KR20070052091A - Natural antioxidant using oltmannsiellopsis unicellularis - Google Patents
Natural antioxidant using oltmannsiellopsis unicellularis Download PDFInfo
- Publication number
- KR20070052091A KR20070052091A KR1020050109714A KR20050109714A KR20070052091A KR 20070052091 A KR20070052091 A KR 20070052091A KR 1020050109714 A KR1020050109714 A KR 1020050109714A KR 20050109714 A KR20050109714 A KR 20050109714A KR 20070052091 A KR20070052091 A KR 20070052091A
- Authority
- KR
- South Korea
- Prior art keywords
- unicellularis
- ability
- oltmannsiellopsis
- natural antioxidant
- oltmansiellopsis
- Prior art date
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Classifications
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/02—Algae
- A61K36/05—Chlorophycota or chlorophyta (green algae), e.g. Chlorella
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/302—Foods, ingredients or supplements having a functional effect on health having a modulating effect on age
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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Abstract
본 발명은 올트맨시엘롭시스 유니셀루러리스( Oltmannsiellopsis unicellularis)를 이용한 천연 항산화제 조성물에 관한 것으로, 더욱 상세하게는, 청정해안으로 알려져 있는 한국의 제주도연안 조수웅덩이에 서식하는 미세조류 중, 우점종으로 존재하는 해양식물성 플랑크톤인 미세녹조류 녹조강(Chlorophyceae)의 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)에 얻어진 분획물과 추출물이 활성산소종 억제능력, 자유 라디칼 NO. 저해능력, 메탈킬레이팅(Metal chelating) 능력, 환원력, 지질과산화 저해능력, TBARS형성 억제능력, CDH형성 억제능력 등이 있음을 증명하고, 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)를 이용한 의학적 약품 및 기능성 식품소재나 건강식품 등의 천연 항산화제 조성물을 제공함으로써, 활성 산소종(Hydrogen peroxide, Superoxide, Hydroxyl radical)과 프리 라디칼(Nitric oxide와 Nitrogen dioxide)에 의한 인체의 세포나 조직 및 식품의 산화적인 손상을 억제할 뿐만아니라, 종래에 항산화를 위하여 사용되던 합성 항산화제의 간 손상과 면역력 감소, 심장병, 암유발 등의 부작용을 방지할 수 있는 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis )를 이용한 천연 항산화제 조성물에 관한 것이다. The invention come teumaen Ciel Rob System Uni cellular multiple leases relates to a natural antioxidant composition with (Oltmannsiellopsis unicellularis), more specifically, of micro-algae that live in Cheju Island coast tide pools of Korea, known as clean shore, dominant species Fractions and extracts obtained from Oltmannsiellopsis unicellularis of marine green plankton, Chlorophyceae, a marine phytoplankton, have the ability to inhibit free radical species and free radical NO. It proved that it has inhibitory ability, metal chelating ability, reducing ability, lipid peroxidation inhibitory ability, TBARS formation inhibitory ability, CDH formation inhibitory ability, and the like, using Oltmansiellopsis unicellularis. By providing natural antioxidant compositions such as medical drugs and functional food materials and health foods, human cells, tissues and foods by active oxygen species (Hydrogen peroxide, Superoxide, Hydroxyl radical) and free radicals (Nitric oxide and Nitrogen dioxide) In addition to inhibiting the oxidative damage of Otmannsiellopsis (Oltmannsiellopsis), which can prevent side effects such as liver damage, decreased immunity, heart disease, cancer, etc. unicellularis ) to a natural antioxidant composition.
미세조류, 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis), 활성산소 제거, 항산화제 Microalgae, Oltmansiellopsis unicellularis, free radicals, antioxidants
Description
도 1은 한국의 제주도의 지도중 샘플링을 한 지점의 표시도. 1 is a display diagram of a sampling point of a map of Jeju Island in Korea.
도 2는 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)용매에 의한 분획과정도. Figure 2 is a fractional process by the solvent Oltmansiellopsis unicellularis (Oltmannsiellopsis unicellularis).
도 3은 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)로부터의 분획물과 추출물들의 DPPH 라디칼 소거 효과결과그래프. 3 is a graph of the results of DPPH radical scavenging effect of fractions and extracts from Oltmannsiellopsis unicellularis.
도 4는 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)의 추출물과 분획물의 슈퍼옥사이드 음이온(Superoxide anion) 소거 활성 효과결과그래프. 4 is a graph showing the results of the superoxide anion scavenging activity of the extracts and fractions of Oltmann siellopsis unicellularis (Oltmannsiellopsis unicellularis).
도 5는 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)의 추출물과 분획물의 하이드로겐 페록사이드(hydrogen peroxide) 소거 활성 효과결과그래프. 5 is a graph showing the results of hydrogen peroxide scavenging activity of extracts and fractions of Oltmann siellopsis unicellularis (Oltmannsiellopsis unicellularis).
도 6은 과산화수소수(H2O2) 에 의해 유도한 쥐림프구의 디엔에이 손상(rat lymphocytes DNA damage)에 대한 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)의 물 추출물의 다른 농도별 인비트로(in vitro)상에서의 억제 효과결과그래프. FIG. 6 shows different concentration-specific in vitro of water extracts of Oltmannsiellopsis unicellularis against rat lymphocytes DNA damage induced by hydrogen peroxide (H 2 O 2). Inhibitory effect on the graph.
도 7은 도6의결과에 따른 쥐 림프구의 코멧이미지(Comet images)의 도시도. FIG. 7 is a diagram of Comet images of rat lymphocytes according to the results of FIG. 6.
도 8은 H2O2 에 의해 유도한 쥐림프구의 디엔에이 손상(rat lymphocytes DNA damage)에 대한 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)의 80% 메탄올 추출물의 다른 농도별 인 비트로(in vitro)상에서의 억제 효과결과그래프FIG. 8 shows different concentrations of 80% methanol extracts of Oltmannsiellopsis unicellularis against rat lymphocytes DNA damage induced by H 2 O 2 . Graph of results of inhibitory effects in vitro
도 9는 도8의 결과에 따른 쥐 림프구의 코멧이미지(Comet images)의 도시도. 9 is a diagram of Comet images of rat lymphocytes according to the results of FIG. 8.
도 10은 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)의 추출물과 분획물의 하이드록실라디칼(Hydroxyl radical) 소거 활성 효과결과그래프. 10 is a graph showing the results of the hydroxyl radical scavenging activity of the extracts and fractions of Oltmann siellopsis unicellularis (Oltmannsiellopsis unicellularis).
도 11은 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)의 추출물과 분획물의 니트릭옥사이드(Nitric oxide) 소거 활성 효과결과그래프. FIG. 11 is a graph showing the results of nitric oxide scavenging activity of extracts and fractions of Oltmansiellopsis unicellularis.
도 12는 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)의 추출물과 분획물의 메탈킬레이팅(Metal chelating) 소거 활성 효과결과그래프. 12 is a graph showing the results of metal chelating scavenging activity of extracts and fractions of Oltmansiellopsis unicellularis.
도 13은 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)의 추출물과 분획물의 리듀싱파워(Reducing power) 소거 활성 효과결과그래프. FIG. 13 is a graph of the results of reducing power scavenging activity of extracts and fractions of Oltmannsiellopsis unicellularis. FIG.
도 14는 헤모글로빈에 의해 유도된 리놀레익(linolecic)산의 과산화에 대한 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)의 추출물과 분획물의 리듀싱파워(Reducing power) 소거 활성 효과결과그래프. 14 is a graph showing the results of reducing power scavenging activity of extracts and fractions of Oltmansiellopsis unicellularis on peroxidation of linolecic acid induced by hemoglobin.
도 15는 60°C의 가속화된 산화 상태의 암실에서 저장동안 리놀레익(linoleic)산에서 TBARS(thiobarbituric acid reactive substances) 의 형성에 대한 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis) 클로로포름 분획물의 효과결과그래프. FIG. 15 shows the Oltmannsiellopsis unicellularis chloroform fraction for the formation of thibarbituric acid reactive substances (TBARS) in linoleic acid during storage in the dark at 60 ° C. Effect result graph.
도 16은 60℃의 가속화된 산화 상태의 암실에서 저장된 리놀레익(linoleic)산에서 콘쥬게이트된 디엔의 하이드로프록사이드(hydroproxide)의 형성에 대한 Oltmannsiellopsis unicellularis의 클로로포름 분획물의 효과결과그래프 . .FIG. 16 is a result graph of the effect of the chloroform fraction of Oltmannsiellopsis unicellularis on the formation of hydroproxide of conjugated dienes in stored linoleic acid in the dark at 60 ° C. in an accelerated oxidation state. .
본 발명은 올트맨시엘롭시스 유니셀루러리스( Oltmannsiellopsis unicellularis)를 이용한 천연 항산화제 조성물에 관한 것으로, 더욱 상세하게는, 청정해안으로 알려져 있는 한국의 제주도연안 조수웅덩이에 서식하는 미세조류 중, 우점종으로 존재하는 해양식물성 플랑크톤인 미세녹조류 녹조강(Chlorophyceae)의 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)에 얻어진 분획물과 추출물이 활성산소종 억제능력, 자유 라디칼 NO.저해능력, 메탈킬레이팅(Metal chelating) 능력, 환원력, 지질과산화 저해능력, TBARS형성 억제능력, CDH형성 억제능력 등이 있음을 증명하고, 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)를 이용한 의학적 약품 및 기능성 식품소재나 건강식품 등의 천연 항산화제 조성물을 제공함으로써, 활성 산소종(Hydrogen peroxide, Superoxide, Hydroxyl radical)과 프리 라디칼(Nitric oxide와 Nitrogen dioxide)에 의한 인체의 세포나 조직 및 식품의 산화적인 손상을 억제할 뿐만아니라, 종래에 항산화를 위하여 사용되던 합성 항산화제의 간 손상과 면역력 감소, 심장병, 암유발 등의 부작용을 방지할 수 있는 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)를 이용한 천연 항산화제 조성물에 관한 것이다.The invention come teumaen Ciel Rob System Uni cellular multiple leases relates to a natural antioxidant composition with (Oltmannsiellopsis unicellularis), more specifically, of micro-algae that live in Cheju Island coast tide pools of Korea, known as clean shore, dominant species Fractions and extracts obtained from Oltmansiellopsis unicellularis of marine green plankton, Chlorophyceae, a marine phytoplankton, were found to inhibit reactive oxygen species, free radical NO. (Metal chelating) ability, reducing ability, lipid peroxidation inhibitory ability, TBARS formation inhibitory ability, CDH formation inhibitory ability, etc., and medical drugs and functional food materials using Oltmansiellopsis unicellularis By providing a natural antioxidant composition, such as health foods, active oxygen species (Hydrogen peroxide, In addition to suppressing oxidative damage to human cells, tissues, and foods caused by superoxide, hydroxy radicals and free radicals (nitric oxide and nitrogen dioxide), it also reduces liver damage and immunity of synthetic antioxidants used for antioxidants. relates to heart disease, Ciel come teumaen which can prevent side effects such as cancer Rob system Uni multiple cellular less natural antioxidant composition with (Oltmannsiellopsis unicellularis).
일반적으로, 활성 산소종(Hydrogen peroxide, Superoxide, Hydroxyl radical)과 프리 라디칼(Nitric oxide와 Nitrogen dioxide)은 의학적인 분야에서는 생체구성 성분들과 반응하여 세포나 조직에산화적 손상을 입히고 이러한 손상들이 축적되어 아테롬성 동맥 경화증, 간경변, 기종, 노화와 암등의 질병을 유발하며, 식품 분야에서는 저장과 제조 과정동안 지질과산화에 의한 식품의 변질과 변색을 일으켜더 나아가 돌연변이와 동맥경화증, 암을 유발한다고 알려져 있다. In general, reactive oxygen species (Hydrogen peroxide, Superoxide, Hydroxyl radical) and free radicals (Nitric oxide and Nitrogen dioxide) in the medical field react with biological components to cause oxidative damage to cells or tissues and accumulate these damages. It causes diseases such as atherosclerosis, cirrhosis, emphysema, aging and cancer.In the food field, it is known that it causes the deterioration and discoloration of food by lipid peroxidation during the storage and manufacturing process, and also causes mutations, atherosclerosis and cancer. .
이러한 많은 문제점들을 해결하기 위해, 합성항산화제인 BHT, BHA, α-tocopherol, propyl gallate(PG)와 tert-butylhydroquinone(TBHQ) 등이 산화적인 손상을 억제해 주기 때문에 의학 분야와 식품 분야에서 사용되어지고 있다. 그러나 이런 합성 항산화제는 인체 내에서 간 손상과 면역력 감소, 심장병, 암유발 등의 많은 부작용을 가지고 있다는 점에서 이용이 규제되어지고 있으며, 천연자원을 이용한 천연항산화제의 개발과 연구에 전 세계의 모든 연구자들이 큰 관심을 갖고 있 는 실정이다. 이런 천연 항산화제들로 각광 받고 있는 것 중에서, 해양에서 생산되는 해조류와 미세조류를 들 수 있다. 이것들은 호기적인 상태에서 햇빛에 노출되어져 있으며, 산화 작용을 억제한다고 알려져 있는 Docosahexaenoic acid (DHA), Eicosapentenoic acid (EPA) 와 같은 불포화 지방산과 많은 폴리 페놀릭 성분을 가지고 있어 활성화된 산소종에 대한 방어능력이 발달한 것으로 알려져 있다. To solve many of these problems, synthetic antioxidants such as BHT, BHA, α-tocopherol, propyl gallate (PG) and tert-butylhydroquinone (TBHQ) are used in the medical and food fields because they inhibit oxidative damage. have. However, these synthetic antioxidants are regulated in that they have many side effects such as liver damage, reduced immunity, heart disease, cancer, etc. in the human body. All researchers are very interested. Among these natural antioxidants, seaweeds and microalgae produced in the ocean are mentioned. They are exposed to sunlight in aerobic conditions and contain many polyphenolic compounds and unsaturated fatty acids, such as Docosahexaenoic acid (DHA) and Eicosapentenoic acid (EPA), which are known to inhibit oxidation. Ability is known to have developed.
이런 해조류에 대해서는 지난 10여년간 많은 항산화 연구가 진행되어 왔고, 진행되어 가고 있는 실정이나, 미세조류에 대한 연구는 많이 필요한 실정이다. 따라서 이번 연구에서는 청정해안으로 알려져 있는 한국의 제주도 연안 조수웅덩이에 서식하는 미세조류 중 우점종으로 존재하는 해양식물성 플랑크톤인 미세녹조류 녹조강(Chlorophyceae)의 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis )는 배양이 매우 쉬울 뿐만 아니라 대량 생산도 가능하기 때문에 기능성 혹은 건강 식품소재로서의 응용될 수 있지만 이에 관한 연구는 현재까지 전혀 이루어지고 있지 않다.Antioxidant research has been conducted on these algae over the last decade, and the current situation is progressing, but much research on microalgae is required. Therefore, this study come teumaen Ciel Rob sheath Uni Cellular multiple lease of micro algae chlorophyceae (Chlorophyceae) of marine phytoplankton present in the dominant species of algae that inhabit the island coastal tide pools of Korea, known as the Clean Coast (Oltmannsiellopsis unicellularis) It can be applied as a functional or health food material because it is not only easy to cultivate but also can be mass-produced, but there is no research on this.
이에, 활성산소종 억제능력, 자유 라디칼 NO 저해능력, 메탈킬레이팅(Metal chelating) 능력, 환원력, 지질과산화 저해능력, TBARS 형성 억제능력, CDH 형성 억제능력을 가지는 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)에 얻어진 분획물과 추출물의 효능을 유용하게 활용할 수 없는 문제점이 있었다. Accordingly, Oltman Cielopsis Unicellluris has the ability to inhibit reactive oxygen species, free radical NO inhibitory ability, metal chelating ability, reducing power, lipid peroxidation inhibitory ability, TBARS formation inhibitory ability, CDH formation inhibitory ability (Oltmannsiellopsis unicellularis) there was a problem that can not effectively utilize the efficacy of the fractions and extracts obtained.
따라서 본 발명의 목적은 상기와 같은 종래의 여러 가지 문제점을 개선시키기 위하여 도출한 것으로, 더욱 상세하게는, 청정해안으로 알려져 있는 한국의 제주도연안 조수웅덩이에 서식하는 미세조류 중, 우점종으로 존재하는 해양식물성 플랑크톤인 미세녹조류 녹조강(Chlorophyceae)의 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)에 얻어진 분획물과 추출물이 활성산소종 억제능력, 자유 라디칼 NO 저해능력, 메탈킬레이팅(Metal chelating) 능력, 환원력, 지질과산화 저해능력, TBARS 형성 억제능력, CDH 형성 억제능력 등이 있음을 증명하고, 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)를 이용한 의학적 약품 및 기능성 식품소재나 건강식품 등의 천연 항산화제 조성물을 제공함으로서, 활성 산소종(Hydrogen peroxide, Superoxide, Hydroxyl radical)과 프리 라디칼(Nitric oxide와 Nitrogen dioxide)에 의한 인체의 세포나 조직 및 식품의 산화적인 손상을 억제할 뿐만아니라, 종래에 항산화를 위하여 사용되던 합성 항산화제의 간 손상과 면역력 감소, 심장병, 암유발 등의 부작용을 방지할 수 있는 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)를 이용한 천연 항산화제 조성물을 제공하는데 그 목적이 있다.Therefore, the object of the present invention was derived in order to improve the various problems as described above, more specifically, the micro-algae in the tidal pools of the Jeju island coast of Korea known as the clean coast, which exist as a dominant species Fractions and extracts of phytoplankton, Chlorophyceae, from Oltmansiellopsis unicellularis, are capable of inhibiting free radical species, free radical NO inhibition, and metal chelating ability. It has been proved to have the ability to inhibit, reduce, peroxidation of lipids, inhibit TBARS formation, and inhibit CDH formation. By providing a natural antioxidant composition, active oxygen species (Hydrogen peroxide, Superoxide, Hydroxyl In addition to inhibiting oxidative damage to human cells, tissues, and foods caused by radicals and free radicals (Nitric oxide and Nitrogen dioxide), they also reduce liver damage, immune immunity, heart disease, It is an object of the present invention to provide a natural antioxidant composition using Oltmansiellopsis unicellularis that can prevent side effects such as cancer.
본 발명은 올트맨시엘롭시스 유니셀루러리스 ( Oltmannsiellopsis unicellularis)를 이용한 천연 항산화제 조성물에 관한 것으로, 더욱 상세하게는, 청정해안으로 알려져 있는 한국의 제주도연안 조수웅덩이에 서식하는 미세조류 중, 우점종으로 존재하는 해양식물성 플랑크톤인 미세녹조류 녹조강(Chlorophyceae)의 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)에 얻어진 분획물과 추출물이 활성산소종 억제능력, 자유 라디칼 NO.저해능력, 메탈킬레이팅(Metal chelating) 능력, 환원력, 지질과산화 저해능력, TBARS형성 억제능력, CDH형성 억제능력 등이 있음을 증명하고, 올트맨시엘롭시스 유니셀루러리스( Oltmannsiellopsis unicellularis )를 이용한 의학적 약품 및 기능성 식품소재나 건강식품 등의 천연 항산화제 조성물을 제공함으로서, 활성 산소종(Hydrogen peroxide, Superoxide, Hydroxyl radical)과 프리 라디칼(Nitric oxide와 Nitrogen dioxide)에 의한 인체의 세포나 조직 및 식품의 산화적인 손상을 억제할 뿐만아니라, 종래에 항산화를 위하여 사용되던 합성 항산화제의 간 손상과 면역력 감소, 심장병, 암유발 등의 부작용을 방지할 수 있는 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis )를 이용한 천연 항산화제 조성물에 관한 것이다.The present invention Oltman Cielopsis Uni Cellular multiple leases relates to a natural antioxidant compositions using (Oltmannsiellopsis unicellularis), more specifically, a marine phytoplankton that of the algae that inhabit the island coastal tide pools of Korea, known as the Clean Coast presence as a dominant species fine Fractions and extracts obtained from Oltmannsiellopsis unicellularis of Chlorophyceae have the ability to inhibit reactive oxygen species, free radical NO., Metal chelating ability, reducing power, lipid peroxidation inhibitory ability, demonstrated that the suppression of such TBARS formation, suppression of CDH formed and all teumaen Ciel Rob system Uni cellular multiple-less (Oltmannsiellopsis By providing a natural antioxidant composition such as medical drugs and functional food materials or health foods using unicellularis ) , cells of the human body by active oxygen species (Hydrogen peroxide, Superoxide, Hydroxyl radical) and free radicals (Nitric oxide and Nitrogen dioxide) as well as to suppress oxidative damage and tissues and products, damage to the immune system decreased liver that was used for the antioxidant to the conventional synthetic antioxidants, heart disease, Rob Ciel come teumaen which can prevent side effects such as cancer. system Uni cellulose It relates to a natural antioxidant composition using Rulis (Oltmannsiellopsis unicellularis ) .
본 발명의 구성을, 실험준비단계를 나타낸 참조예와, 상기의 효과를 증명하기 위한 실험방법을 나타낸 실험예와 해당실험의 결과그래프를 나타낸 도면을 통해 설명하면 다음과 같다. Referring to the configuration of the present invention, a reference example showing the experimental preparation step, an experimental example showing the experimental method for demonstrating the above effect and the result graph of the corresponding experiment will be described as follows.
1. 실험 준비 단계 1. Experiment Preparation
[[ 참조예Reference Example 1] 샘플 수집 1] sample collection
한국의 제주도의 강정 연안의 해수로부터 식물성 플랑크톤은 2004년 11월에 수집하였으며, 수집된 해수 샘플은 1000ml의 폴리에틸렌성 병과 25ml 플라스크에 모아졌고, 환경적인 요소는 수온-염분 측정기(Termo-Salino meter)와 pH 측정기를 이용하여 샘플링 지점에서 측정되었다. 수온, pH와 염분정도는 각각 12.7℃에서 8.22와 35.4‰였다. 샘플들은 즉시 플랜트(plant) 배양 챔버로 옮겨졌고, 분리를 위해 낮과 밤의 주기를 12:12로 20℃에서 보관되어졌다. 배양된 식물플랑크톤의 동정을 위해, 샘플들은 400배율로 상대조 현미경 하에서 관찰되어졌고, 동정은 Carmelo R. Tomas의 도감을 참조하였다(도 1참조). Phytoplankton was collected in November 2004 from seawater off the Gangjeong coast of Jeju Island, Korea. The collected seawater samples were collected in 1000 ml of polyethylene bottles and 25 ml flasks, and the environmental factors were termo-salino meters. And at the sampling point using a pH meter. Water temperature, pH and salinity were 8.22 and 35.4 ‰ at 12.7 ℃, respectively. Samples were immediately transferred to a plant culture chamber and stored at 20 ° C. with 12:12 day and night cycles for separation. For the identification of cultured phytoplankton, samples were observed under relative light microscope at 400 magnification and the identification referred to the illustration of Carmelo R. Tomas (see FIG. 1).
[[ 참조예Reference Example 2] 2] 식물플랑크톤의Phytoplankton 대량배양과 동결건조 Mass culture and freeze drying
식물플랑크톤의 대량 배양은 f/2 영양소가 풍부한 멸균된 인공적인 해수 배지에서 행해졌고, 배양의 조건은 염분, 온도, pH, 낮과 밤의 주기와 광량이 각각 32‰, 22℃, 8.20, 12:12와 450uE였다. 식물 플랑크톤 세포들은 6000 rpm에서 5분간 원심분리 되어졌고, 페트리트 접시로 옮겨져 분리된 후, 동결 건조되어졌다. Mass cultivation of phytoplankton was carried out in sterile artificial seawater medium rich in f / 2 nutrients, and the conditions of the cultivation were salt, temperature, pH, day and night cycles, and light intensity of 32 ‰, 22 ° C, 8.20, and 12, respectively. : 12 and 450uE. Phytoplankton cells were centrifuged at 6000 rpm for 5 minutes, transferred to a Petri dish, separated and lyophilized.
[[ 참조예Reference Example 3] 3] 올트맨시엘롭시스Oltman Cielopsis 유니셀루러리스( Unicelllureless ( OltmannsiellopsisOltmannsiellopsis unicellularisunicellularis )의 추출물과 Extracts) 분획물Fraction 제조 Produce
건조된 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis) 시료들은 정밀한 파우더로 갈아졌고, 5g은 80% 메탄올 1ℓ를 가지고 25℃에서 24시간동안 추출되어졌다. 그 후, Buchner funnel을 이용하여 여과되어졌고, 여기서 얻어진 메탄올 추출물은 농축기를 이용하여 농축되어졌다. 농축된 추출물은 분획 여두를 가지고 n-핵산(n-hexane), 클로로포름(chloroform)과 에틸아세이트(ethyl acetate) 용액 1000ml로 분획되어졌다. 분획물들과 증류수 1ℓ에 파우더 5g을 추출하여 얻은 물 추출물을 농축기를 이용하여 농축하였다. 이렇게 얻어진 분획물들과 추출물의 활성은 합성 항산화제인 메탄올을 가지고 녹인 BHT와 α- tocopherol 의 활성과 비교되어졌다(도 2참조). Dried Oltmannsiellopsis unicellularis samples were ground to a fine powder and 5 g were extracted for 24 hours at 25 ° C. with 1 L of 80% methanol. It was then filtered using Buchner funnel, and the methanol extract obtained was concentrated using a concentrator. The concentrated extract was fractionated with 1000 ml of n-hexane, chloroform and ethyl acetate solution with fractional filter head. Fractions and water extract obtained by extracting 5g of powder into 1L of distilled water were concentrated using a concentrator. The activities of the fractions and extracts thus obtained were compared with those of BHT and α-tocopherol dissolved with methanol, a synthetic antioxidant (see FIG. 2).
[[ 참조예Reference Example 4] 동결 건조된 4] lyophilized 올트맨시엘롭시스Oltman Cielopsis 유니셀루러리스( Unicelllureless ( OltmannsiellopsisOltmannsiellopsis unicellularis)의 unicellularis) 화학적성분Chemical composition
동결 건조된 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)의 화학적 성분은 AOAC 방법에 의해 결정되어졌다. 정제된 지질 성분은 Soxhlet 방법, 단백질 성분은 Kjeldhal 방법, 회분 성분은 550℃의 회화로에서 화소에 의해, 수분은 105℃ 오븐에서 24시간동안 건조하는 방법에 의해서 각각 결정되어졌다. 분획물과 추출물에서, 정제된 단백질 성분은 Lowry의 방법으로 기준으로 혈청 단백질인 알부민을 사용하여 540nm에서 흡광도를 측정함으로써 결정하였고, 당성분은 기준으로 글루코스를 사용하는 phenol-sulfuric acid 반응에 의해 결정되어졌다. The chemical composition of the lyophilized Oltmannsiellopsis unicellularis was determined by the AOAC method. The purified lipid component was determined by Soxhlet method, protein component by Kjeldhal method, ash component by means of pixels in an incinerator at 550 ° C, and moisture by drying in a 105 ° C oven for 24 hours. In fractions and extracts, the purified protein components were determined by measuring absorbance at 540 nm using albumin, a serum protein, by Lowry's method, and the sugar components were determined by phenol-sulfuric acid reaction using glucose as a reference. lost.
[[ 참조예Reference Example 5] 소거 활성/ 5] erase activity / 킬레이트Chelate 능력 계산 Ability calculation
소거 활성/ 킬레이트 능력은 [1-(Ai-Aj)/Ac] *100 식에 의해 결정되었다. Scavenging activity / chelating ability was determined by the formula [1- (Ai-Aj) / Ac] * 100.
Ai - 활성성분과 혼합된 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)샘플의 흡광도 Ai-absorbance of Oltmannsiellopsis unicellularis sample mixed with active ingredient
Aj - 활성성분이없는 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)샘플의 흡광도 Absorbance of Aj-Oltmannsiellopsis unicellularis Samples Without Active Ingredients
Ac - 샘플이 없는 특정 용매의 흡광도 Ac-absorbance of certain solvents without samples
[[ 참조예Reference Example 6] 6] 통계적 분석 Statistical analysis
통계적 분석은 각 3개의 실험 데이터에 대한 SPSS 11.5 버전 소프트웨어 패키지를 가지고 실행되었다. 각 처리의 평균 수치는 다양성의 한 분석방법인 ANOVA와 비교되어졌고, P-value는 0.05보다 낮은 것으로 여겨졌다. Statistical analysis was performed with the SPSS version 11.5 software package for each of the three experimental data. The mean value of each treatment was compared to ANOVA, an analytical method of diversity, and the P-value was considered to be lower than 0.05.
2. 실험 단계 2. Experimental stage
[[ 실험예Experimental Example 1] One] 활성산소종Reactive oxygen species 억제 관련 자유 Restraint-related freedom 라디칼Radical 소거 활성능력 실험 Scavenging activity test
올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)의 자유라디칼 소거 활성은 Brand-Williams의 방법을 수정하여 사용하였으며, 다른 방법보다도 더 널리 사용되는 방법으로 DPPH이 안정한 분자를 형성하기 위해 수소 라디칼이나 전자를 받아들이는데, 이 수소 공여능을 검사하는 것이다. 우선 미세조류 시 료 분획물 2ml는 96well플레이트에서 DMSO로 용해된 DPPH (3X10-5M) 2ml와 혼합하여 1시간동안 암실에서 보관 후, UV분광광도계를 이용하여 517nm에서 흡광도를 측정하였다. The free radical scavenging activity of Oltmannsiellopsis unicellularis was modified by Brand-Williams' method and is more widely used than other methods. It accepts electrons and checks the hydrogen donating ability. First, 2 ml of the microalgal sample fraction was mixed with 2 ml of DPPH (3X10-5M) dissolved in DMSO in 96well plate and stored in the dark for 1 hour, and then absorbance was measured at 517 nm using a UV spectrophotometer.
도 3은 상기 실험의 결과를 나타낸 그래프로서, A는 물 추출물, B는 80% 메탄올 추출물, C는 n-핵산 분획물, D는 클로로포름 분획물, E는 에틸아세테이트 분획물, F는 수용성 잔유물, Toc는 α-Tocopherol, BHT는 Butylated hydroxytoluene를 나타내며, 샘플의 농도는 1 mg/ml이었고, Mean±SE 는 같은 샘플당 3개씩 하였으며, DPPH 라디칼 소거 활성을 측정 한 결과, 80% 메탄올 추출물이 90.6%, n-핵산 분획물이 89%, 물 추출물이 79.9%, 클로로포름 분획물이 76% 순으로 나타내어졌다. Figure 3 is a graph showing the results of the experiment, A is water extract, B is 80% methanol extract, C is n-nucleic acid fraction, D is chloroform fraction, E is ethyl acetate fraction, F is water-soluble residue, Toc is α -Tocopherol and BHT represent Butylated hydroxytoluene, sample concentration was 1 mg / ml, Mean ± SE was 3 per sample, and 80% methanol extract was 90.6%, n- The nucleic acid fraction was 89%, the water extract 79.9%, and the chloroform fraction 76%.
[[ 실험예Experimental Example 2] 2] 활성산소종Reactive oxygen species 억제 관련 Suppression related 슈퍼옥사이드Superoxide 음이온( Negative ions SuperoxideSuperoxide anion)이온 소거 활성 분석실험 ion removal activity assay
슈퍼옥사이드 음이온(Superoxide anion) 소거 활성 분석은 Nagai의 방법에 의해 수행되어졌으며, 반응 혼합물은 0.05M sodium carbonate 완충용액 0.48ml(pH 10.5), 3mM xanthine 0.02 ml, 3 mM EDTA 0.02 ml, 0.15% 보바인 세륨 알부민(bovine serum albumin) 0.02 ml, 0.75 mM NBT 0.02ml와 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis ) 시료 0.02ml를 혼합하여 10분 동안 25℃에서 인큐베이션 시킨 후, 반응은 6mU XOD를 첨가하는 것에 의해 시작되어 졌고, 20분 동안 25℃에서 보관되었다. 반응은 6mM CuCl을 0.02ml 첨가하는 것에 의해 종결되었으며, 560nm에서 흡광도를 측정하였다. Superoxide anion scavenging activity analysis was performed by Nagai's method, and the reaction mixture was 0.48 ml (0.05 ml pH 0.05), 0.05 mM sodium carbonate, 0.02
도 4는 상기 실험의 결과를 나타낸 그래프로서, A는 물 추출물, B는 80% 메탄올 추출물, C는 n-핵산 분획물, D 클로로포름 분획물, E는 에틸아세테이트 분획물, F는 수용성 잔유물, Toc는 α-Tocopherol, BHT는 Butylated hydroxytoluene를 나타내며, 샘플의 농도는 1 mg/ml이었고, Mean±SE 는 같은 샘플당 3개씩 하였으며, 슈퍼옥사이드 음이온(superoxide anion) 소거 활성을 측정한 결과, 물 추출물이 45.6%, 에틸 아세테이트 분획물이 44%, 80% 메탄올 추출물이 26%를 각각 나타내었다. Figure 4 is a graph showing the results of the experiment, A is water extract, B is 80% methanol extract, C is n-nucleic acid fraction, D chloroform fraction, E is ethyl acetate fraction, F is water-soluble residue, Toc is α- Tocopherol and BHT represent Butylated hydroxytoluene, sample concentration was 1 mg / ml, Mean ± SE was 3 per sample, and superoxide anion scavenging activity was measured. 44% ethyl acetate fraction and 26% 80% methanol extract respectively.
[[ 실험예Experimental Example 3] 3] 활성산소종Reactive oxygen species 억제 관련 Suppression related H2O2H2O2 소거 활성 분석실험 Scavenging activity assay
H2O2 소거 활성은 Muller의 방법을 사용하였으며, 96well 플레이트에 올트맨시엘롭시스 유니셀루러리스 ( Oltmannsiellopsis unicellularis ) 시료 80ml와 10mM H2O2 20ul를 0.1M phosphate buffer 100ul(pH 5.0)를 섞어준 후, 37℃ 에서 5분 동안 인큐베이션 시켰다. 1.25mM ABTS 와 1U/ml peroxidase를 각각 30㎕씩 첨가하고 혼합하여, 37℃ 에서 1분 동안 인큐베이션 시킨 후, 405nm에서 흡광도를 측정하였다. H 2 O 2 scavenging activity was using the method of Muller, Rob System Uni multiple cellular Ciel come teumaen in 96well plate-less (Oltmannsiellopsis unicellularis ) 80ml and 20ul 10mM H 2 O 2 20ul 0.1M phosphate buffer 100ul (pH 5.0) was mixed and incubated at 37 ℃ for 5 minutes. 30 μl of 1.25 mM ABTS and 1 U / ml peroxidase were added and mixed, incubated at 37 ° C. for 1 minute, and the absorbance was measured at 405 nm.
도 5는 상기 실험의 결과를 나타낸 그래프로서, A는 물 추출물, B는 80% 메탄올 추출물, C는 n-핵산 분획물, D는 클로로포름 분획물, E는 에틸아세테이트 분획물, F는 수용성 잔유물, Toc는 α-Tocopherol, BHT는 부틸레이트 하이드록시톨루엔(Butylated hydroxytoluene)을 나타내며, 샘플의 농도는 1 mg/ml이었고, Mean±SE 는 같은 샘플당 3개씩 하였으며, H2O2에 대한 소거 활성을 측정한 결과, n-핵산이 28%, 클로로포름 분획물이 24%를 나타내었으나, α-Tocopherol과 BHT의 소거 활성보다는 낮은 활성을 나타내었다. Figure 5 is a graph showing the results of the experiment, A is water extract, B is 80% methanol extract, C is n-nucleic acid fraction, D is chloroform fraction, E is ethyl acetate fraction, F is water-soluble residue, Toc is α -Tocopherol and BHT represent butylated hydroxytoluene, sample concentration was 1 mg / ml, Mean ± SE was 3 per sample, and scavenging activity against H2O2 was measured. 28% of the nucleic acid and 24% of the chloroform fraction showed lower activity than the scavenging activity of α-Tocopherol and BHT.
[[ 실험예Experimental Example 4] 4] 활성산소종Reactive oxygen species 억제 관련 Suppression related 코멧에세이(Comet assay)에In Comet assay 의한 디엔에이 손상(DNA damage) 측정실험 DNA damage measurement experiment
코멧에세이(Comet assay)에 의한 디엔에이 손상(DNA damage) 측정은 Sing의 방법을 사용하였으며, 1% NMPA(nomarl melting point agarose)로 슬라이드를 코팅하여, L-5178 YR cell을 5 x 104 세포수로 맞춰, e-tube에 1ml씩 넣어준다. 5분간 1000RPM에서 원심분리 시켜 준 후, PBS로 씻고, 시료 10ul를 넣어준 후, 다시 37℃ 에서 30분간 배양시킨다. 그 후, PBS와 H2O2를 전체 1ml가 되도록 넣어 5분간 얼음에서 반응시킨 후, 0.7% LMPA( low melting point agarose ) 100ul를 첨가하여 세포를 슬라이드위에 깔아준다. 다시 LMPA 100ul로 코팅하고 lysis buffer 34ml (pH 10)와 1% triton X-100 340 ul가 있는 jar에 코팅된 부분이 겹치지 않게 두개씩 포개어 담거둔 후, 1시간 30분 동안 4℃ 의 암실에서 lysis시킨다. 그 후, Unwinding buffer(pH 8)가 들어있는 jar에서 20분간 냉장 보관한 후, 암실상태에 서, 25V, 300mA으로 20분간 전기영동을 시켜 0.4M Tris buffer 35ml가 들어있는 jar에 슬라이드가 겹쳐지지 않게 넣어 10분간 회 세척한다. 세척 후, 70% EtOH 35ml가 들어있는 jar에 슬라이드를 넣고 5분간 탈수시킨 후, 암실에서 슬라이드를 건조시켜 에씨디움 브로마이드(Ethidium bromide) 50ul를 슬라이드에 첨가하고, 형광현미경과 코멧프로그램(komet program)을 이용하여 세포의 tail을 측정한다. DNA damage was measured by Comet assay using Sing's method, and the slides were coated with 1% nomarl melting point agarose (NMPA) to convert L-5178 YR cells into 5 x 104 cells. Inject 1ml into the e-tube. After centrifugation at 1000RPM for 5 minutes, washed with PBS, put 10ul of the sample, and incubated for 30 minutes at 37 ℃ again. After that, PBS and H2O2 are added to a total of 1ml and reacted on ice for 5 minutes, and then 100% of 0.7% LMPA (low melting point agarose) is added to spread the cells on the slide. Then, coated with 100ul of LMPA, immersed in two jars of lysis buffer 34ml (pH 10) and 1% triton X-100 340ul, overlapping each other so that they do not overlap, and then lysis in a dark room at 4 ℃ for 1
도 6, 도 7, 도 8, 도 9는 상기 실험의 결과를 나타낸 그래프로서, 도 6과 도8의 Mean±SE 는 같은 샘플당 3개씩 하였으며, 도 7의 (a)는 아무것도 처리 하지 않은 것, (b)는 50 lM H2O2 을 처리한 림프구, (c)는 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis) 의 물 추출물 12.5 g/ml + 50 lMH2O2 을 처리한 림프구, (d)는 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis) 의 물 추출물 25 g/ml + 50 lMH2O2 을 처리한 림프구, (E)는 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis) 의 물 추출물 50 g/ml + 50 lMH2O2 를 처리한 림프구를 나타내며, 도 8의 (a)는 아무것도 처리 하지 않은 것 (b)는 50 lM H2O2 을 처리한 림프구, (c)는 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis) 의 80% 메탄올 추출물 12.5 mg/ml + 50 lMH2O2 을 처리한 림프구, (d)는 Oltmannsiellopsis unicellularis 의 80% 메탄올 추출물 25 mg/ml + 50 lMH2O2 를 처리한 림프구, (E)는 올트맨시엘롭시스 유니셀루 러리스(Oltmannsiellopsis unicellularis) 의 80% 메탄올 추출물 50 mg/ml + 50 lM H2O2 를 처리한 림프구를 나타낸다.6, 7, 8, and 9 are graphs showing the results of the above experiments. Mean ± SE of FIGS. 6 and 8 are three per sample, and FIG. 7A shows that nothing is processed. (b) Lymphocytes treated with 50 lM H2O2, (c) Lymphocytes treated with 12.5 g / ml + 50 lMH2O2 water extract from Oltmannsiellopsis unicellularis, (d) Lymphocytes treated with 25 g / ml + 50 lMH 2 O 2 water extract of Tmannciellopsis unicellularis, (E) water extract of Oltmannsiellopsis unicellularis Representing lymphocytes treated with 50 g / ml + 50 lMH 2 O 2 , Figure 8 (a) is not treated anything (b) is 50 lM H 2 O 2 Lymphocytes treated with (c) were 80% methanol extracts from Oltmannsiellopsis unicellularis (12.5 mg / ml + 50 lMH 2 O 2 ), and (d) 80 cells from Oltmannsiellopsis unicellularis. Lymphocytes treated with
[[ 실험예Experimental Example 5] 5] 활성산소종Reactive oxygen species 억제 관련 Suppression related 하이드록실Hydroxyl 라디칼Radical (Hydroxyl radical) 소거 활성 분석실험 (Hydroxyl radical) scavenging activity assay
하이드록실 라디칼(Hydroxyl radical) 소거 활성 분석은 Chung의 방법을 사용하였다. 하이드록실 라디칼(Hydroxyl radical) 은 가장 반응성이 좋은 활성 산소종으로 다양한 생체 분자와 결합할 수 있고 라디칼 생성, 자연 산화 유발, 전자 이동에 관여하는 라디칼로서 이 라디칼을 얼마나 억제할 수 있는 지를 위해 측정하였다 10mM FeSO4. 7H2O, 10mM EDTA, 10mM 2-deoxyribose로 구성된 펜턴(Fenton) 반응 혼합물은 0.1M phosphate buffer 1.2ml(pH 7.4), 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis) 시료와 10mM H2O2 를 각각 200ml씩 첨가하였고 37℃에서 4시간 동안 인큐베이션 시킨 후, 2.8% TCA와 1% TBA를 각각 1ml씩 첨가하였다. 10분 동안 100℃ 물에서 반응시켜 식힌 후, 식혀 5분 동안 395 x g에서 원심분리 시켜 분광광도계를 이용하여 532nm에서 흡광도를 측정하였다. Analysis of hydroxyl radical scavenging activity was carried out using Chung's method. Hydroxyl radicals are the most reactive reactive oxygen species and have been measured for their ability to bind various biomolecules and to inhibit them as radicals involved in radical production, natural oxidation, and electron transfer. 10 mM FeSO 4 . The Fenton reaction mixture, consisting of 7H 2 O, 10 mM EDTA, and 10 mM 2-deoxyribose, contains 1.2 ml of 0.1 M phosphate buffer (pH 7.4), Oltmannsiellopsis unicellularis sample and 10 mM H 2 O. 200 ml of 2 each was added and incubated at 37 ° C. for 4 hours, followed by 1 ml of 2.8% TCA and 1% TBA. After cooling for 10 minutes in water at 100 ℃, cooled and centrifuged at 395 xg for 5 minutes and measured the absorbance at 532nm using a spectrophotometer.
도 10은 상기 실험의 결과를 나타낸 그래프로서, A는 물 추출물, B는 80% 메탄올 추출물, C는 n-핵산 분획물, D는 클로로포름 분획물, E는 에틸 아세테이트 분획물, F는 수용성 잔유물, Toc는 α-Tocopherol, BHT는 Butylated hydroxytoluene 를 나타내며, 샘플의 농도는 1 mg/ml이었고, Mean±SE 는 같은 샘플당 3개씩 하였으며, Hydroxyl radical의 소거 활성을 측정한 결과, 물 추출물이 31.1%, 수용성 잔유물이 24%로 높은 활성을 나타내었다. 그러나, 그 활성 정도는 현재 사용되어지는 합성 항산화제의 활성보다 낮은 소거 활성을 나타내었다. Figure 10 is a graph showing the results of the experiment, A is water extract, B is 80% methanol extract, C is n-nucleic acid fraction, D is chloroform fraction, E is ethyl acetate fraction, F is water-soluble residue, Toc is α -Tocopherol and BHT represent Butylated hydroxytoluene, sample concentration was 1 mg / ml, Mean ± SE was 3 per sample, and water extract was 31.1% and water soluble residue was measured by scavenging activity of Hydroxyl radical. It showed high activity at 24%. However, the degree of activity showed lower scavenging activity than that of synthetic antioxidants currently used.
[[ 실험예Experimental Example 6] 자유 6] freedom 라디칼Radical NO 저해활성 분석실험 NO inhibitory activity assay
NO 라디칼 저해 활성은 Garrat법으로 측정했다. Nitric oxide (NO.)는 물리학적인 측면과 병리학적인 측면에서 중요한 역할을 하는 가스상태의 자유 라디칼이다. 이것은 O2-. 와 결합하여 ONOO- (peroxynitrite) 를 생산하는데 이것은 독성물질로서 단백질 타이로신의 질소화, 지질 과산화와 DNA 변성을 일으킨다고 알려져 있다. NO.는 Sodium nitroprusside가 수용액 상태일 때 물리적인 pH에서 산소와 결합하여 질소 이온을 생성해 자발적으로 전이되고, 이러한 Griess Illosvoy 반응으로 측정한다. 10mM sodium nitroprusside 2ml와 phosphate buffer salin(pH 7.4) 0.5ml를 섞고 난 후, 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis) 0.5 ml 샘플을 처리하여 25℃에서 150분 간 반응 시킨다. 반응물을 0.5ml 취한 후 1.0 ml sulphanilic acid reagent (0.33% in 20% glacial acetic acid)와 섞은 후 상온에서 5분간 반응 시킨다. 최종적으로 1.0 ml naphthylethylenediamine dihydrochloride (0.1 % w/v) 를 섞고 상온에서 30분간 반응한 후 540nm에서 흡광도 값을 읽는다. NO radical inhibitory activity was measured by the Garrat method. Nitric oxide (NO.) Is a free radical in the gaseous state that plays an important role in physical and pathological aspects. This O2 -. In combination with, it produces ONOO- (peroxynitrite), a toxic substance known to cause the nitrification of protein tyrosine, lipid peroxidation and DNA degeneration. NO. Is spontaneously transferred by the formation of nitrogen ions in combination with oxygen at the physical pH when the sodium nitroprusside is in an aqueous solution, and is measured by the Griess Illosvoy reaction. After mixing 2 ml of 10 mM sodium nitroprusside and 0.5 ml of phosphate buffer salin (pH 7.4), 0.5 ml samples of Oltmansiellopsis unicellularis were treated and reacted at 25 ° C. for 150 minutes. 0.5 ml of the reactant is mixed with 1.0 ml sulphanilic acid reagent (0.33% in 20% glacial acetic acid) and allowed to react at room temperature for 5 minutes. Finally, 1.0 ml naphthylethylenediamine dihydrochloride (0.1% w / v) is mixed and reacted at room temperature for 30 minutes, and the absorbance value is read at 540 nm.
도 11은 상기 실험의 결과를 나타낸 그래프로서, A는 물 추출물, B는 80% 메탄올 추출물, C는 n-핵산 분획물, D는 클로로포름 분획물, E는 에틸 아세테이트 분획물, F는 수용성 잔유물, Toc는 α-Tocopherol, BHT는 Butylated hydroxytoluene를 나타내며, 샘플의 농도는 1 mg/ml이었고, Mean±SE 는 같은 샘플당 3개씩 하였으며, Nitric oxide 소거 활성을 측정한 결과, 합성황산화제인 BHT(26%)와 α-Tocopherol(25%)보다 80% 메탄올 추출물(49%)의 소거 활성이 더 높은 활성을 나타내었다. 11 is a graph showing the results of the experiment, A is a water extract, B is 80% methanol extract, C is n-nucleic acid fraction, D is chloroform fraction, E is ethyl acetate fraction, F is water-soluble residue, Toc is α -Tocopherol and BHT represent Butylated hydroxytoluene, sample concentration was 1 mg / ml, Mean ± SE was 3 per sample, and Nitric oxide scavenging activity was measured. Scavenging activity of 80% methanol extract (49%) showed higher activity than α-Tocopherol (25%).
[[ 실험예Experimental Example 7] 7] 메탈킬레이팅Metal chelating (Metal chelating) 능력 측정실험 (Metal chelating) ability measurement experiment
메탈킬레이팅(Metal chelating)능은 Decker와 Welch의 방법을 이용하여 ferrozine Fe2+ 복합체의 형성이 얼마나 억제되는지에 따라 활성을 측정하였다. 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis) 샘플 5ml에 2mM FeCl2를 0.1ml를 넣었다. 이 반응물에 5mM ferrozine 용액을 0.2 ml 첨가하여 상온에서 10분간 교반 하면서 반응 시킨 후 562nm에서 흡광도값을 얻었다. Metal chelating activity was measured according to the inhibition of the formation of ferrozine Fe2 + complex using the method of Decker and Welch. 0.1 ml of 2 mM FeCl 2 was added to 5 ml of the Oltmannsiellopsis unicellularis sample. 0.2 ml of 5 mM ferrozine solution was added to the reactants, followed by stirring at room temperature for 10 minutes to obtain absorbance at 562 nm.
도 12는 상기 실험의 결과를 나타낸 그래프로서, A는 물 추출물, B는 80% 메탄올 추출물, C는 n-핵산 분획물, D는 클로로포름 분획물, E는 에틸 아세테이트 분획물, F는 수용성 잔유물, Toc는 α-Tocopherol, BHT는 Butylated hydroxytoluene을 나타내며, 샘플의 농도는 1 mg/ml이었고, Mean±SE 는 같은 샘플당 3개씩 하였으며, metal chelating 효과를 측정한 결과, 물 추출물이 82.3%, 클로로포름 분획물이 64%, 80% 메탄올 추출물이 40%, 수용성 잔유물이 44%를 나타내었는데, 이것은 BHT(18%)와 α-Tocopherol(25%)의 활성보다 상당히 높은 활성이었다. 12 is a graph showing the results of the experiment, A is a water extract, B is 80% methanol extract, C is n-nucleic acid fraction, D is chloroform fraction, E is ethyl acetate fraction, F is water-soluble residue, Toc is α -Tocopherol and BHT represent Butylated hydroxytoluene, sample concentration was 1 mg / ml, Mean ± SE was 3 per sample, and metal chelating effect was measured. Water extract was 82.3% and chloroform fraction was 64%. , 80
[[ 실험예Experimental Example 8] 환원력 측정 실험 8] Reducing force measurement experiment
환원력의 측정은 Oyaizu의 방법을 사용하였다. 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis) 분획물 2.5ml를 0.2M phosphate 버퍼(pH 6.6) 2.5ml와 섞고, 1% potassium ferricyanide 2.5ml를 첨가 후, 50˚C 수욕조에서 20분간 반응 시켰다. 반응이 끝난 후 재빨리 식힌 후 10 % trichloroacetic acid를 2.5ml 첨가 하고 3000rpm에서 10분 동안 원심분리 했다. 상층액과 증류수를 1:1(5ml:5ml) 비율로 섞은 후 0.1 % ferric chloride 1ml와 반응 시킨 후 700nm에서 10분 동안 흡광도를 측정하였다. 흡광도 값이 높을 수록 환원력이 높음을 나타낸다. Reducing power was measured by Oyaizu's method. 2.5 ml of Oltmannsiellopsis unicellularis fraction was mixed with 2.5 ml of 0.2 M phosphate buffer (pH 6.6), 2.5 ml of 1% potassium ferricyanide was added, and then reacted in a 50˚C water bath for 20 minutes. . After the reaction, the mixture was cooled quickly and 2.5 ml of 10% trichloroacetic acid was added and centrifuged at 3000 rpm for 10 minutes. The supernatant and distilled water were mixed at a ratio of 1: 1 (5ml: 5ml) and reacted with 1ml of 0.1% ferric chloride, and then absorbance was measured at 700 nm for 10 minutes. The higher the absorbance value, the higher the reducing power.
도 13은 상기 실험의 결과를 나타낸 그래프로서, A는 물 추출물, B는 80% 메탄올 추출물, C는 n-핵산 분획물, D는 클로로포름 분획물, E는 에틸 아세테이트 분획물, F는 수용성 잔유물, Toc는 α-Tocopherol, BHT는 Butylated hydroxytoluene를 나타내며, 샘플의 농도는 1 mg/ml이었고, Mean±SE는 같은 샘플 당 3개씩 하였으며, Reducing power 능력을 측정한 결과, BHT와 α-Tocopherol에 비해 모두 낮은 활성을 나타내었으나, 그 중 클로로포름 분획물이 가장 높은 활성을 나타내었다. Figure 13 is a graph showing the results of the experiment, A is water extract, B is 80% methanol extract, C is n-nucleic acid fraction, D is chloroform fraction, E is ethyl acetate fraction, F is water-soluble residue, Toc is α -Tocopherol and BHT represent Butylated hydroxytoluene, the sample concentration was 1 mg / ml, Mean ± SE was 3 per sample, and as a result of measuring the reducing power ability, both activity was lower than that of BHT and α-Tocopherol. Although, the chloroform fraction showed the highest activity.
[[ 실험예Experimental Example 9] 지질과산화 저해관련, 헤모글로빈으로 유도한 9] Hemoglobin-induced lipid peroxidation inhibition linoleiclinoleic acid system에서의 항산화 효과 실험 Antioxidant Effect in Acid System
항산화 활성은 Kuo의 thiocyanate법을 사용하여 측정하였다. 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)추출물 0.1mL와 에탄올에 녹인 0.1m linoleic산 0.025mL를 섞은 후 0.2M phosphate buffer (pH 7.2) 0.075mL를 첨가하였다. 자연적인 산화는 0.08% hemoglobin 0.05mL를 첨가하여 시작되었다. 물 37˚C에서 60분간 반응시킨 후, 지질과산화 반응을 멈추기 위해 0.6% HCl/ethanol 5mL를 첨가하였다. 20mM FeCl2 0.02mL를 첨가한 후, 30% ammonium thiocyanate 0.01mL를 넣고 이 반응물의 과산화값을 얻기 위해 0.2mL를 취해 490nm에서 흡광도 값을 측정하였다. Antioxidant activity was measured using Kuo's thiocyanate method. 0.1 ml of Oltmansiellopsis unicellularis extract was mixed with 0.025 mL of 0.1 m linoleic acid dissolved in ethanol, and then 0.075 mL of 0.2 M phosphate buffer (pH 7.2) was added. Natural oxidation was initiated by adding 0.05 mL of 0.08% hemoglobin. After reacting for 60 minutes at 37 ° C. water, 5 mL of 0.6% HCl / ethanol was added to stop the lipid peroxidation reaction. After adding 0.02 mL of 20 mM FeCl 2 , 0.01 mL of 30% ammonium thiocyanate was added thereto, and 0.2 mL of the reaction product was measured to obtain absorbance at 490 nm.
도 14는 상기 실험의 결과를 나타낸 그래프로서, 퍼옥사이드 값은 thiocyanate 방법에 의해 측정되었으며, A는 물 추출물, B는 80% 메탄올 추출물, C는 n-핵산 분획물, D는 클로로포름 분획물, E는 에틸 아세테이트 분획물, F는 수용성 잔유물, Toc는 α-Tocopherol, BHT는 Butylated hydroxytoluene를 나타내며, 샘 플의 농도는 1 mg/ml이었고, Mean±SE 는 같은 샘플당 3개씩 하였으며, 헤모글로빈에 의해 유도된 linoleic acid system에서의 항산화 측정한 결과, 클로로포름 분획물이 63%, 물 추출물이 52%, n-핵산 분획물이 41%를 나타내었다. Figure 14 is a graph showing the results of the experiment, the peroxide value was measured by the thiocyanate method, A is water extract, B is 80% methanol extract, C is n-nucleic acid fraction, D is chloroform fraction, E is ethyl Acetate fraction, F is water soluble residue, Toc is α-Tocopherol, BHT is Butylated hydroxytoluene, sample concentration was 1 mg / ml, Mean ± SE was 3 per sample, linoleic acid induced by hemoglobin As a result of antioxidant measurement, 63% of chloroform fraction, 52% of water extract, and 41% of n-nucleic acid fraction were found.
[[ 실험예Experimental Example 10] 지질과산화 저해관련, 10] related to lipid peroxidation inhibition, ThiobarbituricThiobarbituric acid-reactive substances ( acid-reactive substances ( TBARSTBARS ) assay 실험) assay experiment
TBARS는 Madsen의 방법에 의해 2일 간격으로 측정하였다. 유기분획물 시료를 0.1% linoleic산 25g에 혼합하였고, 0.01% 추출물과 0.01% α-tocopherol과 BHT도 앞과 동일한 방법으로 TBARS를 측정하였다. 항산화제를 첨가하지 않은 것을 대조구로 사용하였으며, 앞의 샘플을 60℃ 암실에서 11시간 동안 산화반응을 시켰다. 이틀에 한번씩 반응물 1g을 cyclohexane 3.5ml와 TCA-TBA 혼합물(7.5 % TCA와 0.34 % TBA) 4.5ml에 섞어 5분간 교반하고 2780 x g에서 15동안 원심 분리 하였다. TCA-TBA 상를 제거한 후 수욕조에서10분간 끓이고 532nm에서 흡광도를 측정하였다. TBARS were measured every two days by Madsen's method. Organic fraction samples were mixed with 25 g of 0.1% linoleic acid, and TBARS was measured by 0.01% extract, 0.01% α-tocopherol and BHT. The antioxidant was not added as a control, and the previous sample was oxidized for 11 hours in the dark 60 ℃. Once every two days, 1 g of the reaction was mixed with 3.5 ml of cyclohexane and 4.5 ml of a mixture of TCA-TBA (7.5% TCA and 0.34% TBA), stirred for 5 minutes, and centrifuged at 2780 x g for 15 minutes. After removing the TCA-TBA phase was boiled in a water bath for 10 minutes and the absorbance was measured at 532nm.
항산화 능력은 어유(fish oil)의 malonaldehyde equivalents (mol/kg)로 나타내고 농도는 tetraethoxypropane 스텐다드 곡선으로(농도 범위: 1 ~ 20mM) 추정하였다. Antioxidant activity was expressed as malonaldehyde equivalents (mol / kg) of fish oil and concentration was estimated by tetraethoxypropane standard curve (concentration range: 1 ~ 20mM).
도 15는 상기 실험의 결과를 나타낸 그래프로서, TBARS 값은 linoleic산의 말론알데하이드 대응물(mmol/Kg)로서 표현되어졌고, Mean±SE 는 같은 샘플당 3개씩 하였으며, TBARS 형성 억제 효과를 CDH 형성 억제 효과를 0.1%와 0.01% 클로로 포름 분획물과 BHT, α-Tocopherol을 가지고 측정한 결과, 0.01%의 클로로포름 분획물의 TBARS 형성 억제 효과는 BHT, α-Tocopherol의 억제 효과와 비슷하였다. Figure 15 is a graph showing the results of the experiment, TBARS value was expressed as the malonaldehyde counterpart of linoleic acid (mmol / Kg), Mean ± SE was 3 per sample, CDH formation inhibitory effect TBARS formation The inhibitory effect was measured with 0.1% and 0.01% chloroform fractions, BHT and α-Tocopherol. The inhibitory effect of 0.01% chloroform fraction on TBARS formation was similar to that of BHT and α-Tocopherol.
[[ 실험예Experimental Example 11] 지질과산화 저해관련, 11] related to lipid peroxidation inhibition, 콘쥬게이트된Conjugated 디엔Dien H2O2H2O2 ( ( CDHCDH ) 분석실험 Assay
CDH 양의 측정은 Roozen의 방법을 사용하였다. TBARS에서 사용된 각 샘플 50mg을 싸이클로헥산 5ml와 섞어준 후, 234nm에서 흡광도를 측정하였다. Determination of the amount of CDH using Roozen's method. 50 mg of each sample used in TBARS was mixed with 5 ml of cyclohexane, and the absorbance was measured at 234 nm.
도 16은 상기 실험의 결과를 나타낸 그래프로서, 콘쥬게이트된 다엔의 hydroperoxide의 값은 50mg linoleic acid의 234nm에서 흡광도로서 표현되어졌고, Mean±SE 는 같은 샘플당 3개씩 하였으며, CDH 형성 억제 효과를 0.1%와 0.01% 클로로포름 분획물과 BHT, α-Tocopherol을 가지고 측정한 결과, 0.01%의 클로로포름 분획물의 CDH 형성 억제 효과는 BHT, α-Tocopherol의 억제 효과와 0.1%의 억제 효과는 상당히 다른 것을 확인할 수 있었다. FIG. 16 is a graph showing the results of the above experiment, wherein the conjugated diene hydroperoxide value was expressed as absorbance at 234 nm of 50 mg linoleic acid, Mean ± SE was 3 per sample, and the CDH formation inhibitory effect was 0.1. As a result of the measurement of% and 0.01% chloroform fraction, BHT and α-Tocopherol, the inhibitory effect of 0.01% chloroform fraction on CDH formation was significantly different from that of BHT and α-Tocopherol and 0.1%. .
[[ 실험예Experimental Example 12] 총 12] total 페놀릭Phenolic 성분 및 총 플라보노이드 성분 측정실험 And total flavonoid component test
[총 페놀릭 성분 측정실험] [Total Phenolic Component Measurement Experiment]
총 페놀릭 성분은 Chandler와 Dodds의 방법을 사용하였고, 폴리페놀성분은 좋은 전자공여자로서, H2O2 에서 H2O로 전환시킬 수 있는 능력을 가지기에 측정하였다. 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis) 샘플과 95 % 에탄올 1ml, 증류수 5ml와 50% Folin-Ciocalteu 시약 0.5ml를 섞어 만든 혼합물을 5분 동안 반응시켰다. 혼합물에 5% Na2CO3 1ml를 첨가하여 섞어준 후, 암실상태에서 1시간동안 보관하여 분광광도계를 이용하여 725nm에서 흡광도를 측정하였다. The total phenolic component was determined by the method of Chandler and Dodds, and the polyphenolic component was measured as a good electron donor with the ability to convert from H 2 O 2 to H 2 O. An Otmannsiellopsis unicellularis sample, 1 ml of 95% ethanol, 5 ml of distilled water, and 0.5 ml of 50% Folin-Ciocalteu reagent were reacted for 5 minutes. 1 ml of 5% Na 2 CO 3 was added to the mixture, followed by mixing for 1 hour in the dark. The absorbance was measured at 725 nm using a spectrophotometer.
[총 플라보노이드의 성분][Components of Total Flavonoids]
총 플라보노이드의 성분 측정은 colorimetric 방법을 사용하였다. 에탄올에 녹인 2% AlCl3와 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis) 시료를 각각 0.5ml씩 섞어준 후, 상온에서 1시간동안 반응시켜 분광광도계를 사용하여 420nm에서 흡광도를 측정하였다. Component measurement of total flavonoids was performed using the colorimetric method. 0.5% each of 2% AlCl 3 dissolved in ethanol and Oltmannsiellopsis unicellularis samples were mixed, and then reacted at room temperature for 1 hour to measure absorbance at 420 nm using a spectrophotometer.
표 1은 상기 실험의 결과를 나타낸 것으로서, 한국의 제주도 연안에서 수집된 녹조류인 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)의 정제되지 않은 지질, 단백질, 회분, 수분과 당 성분을 측정한 결과, 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)의 지질은 3.4%, 단백질은 4.3%, 회분은 18.0%, 수분은 3.1%, 당은 50.1%를 나타내었다. 그 분획물을 가지고 폴리페놀 성분과 플라보노이드 성분 함양 정도를 측정한 결과, n-핵산 분획물이 각각 3731mg/100g와 4833mg/100g로 가장 높은 정도를 나타내었으며, 그 다음으로 클로로포름 분획물이 각각 585mg/100g과 4206mg/100g로 높게 나타내었다. Table 1 shows the results of the above experiments, and measured unrefined lipids, proteins, ash, moisture and sugar components of Oltmansiellopsis unicellularis, a green algae collected from the coast of Jeju Island, Korea. As a result, the lipid of Oltmannsiellopsis unicellularis showed 3.4% of lipid, 4.3% of protein, 18.0% of ash, 3.1% of moisture, and 50.1% of sugar. The fractions of polyphenols and flavonoids were measured with the fractions. The n-nucleic acid fractions showed the highest levels of 3731 mg / 100 g and 4833 mg / 100 g, respectively, followed by the chloroform fractions of 585 mg / 100 g and 4206 mg, respectively. It was shown as high as / 100g.
표 1. 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)로부터의 분획물과 다른 추출물의 총 페놀릭, 플라보노이드, 당과 단백질 성분Table 1. Total phenolic, flavonoids, sugars and protein components of fractions from Oltmannsiellopsis unicellularis and other extracts
상기와 같이 한국의 청정해안으로 알려진 강정의 조수웅덩이에서 우점종으로 서식하고 있는 해양 미세 녹조류인 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)는 배양이 쉽고 대량생산이 가능하여 천연 기능성 식품소재나 건강식품으로 응용할 수 있다. 따라서 우선 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)를 잠재적인 천연 항산화제로 개발하고자 물 추출물, 메탄올 추출물과 다른 유기용매의 의한 분획물들에 대하여 DPPH radical, metal chelating, 항산화(hemoglobin-induced linoleic acid system)와 슈퍼옥사이드 음이온(superoxide anion) 소거을 측정해 본 결과 높은 활성을 갖고 있다는 사실과 활성 성분들이 친수성과 소수성의 성질을 모두 갖고 있다는 점을 확인할 수 있었다. 이런 결과들을 종합해 볼 때, 이전 연구에서 한번도 연구되어지지 않은 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)의 높은 항산화 능력이 전세계의 식품 분야와 의학 분야에서 많은 부작용을 가지는 합성 항산화제의 대안책으로서 잠재적인 천연 자원의 항산화제로 이용될 수 있다는 점에서 큰 가치를 갖는다. As described above, Oltmannsiellopsis unicellularis, a marine microalgae that lives as a dominant species in the tidal pool of Gangjeong, known as the clean coast of Korea, is easy to culture and can be mass-produced. It can be applied as a health food. Therefore, in order to develop Oltmannsiellopsis unicellularis as a potential natural antioxidant, DPPH radical, metal chelating, and antioxidant (hemoglobin-induced linoleic) fractions from water extracts, methanol extracts and other organic solvents The measurement of acid system and superoxide anion scavenging showed that it has high activity and that the active ingredients have both hydrophilic and hydrophobic properties. Taken together, these results suggest that the high antioxidant capacity of Oltmannsiellopsis unicellularis, which has never been studied in previous studies, has been shown to have a number of adverse effects in the food and medicine world. This is of great value in that it can be used as an antioxidant of potential natural resources.
이상에서와 같이 본 발명은 비록 상기의 참조예 및 실험예에 한하여 설명하였지만 반드시 여기에만 한정되는 것은 아니며 본 발명의 범주와 사상을 벗어나지 않는 범위 내에서 다양한 변형실시가 가능함은 물론이다. As described above, although the present invention has been described with reference to the above-described examples and experimental examples, it is not necessarily limited thereto, and various modifications may be made without departing from the scope and spirit of the present invention.
전술한 구성 및 작용에 의한 효과를 상세하게 설명하면 다음과 같다. Referring to the effects of the above-described configuration and operation in detail as follows.
본 발명의 올트맨시엘롭시스 유니셀루러리스(Oltmannsiellopsis unicellularis)를 이용한 천연 항산화제 조성물은 활성 산소종(Hydrogen peroxide, Superoxide, Hydroxyl radical)과 자유 라디칼(Nitric oxide와 Nitrogen dioxide)에 의한 인체의 세포나 조직 및 식품의 산화적인 손상을 억제할 뿐만아니라, 종래에 항산화를 위하여 사용되던 합성 항산화제의 간 손상과 면역력 감소, 심장병, 암유발 등의 부작용을 방지할 수 있는 등의 효과를 제공한다. Natural antioxidant composition using the Oltmannsiellopsis unicellularis of the present invention is a cell of the human body by free radicals (Hydrogen peroxide, Superoxide, Hydroxyl radical) and free radicals (Nitric oxide and Nitrogen dioxide) In addition to inhibiting oxidative damage of tissues and foods, it also provides the effect of preventing the side effects such as liver damage and immunity reduction, heart disease, cancer-causing of the synthetic antioxidants used in the past for antioxidants.
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| KR100943916B1 (en) * | 2007-12-21 | 2010-02-24 | 부경대학교 산학협력단 | A composition of antioxidative and antihypertensive peptides derived from marine zooplankton |
| KR20190016295A (en) * | 2017-08-08 | 2019-02-18 | 한국해양과학기술원 | Composition for Preventing or Treating Cancer Containing Extract of chloromonas sp. |
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| KR100943916B1 (en) * | 2007-12-21 | 2010-02-24 | 부경대학교 산학협력단 | A composition of antioxidative and antihypertensive peptides derived from marine zooplankton |
| KR20190016295A (en) * | 2017-08-08 | 2019-02-18 | 한국해양과학기술원 | Composition for Preventing or Treating Cancer Containing Extract of chloromonas sp. |
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