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Ⅳ. 연구개발결과(필요에 따라 제목을 달리할 수 있음)
제1세부 해조류로부터 혈전분해 활성 물질정제 및 특성분석(조선대 김성준)
1. 선별된 해양식물로부터 혈전분해효소 정제, 특성 및 혈전형성예방 기작분석
○ 선별된 해조류 : Codium fragile, Undaria pinnatifida, Ulva pertusa Costaria costata
○ Codium fragile(청각)
- 정제된 혈전분해효소의 총 활성 : 246unit, 수율:7.8%, size: 48.9kDa
- Fe²⁺

Ⅳ. 연구개발결과(필요에 따라 제목을 달리할 수 있음)
제1세부 해조류로부터 혈전분해 활성 물질정제 및 특성분석(조선대 김성준)
1. 선별된 해양식물로부터 혈전분해효소 정제, 특성 및 혈전형성예방 기작분석
○ 선별된 해조류 : Codium fragile, Undaria pinnatifida, Ulva pertusa Costaria costata
○ Codium fragile(청각)
- 정제된 혈전분해효소의 총 활성 : 246unit, 수율:7.8%, size: 48.9kDa
- Fe²⁺ 이온에 의해 활성이 감소되고 Zn²⁺, Cu²⁺ 이온에 의해 활성이 증가됨. plasmin-like serine protease 계열의 혈전분해효소임.
- 35℃와 pH 5.0에서 최적활성을 나타냄.
- Amydolytic activity : plasmin substrate, t-PA substrate에서 activity를 확인
- N-말단 아미노산 서열을 분석한 결과, APKASTDQTLPL의 아미노산을 얻었고 serine 계열의 protease와 50%정도 상동성을 보임.
- FTIR assay : fibrin, fibrinogen의 α chain과 β chain이 분해되었음을 확인함.
- fibrinolysis : α chain(66 kDa)을 30분 내에 가장 먼저 완전히 분해시켰고 γ-γ chain(97kDa), β chain(55 kDa) 순서로 대부분 분해시킴.
- fibrinogenolysis : Aα chain(66kDa)을 30분 내에 가장 먼저 완전히 분해시켰고 γ chain(48 kDa), Bβ chain(55kDa) 순서로 대부분 분해시킴
- blood clot를 이용한 in vitro 실험 결과, blood에서 형성된 clot을 직접적으로 분해하는 것을 확인함.
- anticoagulant assay : 외인성 경로에서 혈전을 형성시키는 응고인자들(Ι,Ⅱ,Ⅴ,Ⅶ,Ⅹ)과 반응하여 혈액응고를 지연시켰음을 확인함. fibrin 형성을 지연시키지는 못함.
- antiplatelet activity : 혈소판이 응집되는 시간을 상당히 지연시켰음을 확인함.
- in vivo antithrombotic activity
① Carrageenan-induced thrombosis model의 꼬리정맥에 유도된 혈전을 제거 또는 억제하는 활성이 존재하는 것을 확인
② bleeding time 측정에서는 control인 saline과 유사한 지혈시간을 나타내며. u-PA보다도 짧음. 실험동물 내로 투여 시 지혈반응에 대한 안전성 확인
○ Undaria pinnatifida(미역귀)
- 정제된 혈전분해효소의 총 활성 : 19unit, 수율:8.4%, size:50.12kDa
- Co²⁺이온에 의해 활성이 감소되고 Zn²⁺이온에 의해 활성이 증가됨. trypsin-like serine and metalloprotease 계열의 혈전분해효소임.
- 50℃와 pH 7.0에서 최적활성을 나타냄.
- substrate s-2251을 이용한 kinetics는 다음과 같은 결과를 얻음. Vmax (mU/min/ml) : 113.0, Km(μM):380, Kcat(s-a):2.83, Kcat/Km(nM-1s-a):7.45
- N-말단 아미노산 서열을 분석한 결과, LTATTCEELAAAPTD의 아미노산을 얻었고 serine 계열의 protease와 상동성을 보임.
- FTIR assay : fibrin, fibrinogen이 분해되었음을 확인함.
- fibrinolysis : fibrin의 α, β, γγ chain를 10분내에 모두 분해시키는 분해패턴을 확인하였다.
- fibrinogenolysis : fibrinogen의 Aα, Bβ, γ chain을 4시간에 걸쳐 γ-chain을 제외하고 거의 분해시키는 것을 확인하였고 특히 10분 내에 Aα, Bβ-chain을 모두 분해시켰다.
- blood(plasma) protein 중 globulin, albumin에 대해 영향을 미치지 않음.
- mouse blood clot과 human plasma clot를 이용한 in vitro 실험 결과, blood에서 형성된 clot을 직접적으로 분해하는 것을 확인함.
- anticoagulant assay : 외인성 경로에서 혈전을 형성시키는 응고인자들(Ⅰ,Ⅱ,Ⅴ,Ⅶ,Ⅹ)과 반응하여 혈액응고를 지연시켰음을 확인함. fibrin 형성을 지연시키지는 못함.
- antiplatelet activity : 혈소판이 응집되는 시간을 상당히 지연시켰음을 확인함.
- in vivo antithrombotic activity
① Collagen/Epinephrine에 의해 유도되는 thromboembolism에 대한 보호효과를 확인함.
② Carrageenan-dinsuced thrombosis model의 꼬리정맥에 유도된 혈전을 제거 또는 억제하는 활성이 존재하는 것을 확인.
③ Factory assay를 통한 기전분석 : thrombin, plasmin, factor Xa, plasminogen, t-PA의 활성이 억제되는 것을 확인함, u-PA의 활성을 억제시키지 못함.
○ Costaria costata(쇠미역)
- 정제된 혈전분해효소의 총 활성 : 915unit, 수율 : 6.9%, size:60.55 kDa
- Mg²⁺, Ca²⁺ 이온에 의해 활성이 감소되고 Co²⁺, Mn²⁺, Fe²⁺,Fe²⁺,Cu²⁺, Zn²⁺ 이온에 의해 활성이 증가됨. serine protease 계열의 혈전분해효소임.
- 25~37℃와 pH 6.0에서 최적활성을 나타냄.
- Amydolytic activity : plasmin substrate에서 activity를 확인
- N-말단 아미노산 서열을 분석한 결과, SCNSCLDKVDADGLN의 아미노산을 얻었고 serine 계열의 protease와 상동성을 보힘.
- FTIR assay : fibrin, fibrinogen의 구조변화 확인.
- fibrinolysis : 30분 반응 결과 Aα chain와 Bβ chain은 완전히 분해된 것을 알 수 있고, γ-γ chain은 4시간 반응 후에도 한 개의 체인만 잘린 것을 알 수 있었음.
- fibrinogenolysis : Aα chain와 γ-γ chain은 30분 반응 결과 모두 분해가 되었다는 것을 알 수 있었고, Bβ chain은 2시간 후에 모두 분해 됨.
- Fluorescence microscope를 통해 fibrin clot에 대해 fibrinolysis 효과를 확인 함.
- blood clot를 이용한 in vitro 실험 결과, blood에서 형성된 clot을 직접적으로 분해하는 것을 확인함.
- anticoagulant assay : 외인성 경로에서 혈전을 형성시키는 응고자인들(Ⅰ,Ⅱ,Ⅴ,Ⅶ,Ⅹ)과 반응하여 혈액응고를 지연시켰음을 확인함. fibrin 형성을 지연시키지는 못함.
- antiplatelet activity : 혈소판이 응집되는 시간을 상당히 지연시켰음을 확인함.
- in vivo antithrombotic activity
① Carrageenan-induced thrombosis model의 꼬리정맥에 유도된 혈전을 제거 또는 억제하는 활성이 존재하는 것을 확인.
② bleeding time 측정에서는 control(saline)보다는 느리지만, u-PA보다는 짧은. 실험동물 내로 투여 시 지혈반응에 대한 안전성 확인.
○ Ulva pertusa(구멍갈파래)
- 정제된 혈전분해효소의 총 활성 : 245unit, 수율 : 2.5%, size:55kDa Mg²⁺,Cu²⁺,Na²⁺,Fe²⁺ 이온에 의해 활성이 억제됨. chymotypsin like serine protease 계열의 혈전분해효소임.
- 40℃와 pH 5.0에서 최적활성을 나타냄.
- Amydolytic ativity : t-PA like substrate에서 activity를 확인
- N-말단 아미노산 서열을 분석한 결과, NYDAATLPEELYF의 아미노산을 얻었고 serine 계열의 protease와 상동성을 보임.
- fibrinogenolysis : Aα chain, Bβ chain은 4시간 후 완전히 분해되지만 γ chain은 72시간 이후에도 분해되지 않았음.
- mouse blood clot과 human fibrin clot를 이용한 in vitro 실험 결과, blood에서 형성된 clot을 직접적으로 분해하는 것을 확인함.
- anticoagulant assay : 외인성 경로에서 혈전을 형성시키는 응고자인들(Ⅰ,Ⅱ,Ⅴ,Ⅶ,Ⅹ)과 반응하여혈액응고를 지연시켰음을 확인함. fibrin 형성을 지연시키지는 못함.
- antiplatelet activity : 혈소판이 응집되는 시간을 상당히 지연시켰음을 확인함.
- in vivo antithrombotic activity
① Collagen/Epinephrine에 의해 유도되는 thromboembolism에 대한 보호효과를 확인함.
② Carrageenan-induced thrombosis model의 꼬리정맥에 유도된 혈전을 제거 또는 억제하는 활성이 존재하는 것을 확인
③ bleeding time 측정에서는 u-PA보다 지혈시간이 짧음. 실험동물 내로 투여 시 지혈반응에 대한 안전성 확인
2. 선별된 해조류를 이용한 anti-hyperlipidemic activity 분석
- total cholesterol(총콜레스테롤), HDL-cholesterol(고밀도 지단백 콜레스테롤), LDL-cholesterol(저밀도 지단백 콜레스테롤),VLDL-cholesterol(초저밀도 지단백 콜레스테롤), TG(중성지방) 측정함.
- 고지혈증을 유발시킨 Rat에 해조류 mix. 분말을 첨가한 사료를 14일 동안 식이를 시킨 결과, 6 종류 시료 중 Algae mixture sample 5 시료가 5가지 모든 항목의 결과에서 혈행개선 효과를 확인함.
- 동맥경화지수, Total cholesterol/HDL ratio와 HDL/LDL ratio의 수치에서도 항고지혈효과의 유의성을 확인.
제1협동 해조류추출물의 신경보호효과 및 다양한 기능성 분석(광주대 김승)
1. 선별된 해조류에 대한 안전성 및 기능성 분석
○ 항산화 활성
- DPPH : 녹조류인 파래와 미생이가 갈조류, 홍조류들 보다 높은 radical 제거소거능을 나타냄
- 총 페놀 함량 및 총 플라보노이드 함량 : 파래가 가장 높게 나타냄
- 아질산염 소거능 : 미역에서 가장 높은 소거능을 나타냄
○ 세포 독성 : 모든 추출물에서 10μg/ml까지는 세포 생존율에서 큰 변화를 나타내지는 않음. 대부분의 세포주에서 100μg/ml 이상의 농도에서 세포 생존율 감소가 나타남.
○ 미백 효과
- UVA/B/C 모두에 대해 흡수도가 높지 않음.
- 모든 추출물에서 mushroom tyrosinase 억제효과를 나타냄.
- tyrosinase 활성과 멜라닌 생성 억제에서 톳 추출물이 높은 효과를 나타냄
○ 염증 및 알레르기 억제 효과
- β-hexosaminidase 분비 억제에서는 톳 추출물이 높은 효과를 나타냄
- NO 분비량 억제에서는 톳, 미역 추출물이 높은 효과를 나타냄.
2. 신경퇴행성질병예방 관련 유전자의 유도발현 분석
○ 신경세포 보호 효능 분석
- 6-OHDA를 처리하였을 때는 톳에서 가장 높은 세포 보호효과를 나타냄. Rotenone을 처리하였을 때도 역시 미역에서 가장 높은 세포 보호효과를 나타냄
- Retenone, 6-OHDA에 의한 활성산소종의 발생은 톳과 미역에서 가장 높은 감소율을 보여줌.
- 6-OHDA에 의한 산화적스트레스에 대한 fucoxanthin의 세포사멸 억제 효과
① 6-OHDA에 의한 SH-SY5Y 세포의 사멸이 fucoxanthin에 의해 억제됨
② ROS 생성, mitochondria memebrane potential 감소, intracellular Ca²⁺농도 증가 억제
③ 세포 핵 형태변화 억제
④ anti-apoptotic factor인 Bcl-2의 발현 감소 억제, apoptotic factor인 Bax, caspase-3의 발현 및 활성화 억제
⑤ 6-OHDA에 의한 endoplasmic reticulumn stress와 관련된 인자(ATF6, CHOP, GRP78)의 발현 억제
⑥ 6-OHDA에 의한 ERK, p38, AKT의 인산화 및 NFk-B의 핵 내 이동을 억제시킴
- 뇌소교세포의 염증반응에 대한 fucoxanthin의 억제 효과
① fucoxanthin((0,5,10,20 μM))과 LPS 처리 후 세포 생존율 변화가 나타나지 않음.
② LPS에 의한 No 생성, ROS 생성 및 phagocytosis 감소
③ LPS에 의한 PGE2 및 proinfammatory cytokine(IL-1β와 TNF-α) 생성량 감소
④ LPS에 의한 iNOS와 COX-2의 발현 증가 억제 및 MMP-3의 발현 증가 억제
○ 혈전분해활성물질의 베타 아밀로드이드 분해 활성
- Thioflavin assay : 청각에서 정제된 분해효소에 의해 fluorescence intensity 감소가 가장 높게 나타남.
- 정제된 분해효소의 세포 독성은 대부분은 100㎍/㎖ 이상에서 세포 생존율의 감소가 나타남.
- 청각에서 정제된 단백질을 처리하였을 때 베타 아밀로이드에 의한 세포의 생존율 감소 억제됨.
제 2협동 해조류의 기능성 성분 및 이를 이용한 제품화(전북대 김명곤)
1. 미역에 대한 용매조건 및 분획물 제조, 항염증 활성 분석
- 미역에 대한 용매조건에 따른 항산화 효과(DPPH radical scavenging activity)는 Haxane보다 MeOH에서 높았음.
- Silica-gel column chromatography을 이용하여 Hexane : Ethylacetate=10:1 조건에서 주요활성(DPPH radical scavenging activity)을 갖는 분획물이 용출되었음.
(1) 미역의 세포독성 및 뇌소교세포에서의 항염증 활성
- 5 ㎍/㎖까지는 모든 세포에서 모든 농도에서 95% 이상의 생존율을 보임.
- Nitric oxide 생성 억제 효과, LPS 처리군의 경우 NO의 농도는 45.30±4.44 μM로 나타났지만, 분획물의 농도가 증가 할수록, 39.07±1.94 μM, 33.53±1.35 μM, 21.78±1.0 μM의 농도로 감소됨을 확인.
- Prostagladin E2(PGE2) 생성 억제 효과, LPS 처리군의 경우 PGE2의 농도는 1400±1.55 pg/ml 로 나타났지만, 분획물의 농도가 증가 할수록, 891.64±3.69 pg/ml, 685.15±6.94 pg/ml, 200.45±02 pg/ml 의 농도로 감소됨을 확인.
- Tumor necrosis factor-α(TNF-α) 생성 억제 효과, LPS 처리군의 경우 TNF-α의 농도는 3801.45±3.51 pg/ml 로 나타났지만, 분획물의 농도가 증가 할수록, 3612.53±1.09 pg/ml, 2902.51±2.84 pg/ml, 2064.84±3.42 pg/ml 의 농도로 감소됨을 확인.
(2) 미역으로부터 fucoxanthin 분리 및 구조분석
- 활성성분은 미역을 대상으로 하여 용매 추출 및 예비분획, 실리카 겔 칼럼크로마토그래피를 반복하여 98.5% 이상의 순도를 가지는 단일 성분을 분리하였음. 분리된 성분은 446.5 nm(acetone)에서 최대 흡수파장(λmax)을 나타내었고, LC-API-MS(positive mode)에 의한 molecular ion은 분자량이 658인 것을 확인함.
- 분리한 성분은 13C-NMR에서 42개의 carbon signal이 검출되었고 1H-NMR spectrum에서 분자냐에 1개의 acetyl 기, 1개의 conjugated keone 기, 2개의 quaternary germinal dimethyl 기, 2개의 quaternary germinal oxygen methyl 기, 4개의 olefinic methyl 기 및 allene 기를 지니고 있는 polyene 계열 화합물인 것으로 추정되었으며, 13C-NMR, DEPT 및 1H-NMR spectrum에서 이 성분은 갈조류에 함유되어 있는 색소성분인 all-trans-fucoxanthin 인 것으로 확인.
(3) 갈조류에서 생물학적 활성을 갖는 주요 compound 및 구성물질 분석
- 갈조류 중 다시마에서의 fucoxanthin 함량은 1.10 mg/g 수준인데 비하여 미역과 톳에서의 함량은 각각 1.83 및 1.87 mg/g 으로서 다시마보다는 미역과 톳에서 함유량이 높았음을 확인함.
- Total polyphenol 함량은 톳에서 건물 중량으로 543.8 mg/g 으로 가장 높았고, 그 다음이 미역에서 높았으며, 다시마와 김에서의 함량은 톡이나 미역에 비하여 낮았음을 확인함.
- DPPH법을 통한 전자공여능은 톳, 감태, 미역, 미역귀, 돌미역, 김, 다시마 순 이었음.
- 다양한 해조류(김, 미역, 미역귀, 톳, 감태, 파래, 미생이)에 대한 polyphenol, flavonoid, polysaccharide의 함량을 분석하였음.
- 구성 아미노산 총 함량은 김>파래>감태>매생이>미역>톳>다시마 순으로 김에서 높은 함량을 보임.
- γ-Amminobutyric acid(GaBa) 함량은 15~80mg % 였으며, 톳, 김, 감태=다시마, 미역, 매생이=파래 순으로 톳과 김에서 우수하였으며, taurine 은 김과 톳에서 존재하고 있었으며, 그 함량은 김에서 월등히 높았음.
- 유리아미노산 함량은 해조류의 종류에 따라 42-935mg %의 함량을 보였으며 김, 파래, 미역, 톳, 감태, 매생이의 순으로 김에서 유리아미노산의 총량이 높았음.
(4) Fucosterol의 분리 및 구조 동정
- 미역 겆노 분말시료를 유기용매로 추룰한 다음 silica gel column chromatography 및 Lichroprep RP-18을 이용한 column chromatography 를 반복하여 건조한 미역시료 800 g으로부터 백색 분말상의 물질로서 약 272 mg 을 얻었음. HPLC-MS(positive API mode)로 분석한 결과 molecular ion으로서 m/z 412(C29H480) 임을 확인함. 또한 이 성분을 TMS 유도체화한 후 GC-MS(EI-MS)로 분석한 결과 melecular ion으로서 m/z 412 임을 증명함.
- 1H-NMR(600 MHz, CDCI3) spectrum에서는 δH(ppm) : 5.28(1H, br, d, H-6), 5.11(1H, q, H-28), 3.38(1H, m, H-3), 1.78(3H, m, H-29),0.94(3H, s, H-19), 0.93(3H, d, H-21), 0.92(3H, d, H-27), 0.91(3H, s, H-26), 0.90(3H, s, H-18)에서 signal 이 확인됨.
- 13C-NMR spectrum 에서는 29개의 carbon signal들이 검출되었고, 갈조류에서 많이 발견되는 fucosterol(5,24(28)-stigmastadien-3βo1)로 판명됨.
(5) 해조류 중 sterol 함량 분석
- 채취시기에 따라 fucosterol 함량에 많은 차이가 있음을 확인함.
- 해조류에서 fucosterol을 분리하여 활용할 경우는 홍조류나 녹조류보다는 미역, 톳, 및 지충이 등과 같은 갈조류가 바람직한 생물자원일 것으로 판단됨.
(6) antheraxanthin 분리 및 구조동정
- 동결 건조한 톳(Hijikia fursiforms) 분말시료를 petroleum ether : methanol : ethyl acetate petroleum ether : methanol : ethyl acetate(1:1:1, v/v) 혼합액으로 추출하여 농축한 다음한 다음 silica gel column chromatography 를 반복하여 TLC에서 단일 spot를 나타내는 암적색의 분말상 물질(UPS-2)을 얻음.
- 이성분의 UV spectrum은 λmax(CH2CI2)가 445, 455 ,nm 로서 carotenoid계 색소인 antheraxanthin과 유사한 UV spectrum을 나타내었고 HPLC-MS(positive ESI mode)로 분자량을 측정한 결과 m/z 585.4[M+H] +이 검출되어 분자량이 m/z 584.4(C40H56O3)
- 1H-NMR(600 MHz, CDCI3) spectrum에서 δ:0.84(s, 3H, H-16'), 1.07(s, 3H, H-17'), 1.20(s, 3H, H-18'), 1.25(s,3H, H-17), 1.26(dd, 1H, J=13.0, 14.0Hz, H-20), 1.37(s, 3H, H-16), 1.73(s, 1H), 1.83(d, 1H, J=5.4 Hz,H-2), 1.95(s, 3H, H-19'), 1.97(s, 3H, H-18), 1.99(s, 3H, H-20'), 2.04(s,3H, H-20), 2.96(dd, 1H, J=9.0,14.1 Hz), 4.03(m, 1H, H-3'), 4.13(dd,1H, J=6.6, 14.4 Hz, H-3), 6.15(d, 1H, J=12.4 Hz, H-7'), 6.26(d, 1H, J=12.0 Hz, H-10'), 6.44(d, J=15.0 Hz, H-8'), 6.54(d, 1H, J=16.8 Hz, H-8), 6.90(m, 1H, H-15) 에서 signal 들이 확인됨.
- 13C-NMR sepectrum에서는 40여개의 carbon signal 들이 검출됨.
(7) 해조류 중 antheraxanthin 함량 분석
- antheraxanthin은 2012년산 미역, 2013년산 톳, 2012년산 톳, 지충이, 2013년 산 미역, 다시마 순으로 높았으며 Lutein은 파래와 슈퍼 김에서, Zeaxanthin은 파래와 슈퍼 김에서, a-Carotene은 2012년 산 미역에서, b-Carotene은 슈퍼 김에서 각각 높은 함량을 보임.

Abstract ▼

The lead institution : Purification and characterization of fibrinolytic agents from algae(Chosun University, Kim Sung-Jun)
1. Purification and characterization, mechanism analysis of fibrinolytic agents from selected algae with anti-thrombotic activity
- The selected alga : Codium fragile, Un

The lead institution : Purification and characterization of fibrinolytic agents from algae(Chosun University, Kim Sung-Jun)
1. Purification and characterization, mechanism analysis of fibrinolytic agents from selected algae with anti-thrombotic activity
- The selected alga : Codium fragile, Undaria pinnatifida, Ulva pertusa Costaria costata
(1) Codium fragile
- Total fibrinolytic activity of purified agents : 246 unit,yield : 7.8%, molecular weight : 48.9 kDa.
- Incubation of this enzyme with Fe²⁺ metal ion induced to decrease this enzyme's activity, but incubation of this enzyme with Zn²⁺ and Cu²⁺ metal ion induced to increase this enzyme's activity.
- This enzyme is plasmin-like serine protease that showed optimum activity at 35 and pH 5.0.
- Amidolytic activity of this enzyme was highest on S-2251 substrate(plasmin), following S-2288 substrate(u-PA).
- N-terminal sequence is APKASTDQTLPL that showed 50% of indentification(%) to serine like protease.
- In FTIR assay, the αchain and βchain of fibrin, fibrinogen were changed that structures by this enzyme.
- This enzyme lysed completely αchain(66 kDa) of fibrin within 30 min first, following lysed γ-γ chain(97 kDa) of fibrin and β chain(55 kDa) of fibrin.
- This enzyme lysed completely Aαchain(66 kDa) of fibrinogen within 30 min first, following lysed γchain(48 kDa) of fibrinogen and Bβ chain(55 kDa) of fibrinogen.
- in vitro using blood clot, this enzyme can lyse directly blood clot.
- In anticoagulant assay, this enzyme can delay blood coagulation tiome with coagulant factors(Ⅰ,Ⅱ,Ⅴ,Ⅶ,Ⅹ) this enzyme can delay blood coagulation time with coagulant factors(Ⅰ,Ⅱ,Ⅴ,Ⅶ,Ⅹ) that form thrombus.
- In antiplatelet activity assay, This enzyme delay strongly closure time.
- in vivo antithrombotic assay, this enzyme has antithrombotic activity(about 100% inhibition at 400 μg) against tail thrombosis in carrageenan-induced thrombosis model, safety in animal body for hemostasis.
(2) Undaria pinnatifida
- Total fibrinolytic activity of purified agents : 19 unit, yield : 8.4%, molecular weight : 50.12 kDa
- Incubation of this enzyme with Co²⁺ metal ion induced to decrease this enzyme's activity, but incubation of this enzyme with Zn²⁺ metal ion induced to increase this enzyme's activity.
- This enzyme is trypsin-like serine protease and metalloprotease that showed optimum activity at 50 and pH 7.0.
- Amidolytic activity of this enzyme was highest on S-2251 substrate(plasmin), its showed following results that have kinetics of Vmax(mU/min/ml) : 113.0, Km(μM):380, Kcat(s-1):2.83, Kcat/Km(nM-1s-1) : 7.45.
- N-terminal sequence is LTATTCEELAAAPTD that showed 60% of indentification(5) to serine like protease.
- In FTIR assay, the αchain and βchain of fibrin, fibrinogen were changed that structures by this enzyme.
- This enzyme lysed completely αchain(66 kDa), βchain(55kDA), γ-γchain(97kDA) of fibrin within 10 min first.
- This enzyme lysed completely Aαchain(66kDa), Bβchain(55kDa) of fibrinogen within 10 min first.
- This enzyme lysed completely Aαchain(66 kDa), Bβchain(55 kDa) of fibrinogen within 10 min first, following lysed γchain(48 kDa) of fibrinogen.
- The proteolysis effect of this enzyme on blood(plasma) protein, globulin and albumin, was not showed, however, incubation of this enzyme with fibrinogen result in to induce proteolysis effect(about 80% of protein lysis)
- in vitro using mouse blood clot and human plasma clot, this enzyme can lyse directly blood clot.
- In anticoagulant assay, this enzyme can delay blood coagulation time with coagulant factors(Ⅰ,Ⅱ,Ⅴ,Ⅶ,Ⅹ) that form thrombus.
- In antiplatelet activity assay, This enzyme delay strongly closure time.
- In vivo antithrombotic assay, this enzyme has protective effect(about 100% inhibition at 200 μg) against thromboembolism in collagen/epinephrine-induced thromboembolism model, safety in animal body for hemostasis.
- In mechanism assay on coagulant factors and fibrinolysis factors, these results showed this enzyme inhibited each factors activity(thrombin, plasmin, factor X activated, plasminogen, t-PA), however, inhibitory effect of this enzyme on u-PA not revealed.
(3) Costaria costata
- Total fibrinolytic activity of purified agents : 915unit, yield : 6.9%, molecular weight : 60.55 kDa
- Incubation of this enzyme with Mg²⁺, Ca²⁺ metal ion induced to decrease this enzyme's activity, but incubation of this enzyme with Co²⁺, Mn²⁺, Fe²⁺,Fe²⁺,Cu²⁺, Zn²⁺ metal ion induced to increase this enzyme's activity.
- This enzyme is plasmin-like serine protease that showed optimum activity at 25~37 and pH 6.0.
- Amydolytic activity of this enzyme was highest on S-2251 substrate(plasmin).
- N-terminal sequence is SCNSCLDKVDADGLN that showed 53% identification(5) to serine protease.
- In FTIR assay the α chain and Bβ chain of fibrin, fibrinogen were changed that structures by this enzyme.
- This enzyme lysed completely α chain and Bβ chain(66 kDa), Bβ chain(55 kDa) of fibrin within 30min first, after 4 h proteolysis effect of γ-γchain(97 kDa) was revealed.
- This enzyme lysed completely Aαchain(66 kDa), γchain(48 kDa) of fibrinogen within 30 in first, following lysed Bβ chain(55 kDa) of fibrinogen after 2 h.)
- In fluorescence microscope, this enzyme showed fibrinolysis effect on fibrin clot using fibrinogen conjugate.
- in vitro using mouse blood clot and human fibrin clot, this enzyme can lyse directly blood clot.
- In anticoagulant assay this enzyme can dealy blood coagulation time with coagulant factors(Ⅰ,Ⅱ,Ⅴ,Ⅶ,Ⅹ) that form thrombus.
- In antiplatelet activity assay, This enzyme delay strongly closeure time
- in vivo antithrombotic assay, this enzyme has antithrombotic activity(60% protection at 0.156 M/kg) against tail thrombosis in carrageenan-induced thrombosis model, safety in animal body for hemostasis.
(4) Ulva pertusa
- Total fibrinolytic activity of purified agents : 245 unit, yield : 2.5%, molecular weight :55 kDa
- Incubaton of this enzyme with Mg²⁺,Cu²⁺,Na²⁺,Fe²⁺ metal ion induced to decrease this enzyme's activity.
- This enzyme is chymotrypsin-like serine protease that showed optimum activity at 40 and pH 5.0.
- Amydolytic ativity of this enzyme was highest on S-2288 substrate(t-PA), following substrate for u-PA.
- N-terminal sequence is NYDAATLPEELYF that showed 53 % of identification(%) to serine protease.
- This enzyme lysed completely Aα chain(66 kDa), Bβ chain(55 kDa) of fibrinogen within 4 h first 72 proteolysis effect of γ chain(97 kDa) was not revealed.
- in vitoro using blood clot and human fibrin clot, this enzyme can lyse directly blood clot.
- In anticoagulant assay, this enzyme can delay blood coagulation time with coagulant factors(Ⅰ,Ⅱ,Ⅴ,Ⅶ,Ⅹ) that form thrombus.
- In antiplatelet activity assay, This enzyme delay strongly closure time.
- in vivo antithrombotic assay, this enzyme has protective effect (90% inhibition at 200 μg/ml) against thromboembolism in collagen/epinephrine-induced thromboembolism model, antithrombotic activity(80% protection at 200 μg/kg) against tail thromboembolism model, antithrombotic activity(80% protection at 200 μg/kg) against tail thrombosis in carrageenan-induced thrombosis model, safety in animal body for hemostasis.
2. Analysis of selected algae with anti-hyperlipidemic activity
- In this assay, total 4 types of biochemical properties were analyzed(Total cholesterol, HDL,LDL,VLDL,TG)
- Among 6 samples containing algae-derived powder for feeding to animal, 5 samples showed that significant inhibition effect against hyperlipidemia in high cholesterol.
- induced hyperlipidemic model, also, remains of sample showed its inhibition effect against hyperlipidemia.
- Through AI(Atherogenic index), total cholesterol/HDL ratio and HDL/LDL ratio, inhibitory effect of all samples was confirmed significantly.
The first cooperative institution : Analysis of algae extracts with neuroprotective effects and various biological activities(Gwangju University, Kim Seung)
1. The safety and biological activities analysis of selected algae.
○ Anti-oxidant activity
- The DPPH radical scavenging activity of green algae, Enteromorpha and Capsosiphon fulvescens was higher than that activity of brown algae and red algae.
- The contents of total polyphenol and total flavonoid were hghest in Enteromorpha.
- The nitrite inhibitory activity was highest in Undaria pinnatifida.
(2) Cytotoxicity
- Up to 10 μg/ml, the survival percentage of cell was not changed at all algae extracts, significantly.
- At 100 μg/ml of all algae extracts over, decrease of the servival percentage of most cells was revealed.
(3) Whitening effect
- The absorbance at UV A,B and C was not high.
- The mushroom tyrosinase inhibitory effect of all algae extracts was existed.
- Hizikia fusiformis extracts have high inhibition activity against both tyrosinase activity and melanin production.
(4) Anti-inflammatoryand anti-allergy activity
- The inhibition activity against β-hexosaminidase secretory was highest in Hizikia fusiformis extract.
- The inhibition activityagainst NO secretory was higher in Hizikia fusiformis extract and Undaria pinnatifida extract than others.
2. Anaysis of induction and expression of neurodegenerative diseases-related gene for provention
(1) Neuroprotective effects
- When 6-OHDA was treated to related cell, neuroprotective effect of Hizikia fusiformis extract was higher than others's effect. Also, when rotenoe treated to related cell, neuroprotective effect of Undaria pinnatifida extract was higher than others's effect.
- ROS induction by rotenone and 6-OHDA was decreased strongly by Hizikia fusiformis extract and Undaria pinnatifida extract.
a. Anti-apoptosis effect of fucoxanthin against 6-OHDA-induced oxidant stress and apoptosis.
- 6-OHDA-induced apoptosis in SH-SY5Y cell was inhibited by fucoxanthin
- Following effects were revealed : inhibition of ROS induction, decrease of mitochondria memebrane potential, increase of intracellular Ca²⁺ concentration.
- The change of nucleic conformation was inhibited.
- The devrease of Bcl-2, anti-apoptotic factor was inhibited, also, the expression and activation of Bax, caspase-3 were inhibited.
- The expression of 6-OHDA-induced endoplasmic reticulumn stress-related factors(ATF6, CHOP, GRP78) was inhibited.
- 6-OHDA-induced phosphorylation of ERK, p38, AKT and translocaton of NF k-B were inhibited.
b. The inhibitory effects of fucoxanthin against inflammatory response in microglial cell
- After treated fucoxanthin((0,5,10,20 μM)) and LPS to microglia, any change of survival percentage was not revealed.
- In LPS-treated microglia, No induction, ROS induction and phagocytosis were decreased.
- In LPS-treated microglia, PGE2 inducton and pro-inflammatory cytokines(IL-1β, TNF-α) induction were decreased.
- In LPS-treated microglia, expression increase of iNOS, COX-2 and MMP-3 were decreased.
(2) The β-amyloid degradation activity by fibrinolytic agents
- In Thioflavin T assay, fibrinolytic agent of Codium fragile was showed that devrease effects by that agents's degradative action.
- Cytotoxicity of purified agent was revealed that survival percentage was decreased at over 100㎍/㎖.
- When Codium fragile-derived agent was treated to cell, decrease of survival percentage by β-amyloid was inhibited.
The second cooperative institution : Analysis of bio-active compound and productization (Chonbuk National University, Kim Myung-Kon)
1. Anti-inflammatory activity analysis, fraction preparation and solvent condition about Undaria pinnatifida
- Following this solvent condition, DPPH radical scavenging activity of Undaria pinnatifida was higher at Haxane solvent than MeOH solvent.
- The main fraction was eluted at Hexane:Ethylacetate=10:l using Silica-gel column chromatography
(1) Cytotoxicity and Anti-inflammatory activity on microglial cell of Undaria pinnatifida
- Up to 5 μg/ml of concentration, all cells were survived over 95%.
- Inhibitory effect of nitric oxide production: In LPS-treated group(control), NO concentration was 45.30±4.44μM. In each higher concentration of active compound, 39.07 ± 1.94 μM, 33.53 ± 1.35 μM, 21.78 ± 1.0 μM, NO concentration has decreased by active compound.
- Inhibitory effect of prostagladin E2 production: In LPS-treated group(control), PGE2 concentration was 1400 ± 1.55 μM. In each higher concentration of active compound, 891.64 ± 3.69 μM, 685.15 ± 6.94 μM, 200.45 ± 4.0 μM, PGE2 concentration has decreased by active compound.
- Inhibitoly effect of TNF-α production : In LPS-treated group(control), TNF-α concentration was 3801.45 ± 3.51 μM, In each higher concentration of active compound, 3612.53 ± 1.09 μM, 2902 ± 2.84 μM, 2064.84 ± 3.42 μM, TNF-α concentration has decreased by active compound.
(2) Isolation and structure study of fucoxanthin from Undaria pinnatifida
- The purity of active fraction that have single band was 98.5 % over.
- The isolated compound was Amax at 446.5 nm, 658 molecular weight by LC-API -MS(positive mode).
- In 13C-NMR 뭄lysis, 42 carbon signals were detected. In IH-NMR spectrum, 1 acetyl resine, 1 conjugated keone resine, 2 quaternary germinal dimethyl resine, 4 olefinic methyl resine, allene resine were detected in molelecule, that compound was presumed polyene like chemicals.
- Therefore, that isolated compound is all-trans-fucoxanthin.
2. Analysis of material composition and main compound with biological activity from brown algae
- Among a brown algae, fucoxanthin content of kelf (Laminaria japonica) was 1.10 mg/g, fucoxanthin contents of kelf (Undaria pinnatifida) and Hizikia fusiformis were 1.83 and 1.87 mg/g, these contents were higher than Laminaria japonica's content.
- Total polyphenol content as dried materials of Hizikia fusiformis was highest 543.8 mg/g, following by Undaria pinnatifida, Laminaria japonica, Porphyra tenera.
Electron-donating ability by DPPH method was highest on Hizikia fusiformis, follwing Ecklonia cava, Undaria pinnatifida, sporophyll of Undaria pinnatifida, Undaria pinnatificla from market, Porphyra tenera, Laminaria japonica.
- Polyphenol, flavonoid, polysaccharide contents of various algae (Porphyra tenera, Undaria pinnatifida, sporophyll of Undaria pinnatificla, Hizikia fusiformis, Ecklonia cava, Enteromorpha, Capsosiphon fulvescens) were analyzed.
- Total amino acid contents of Porphyra tenera were highest, following Enteromorpha, Ecklonia cava, Capsosiphon fulvescens, Unciaria pinnatifida, Hizikia fusiformis.
- γ-Amminobutyric acid(GABA) contents were 15-80mg%, content of Hizikia fusiformis was highest, following Porphyra tenera, Ecklonia cava=Laminaria japonica, Undaria pinnatifida, Capsosiphon fuivescens=Enteromorpha.
- Taurine was contained in Porphyra tenera and Hizikia fusiformis, that contents of Porphyra tenera were highest.
- Free amino acid contents of various algae were 42-935 mg%, in Porphyra tenera highest contet was detected, following Enteromorpha, Undaria pinnatifida, Hizikia fusiformis, Ecklonia cava, Capsosiphon fulvescens.
3. Isolation and structure study of fucosterol
- Dried-powder material of Undaria pinnatifida was extracted using organic solvent, then, white color-powder material (272 mg) was isolated from dried Undaria pinnatifida(800 g) using column chromathgraphy as silica gel column and Lichroprep RP-18 repeatedly.
- The molecular weight of this compound was 412 (C2911480) by HPLC-MS (positive API mode) and GC-MS (EI-MS).
- 1H-NMR(600 MHz, CDCI3) :δH(ppm): 5.28 (1H, br, d, H-6), 5.11 (1H, q, H-28), 3.38 (lH, m, H-3), 1.78(3H, m, H-29), 0.94(3H, s, H-19), 0.93(3H, d, H-21), 0.92(3H, d, H-27), 0.91 (3H, s, H-26), 0.90 (3H, s, H-18)
- 13C-NMR spectrum : 29 carbon signals were detected, finally, This compound was fucosterol(5, 24(28)-stigmastadien-3β-ol)
4. Analysis of sterol contents
- Depending on the time of collection, fucosterol contents were changed.
- For usage and isolation of fucosterol, brown algae (Undaria pinnatifida, Hizikia fusiformis, Sargassum thunbergii) was the most appropriate samples than red algae or green algae.
5. Isolation and structure study of antheraxanthin
- Powder material of freez-dried Hizikia fusiformis was extracted using petroleum ether : methanol: ethyl acetate petroleum ether: methanol: ethyl acetate (1:1:1, v/v) solvent, then, was concentrated, black-red color powder material was isolated by silica gel column chromatography repeatedly.
- The isolated compound has λmax at 445 and 455 nm like carotenoid-antheraxanthin, 584.4 molecular weight (C40H5603) by HPLC-MS (positive ESI mode).
- 1H-NMR(600 MHz, CDCI3) spectrum: δ : 0.84(s, 3H, H-16'), 1.07(s, 3H, H-17'), 1.20(s, 3H, H-18'), 1.25(s,3H, H-17), 1.26(dd, 1H, J=13.0, 14.0Hz, H-20), 1.37(s, 3H, H-16), 1.73(s, 1H), 1.83(d, 1H, J=5.4 Hz,H-2), 1.95(s, 3H, H-19'), 1.97(s, 3H, H-18), 1.99(s, 3H, H-20'), 2.04(s,3H, H-20), 2.96(dd, 1H, J=9.0,14.1 Hz), 4.03(m, 1H, H-3'), 4.13(dd,1H, J=6.6, 14.4 Hz, H-3), 6.15(d, 1H, J=12.4 Hz, H-7'), 6.26(d, 1H, J=12.0 Hz, H-10'), 6.44(d, J=15.0 Hz, H-8'), 6.54(d, 1H, J=16.8 Hz, H-8), 6.90(m, 1H, H-15)
- 13C-NMR sepectrum: about 40 carbon signals were detected.
6. Analysis of antheraxanthin contents in algae
- Antheraxanthin contents were highest in Undaria pinnatifida from 2012, following in Hizilda fusiformis from 2013, in Hizikia fusiformis from 2012, in Sargassum thunbergii, in Undaria pinnatifida from 2013, Laminaria japonica.
- Lutein contents were highest in Enteromorpha and Undaria pinnatifida from market.
- Also, Zeaxanthin contents were highest in Enteromorpha and Undaria pinnatifida from market.
- α-Carotene contents were highest in Undaria pinnatificla from 2012. β-Carotene contents were highest in Undaria pinnatifida from market.