviolet rays are divided into long wave UV-A (320-400 ㎚), medium-wave ultraviolet (UV-B) and short-wave ultraviolet light (UV-C: 200-250 ㎚) It induces sunburn, pigmentation, photoaging, skin cancer, etc., and causes various skin damage depending on wavelength.
단파 자외선은 살균 작용 및 세포 사멸을 일으키나, 대부분 오존층에서 흡수되어 피부에 도달하기 어렵고, 중파 자외선은 표피 기저층과 진피 상부까지 도달하여 피부 각화를 지속시키고 홍반, 수포 등의 일광화상과 색소 침착을 유발하며 세포막 손상, 피부암을 유발할 뿐만 아니라, 햇볕으로 인한 피부 화상(sunburn, 일광화상)의 원인이 된다. 나아가, 피부 세포의 DNA에 직접적으로 손상을 입혀 피부 재생 기전에 직접적인 영향을 주기도 한다.Although short-wave ultraviolet rays cause bactericidal action and apoptosis, most of them are absorbed in the ozone layer and hard to reach the skin. Medium-wave ultraviolet rays reach the epidermal basal layer and the upper part of the dermis to sustain skin keratinization and cause sunburn and pigmentation such as erythema It causes cell membrane damage and skin cancer, as well as causing sunburn (sunburn). Furthermore, it directly damages the DNA of skin cells and directly affects the skin regeneration mechanism.
특히, 장파 자외선은 진피까지 도달하여 즉각적인 색소 침착과 자연홍반, 광독성, 광알레르기 반응, 유리기(free radical) 생성으로 인한 만성적 광노화를 유발할 수 있고, 진피의 탄력 섬유와 교원 섬유를 변성시켜 피부의 탄력을 감소시키고, 주름, 피부 위축현상을 유발한다. 또한, 피부 세포의 DNA에 간접적으로 영향을 주어 피부암의 위험을 증가시키기도 한다.In particular, the long-wave ultraviolet light reaches the dermis and can induce chronic photoaging due to immediate pigmentation, natural erythema, phototoxicity, photoallergic reaction, free radical generation, and elasticity and collagen fibers of the dermis, And causes wrinkles and skin atrophy. It also indirectly affects the DNA of skin cells, increasing the risk of skin cancer.
최근 들어, 자외선으로부터 피부를 보호할 수 있는 많은 물질에 대한 연구와 실험이 진행되고 있으며, 그 결과 천연식품이나 한약재료로부터 추출한 성분을 함유한 다양한 건강보조식품이 이와 관련되어 개발되어 있는 실정이다. 이러한 연구의 일환으로, 대한민국 등록특허 제10-1549148호는 천연 추출물을 포함하는 자외선에 의한 피부세포 보호용 및 치료용 조성물에 관해 기재하고 있다.
In recent years, researches and experiments have been carried out on many substances that can protect skin from ultraviolet rays, and as a result, various health supplements containing ingredients extracted from natural foods and herbal medicine materials have been developed in connection therewith. As a part of this research, Korean Patent No. 10-1549148 discloses a composition for protecting and treating skin cells by UV rays containing a natural extract.
한편, 세리포리아 락세라타는 백색 부후균의 일종으로서, 생태계에서 셀룰로오스, 헤미 셀룰로오스, 기타 다당류 및 글리세롤 등의 탄소원을 이용하기 위하여 리그닌 분해라는 공동대사(co-metabolism)를 수행하는 것으로 알려져 있다.On the other hand, Sellapora lacerata is a kind of white rot fungus, and it is known that it performs co-metabolism called lignin decomposition in order to utilize carbon sources such as cellulose, hemicellulose, other polysaccharides and glycerol in an ecosystem.
세리포리아 락세라타를 이용한 의학적 치료 용도와 관련하여, 본 발명자들에 의해 출원된 대한민국 등록특허 제10-1031605호에 세리포리아 락세라타 배양액 추출물의 당뇨 치료 용도만이 알려져 있을 뿐, 세리포리아 락세라타를 이용한 자외선에 대한 피부 보호 용도는 아직까지 보고된 바 없다.Regarding the use of medicinal treatment using ceraplora lucerata, Korean Patent No. 10-1031605 filed by the inventors of the present invention discloses only the treatment of diabetes treatment of the extract of the culture liquid of ceriplora lacercata, Skin protection against ultraviolet rays using Lipitor Laceratta has not been reported yet.
이에, 본 발명자들은 세리포리아 락세라타로부터 분리한 세포외다당체; 상기 세포외다당체를 포함하는 세리포리아 락세라타의 균사체 배양액; 상기 균사체 배양액의 건조분말; 또는 상기 균사체 배양액의 추출물이 자외선에 대한 피부 보호 효과를 나타낸다는 것을 확인함으로써 본 발명을 완성하였다.
Accordingly, the present inventors have found that an extracellular polysaccharide separated from a cerporolactacera; A mycelial culture solution of ceriplora lacerata containing the extracellular polysaccharide; A dry powder of the mycelial culture liquid; Or the extract of the mycelium culture liquid shows a skin protecting effect against ultraviolet rays, thereby completing the present invention.
본 발명의 목적은 세리포리아 락세라타에 의해 생산되는 활성성분을 함유하는 자외선에 대한 피부 보호용 조성물, 및 이를 포함하는 약학 조성물, 화장료 조성물 또는 건강기능식품을 제공하는 것이다.
It is an object of the present invention to provide a composition for protecting skin against ultraviolet rays containing an active ingredient produced by ceriplora lactaclorata, and a pharmaceutical composition, cosmetic composition or health functional food containing the same.
상기 목적을 달성하기 위하여, 본 발명은 세리포리아 락세라타(Ceriporia lacerata)에 의해 생산되는 세포외다당체; 상기 세포외다당체를 포함하는 세리포리아 락세라타의 균사체 배양액; 상기 균사체 배양액의 건조분말; 또는 상기 균사체 배양액의 추출물을 유효성분으로 함유하는, 피부 보호용 조성물을 제공한다.In order to achieve the above object, the present invention relates to an extracellular polysaccharide produced by Ceriporia lacerata ; A mycelial culture solution of ceriplora lacerata containing the extracellular polysaccharide; A dry powder of the mycelial culture liquid; Or an extract of said mycelial fluid as an active ingredient.
상기 목적을 달성하기 위하여, 본 발명은 본 발명에 따른 조성물을 유효성분으로 포함하는 약학 조성물, 화장료 조성물 또는 건강기능식품을 제공한다.
In order to achieve the above object, the present invention provides a pharmaceutical composition, a cosmetic composition or a health functional food comprising the composition according to the present invention as an active ingredient.
본 발명에 따른 세리포리아 락세라타에 의해 생산되는 세포외다당체; 상기 세포외다당체를 포함하는 세리포리아 락세라타의 균사체 배양액; 상기 균사체 배양액의 건조분말; 또는 상기 균사체 배양액의 추출물을 유효성분으로 함유하는 자외선에 대한 피부 보호용 조성물은 자외선에 노출된 피부 각질세포의 보호 효과가 있으므로, 자외선에 의한 피부 손상 예방, 개선 또는 치료용 약학 조성물, 화장료 조성물 또는 건강기능식품으로 유용하게 사용될 수 있다.
An extracellular polysaccharide produced by a cellulolytic enzyme according to the present invention; A mycelial culture solution of ceriplora lacerata containing the extracellular polysaccharide; A dry powder of the mycelial culture liquid; Or a composition for skin protection against ultraviolet rays containing an extract of the mycelial body as an active ingredient has a protective effect on skin keratinocytes exposed to ultraviolet rays and is therefore useful as a pharmaceutical composition for preventing, improving or treating skin damage due to ultraviolet rays, It can be used effectively as a functional food.
도 1은 세리포리아 락세라타 세포외다당체(CL01)를 처리한 후, UV-B를 조사하였을 때 인간 각질세포(HaCaT)의 세포생존율을 나타내는 그래프이다.FIG. 1 is a graph showing cell viability of human keratinocyte (HaCaT) upon irradiation with UV-B after treatment of the cellulolytic theraser extracellular polysaccharide (CL01).
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 세리포리아 락세라타(Ceriporia lacerata)에 의해 생산되는 세포외다당체; 상기 세포외다당체를 포함하는 세리포리아 락세라타의 균사체 배양액; 상기 균사체 배양액의 건조분말; 또는 상기 균사체 배양액의 추출물을 유효성분으로 함유하는, 피부 보호용 조성물을 제공한다.The present invention relates to an extracellular polysaccharide produced by Ceriporia lacerata ; A mycelial culture solution of ceriplora lacerata containing the extracellular polysaccharide; A dry powder of the mycelial culture liquid; Or an extract of said mycelial fluid as an active ingredient.
본 명세서에서 사용된 용어 "세포외다당체(extracellular polysaccharide, EPS)"란 균류 등 미생물의 세포벽의 일부로서, 다당류가 세포 외로 분비되어 그 주위에 협막을 형성하거나 점질물로서 세포주위나 배지로 분비되는 물질을 의미한다. 상기 세포외다당체는 미생물이 항체, 독성물질, 원생동물 및 박테리오파지 등의 외부환경으로부터 자신을 보호하기 위해 분비된다.As used herein, the term "extracellular polysaccharide (EPS)" refers to a part of the cell wall of a microorganism such as fungi. The polysaccharide is secreted extracellularly to form a cavernous membrane or secreted as a viscous substance on a cell line or medium it means. The extracellular polysaccharide is secreted by the microorganism to protect itself from the external environment such as antibodies, toxins, protozoa, and bacteriophages.
상기 세포외다당체는 40 내지 60 중량%의 당과 30 내지 40 중량%의 단백질, 40 내지 50 중량%의 당과 32 내지 38 중량%의 단백질, 43 내지 47 중량%의 당과 33 내지 36 중량%의 단백질, 또는 약 45 중량%의 당과 약 34 중량%의 단백질을 포함할 수 있다.Wherein the extracellular polysaccharide comprises 40 to 60 wt% sugar, 30 to 40 wt% protein, 40 to 50 wt% sugar, 32 to 38 wt% protein, 43 to 47 wt% sugar, 33 to 36 wt% Of the protein, or about 45 wt% sugar and about 34 wt% protein.
상기 당은 만노오스, 갈락토오스 및 글루코오스를 함유할 수 있다.The sugar may contain mannose, galactose and glucose.
상기 세포외다당체는 100 내지 150 kDa, 110 내지 140 kDa 또는 115 내지 125 kDa의 분자량을 가질 수 있고, 보다 구체적으로는 약 120 kDa의 분자량을 가질 수 있다.The extracellular polysaccharide may have a molecular weight of 100 to 150 kDa, 110 to 140 kDa or 115 to 125 kDa, and more specifically about 120 kDa.
본 명세서에서 사용된 용어 "피부 보호"란, 외부 자극 또는 환경에 의한 피부 손상으로부터 피부를 보호하는 것을 의미하며, 노화에 의한 피부 변화도 포함할 수 있다. 피부를 손상시킬 수 있는 외부 자극 중의 하나는 자외선이 있으며, 자외선에 의한 피부 손상에는 일광화상, 색소침착, 광노화, 자연홍반, 탄력감소, 주름, 피부암 등을 포함한다.
As used herein, the term "skin protection" means protecting the skin from external damage or environmental damage, and may also include skin changes due to aging. One of the external stimuli that can damage the skin is ultraviolet rays. Skin damage caused by ultraviolet rays includes sunburn, pigmentation, photoaging, natural erythema, reduced elasticity, wrinkles and skin cancer.
본 발명의 일 구현예로서, 상기 세포외다당체는, (a) 세리포리아 락세라타 균사체를 액체 배양하여 세리포리아 락세라타 균사체 배양액을 제조하는 단계; (b) 상기 세리포리아 락세라타 균사체 배양액을 건조시켜 분말화하는 단계; 및 (c) 세리포리아 락세라타 균사체 배양액 분말을 용매로 추출한 후 이를 여과하고 감압농축하는 단계를 포함하는 제조방법에 의해서 제조될 수 있다.As an embodiment of the present invention, the extracellular polysaccharide may be prepared by: (a) liquid culturing mycelia lacticera mycelium to prepare a culture medium of a seriposita lactamera mycelium; (b) drying and cultivating the culture medium of the above-mentioned sera lipolactacera mycelium; And (c) a step of extracting the culture medium of the cultivated mycelium lacticera mycelium with a solvent, followed by filtration and concentration under reduced pressure.
상기 (a) 단계에서의 세리포리아 락세라타 균사체의 액체 배양을 위한 배지는 설탕, 포도당, 전분, 수수분, 대맥분, 대두분, 황산마그네슘(MgSO4), 1인산칼륨(KH2PO4), 2인산칼륨(K2HPO4) 및 물을 포함할 수 있고, 수소이온농도(pH)가 4.5 내지 6.0인 것일 수 있다.The medium for liquid culture of the mycelium of seripositive Lactacera in the step (a) is selected from the group consisting of sugar, glucose, starch, water, barley, soybean powder, magnesium sulfate (MgSO 4 ), potassium monophosphate (KH 2 PO 4 ), potassium diphosphate (K 2 HPO 4 ), and water, and may have a hydrogen ion concentration (pH) of 4.5 to 6.0.
구체적으로, 상기 배지는, 설탕 0.2 내지 3 중량%, 포도당 0.2 내지 3 중량%, 전분 0.2 내지 4 중량%, 수수분 0.1 내지 0.5 중량%, 대맥분 0.1 내지 0.5 중량%, 대두분 0.2 내지 3 중량%, 황산마그네슘(MgSO4) 0.05 내지 0.1 중량%, 1인산칼륨(KH2PO4) 0.05 내지 0.25 중량%, 2인산칼륨(K2HPO4) 0.05 내지 0.25 중량% 및 잔량의 물을 포함할 수 있다.Specifically, the culture medium contains 0.2 to 3% by weight of sugar, 0.2 to 3% by weight of glucose, 0.2 to 4% by weight of starch, 0.1 to 0.5% by weight of water, 0.1 to 0.5% 0.05 to 0.15% by weight of magnesium sulfate (MgSO 4 ), 0.05 to 0.25% by weight of potassium monophosphate (KH 2 PO 4 ), 0.05 to 0.25% by weight of potassium diphosphate (K 2 HPO 4 ) .
상기 (a) 단계에서의 액체 배양은 청색 LED 광원 하에서 수행될 수 있고, 이산화탄소의 농도를 1,000 내지 2,000 ppm으로 유지하여 수행될 수 있다.The liquid culture in the step (a) may be performed under a blue LED light source, and may be performed by maintaining the concentration of carbon dioxide at 1,000 to 2,000 ppm.
상기 액체 배양은 예를 들어, 20 내지 28℃에서, 수소이온농도는 4.5 내지 6.0, 광원은 청색 LED, 조도는 0.1 내지 0.8 LUX를 유지하며 공기는 0.5 내지 2.0 kgf/㎠으로 주입하고, 이산화탄소의 농도는 1,000 내지 2,000 ppm으로 유지하면서 8 내지 13일간 수행될 수 있다. 구체적으로, 20 내지 25℃, pH 4.5 내지 6.0, 0.5 내지 2.0 kgf/㎠의 공기 주입 및 1,000 내지 2,000 ppm의 이산화탄소 농도의 조건에서 5 내지 15일간 수행될 수 있다. 상술한 바와 같은 조건으로 액체 배양할 경우, 세포외다당체의 함량이 높으므로 바람직하다.The liquid culture is, for example, carried out at 20 to 28 ° C with a hydrogen ion concentration of 4.5 to 6.0, a light source of a blue LED, an illuminance of 0.1 to 0.8 LUX, air is injected at 0.5 to 2.0 kgf / cm 2, The concentration can be carried out for 8 to 13 days while maintaining the concentration at 1,000 to 2,000 ppm. Specifically, it can be carried out at a temperature of 20 to 25 占 폚, a pH of 4.5 to 6.0, an air injection of 0.5 to 2.0 kgf / cm2 and a carbon dioxide concentration of 1,000 to 2,000 ppm for 5 to 15 days. When liquid culture is carried out under the above-described conditions, the content of extracellular polysaccharide is high, which is preferable.
상기 (a) 단계 모균주는 PDA(potato dextrose agar) 배지 상태로 1 내지 5℃에 보관중인 우량 균주를 삼각플라스크에 PDB(potato dextrose broth) 배지를 사용하여 진탕 배양기에서 25℃의 항온을 유지하며 7 내지 9일간 배양과정을 거친 후 사용할 수 있다. 또한, 이와 같이 모균주를 배양한 후 배양액 또는 수득한 균사체를 접종원으로 이용할 수 있다. 이때, 접종원으로 투입될 균사체의 양은 배양할 용액의 양을 기준으로 0.5%(w/v) 정도인 것이 바람직하다. 균사체량(%/100 ㎖, w/v)이 많다고 세포외다당체의 함량도 같이 높아지는 것이 아니므로 배지 조성은 균사체의 성장에 가장 양호한 영양 비율 및 환경조건이 아닌, 세포외다당체의 함량을 가장 높게 형성시키는 선택적 배양조건을 적용하는 것이 바람직하다.In step (a), the parent strain in the PDA (potato dextrose agar) medium is maintained at 25 ° C. in a shaking incubator using PDB (potato dextrose broth) medium in an Erlenmeyer flask And then cultured for 7 to 9 days. In addition, the culture broth or obtained mycelium can be used as an inoculum after the parent strain is cultured as described above. At this time, the amount of the mycelium to be added to the inoculation source is preferably about 0.5% (w / v) based on the amount of the solution to be cultured. Since the amount of extracellular polysaccharide is not so high as much as the amount of mycelium (% / 100 ml, w / v), the composition of the medium is not the best nutritional ratio and environmental condition for growth of the mycelium, It is preferable to apply selective culture conditions in which the cells are formed.
상기 배양액은 균사체와 수용액으로 분리 정제할 수 있다. 구체적으로, 상기 분리 정제는 원심분리기로 균사체를 제거한 용액을 다중필터프레스(multi-sheet filter press)와 진동원심막분리기(PALLSEP)로 반복하여 정제한 후, 1분간 자외선(UV)을 조사할 수 있다. 또한, 상기 배양액은 산소를 제거한 후 밀봉하여 보관할 수 있으며, 이는 배양액 속에 균사가 존재할 경우 균사의 성장으로 유효성분의 함량에 변화를 가져올 수 있다.
The culture solution can be separated and purified into mycelium and an aqueous solution. Specifically, the separated tablets may be purified by repeatedly removing the mycelium from the mycelium with a centrifugal separator using a multi-sheet filter press and a vibrating centrifugal separator (PALLSEP), and then irradiating ultraviolet rays (UV) for 1 minute have. In addition, the culture solution can be kept sealed after removing oxygen. If the mycelium is present in the culture solution, the content of the active ingredient may be changed by growing mycelium.
상기 (b) 단계에서는 상기 (a) 단계에서 제조된 균사체 배양액을 건조시켜 분말화할 수 있다. 상기 건조는 유효물질의 소멸을 방지하기 위해 40℃ 이하의 온도, 보다 구체적으로는 30℃ 이하의 온도에서 48 내지 96시간 동안 수행될 수 있다. 또한, (b) 단계의 건조는 증발온도를 상대적으로 높게 설정하는 진공건조기보다 진공동결건조기를 사용하는 것이 유효물질 함량 변화가 최소화되므로 바람직하다.
In the step (b), the mycelial culture liquid prepared in the step (a) may be dried and pulverized. The drying may be carried out at a temperature of 40 DEG C or less, more specifically, at a temperature of 30 DEG C or less for 48 to 96 hours to prevent the disappearance of the active material. Also, in the step (b), it is preferable to use a vacuum freeze dryer rather than a vacuum dryer in which the evaporation temperature is set to be relatively high, since the change in effective substance content is minimized.
상기 (c) 단계에서는 (b) 단계에서 얻은 균사체 배양액의 건조분말을 용매로 추출한 후, 본 발명에 따른 조성물의 유효성분인 세포외다당체를 분리한다.In step (c), the dry powder of the mycelial liquid obtained in step (b) is extracted with a solvent, and the extracellular polysaccharide, which is an active ingredient of the composition according to the present invention, is isolated.
구체적으로, 균사체 배양액의 건조 분말 3 내지 10 g에 증류수 100 ㎖를 첨가하여 잘 현탁한 후, 5,000 내지 10,000 rpm으로 10 내지 30분 동안 원심분리하여 상등액을 수득하고, 상기 상등액에 그 양의 2 내지 3배에 해당하는 추출 용매를 첨가한 후, 1 내지 5℃의 냉장고에 넣어 10 내지 15시간 정치시킬 수 있다. 상기 정치물에서 상등액만을 다시 5,000 내지 10,000 rpm으로 10 내지 30분 동안 원심분리한 후, 침전물을 회수하여 조(crude) 세포외다당체를 제조할 수 있다. 상기 조 세포외다당체를 30℃ 이하에서 진공동결건조하여 세포외다당체를 수득할 수 있다.Specifically, 100 ml of distilled water is added to 3 to 10 g of the dried powder of the mycelial culture liquid, and the suspension is well suspended. After centrifuging at 5,000 to 10,000 rpm for 10 to 30 minutes, a supernatant is obtained. Three times as much extraction solvent may be added, and the mixture is allowed to stand in a refrigerator at 1 to 5 DEG C for 10 to 15 hours. After centrifuging the supernatant alone at 5,000 to 10,000 rpm for 10 to 30 minutes in the supernatant, the precipitate may be recovered to produce a crude extracellular polysaccharide. The extracellular polysaccharide may be lyophilized and dried at 30 DEG C or lower to obtain an extracellular polysaccharide.
상기 추출 용매는 물, 탄소수 1 내지 4의 저급 알코올, 아세톤, 에테르, 클로로포름 및 에틸아세테이트로 이루어진 군에서 선택되는 용매 또는 이들의 혼합용매일 수 있고, 보다 구체적으로는, 물, 메탄올, 에탄올, 부탄올, 아세톤 및 에틸아세테이트로 이루어진 군에서 선택되는 용매 또는 이들의 혼합용매일 수 있으며, 더욱 바람직하게는, 물 또는 50 내지 99%(v/v)의 에탄올 수용액일 수 있다.
The extraction solvent may be a solvent selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms, acetone, ether, chloroform and ethyl acetate, or a mixture thereof. More specifically, water, methanol, ethanol, , Acetone and ethyl acetate, or a mixture thereof, and more preferably water or an aqueous solution of 50 to 99% (v / v) of ethanol.
본 발명은 세리포리아 락세라타에 의해 생산되는 세포외다당체; 상기 세포외다당체를 포함하는 세리포리아 락세라타의 균사체 배양액; 상기 균사체 배양액의 건조분말; 또는 상기 균사체 배양액의 추출물을 포함하는 피부 보호용 조성물을 제공한다. 이때, 상기 피부 보호용 조성물은 자외선에 대한 피부손상 예방, 개선 또는 치료를 위한 목적으로 약학 조성물, 화장료 조성물 또는 건강기능식품 등에 첨가제로 사용될 수 있으며, 이때 사용량 및 사용 형태는 제조 목적에 따라 적절히 조절할 수 있다. 상기 자외선에 의한 피부손상은 일광화상, 색소침착, 광노화, 자연홍반, 탄력감소, 주름 또는 피부암 등을 포함할 수 있다.The present invention relates to an extracellular polysaccharide produced by a cellulolytic enzyme; A mycelial culture solution of ceriplora lacerata containing the extracellular polysaccharide; A dry powder of the mycelial culture liquid; Or an extract of the mycelial culture liquid. At this time, the skin protecting composition may be used as an additive for pharmaceutical composition, cosmetic composition or health functional food for the purpose of prevention, improvement or treatment of skin damage to ultraviolet rays. In this case, have. Skin damage by ultraviolet rays may include sunburn, pigmentation, photoaging, natural erythema, reduced elasticity, wrinkles or skin cancer.
상기 세포외다당체는 피부 보호용 조성물의 총 중량 대비 0.1 내지 80 중량%, 또는 0.1 내지 50 중량%로 포함될 수 있으며, 세리포리아 락세라타의 균사체 배양액, 이의 건조분말 또는 상기 균사체 배양액의 추출물은 상기 세포외다당체의 함량에 해당하는 양으로 적절히 포함될 수 있다. 그러나 보다 구체적으로, 세포외다당체; 이를 포함하는 배양액; 상기 배양액의 건조분말; 또는 상기 배양액의 추출물의 유효 함량은 피부 보호용 조성물의 사용방법 및 목적에 따라 적절히 조절될 수 있다.
The extracellular polysaccharide may be contained in an amount of 0.1 to 80% by weight, or 0.1 to 50% by weight, based on the total weight of the composition for skin protection, and the extract of the mycelium culture medium, the dried powder thereof or the mycelium culture solution of the cellulolytic enzyme, It may be included in an amount corresponding to the content of the exopolysaccharide. More specifically, however, an extracellular polysaccharide; A culture fluid containing the same; A dry powder of the culture solution; Or the effective amount of the extract of the culture liquid may be suitably adjusted according to the method and purpose of use of the skin protection composition.
또한, 본 발명은 상술한 바와 같은 피부 보호용 조성물을 포함하는 자외선에 의한 피부손상 치료용 약학 조성물을 제공한다.The present invention also provides a pharmaceutical composition for treating skin damage caused by ultraviolet rays, which comprises the composition for skin protection as described above.
상기 약학 조성물은 세리포리아 락세라타에 의해 생산되는 세포외다당체; 이를 포함하는 세리포리아 락세라타의 균사체 배양액; 상기 균사체 배양액의 건조분말; 또는 상기 균사체 배양액의 추출물을 유효성분으로 함유하는 것 이외에 통상적으로 사용되는 적절한 담체, 부형제 및 희석제가 더 포함될 수 있다.Wherein said pharmaceutical composition is an extracellular polysaccharide produced by three lipolactaceratas; A culture solution of mycelium of Sellapora lacera rata containing the same; A dry powder of the mycelial culture liquid; Or an appropriate carrier, excipient and diluent which are conventionally used in addition to those containing the extract of the mycelial liquid as an active ingredient.
본 발명에 따른 약학 조성물은 각각 통상의 방법에 따라 제형화하여 사용될 수 있다. 적합한 제형으로는 정제, 환제, 산제, 과립제, 당의정, 경질 또는 연질의 캡슐제, 용액제, 현탁제 또는 유화액제, 주사제, 좌제 등이 있으나, 이에 한정되는 것은 아니다.The pharmaceutical composition according to the present invention can be formulated according to a conventional method. Suitable formulations include, but are not limited to, tablets, pills, powders, granules, dragees, hard or soft capsules, solutions, suspensions or emulsions, injections, suppositories and the like.
본 발명에 따른 약학 조성물은 약학적으로 불활성인 유기 또는 무기 담체를 이용하여 적합한 제형으로 제조할 수 있다. 즉, 제형이 정제, 코팅된 정제, 당의정 및 경질 캡슐제인 경우, 락토스, 수크로스, 전분 또는 그 유도체, 탈크, 칼슘 카보네이트, 젤라틴, 스테아르산 또는 그 염을 포함할 수 있다. 또한, 제형이 연질 캡슐제인 경우, 식물성 오일, 왁스, 지방, 반고체 및 액체의 폴리올을 포함할 수 있다. 나아가, 제형이 용액 또는 시럽 형태인 경우, 물, 폴리올, 글리세롤, 식물성 오일 등을 포함할 수 있다.The pharmaceutical composition according to the present invention can be prepared into a suitable formulation using a pharmaceutically inert organic or inorganic carrier. That is, when the formulations are tablets, coated tablets, dragees and hard capsules, they may include lactose, sucrose, starch or derivatives thereof, talc, calcium carbonate, gelatin, stearic acid or a salt thereof. Also, when the formulation is a soft capsule, it may include vegetable oils, waxes, fats, semi-solid and liquid polyols. Furthermore, if the formulation is in the form of a solution or syrup, it may contain water, polyols, glycerol, vegetable oils and the like.
본 발명에 따른 약학 조성물은 상기의 담체 외에도 보존제, 안정화제, 습윤제, 유화제, 용해제, 감미제, 착색제, 삼투압 조절제, 산화방지제 등을 더 포함할 수 있다.The pharmaceutical composition according to the present invention may further contain a preservative, a stabilizer, a wetting agent, an emulsifier, a solubilizer, a sweetener, a colorant, an osmotic pressure regulator, an antioxidant and the like in addition to the above carrier.
본 발명에 따른 약학 조성물의 투여방법은 제형에 따라 용이하게 선택될 수 있으며, 경구 또는 비경구 투여될 수 있다. 투여량은 환자의 나이, 성별, 체중, 병증의 정도, 투여경로에 따라 달라질 수 있으나, 일반적으로 유효성분인 세포외다당체를 기준으로 5 내지 1,000 ㎎/㎏체중의 양, 구체적으로는 10 내지 600 ㎎/㎏체중의 양을 1일 1회 내지 3회로 나누어 투여할 수 있다. 그러나, 상기 투여량은 본 발명의 범위를 한정하는 것은 아니다.The method of administering the pharmaceutical composition according to the present invention can be easily selected according to the formulation, and can be administered orally or parenterally. The dose may vary depending on the patient's age, sex, weight, degree of pathology, and route of administration, but is generally in the range of 5 to 1,000 mg / kg body weight based on the extracellular polysaccharide, Mg / kg body weight may be administered once or three times a day. However, the dose does not limit the scope of the present invention.
본 발명에 따른 약학 조성물은 우수한 피부 보호 효과를 제공할 뿐만 아니라, 약물에 의한 독성 및 부작용도 거의 없어 피부 보호의 목적으로 장기간 복용시에도 안심하고 사용할 수 있다.
The pharmaceutical composition according to the present invention not only provides excellent skin protection effect but also has little toxicity and side effects caused by drugs, so that it can be safely used for long-term use for protection of skin.
또한, 본 발명은 상술한 바와 같은 피부 보호용 조성물을 포함하는 자외선에 의한 피부손상 예방 및 개선용 화장료 조성물을 제공한다.In addition, the present invention provides a cosmetic composition for prevention and improvement of skin damage caused by ultraviolet rays, which comprises the skin protecting composition as described above.
본 발명의 화장료 조성물은 피부손상의 예방 또는 개선을 목적으로 피부에 직접 적용될 수 있다. The cosmetic composition of the present invention can be directly applied to the skin for the purpose of preventing or improving skin damage.
상기 화장료 조성물은 통상적으로 제조되는 화장료 제형으로 제제화될 수 있다. 상기 화장료 조성물의 예는 용액, 외용연고, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클렌징, 오일, 파운데이션 및 스프레이 등이 있다. 보다 구체적으로, 상기 화장료 조성물은 화장수, 영양크림, 마사지 크림, 에센스, 아이크림, 클렌징폼, 클렌징 워터, 팩, 스프레이 또는 파우더의 제형으로 제제화될 수 있다.The cosmetic composition can be formulated into cosmetic formulations that are conventionally prepared. Examples of the cosmetic composition include solutions, external ointments, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansers, oils, foundations and sprays. More specifically, the cosmetic composition may be formulated into a formulation of a lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.
본 발명의 화장료 조성물은 일반 피부 화장료에 배합되어 허용가능한 담체를 1종 이상 추가로 포함할 수 있으며, 통상의 성분으로 예를 들면, 유분, 물, 계면활성제, 보습제, 저급 알콜, 증점제, 킬레이트제, 색소, 방부제, 향료 등을 적절히 배합할 수 있으나, 이에 한정되는 것은 아니다.The cosmetic composition of the present invention may further contain at least one acceptable carrier in combination with an ordinary skin cosmetic composition. Examples of the conventional ingredients include oil, water, a surfactant, a moisturizer, a lower alcohol, a thickener, , A coloring agent, a preservative, a perfume, and the like may be appropriately compounded, but the present invention is not limited thereto.
본 발명의 화장료 조성물의 제형이 페이스트, 크림 또는 겔인 경우에는 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라가칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크, 산화아연 및 이의 혼합물로 이루어진 군에서 선택되는 담체 성분을 포함할 수 있다.When the formulation of the cosmetic composition of the present invention is a paste, a cream or a gel, it may be an animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, , ≪ / RTI > and the like.
본 발명의 화장료 조성물의 제형이 파우더 또는 스프레이인 경우에는 락토스, 탈크, 실리카, 알루미늄 하이드록시드, 칼슘 실리케이트, 폴리아미드 파우더 및 이의 혼합물로 이루어진 군에서 선택되는 담체 성분을 포함할 수 있으며, 특히 스프레이인 경우에는 추가적으로 클로로플루오로하이드로카본, 프로판/부탄 또는 디메틸에테르 등을 더 포함할 수 있다.When the formulation of the cosmetic composition of the present invention is a powder or a spray, it may contain a carrier component selected from the group consisting of lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder and mixtures thereof, , It may further include chlorofluorohydrocarbons, propane / butane, dimethyl ether, and the like.
본 발명의 화장료 조성물의 제형이 용액 또는 유탁액인 경우에는 용매, 용매화제, 유탁화제 및 이의 혼합물로 이루어진 군에서 선택되는 담체 성분을 포함할 수 있다. 이의 예로는, 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌 글리콜, 1,3-부틸글리콜오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜, 소르비탄 지방산 에스테르, 및 이의 혼합물 등을 들 수 있다.When the formulation of the cosmetic composition of the present invention is a solution or an emulsion, it may contain a carrier component selected from the group consisting of a solvent, a solubilizing agent, an emulsifying agent and a mixture thereof. Examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, sorbitan fatty acid ester, .
본 발명의 화장료 조성물의 제형이 현탁액인 경우에는 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미세결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가, 트라가칸트 및 이의 혼합물로 이루어진 군에서 선택되는 담체 성분을 포함할 수 있다.When the formulation of the cosmetic composition of the present invention is a suspension, it may be diluted with water, a liquid diluent such as ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Hydroxypropylmethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose,
본 발명의 화장료 조성물의 제형이 계면-활성제 함유 클렌징인 경우에는 지방족 알코올 설페이트, 지방족 알코올 에테르 설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 글리세롤 지방산 에스테르 및 이의 혼합물로 이루어진 군에서 선택되는 담체 성분으로 포함할 수 있다.When the formulation of the cosmetic composition of the present invention is an interfacial-active agent-containing cleansing, it is preferable to use an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, an isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, Ether sulfates, alkylamido betaines, aliphatic alcohols, glycerol fatty acid esters, and mixtures thereof.
본 발명의 화장료 조성물은 담체 성분 이외에도, 항산화제, 안정화제, 용해화제, 보습제, 안료, 향료, 자외선 차단제, 발색제, 계면 활성제 및 이의 조합으로 이루어진 군에서 선택되는 보조제를 추가로 포함할 수 있다. 상기 보조제는 화장료 조성물의 제조에 통상적으로 사용되는 보조제라면 사용에 제한이 없다.
The cosmetic composition of the present invention may further comprise, in addition to the carrier component, an adjuvant selected from the group consisting of an antioxidant, a stabilizer, a solubilizer, a moisturizer, a pigment, a fragrance, a sunscreen, a colorant, a surfactant and a combination thereof. The adjuvant is not limited as long as it is an adjuvant commonly used in the production of a cosmetic composition.
나아가, 본 발명은 상술한 바와 같은 피부 보호용 조성물을 포함하는 자외선에 의한 피부 손상 예방 및 개선용 건강기능식품을 제공한다.Further, the present invention provides a health functional food for prevention and improvement of skin damage caused by ultraviolet rays, which comprises the skin protecting composition as described above.
본 발명에 따른 건강기능식품은 분말, 과립, 정제, 캡슐 또는 음료의 형태일 수 있고, 캔디, 초콜릿, 껌, 차, 비타민복합체, 건강보조식품 등일 수 있다.The health functional food according to the present invention may be in the form of a powder, a granule, a tablet, a capsule or a drink, and may be a candy, a chocolate, a gum, a tea, a vitamin complex,
이때, 상기 건강기능식품 중에 포함되는 본 발명에 따른 세포외다당체; 이를 포함하는 균사체 배양액; 상기 균사체 배양액의 건조분말; 또는 상기 균사체 배양액의 추출물의 함량은 통상적으로 전체 식품 중량의 0.01 내지 50 중량%, 또는 0.1 내지 20 중량%로 포함될 수 있다. 또한, 건강 음료 조성물의 경우, 건강 음료 조성물 100 ㎖를 기준으로 0.02 내지 10 g, 또는 0.3 내지 1 g의 함량으로 포함될 수 있다.Herein, the extracellular polysaccharide according to the present invention contained in the health functional food; A mycelial culture fluid containing the same; A dry powder of the mycelial culture liquid; Or the content of the extract of the mycelial culture liquid may be usually 0.01 to 50% by weight, or 0.1 to 20% by weight of the total food weight. Also, in the case of a health drink composition, it may be contained in an amount of 0.02 to 10 g, or 0.3 to 1 g, based on 100 ml of the health drink composition.
상기 식품은 본 발명의 세리포리아 락세라타의 세포외다당체; 이를 포함하는 균사체 배양액; 상기 균사체 배양액의 건조분말; 또는 상기 균사체 배양액의 추출물과 함께 식품학적으로 허용가능한 식품 보조 첨가제를 더 포함할 수 있다.
The food may be an extracellular polysaccharide of the cellulolytic enzyme of the present invention; A mycelial culture fluid containing the same; A dry powder of the mycelial culture liquid; Or a food-acceptable food-aid additive together with the extract of the mycelial culture broth.
이하, 본 발명을 하기 실시예에 의해 보다 상세히 설명한다. 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 for illustrative purposes only and are not intended to limit the scope of the present invention.
제조예Manufacturing example 1. One. 세리포리아Three Li Pori 락세라타Lacerata 배양액의 제조 Production of culture medium
경북 상주시에서 채취한 참나무 변제부 속에서 분리한 세리포리아 락세라타 균사체를 계대배양을 통해 육성한 모균을 -80℃에 냉동 보관하였다. 상기 냉동 보관된 균주를 PDA(potato dextrose agar) 배지(87 플라스틱 배양구; Difco, Becton Dickinson and Company)에서 2 내지 3회 계대 후 균주(이하 "PDA 배양균주"라 함)를 4℃ 냉장고에 보관하여 사용하였다. 또한, 삼각플라스크에 상기 PDB(potato dextrose broth) 배지(Difco, Becton Dickinson and Company) 600 ㎖를 조성한 후, 여기에 PDA 배양 균주 1개를 넣고 8일간 진탕 배양하여, PDB 배양 균주를 수득하였다.The mycelium which was cultivated through subculture was stored frozen at -80 ℃. The cryopreserved strains were stored in a potato dextrose agar (PDA) culture medium (87 plastic culture medium; Difco, Becton Dickinson and Company) after 2 to 3 replications (hereinafter referred to as "PDA culture strains") in a refrigerator at 4 ° C Respectively. 600 ml of the above-mentioned PDB (Difco, Becton Dickinson and Company) was added to the Erlenmeyer flask. One PDB culture was added to the flask, followed by shake culture for 8 days to obtain a PDB culture.
한편, 균주의 배양을 위해 설탕 1.5 중량%, 포도당 0.5 중량%, 감자전분 0.5중량%, 수수분 0.25 중량%, 대맥분 0.25 중량%, 대두분 0.75 중량%, 황산마그네슘(MgSO4) 0.05 중량%, 1인산칼륨(KH2PO4) 0.05 중량%, 2인산칼륨(K2HPO4) 0.05 중량% 및 잔량의 물을 포함하는 액체 배양 배지를 800 ℓ 발효조에서 121℃에서 공기를 1.5 kgf/㎠로 주입하여 20분간 멸균하였다. 이를, 23℃로 냉각한 상태에서 스타터로 상기 PDB 배양 균주 600 ㎖를 접종하고, 공기를 0.5 내지 1.5 kgf/㎠로 통기시키면서, 광원은 청색 LED, 조도는 0.5 LUX를 유지하며, 이산화탄소의 농도는 2,000 ppm으로 세리포리아 락세라타 균사체를 23℃의 항온에서 10일간 액체 배양함으로써 세리포리아 락세라타 균사체 배양액을 제조하였다.
On the other hand, 1.5% by weight of sugar for the culture of the strain, glucose 0.5% by weight, potato starch 0.5% by weight, can water 0.25 weight%, barley minutes, 0.25% by weight, soy flour 0.75% by weight, magnesium sulfate (MgSO 4) 0.05% by weight , 0.05% by weight of potassium phosphate monobasic (KH 2 PO 4 ), 0.05% by weight of potassium diphosphate (K 2 HPO 4 ) and the balance of water was fed to an 800 L fermenter at a rate of 1.5 kgf / And sterilized for 20 minutes. 600 ml of the PDB culture strain was inoculated with a starter while being cooled to 23 캜 and air was supplied at 0.5 to 1.5 kgf / cm 2, while the light source was a blue LED, the illuminance was maintained at 0.5 LUX, the concentration of carbon dioxide The mycelium of ceriplora lactacerata at 2,000 ppm was subjected to liquid culture at a constant temperature of 23 캜 for 10 days to prepare a culture medium of Selegiolia lactaceras mycelia.
제조예Manufacturing example 2. 2. 세리포리아Three Li Pori 락세라타Lacerata 배양액의 건조분말의 제조 Preparation of dry powder of culture medium
상기 제조예 1에서 제조된 세리포리아 락세라타 균사체 배양액을 진공동결건조기를 이용하여 25℃에서 72시간 동안 진공동결건조시켜 분말화함으로써, 세리포리아 락세라타 균사체 배양액의 건조분말을 제조하였다.
The culture of the mycelia of the ceriplora lactaceras prepared in Preparation Example 1 was vacuum-freeze-dried at 25 ° C for 72 hours using a vacuum freeze dryer to obtain a dry powder of the culture medium of the seriposita lactamera mycelium .
제조예Manufacturing example 3. 3. 세리포리아Three Li Pori 락세라타Lacerata 배양액 추출물의 제조 Preparation of culture extract
상기 제조예 2에서 제조된 세리포리아 락세라타 균사체 배양액의 건조분말 5 g에 증류수 100 ㎖를 첨가하여 잘 현탁한 후, 8,000 rpm으로 20분 동안 원심분리하였다. 이의 상등액에 그 양의 2 내지 3배에 해당하는 에탄올을 첨가하고 4℃에서 12시간 정치시켰다. 이후, 정치물에서 상등액을 취해 세리포리아 락세라타 균사체 배양액의 추출물을 수득하였다.
100 ml of distilled water was added to 5 g of the dry powder of the cultured Mycelium lacticcerus cultured product prepared in Preparation Example 2, suspended well, and then centrifuged at 8,000 rpm for 20 minutes. Ethanol corresponding to 2 to 3 times its amount was added to its supernatant and allowed to stand at 4 캜 for 12 hours. Then, the supernatant was taken out from the column to obtain an extract of the cultured Mycelium lacticera mycelium.
제조예Manufacturing example 4. 4. 세리포리아Three Li Pori 락세라타Lacerata 배양액으로부터 세포외다당체( The extracellular polysaccharide ( exopolysaccharideexopolysaccharide ; 이하, "EPS"라 함)의 제조; Hereinafter referred to as "EPS")
상기 제조예 3에서 수득된 세리포리아 락세라타 균사체 배양액의 추출물을 다시 8,000 rpm으로 20분 동안 원심분리하였다. 이후, 침전물을 회수하여 조(crude) EPS를 얻었다. 상기 조 EPS를 진공동결건조기를 이용하여 25℃에서 72시간 진공동결건조시켜 세리포리아 락세라타에 의해 생산되는 EPS를 획득하였다.
The extract of the culture medium of the cellulolyticase obtained in Preparation Example 3 was further centrifuged at 8,000 rpm for 20 minutes. Thereafter, the precipitate was recovered to obtain crude EPS. The crude EPS was vacuum-lyophilized in a vacuum freeze dryer at 25 ° C for 72 hours to obtain an EPS produced by Sera lipolactacrata.
실시예Example
1. EPS의 특성 평가 1. Characterization of EPS
1.1. 겔 투과 크로마토그래피(gel permeation chromatography, 1.1. Gel permeation chromatography, GPCGPC )를 이용한 EPS의 분자량 측정Molecular weight measurement of EPS using
상기 제조예 4에서 제조한 EPS를 0.1 M Na2SO4/0.05 M NaN3[빙초산(glacial acetic acid)으로 pH를 4로 조정] 용액에 1%(w/v)가 되도록 녹인 다음, 4,000 rpm으로 0.5시간 동안 원심분리한 후, 상층액만을 0.45 ㎛ 시린지 필터(syringe filter)로 여과하여 GPC로 분석하였다.The EPS prepared in Preparation Example 4 was dissolved in a solution of 0.1 M Na 2 SO 4 /0.05 M NaN 3 (pH adjusted to 4 with glacial acetic acid) to a concentration of 1% (w / v) . After centrifugation for 0.5 hour, the supernatant was filtered with a 0.45 탆 syringe filter and analyzed by GPC.
GPC 분석을 위한 조건은 검출기로 굴절지수를 이용하였으며, GPC 컬럼은 OHpak SB 805 HQ(Shodex, Japan)를 이용하였고, 이동상은 0.1 M Na2SO4/0.05 M NaN3[빙초산(glacial acetic acid)으로 pH를 4로 조정]을 사용하였으며, 이동상의 유속은 0.1 ㎖/분의 속도로 흘려주었다. 표준곡선은 각기 다른 분자량(130 kDa, 400 kDa, 770 kDa 또는 1,200 kDa)을 가진 덱스트란(American Polymer Corporation, USA)을 이용하여 작성하였다. 굴절지수(refractive index, RI) 측정기 Knauer K-2310(Germany)을 이용하여 EPS의 분자량을 측정하였다. 측정 조건은 하기 표 1과 같다.The GPC column used was OHpak SB 805 HQ (Shodex, Japan) and the mobile phase was 0.1 M Na 2 SO 4 /0.05 M NaN 3 [glacial acetic acid, ] Was used, and the flow rate of the mobile phase was flowed at a rate of 0.1 ml / min. Standard curves were generated using dextran (American Polymer Corporation, USA) with different molecular weights (130 kDa, 400 kDa, 770 kDa or 1,200 kDa). The molecular weight of the EPS was measured using a refractive index (RI) meter Knauer K-2310 (Germany). The measurement conditions are shown in Table 1 below.
그 결과, 본 발명의 EPS 분자량은 약 120 kDa으로 나타났다.
As a result, the EPS molecular weight of the present invention was about 120 kDa.
1.2. EPS의 당 및 단백질 함량 측정1.2. Determination of sugar and protein content of EPS
상기 제조예 4에서 제조한 EPS를 2차 정제하고, 단백질 가수분해 효소를 처리하여 당 및 단백질 함량을 측정하였다.The EPS prepared in Preparation Example 4 was subjected to second purification and treated with a protein hydrolyzing enzyme to measure sugar and protein content.
구체적으로, 1차 정제된 EPS(제조예 4에서 제조한 EPS)를 증류수에 녹이고 8,000 rpm으로 20분 동안 원심분리하여 상등액을 분리하였다. 분리된 상등액에 그 양의 2 내지 3배에 해당하는 에탄올을 첨가하고 4℃ 냉장고에 넣어 12시간 정치시켰다. 그 후, 정치물에서 상등액만을 취해 이를 다시 8,000 rpm으로 20분 동안 원심분리하고 침전물을 회수하여 2차 정제된 EPS를 획득하였다. 상기 2차 정제된 EPS를 증류수에 용해하고, 단백질 가수분해 효소인 알칼레이즈(alcalase)를 0.5%(w/v)의 농도로 50℃에서 30분간 처리하였다.Specifically, the first purified EPS (EPS prepared in Preparation Example 4) was dissolved in distilled water and centrifuged at 8,000 rpm for 20 minutes to separate the supernatant. Ethanol corresponding to 2 to 3 times its amount was added to the separated supernatant, and the mixture was placed in a refrigerator at 4 캜 for 12 hours. Thereafter, the supernatant was taken from the column, centrifuged again at 8,000 rpm for 20 minutes, and the precipitate was recovered to obtain a second purified EPS. The second purified EPS was dissolved in distilled water and the protein hydrolyzing enzyme alcalase was treated at a concentration of 0.5% (w / v) at 50 캜 for 30 minutes.
당 함량은 페놀-황산법(phenol-sulfuric acid method)에 의해 측정하였다. 구체적으로, 농도별로 희석한 시료 1 ㎖에 80%(w/v) 페놀을 25 ㎕ 첨가한 후, 황산 2.5 ㎖를 첨가하고 실온으로 냉각하였다. 이를 465 ㎚의 파장에서 흡광도를 측정하여 당 함량을 계산하였다.The sugar content was measured by the phenol-sulfuric acid method. Concretely, 25 μl of 80% (w / v) phenol was added to 1 ml of the sample diluted by concentration, and then 2.5 ml of sulfuric acid was added and the mixture was cooled to room temperature. The sugar content was calculated by measuring the absorbance at a wavelength of 465 nm.
또한, 단백질 함량은 BCA 방법(Smith PK et al., Analytical Biochemistry, 150(1):76-85, 1985)으로 측정하였고, 표준품으로 소혈청알부민을 사용하였다.Protein content was also measured by BCA method (Smith PK et al. , Analytical Biochemistry, 150 (1): 76-85, 1985) and bovine serum albumin was used as a standard.
상술한 바와 같이 측정한 당 및 단백질 함량을 하기 표 2에 나타내었으며, EPS의 당 함량은 45 내지 51 중량%, 단백질 함량은 33 내지 34 중량%인 것으로 나타났다.The sugar and protein contents measured as described above are shown in Table 2 below, and the sugar content of EPS was 45 to 51% by weight and the protein content was 33 to 34% by weight.
*효소 처리: 알칼레이즈 0.5%, 50℃, 30분. * Enzyme treatment: Alkaline 0.5%, 50 ℃, 30 minutes.
각 수치는 평균±SE(n≥3)임.
Each value is mean ± SE (n ≥ 3).
또한, EPS의 당 구성성분 분석 결과, EPS는 주로 만노오스, 갈락토오스 및 글루코오스를 함유하고 있는 것으로 나타났다.
Also, as a result of analyzing sugar components of EPS, it was found that EPS mainly contains mannose, galactose and glucose.
실시예Example 2. 자외선에 대한 피부 보호효과 확인 2. Confirm skin protection effect against UV rays
상기 제조예 4에서 제조한 EPS의 자외선에 대한 피부 보호 효과를 확인하기 위해 MTT 분석을 하여 세포 생존율을 측정하였다.The cell survival rate of the EPS prepared in Preparation Example 4 was measured by MTT analysis to confirm skin protection effect against ultraviolet rays.
먼저, 인간 각질세포주인 HaCaT 세포주(CLS, 독일)는 10% 소태아혈청, 100 ㎍/㎖ 스트렙토마이신 및 100 U/㎖ 페니실린이 첨가된 DMEM 배지로 37℃, 5% CO2 조건하에서 배양하였다. 상기 배양된 세포를 2x104 cell/㎖이 되도록 96-웰 플레이트에 각각 100 ㎕씩 분주하여 24시간 동안 배양하였다. 여기에 상기 EPS를 25 또는 50 ㎍/㎖의 농도가 되도록 첨가하였다. 첨가 2시간 후, 상기 세포주에 UV-B 램프 8개를 20 ㎝ 거리에서 30초간 조사하여 20 mJ/㎠의 강도로 자외선을 조사하였다.First, the human keratinocyte cell line HaCaT cell line (CLS, Germany) was cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 μg / ml streptomycin and 100 U / ml penicillin at 37 ° C. and 5% CO 2 . 100 [mu] L of each of the cultured cells was divided into 96-well plates so as to have a concentration of 2 x 10 < 4 > cells / mL and cultured for 24 hours. The EPS was added thereto to a concentration of 25 or 50 占 퐂 / ml. Two hours after the addition, eight UV-B lamps were irradiated to the cell line at a distance of 20 cm for 30 seconds to irradiate ultraviolet rays at an intensity of 20 mJ / cm 2.
UV-B를 조사하고 24시간 후, 각 웰에 PBS에 녹인 MTT(1 ㎎/㎖)를 10 ㎕씩 첨가하여 4시간 동안 반응시켰다. 4시간 후, 포르마잔 형성을 확인하고, 포르마잔이 흩어지지 않게 상등액을 완전히 제거한 다음, 100 ㎕의 DMSO를 첨가하여 녹인 후, ELISA 리더를 이용하여 570 ㎚ 파장에서 흡광도를 측정하였다.Twenty-four hours after irradiation with UV-B, 10 쨉 l of MTT (1 mg / ml) dissolved in PBS was added to each well, followed by reaction for 4 hours. After 4 hours, formation of formazan was confirmed, and the supernatant was completely removed so that the formazan was not dispersed. Then, 100 μl of DMSO was added to dissolve it, and the absorbance at 570 nm was measured using an ELISA reader.
결과 값은 EPS와 UVB를 모두 처리하지 않은 음성 대조군의 값을 100%로 하였을 때 상대적인 값으로 계산하여 도 1에 나타내었다.The results are shown in FIG. 1 as a relative value when the value of the negative control group not treated with both EPS and UVB is taken as 100%.
그 결과, 도 1에 나타난 바와 같이 UVB를 조사한 경우 HaCaT 세포주의 생존율은 약 60%이었으나, EPS를 처리하고 UVB를 조사한 경우에는 세포 생존율이 약 80%까지 증가하였다.As a result, as shown in FIG. 1, the survival rate of HaCaT cell line was about 60% when UVB was irradiated, but the cell survival rate was increased to about 80% when EPS was treated and UVB was irradiated.
이로부터 본 발명에 따른 EPS뿐만 아니라, 상기 EPS를 포함하는 세리포리아 락세라타의 균사체 배양액, 상기 균사체 배양액의 건조분말, 및 상기 균사체 배양액의 추출물도 자외선에 의한 손상으로부터 피부를 보호하는 효과가 있음을 알 수 있다.From this, not only the EPS according to the present invention but also the culture medium of the mycelium of the cellulolytic enzyme, including the EPS, the dried powder of the mycelium culture liquid and the extract of the mycelial culture liquid have an effect of protecting the skin from damage by ultraviolet rays .
Claims (12)
An extracellular polysaccharide produced by Ceriporia lacerata ; A mycelial culture solution of ceriplora lacerata containing the extracellular polysaccharide; A dry powder of the mycelial culture liquid; Or an extract of said mycelial liquid as an active ingredient.
The composition of claim 1, wherein the extracellular polysaccharide comprises from 40 to 60% by weight of sugar and from 30 to 40% by weight of protein and has a molecular weight of from 100 to 150 kDa.
3. The composition of claim 2, wherein the extracellular polysaccharide comprises 43 to 47% by weight of sugar and 33 to 36% by weight of protein and has a molecular weight of 115 to 125 kDa.
The skin protecting composition according to claim 2, wherein the sugar contains mannose, galactose and glucose.
(a) 세리포리아 락세라타 균사체를 액체 배양하여 세리포리아 락세라타 균사체 배양액을 제조하는 단계;
(b) 세리포리아 락세라타 균사체 배양액을 건조시켜 분말화하는 단계; 및
(c) 세리포리아 락세라타 균사체 배양액 분말을 용매로 추출한 후, 이를 여과하고 감압 농축하는 단계를 포함하는 제조 방법에 의해 제조된, 피부 보호용 조성물.
2. The composition of claim 1, wherein the extracellular polysaccharide is selected from the group consisting of:
(a) liquid culturing the mycelium of ceriplora lactaclora to prepare a culture medium of a seriposita lactamera mycelium;
(b) drying and cultivating the culture medium of the mycelium of ceriplora lactaclora; And
(c) extracting the culture medium of the mycelium of Cerporaria lacerata with a solvent, filtering it, and concentrating under reduced pressure.
The method of claim 5, wherein the medium is a sugar, glucose, starch, be water, barley minutes for the liquid culture, soy flour, magnesium sulfate (MgSO 4), 1 potassium phosphate (KH 2 PO 4), 2 potassium phosphate (K 2 HPO 4 ) and water, and has a hydrogen ion concentration (pH) of 4.5 to 6.0.
6. The composition of claim 5, wherein the liquid culture is performed under a blue LED light source.
6. The skin protection composition according to claim 5, wherein the liquid culture is performed by maintaining the concentration of carbon dioxide at 1,000 to 2,000 ppm.
9. A pharmaceutical composition for treating skin damage caused by ultraviolet rays, comprising the composition according to any one of claims 1 to 8 as an active ingredient.
A cosmetic composition for preventing and improving skin damage caused by ultraviolet rays, comprising the composition according to any one of claims 1 to 8 as an active ingredient.
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| KR20190003404A (en) * | 2017-06-30 | 2019-01-09 | (주)퓨젠바이오 | Pharmaceutical composition for treating scar comprising culture medium of ceriporia lacerata |
| KR20200133063A (en) | 2019-05-15 | 2020-11-26 | 김병천 | A cosmetic composition comprising a culture of Ceriporia lamaritus for skin improvement |
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| KR101031605B1 (en) | 2010-11-11 | 2011-04-27 | 김병천 | Manufacturing method of culture extract of ceriporia lacerata for the therapy of diabetic diseases and culture extract of ceriporia lacerata using the same |
| WO2014112665A1 (en) * | 2013-01-18 | 2014-07-24 | (주)월드바이오텍 | Pharmaceutical composition and health functional food for preventing or treating steroid-induced diabetes |
| KR101522415B1 (en) * | 2015-02-26 | 2015-05-21 | 한상선 | Cosmetic composition including an acanthus extract of ceriporia lacerata and cosmetics using the same |
| KR101549148B1 (en) | 2013-10-29 | 2015-09-03 | 주식회사 세바바이오텍 | Composition for protecting and curing human keratinocyte against UV-B comprising Phryma leptostachya var. asiatica Hara ethanol extract |
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| JP2004339138A (en) * | 2003-05-15 | 2004-12-02 | Kanebo Ltd | Skin cosmetic |
| JP2005015348A (en) * | 2003-06-24 | 2005-01-20 | Asahi Denka Kogyo Kk | Skin cosmetic |
| KR102073943B1 (en) * | 2012-12-21 | 2020-02-06 | 주식회사 코씨드바이오팜 | A skin-care agent or cosmetics containig Lyophyllum ulmarium fruit body extract or Lyophyllum ulmarium mycelium extract |
| US20140193454A1 (en) * | 2013-01-09 | 2014-07-10 | Byoung Cheon KIM | Method from preparing ceriporia lacerata culture extract and pharmaceutical composition for prevention or treatment of diabetes and diabetic complications comprising ceriporia lacerata culture extract as active ingredient |
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- 2015-11-26 KR KR1020150166466A patent/KR101682101B1/en active IP Right Grant
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- 2016-11-23 WO PCT/KR2016/013531 patent/WO2017090970A1/en active Application Filing
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| KR101031605B1 (en) | 2010-11-11 | 2011-04-27 | 김병천 | Manufacturing method of culture extract of ceriporia lacerata for the therapy of diabetic diseases and culture extract of ceriporia lacerata using the same |
| WO2014112665A1 (en) * | 2013-01-18 | 2014-07-24 | (주)월드바이오텍 | Pharmaceutical composition and health functional food for preventing or treating steroid-induced diabetes |
| KR101549148B1 (en) | 2013-10-29 | 2015-09-03 | 주식회사 세바바이오텍 | Composition for protecting and curing human keratinocyte against UV-B comprising Phryma leptostachya var. asiatica Hara ethanol extract |
| KR101522415B1 (en) * | 2015-02-26 | 2015-05-21 | 한상선 | Cosmetic composition including an acanthus extract of ceriporia lacerata and cosmetics using the same |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20190003404A (en) * | 2017-06-30 | 2019-01-09 | (주)퓨젠바이오 | Pharmaceutical composition for treating scar comprising culture medium of ceriporia lacerata |
| WO2019004785A3 (en) * | 2017-06-30 | 2019-02-28 | (주)퓨젠바이오 | Pharmaceutical composition for wound healing comprising ceriporia lacerata culture medium |
| JP2020525405A (en) * | 2017-06-30 | 2020-08-27 | フジェンビオ カンパニー, リミテッドFugenbio Co., Ltd. | Wound healing pharmaceutical composition containing Ceripolia laserata culture |
| KR102150595B1 (en) * | 2017-06-30 | 2020-09-01 | (주)퓨젠바이오 | Pharmaceutical composition for treating scar comprising culture medium of ceriporia lacerata |
| KR20200133063A (en) | 2019-05-15 | 2020-11-26 | 김병천 | A cosmetic composition comprising a culture of Ceriporia lamaritus for skin improvement |
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| WO2017090970A1 (en) | 2017-06-01 |
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