최 종 연구보고서
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Chapter 1. Outline of research development project...12 1. Aims of research development...12 2. Necessity and categories of research development...12 Chapter 2. The present conditions of domestic or outside the country for research development...17 Chapter 3. Contents and results of research project...17 1. Preface...19 2. Antimicrobial activity of extract of Stachys sieboldii....19 1) Material and method...20 2) Extraction of sample...21 3) Minimum inhibition concentration(mic) of fraction...21 4) Is antibacterial activity of extract...21 5) Measurement of β-galactosidase activity...23 3. Scavenging activity of active oxygen by superoxide dismutase(sod)...24 1) Material and method...25 a) Material...25 b) Animal experiment and composition of diets...25 c) Measurement of superoxide dismutase(sod)...25 d) Measurement of peroxide value(pov)...25 e) Measarement of lipid peroide...26 4. Inhibitory effect of oxidative stress by extraction of Stachys sieboldii..26 1) Materials and method...27 a) Composition of diets...27 b) Measurement of cholesterol level...27 c) Measuremtnt of cell menbrame fluidity...28 d) Quantative test of basic and derived free oxygen radical...28 e) Analysis of oxidative stress...28 5. Antioxidative activity of Stachys sieboldii extracts for human LDL...29 1) Antioxidative activity of LDL...29 a) Seperation of human LDL...29 b) Cultivation of macrophage...29-6 -
c) LDL oxidation by mediated metal...30 d) Measurement of thiobarbituric acid reacting substance( TBARS )...30 6. Research result of antimicrobial activity by Stachys sieboldii...31 1) Fraction yield of organic solvent fraction obtained from Stachys sieboldii...31 2) Screening test of ethanol fraction...31 3) Screening test of fractions...31 4) Minimum inhibition concentration of microbials...33 5) Inhibition of growth spectrum...,,,,,...33 6) Antimicrobial activity of Ist column chromatography fraction...34 7) Antimicrobial activity of 2nd column chromatography fraction...35 8) Antimicrobial activity of 3rd fraction...38 9) Cell changes of cell morphology...40 10) Activity of β-galactosidase...40 11) Stability of thermal and ph...40 7. Result of SOD activity of Stachys sieboldii...43 1) SOD activity...43 2) Inhibition of lipid peroxide...43 3) Activity of antioxidative enzyme system...45 8. Inhibition effects of oxidative stress...50 1) changes of contents of liver cholesterol...50 가 ) Estimation of cell menbrane fluidity...51 (1) Memberane fluidity...51 (2) Estimation of basic or derived oxygen free radical...52 (3) Inhibition effect of lipid peroxide...53 9. Antioxidative activity of human LDL...55 1) Antioxidative effect of human LDL...55 2) Electrophoretic mobility of LDL...56 3) Comparision of antioxidative activity...57 10. Antitumor effect of Stachys sieboldii...57 1) Prefaces...59 2) Materials and method...60 3) Material...60 4) Preparation of Stachys sieboldii extraction...61 5) Animal experiment...61-7 -
6) Animal diets...61 7) Measurement of growth of normal cells...61 8) Cytotoxicity of cancer cell or normal cells...61 9) T-lymph...61 10) Measurement of nitric oxide...61 11) measurement of tumor necrosis factor...61 12) Measurement of fluorrasent microbials...61 13) Tumor respiratory test...61 11. Antioxidative activity of root of Stachys sieboldii...62 1) Material and Method...62 2) Material...63 3) Extraction of Stachys sieboldii...63 4) Measurement of total phenols...63 5) Hydrogen donor by DPPH...63 6) Measurement of inhibition of lipid peroxide...64 7) Measurement of nitrite scavenge...64 8) Isolation by columne chromatography...64 12. Results of anticancer or immuno modulator by Sachys sieboldii...65 1) Effect of cell growth...65 2) Effect of T-lymph...67 3) Effect of No ; TNF-α production...68 4) Effect of tumor respiration...69 13. Results of antioxidation...70 1) Yield of extraction of Stachys sieboldii...70 2) Antioxidatively activity by DPPH...71 3) Inhibition effect of lipid peroxide...72 4) Scavenging effect of nitrite...73 5) Seperation of firaetion by column chromatography...74 6) Antioxidative activity of substanceby column chromatography...75 7) Chemical struction of fraction...79 Chapter 4. Achievement degree of aims and relative contribution...85 Chapter 5. Utilization plan of research results....87-8 -
Chapter 6. Abroad scientific information by research development process..87 Chapter 7. References...88-9 -
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본연구는국민의식생활및의식의급격한변화와 WTO 출범등국제환경의변화 에대처하고국민보건증진이라는궁극적목표를달성하기위한목표의일환으로 세계적으로급속하게연구가진행되고있는신기능식품소재를이용한생활습관병의 예방과치료를목적으로하는신기능성건강보조식품을개발하고자한다. 지난수 십년간고도경제성장과산업사회로의급속한진전은우리의식생활패턴을크게변 화시키고있다. 이러한변화는식품소비구조의전환을초래하였고식품소비성향 도고급화, 다양화되고편의성, 안정성에대한선호도가증가하고있으며건강지향 성으로의식변화가일어나고있다. 건강보조식품의개발도 1970 ~ 1980년에는 옛날부터전래되어오는경험을이용한식품성분자체를가공한건강보조식품이많 았으나 1990년대부터는생명공학의기법을이용한새로운기능을가진생리활성물 질로고혈압, 당뇨병, 암, 동맥경화등생활습관병은물론이고각종질병의예방식품 이개발되고있어이러한건강식품산업은경제적으로차지하는비중이높다. 우리 나라의식품산업은년간매출량의 3위이며미국의경우에는국민총생산량의약 2 0 % 에해당하는거대시장을가지고있는분야이다. 그중건강보조식품의시장규모 는 1999년을기준으로매출액은우리나라는 7 천억, 일본은 6천억엔그리고미국의 경우 45억달러로그신장율은매년 30 % 이상증가하고있다. 인간은산소를이용해서생명활동에필요한에너지를얻어살아가고있다. 산소는 사람을포함한호기성생물의생존에필요불가결한요소로경우에따라반응성이 높은활성산소를생성한다. 일상생활이나그릇된식습관으로산화 stress(oxidative stress) 을받아과잉으로발생한활성산소가생체방어계에의하여소거되지않은 경우에는활성산소에의하여장해가발생한다. 즉, 산화적스트레스로활성산소유리 기가발생하여발암, 동맥경화, 당뇨병등각종생활습관병이나노화라는질환이발생 한다. 현재우리가살아가고있는현대사회에서모든사람들은스트레스를받고살아가 고있다. 많은의학전문가들은모든질병의원인은산화적스트레스에의하여발생 한다고주장하고있다. 우리인체는방어시스템에의하여활성산소소거기능이있다지만스트레스에의해 - 14 -
발생하는활성산소의폐해를예방하기위해서는비타민 E 나카로테노이드, 플라보노 이드등이풍부히들어있는황록색야채를충분히섭취하면활성산소가소거된다. 이러한성분은생체내항산화물질로서활성산소의생성을억제하는작용을한다 이러한배경에서특히주목되고있는것이식물성분중산화적스트레스로인한 활성산소의생성을억제하고소거기능을가진신기능성물질이다. 지금까지대부분 의연구는비타민 A, C, E 에집중되었으나, 최근에이르러 in vitro, in vivo 실험을 통하여야채, 다류, 적포도주에함유된 flavonoides, 특히 flavon-(3)-ol 등그유사 물질이생체내에서강력한활성산소소거작용이있다는것이알려졌다. 본연구는농민의소득증대를극대화하고, WTO 개방시대에식품산업의국제경쟁 력확보와국민의건강증진을최종목표로정하여신기능성성분을다량함유하고 있는유용식물을재배하여식품의신소재로개발하고져한다. 유용식물중재배가 쉽고효능이뛰어난초석잠( 草石蠶, 일명식물의동충하초) 을원료로사용하였다. 초 석잠은 1년생풀로서동충하초와모양이비슷하고약효도비슷하여식물의동충하초 라고불리기도하며우리나라남부지방에서자생하는특용작물이지만잘알려지지 않아방치되고있는실정이다. 초석잠은중국의중약편에의하면뇌경색, 기억력증 진, 노인성치매와장을강화하는장수채( 長壽菜 ) 로서옛날부터애용해왔다. 일본에 서도정월요리에귀하게쓰이기도하고여러가지생활습관병과만성병치료에유 용하게쓰이기도하였다. 초석잠의성분중탄수화물은감자와같은전분이아니라 올리고당으로장속의유익세균의생육을도와장의기능을촉진하기도한다. 중국서적에소개된초석잠의효능을보면맛이달며담백하고독이없어풍을쫓 고어혈, 적혈을풀며기를내리게하고신체의조화를이루게하여무병장수에기 여한다고기록되어있다. 또사지무력증, 마비증, 전신골절통, 관절염, 신경통등을 치유하고초석잠즙액으로눈병을낫게하고간을좋게하는작용이있어황달을낫 게한다. 본연구의개발목표는초석잠을이용하여산화적스트레스로인하여발생하는유해 한활성산소를제거하는신기능성식품소재를개발하는것으로궁극적으로는농가 소득증대를보장하는것이다. 이목표를달성하기위하여아래와같이 5가지의내 용를세웠다. 유해한활성산소소거능, 항균, 항암효과가있는성분을초석잠으로부터분리확인하 여선도물질(leading substances) 을도출하였다. 초석잠에서도출된선도물질을신 기능성식품소재로개발하여리딩상품(leading goods) 으로기능성식품의판매시장 에정착시킨다. 초석잠을이용한신기능성고부가가치의제품으로자원화활용시스 템을구축 한다. 초석잠의이용극대화로농촌에서쉽게재배하여이를활용하여실 용화하므로써농가의수익을최대한보장할수있도록한다. 초석잠은우리나라산야에쉽게재배가가능하므로전국농가에서재배하여농가소 - 15 -
득을증대시킬수있고원료를쉽게구입할수있는잇점이있다. 초석잠을이용하여가공적성을알아보고이를이용하여산화적스트레스로인하여발생하는유해한활성산소를소거하는소거제(super oxygen dismutase (SOD)) 로서신기능신소재와천연항균제, 항암효과및안정성도검토하여식용가능성을확인하였다. - 16 -
생체내에서산화스트레스로생산되는활성산소, 즉일중항산소( 一重項酸素 ) super- oxide OH radical등의활성산소종은최근주목받고있는 NO 유래의 ONOO 도함유하는넓은의미의 free radical 이생체손상의원인으로알려졌다. 산화스트레스에의하여발생하는활성산소는체내에서과잉으로생성되면반응 성이높은유리기가생성되어세포벽에과산화지질이생성된다. 생성된과산화지질 은세포의유동성이변화되고막기능이저화되어세포막의효소의활성이감소된다. 결국세포막이파괴되고세포로부터방출된지질과산화물은혈청내과산화지질의 농도가급격히증가하여생체성분인지질, 단백질및핵산등이손상을받게된다. 손 상받은세포가회복되지못하고축적되어한계에달하면산화스트레스에의하여 발생하는활성산소때문에생활습관병이라는질병의원인이된다. 문제는세포막의주요한구성성분에는인지질, 과산화하기쉬운불포화지방산이 있다. 활성산소등유리기가연쇄반응을일으켜 생체지질과산화반응 이진행 되어반응성이높은 free radical 이단백질, 효소, 핵산등의생체구성성분을계속 적으로공격해서산화적손상에의하여최종적으로는암, 동맥경화, 허혈성심장질 환, 치매및파킨스병, 면역력기능저하등질병이발병하며최근에는당뇨병성합 병증도발병한다. 산화적스트레스에의한활성산소를소거하기위하여유용식물을응용하거나섭취함 으로써유용물질들이 lipid peroxide나유리기들에의한 tissue damage를예방할 것이라고여겨졌다. 그러나 α-tocopherol, ascorbic acid, β-carotene와같은천연 항산화제들을매일복용하여도유리기로유발되는질병을예방하는데에는큰효과 를바라긴어렵다. 그러므로, 더새롭고효과적인 antioxidant들을찾는것이시급하 다. 그반면에식물에들어있는특수성분들은활성산소소거능력이 in vitro와 in vivo 실험들로증명되고, isoflavonoids가강력한항산화성을갖는대사물을생산하 는것으로알려졌다. 다양한 in vitro와 in vivo 실험에서 polyphenol의항산화효과 를실험한보고가있다. 예를들어, 초기실험을통하여 polyphenol 화합물들이 ADP-ascorbic acid와 ADP-NADPH이각각실험쥐의간미토콘드리아와마이크 로솜에서 lipid peroxidation 을매우억제하는것을알수있었다. 대부분의 polyphenol 들이 α-tocopherol 보다더우세한억제효과를나타내었다. 2-azo-bis-isobutylonitrile을 photo-irradiation하여 methyl linoleate를 autooxidation 시키는동역학연구를통하여 polyphenol의억제효과가 ascorbic acid나 α-tocopherol 보다더오래지속되는것을알수있었다. Isoflavonoids등은 생체내에서의기능성에관한연구는많이없으나최근에는많은연구가진행되고있 으며식품내의각종유기물은섭취후생체내의동향에관하여분자수준에서연구 - 17 -
가요구된다. 요즘임상적으로응용하는항산화식품의분자설계가일부이루워지고 있으나식품이라는개념의범위내에서기능성을갖는중요성을과학적으로증명하여 매일식생활로일어나는산화적손상으로부터우리몸을보호하여산화스트레스의 원인이되는질병인암, 동맥경화, 당뇨병합병증, 허혈성심장질환및알츠하이머증 등은신기능성신소재식품으로예방할수있을것으로기대된다. 기능성건강식품에대한관심고조, 재배기술의향상그리고국산재료의선호도 증가등으로이들제품의생산및유통은앞으로크게증가할것으로예측하고있 다. 현재건강식품의시장은 7,000 역규모이나앞으로계속신장할것이다. - 18 -
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Stalks of stachys sieboldii MIQ : stalks(580g) 99% methanol 4L 3time for 15 hours Methanol Extract Hexane:Aqueous(1:1) 3time for 3hours Hexane layer Aqueous layer Evaporation Chloroform:Aqueous(1:1) 3time for 3hours Hexane Fr. Chloroform layer Evaporation Aqueous layer Ethylacetate:Aqueous (1:1 3time for 3hours) Chloroform Fr. Ethylacetate Aqueous layer Evaporation Butanol:Aqueous (1:1) 3time for 3hours Ethylacetate Fr. Butanol layer Evaporation Evaporation Butanol Fr. Aqueous layer Fig. 1. Flow chart for solvent fractionation of Stachys - 22 -
Microoraganisns taste Media used Listeria monocytogenes ATCC 15313 Listeria monocytogenes ATCC 19111 Listeria monocytogenes ATCC 19113 Listeria monocytogenes ATCC 19114 Listeria monocytogenes ATCC 43256 Salmonella typhimurium ATCC 14028 Bacillus cereus ATCC 33844 Esherichia coli BHR-12 Staphylococcus aureus ATCC 25923 Pseudomonas aeruginosa ATCC 27853 TSB & TSA TSB & TSA TSB & TSA TSB & TSA TSB & TSA NB & NA NB & NA TSB & TSA TSB & TSA NB & NA Table 1. List of strains and media used for antimicrobial experiment TSB & TSA : Tryptic soy broth/agar(difco) NB & NA : Nuritent broth/agar(difco) - 23 -
β β β β - 24 -
β μ μ μ μ - 25 -
μ μ - 26 -
μ μ μ μ - 27 -
- 28 -
μ - 29 -
μ - 30 -
- 31 - μ
Table 2. The fraction yield of solvent fractions extracted from ethanol extracts of Stachys sieboldii. Solvents Hexane Chloroform Ethylacetate Butanol Water Total Yield(%, w/w) 9.46 4.53 5.74 5.33 26.42 51.48 Table 3. Antimicrobial activity of ethanol extract of Stachys sieboldii leaf on microorganisms Microorganisms 1) Listeria monocytogenes ATCC 19111 Listeria monocytogenes ATCC 43256 Listeria monocytogenes ATCC 15313 Listeria monocytogenes ATCC 19113 Listeria monocytogenes ATCC 15114 Salmonella typhimurium ATCC 14028 Bacillus cereus ATCC 13720 Esherichia coli BHR-12 Pseudomonas aeruginosa ATCC 27853 Clear zone(mm) 20 21 18 20 18 19 15 15 15 1) Final cells concentration for each bacterium was approximately 1.0x10 6 cells/ml. - 32 -
Table 4. Antimicrobial activities 1) Stachys sieboldii leaf on test microorganisms Microorganisms B. cereus ATCC 13720 S. cereus ATCC 25923 L. monocytogenes ATCC 19111 L. monocytogenes ATCC 43256 L. monocytogenes ATCC 15113 L. monocytogenes ATCC 15114 L. monocytogenes ATCC 15313 S. typhimurium ATCC 14028 E. coli BHR-12 P. aeruginosa ATCC 27853 of different solvent fractions obtained from 1) Five hundred μg of solvent extracts was absorved into paper disc ( φ 8mm) and Inhibition zone (mm) Solvents Hexan Chlorofor Ethylacetat Butan Water e m e ol the diameter (mm) of clear zone was measured. 14 14 14 14 12 14 13 12 12 10 9 ND 10 10 ND ND 9 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND μ μ μ μ - 33 -
μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ - 34 -
μ μ μ - 35 -
Table 5. Minimal inhibitory concentrations(mic) of different solvent fractions obtained from Stachys sieboldii leaf on test microorganism Solvents fraction MICs( μg/ml) Microorganisms 2) Hexane Chloroform Ethylacetate Butanol 1) B. cereus ATCC 13720 S. cereus ATCC 25923 L. monocytogenes ATCC 19111 L. monocytogenes ATCC 43256 L. monocytogenes ATCC 15113 L. monocytogenes ATCC 15114 L. monocytogenes ATCC 15313 S. typhimurium ATCC 14028 E. coli BHR-12 P. aeruginosa ATCC 27853 250 500 250 250 500 500 250 500 500 500 500 500< 500 500 500< 500< 500< 500< 500< 500< 500< 500< 500< 500< 500< 500< 500< 500< 500< 500< 500< 500< 500< 500< 500< 500< 500< 500< 500< 500< The MICs represents the lowest concentration of antimicrobials activities that showed no growth after 24hrs incubation at 35. Table 6. Minimum inhibitory concentration (MIC) of the 1st column chromatography fraction( μg/ml) collected from n-hexane extract of Stachys sieboldii leaf Listeria monocytogenes Listeria monocytogenes Fraction Yield(g) A T C C A T C C A T C C A T C C A T C C No. 15313 19111 19113 19114 43256 F1 2.18 100< 100< 100< 100< 100< F2 5.45 100< 100< 100< 100< 100< F3 6.80 50<100 50<100 50<100 50<100 50<100 F4 2.00 50<100 50<100 50<100 50<100 50<100 F5 0.41 50<100 50<100 50<100 50<100 50<100 F6 1.00 50 50 50 50 50 F7 7.20 50 50 50 50 50-36 -
Gram(+) bacteria Gram(-) bacteria 1 B. cereus ATCC 13720 1 E. coli BHR-12 O.D at 600nm 0.8 0.6 0.4 O.D at 600nm 0.8 0.6 0.4 0.2 0.2 0 0 8 16 24 32 40 48 0 0 8 16 24 32 40 48 1 S. aureus ATCC 25923 1 S. typhimurimum ATCC 14028 O.D at 600nm 0.8 0.6 0.4 O.D at 600nm 0.8 0.6 0.4 0.2 0.2 0 0 8 16 24 32 40 48 0 0 8 16 24 32 40 48 1 L. monocytogenes ATCC 19111 1 P. aeruginosa ATCC 27853 O.D at 600nm 0.8 0.6 0.4 O.D at 600nm 0.8 0.6 0.4 0.2 0.2 0 0 8 16 24 32 40 48 0 0 8 16 24 32 40 48 Incubation times(hr) Incubation times(hr) Fig 2. Growth inhibition effect of n-hexane fraction from on various pathogenic bacteria - : 500 μg/ml, - : 250 μg/ml, - : 150 μg/ml, - : 100 μg/ml, - : control - 37 -
μ μ μ μ - 38 -
Table 7. Minimum inhibitory concentration (MIC) of the 2nd column chromatography fraction( μg/ml) collected from n-hexane extract of Stachys sieboldii leaf on Listeria monocytogenes Listeria monocytogenes Fraction No. Yield(g) A T C C A T C C A T C C A T C C A T C C 15313 19111 19113 19114 43256 F7-1 0.38 25<50 25<50 25<50 25<50 25<50 F7-2 2.83 25<50 25<50 25<50 25<50 25<50 F7-3 0.46 25<50 25<50 25<50 25<50 25<50 F7-4 0.45 25<50 25<50 25<50 25<50 25<50 F7-5 0.42 25<50 25<50 25<50 25<50 25<50 F7-6 0.14 50< 50< 50< 50< 50< F7-7 0.35 50< 50< 50< 50< 50< F7-8 0.40 50< 50< 50< 50< 50< Fig 8. Minimum inhibitory concentration of the preparative thin layer chromatography fraction collected n-hexane extract of on Listeria monocytogenes. Listeria monocytogenes Fraction No. Yield(g) A T C C A T C C A T C C A T C C A T C C 15313 19111 19113 19114 43256 F7-2-1 5.1 25 25 25 25 25 F7-2-2 5.4 25 25 25 25 25 F7-2-3 4.0 25 25 25 25 25 F7-2-4 5.3 25 25 25 25 25 F7-2-5 18.0 25 25 25 25 25-39 -
μ β β β μ μ - 40 -
(A) (B) Fig. 3 Transmission electron microscope of E. coli BHR-12 A: Normal cells (Control) B, cells treated with F7-2-5 obtained from Stachys sieboldii (A) (B) Fig. 4 Transmission electron microphage of Listeria monocytogenes ATCC (A) Normal cells control B, cells treated with F7-2-5 obtained from Stachys sieboldii - 41 -
Relative activity of a-glgctosidase 100 90 80 70 60 50 40 30 20 10 0 toluene hexane S. R. Extract Fig 5. Membrane perturbation effect by extract Stachys sieboldii leaf on E. coli cells (A) Fig 6. Thermal stability (a) and PH stability of Stachys sieboldii extract on E.coli plates (A) Thermal drgree, a: 40 b: 60 c: 80 d: 100 e: 120 f: 180 (B) ph, a: ph4 b: ph,6, c: ph7 d: ph8 e: ph9 (B) - 42 -
제 7 절초석잠추출물의 SOD 활성의결과 - 43 -
Table 9. Effect of ethanol fraction of Stachys sieboldii (S.S) on superoxide dismutase(sod) relative activity DMSO 0 Catechin (50 μg/ml) 48.96±1.23 Quercetin (50 μg/ml) 57.24±1.74 Stachy sieboldii (50 μg/ml) 47.53±1.60 (unit /mg, protein) Table 10. Inhibition effect of Stachys sieboldii (S.S) on peroxide value (POV) from rat liver microsome induced by Fe 2+ -ascorbate system sample POV (meq/kg) Inhibition (%) Control 1,205.7 - Catechin (50 μg) 224.7 81.36 Quercetin (50 μg) 216.6 82.1 Stachy sieboldii (50 μg) 248.8 79.4 Inhibition Each value represents the mean value of duplicate determination. (%) = [1-(sample POV/control POV)] 100 In vivo 에서간의 microsome 분획에대한지질과산화에대하여 malondialdehyde (MDA) 의결과를 Table 11. 에나타내었다. 지질과산화에대한 실험결과대조군은 10.53 nmol/mg protein 이었으나, 항산화활성이높은것으로 알려진초석잠추출물 50 μg첨가하였을때 2.80 nmole/mg, protein으로 73.4 % 의 억제효과를볼수있었다. Catechin를 50 μg첨가한경우 2.64 nmole/mg protein 으로 74.9 % 의억제효과를나타내었고, quercetin의경우 2.10 nmole/mg, protein으로 79.3 % 의억제효과를나타내었다. 이상의실험결과로보아초석잠 추출물의지질과산화억제효과는상당히높은것으로사료된다. - 44 -
Table 11. Inhibition effects of Stachys sieboldii on lipid peroxidation from rat river microsome induced by Fe 2+ -ascorbate system Sample MDA (nmol/mg, protein) Inhibition Control 10.53 - Catechin(50 μg) 2.64 74.9 Quercetin (50 μg) 2.18 79.3 Stachy sieboldii (50 μg) 2.80 73.4 Each value represents the mean value of duplicate determination. Inhibition(%) = [1-(sample MDA/control MDA)] 100 MDA means malonaldehyde. (%) - 45 -
Fig. 7. Effects of extract of Stachys sieboldii on superoxide dismutase activity (SOD) in rat liver microsome S.S.: Stachys sieboldii : p<0.05, significantly difference from normal control : p<0.05, significantly difference from CCl 4 treatment Each group represents the mean ± SD 한편, Fig. 9는초석잠추출물을쥐에 2주일간투여한후쥐간의 microsome 획분 에존재하는 catalase 활성을비교하였다. 정상사육한대조군의 catalase 활성은 19.05±1.26 U/mg, protein이였으나 CCl 4 처리군에서는 9.14±1.41 U/mg, protein 으로 CCl 4 대조군에비하여 54.1 % 감소하였으나 200 mg/kg 첨가군에서는 12.43±1.17 U/mg, protein으로 CCl 4 처리군에비하여 35.9 % 증가하였고 500 mg/kg 첨가군에서는 15.07±1.28 U/mg, protein으로 72.8 % 의증가를나타내어유 의성이있었다. Fig. 9. 은초석잠추출물을사료에첨가하여 2주간사육한후 glutathion peroxidase 의활성을측정하였다. 대조군에서는 9.80±0.25 IU/g.protein 이였으나, CCl 4 처리군에서는 2.46±0.20 IU/g.protein 으로대조군에비하여 78.7% 감소하였 다. 그러나대두발효를 200 mg/kg 투여한경우 5.37±0.13 IU/g.protein 으로대조 군에비하여 glutathion peroxidase의활성이 23.54% 증가하였고, 500 mg/kg 투여 군에서는 8.08±0.23 IU/g.protein으로 35.03% 증가함을볼수있었다. - 46 -
Fig. 8. 은 in vivo에서초석잠추출물식이가주간의 microsome 획분의지질과산 화의생성을나타내었다. 정상사육한대조군의과산화지질의지표인 malondialdehyde(mda) 는 1.64±0.16 nmole/mg, protein이었으나 CCl 4 처리군에서 는 3.34±0.24 nmole/mg, protein 이였으나, 200mg/kg 첨가군에서는 2.27±0.11 nmole/mg, protein으로 32.4 % 감소하였고, 500 mg/kg 첨가군에서는 1.74±0.15 nmole/mg, protein으로 48.7 % 감소하여유의성이인정되었다. Fig. 8. Effects of extract of Stachys sieboldii on catalase activity (CAT) in rat liver microsome S.S.: Stachys sieboldii : p<0.05, significantly difference from normal control : p<0.05, significantly difference from CCl 4 treatment Each group represents the mean ± SD - 47 -
Fig. 9. Effects of extract of Stachys sieboldii on glutathione peroxide activity (GTHPx) in rat liver microsome. S.S.: Stachys sieboldii : p<0.05, significantly difference from normal control : p<0.05, significantly difference from CCl4 treatment Each group represents the mean ± SD 모든생물체는유산소호흡을하는대사과정에서활성산소를생성하는데적정량의 활성산소는생체내면역기능에관계된대식세포의살균작용등이있으나그양이 많으면활성산소가산화제로서생체막의지질변화, DNA 변이등산화적스트레스 를발생시킨다. 따라서생물체는이러한활성산소를제거하여생체를보호하기위 하여 superoxide dismutase (SOD), catalase, glutathion peroxidase 등항산화효 소를가지고있다. 유리기를제거하는 SOD는활성산소를 OH - 와산소로전환시키 는데효소작용을함으로써활성산소유리기에의해서생기는생체의지질과산화 를방어한다 [6,17]. SOD는생체내에서비교적널리분포된효소로서포유류에는 3종류의 SOD가알 려져있다. Cu, Zn-SOD는각각세포질과세포외액에존재하나 Mn-SOD는주로 mitochondria 에존재한다. Mn-SOD 는노화과정, 발암기전에일부역할을하고산화 - 48 -
스트레스에의한각종생활습관병발생에의한저항성을갖게한다. 활성산소에의하여산화되는동안발생하는 superoxide anion radical(o - 2 ) 은 SOD 에의하여활성이소실되지만이들유리기들이세포막에손상을일으킬것으로사료 된다. 본실험결과발효대두는과산화지질의억제효과가상당히높게나타났다. 그리 고쥐를이용한 Fe 2+ -ascorbate system에서도 catechin 과 quercetin간의대조구 로하여 microsome의과산화지질의억제효과를실험한결과이를대표적인항산 화제와비슷한효과를나타내었다. 활성산소소거활성에서는 SOD의활성이항산 화제로알려진 catechin과 quercetin의산화억제효과보다약간낮은수준의효과 를나타내었다. 초석잠추출물의동물실험결과쥐의간의 microsome의 SOD, catalase, glutathion peroxidase 및지질과산화물의생성억제효과를항산화효소의작용으 로, 생물체의세포막을보호하여노화등을예방할것으로생각된다. Fig. 10. Effects of Stachys sieboldii on lipid peroxidation activity in rat liver microsome. S.S. : Stachys sieboldii : p<0.05, significantly difference from normal control : p<0.05, significantly difference from CCl 4 treatment Each group represents the mean ± SD - 49 -
한편, CCl 4 를투여하여쥐간에손상을일으킨후발효대두를첨가한식이군에서는 항산화관련효소및 SOD와 catalase, glutathion peroxidase 활성이 CCl 4 처리군 보다증가하는경향은발효대두가항산화관련효소의활성과관련이있는것으로 판단된다. 그리고 CCl 4 로간에손상을일으킨쥐에발효대두를첨가한식이를급여 한군에서 성을볼수있었다. SOD, catalse, glutathion peroxidase의활성을억제시켰으며이때유의 이러한실험결과는생체내에서초석잠추출물이 CCl 4 에의하여손상되었든간에 SOD와 catalase의활성이회복되는것을볼수있었고과산화지질의생성은초석잠 추출물에대하여억제되었다. 본실험결과에서초석잠은 in vitro 에서 를억제하였다. SOD의활성을증가시키면서지질과산화 따라서, 초석잠은항산화관련효소의활성을높여주고지질과산화를억제하므 로서노화등생활습관병예방에기여할것으로생각되어진다. - 50 -
Control(7) 200mg/kg 500mg/kg Mitochondria 129.45±1.26 131.41±2.43 131.62±6.42 Microsome 87.83±2.42 86.63±3.41 85.43±3.62-51 -
Control 200mg/kg 500mg/kg Mitochondria 2.65±0.43 a 2.98±0.12 ** 3.32±0.21 *** Microsome 6.2±0.13 7.43±0.12 *** 8.01±0.21 *** - 52 -
Oxygen radical formation Group (nmol/mg protein/min) Mitochondria Microsome Basal oxygen radical(bor) Control 2.33±0.14 a - 2.76±0.23-200mg/kg 1.85±0.16 ** 2.53±0.13 ** 500mg/kg 1.70±0.21 *** 2.48±0.16 *** Induced oxygen radical(ior) Control 13.10±0.93-14.50±2.10-200mg/kg 12.83±0.82 12.25±1.58 ** 500mg/kg 11.56±0.95 * 12.17±1.39 ** - 53 -
(A) Mitochondria Malondialdehyde content (nmol/mg protein) 24 20 16 12 8 4 0 Control S.S 200mg S.S 500mg (B) Microsome Malondialdehyde content (nmol/mg protein) 20 16 12 8 4 0 Control S.S 200mg S.S 500mg - 54 -
- 55 - μ
TBARS, nmole MDA/dish 30 25 20 15 10 5 0 Native LDL Control 20 40 60 80 Extracts of Stachys siebodii ( μg /ml) μ α - 56 -
Incubation Condition Relative mobility electrophoretic P Native LDL 1.0 LDL + macrophages 1.88±0.24 <0.05 LDL + macrophages + S.S 60 μg/ml 1.23±0.14 <0.01 LDL + macrophages + S.S 80 μg/ml 1.36±0.10 <0.01 α - 57 -
α α 30 TBARS (nmol MDA/mL) 25 20 15 10 5 0 Native LDL Control S.S Tocopherol Vit. C Concentration 80 ( μg /ml) μ α μ α μ μ μ - 58 -
- 59 -
- 60 -
β μ μ μ - 61 -
μ - 62 -
Φ α - 63 -
μ μ μ μ α - 64 -
- 65 -
Stachys CPM * sieboldii( μg/ml) Spleen cells FM3A/S b P383/Sc U937/S 0 3081 6769 6564 6790 250 100,134 5230 4401 4875 500 100,360 3890 3843 2330 1000 3864 1060 1034 979-66 -
Stachys sieboldii( μg/ml) Viability(%) FM3A/S b P383/Sc U937/S 0 46.8 46.6 47.2 100 52.8 50.9 53.8 250 48.6 48.7 50.7 500 51.4 46.0 50 1000 43.9 40.4 Percent of single positive cells Ratio CD4 + CD8 + (L3T + ) CD4 + CD8 + (Lyt2 + ) ofcd4 + CD8 + Nomal control 20.4 5.5 4.3 Stachys sieboldii-fed 30.2 7.4 4.7-67 -
α α α α TNF-a concentration(pg/ml 600 400 200 0 Normal S. Sieboldii α α - 68 -
Stimuli a) NO 2 concertration(um) Media only 12±0.4 Stachys sieboldii 28±0.6 LPS+ γ-ifn 38±0.3 LPS+ γ-ifn+ Stachys sieboldii 37.3±0.4 γ S. sieboldii( μg/ml) NO 2 concertration(um) 0 4.57±0.3 50 12.4±0.2 250 16.0±0.1 500 22.0±0.2 1000 17.0±0.2 μ - 69 -
α γ 90 Colony number/ung 60 30 0 Normal S. Sieboldii - 70 -
Table 6. Amounts of polyphenols and flavonoids extracted with methanol from Stachys sieboldii. polyphenols flavonoids Contain volume(%) 3.02 1.97 80 70 69.19 60 Yield(%) 50 40 30 20 10 0 12.40 11.34 3.31 3.79 1.42 Methanol Hexane Chloroform Ethylacetate Butanol Water Solvent Fig. 3. Relative amounts of the extracted fractions from Stachys sieboldii.. - 71 -
225 200 200 175 150 Weight( μg ) 125 100 75 100 80 100 50 25 0 Hexane Chloroform 2.8 Ethylactate 10 Butanol Water Metanol 25 21.8 α-tocophenol BHT 20.6 BHA Solvent Fig. 4. Antioxidant activity of the extracted fractions from stachys sieboldii by DPPH method. α - 72 -
α 0.6 0.5 Absorbance at 500nm 0.4 0.3 0.2 0.1 0 0 1 2 3 4 5 6 7 8 9 10 Time(Day) Control BHA BHT α- Tocopherol Methanol Hexane Chloroform Ethyl acetate Butanol Water Fig 5. Antioxidant activity of the extracted fractions from Stachys sieboldii on the autooxidant of linoleic acid by ferric thiocyanate method. - 73 -
100 90 80 70 Inhibitor(%) 60 50 40 30 20 1.20 Methanol Hexane Chloroform Ethyl acetate Butanol Water 3.03 5.97 Ph Solvent Fig 6. Nitrite scavenging ability of the extracted fractions from Stachys seboldii. - 74 -
Ⅰ Ⅱ Ⅲ Ⅳ Ⅴ Ⅰ Ⅱ Ⅲ Ⅳ Ⅴ Fig. 7. TLC of the fraction separated from the silica gel column chromatography of the ethyl acetate extract from Stachys sieboldii. (A) under Ce(SO 4 )/10%H 2 SO 4 ; (B) under UV light 254nm. - 75 -
- 76 -
Table 6. UV/VIS spectral data for the fractions of ethyl acetate from Stachys sieboldii by silica gel column chromatography fraction λ max (nm) ES-R1 284,315 ES-R2 317,284 ES-R3 326,287 ES-R4 322,296 ES-R5 325,289, ES-R6 289,330-77 -
2.5 2 ES-R1 ES-R2 ES-R3 ES-R4 ES-R5 ES-R6 Absorabance 1.5 1 0.5 0 250260 270280290 300 310 320330 340350360 370 380390400 410 420 430440 450460470 480 490 500 Wavelength(nm) Fig. 8. UV/VIS spectral scan for the fractions of ethylacetate from Stachys sieboldii MIQ. roots by silica gel column chromatography. - 78 -
δ δ δ δ δ δ δ δ - 79 -
0.40 0.35 0.30 Absorbance at 517nm. 0.25 0.20 0.15 0.10 0.05 0.00 0 1 2 3 4 5 6 7 8 9 10 11 Time(min) control ES-R1 ES-R2 ES-R3 ES-R4 ES-R5 ES-R6 Fig. 9. Antioxidant activity of the isolated fraction of ethylacetate from Stachys sieboldii MIQ. roots by silica gel column by DPPH method. - 80 -
- 81 -
- 82 -
Fig. 11 IR spectrum of the fraction - 83 -
he fraction - 84 -
Fig. 13 1-H NMR spectrum of the fraction - 85 -
spectrum of the fraction - 86 -
α α - 87 -
- 88 -
- 89 -
α - 90 -
- 91 - α
- 92 -
- 93 -