저작자표시 - 비영리 - 변경금지 2.0 대한민국 이용자는아래의조건을따르는경우에한하여자유롭게 이저작물을복제, 배포, 전송, 전시, 공연및방송할수있습니다. 다음과같은조건을따라야합니다 : 저작자표시. 귀하는원저작자를표시하여야합니다. 비영리. 귀하는이저작물을영리목적으로이용할

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1 저작자표시 - 비영리 - 변경금지 2.0 대한민국 이용자는아래의조건을따르는경우에한하여자유롭게 이저작물을복제, 배포, 전송, 전시, 공연및방송할수있습니다. 다음과같은조건을따라야합니다 : 저작자표시. 귀하는원저작자를표시하여야합니다. 비영리. 귀하는이저작물을영리목적으로이용할수없습니다. 변경금지. 귀하는이저작물을개작, 변형또는가공할수없습니다. 귀하는, 이저작물의재이용이나배포의경우, 이저작물에적용된이용허락조건을명확하게나타내어야합니다. 저작권자로부터별도의허가를받으면이러한조건들은적용되지않습니다. 저작권법에따른이용자의권리는위의내용에의하여영향을받지않습니다. 이것은이용허락규약 (Legal Code) 을이해하기쉽게요약한것입니다. Disclaimer

2 약학박사학위논문 약물대사체학을이용한진세노사이드 Rk 1 의항혈소판작용기전관련대사체규명 Metabolomic Investigation of the Anti-Platelet Aggregation Activity of Ginsenoside Rk 1 Reveals Attenuated 12-HETE Production 2013 년 2 월 서울대학교대학원 약학과약품분석전공 주현경

3 국문초록 혈소판은혈관손상및출혈이생기는경우활성화되어지혈작용에중요한역할을하는세포로서, 과도한지혈작용에의해생긴혈전 (thrombus) 으로부터혈액순환장애및심근경색증, 뇌졸중등다양한질환을유발한다. 최근심혈관질환발병율이증가하고있으며, 혈소판의응고기전을억제시키는약물의개발및기전연구는심혈관계질환치료제개발에매우유용하다고할수있다. 약물대사체학 (Pharmacometabolomics) 은세포, 조직, 혹은생체액속에포함되어있는저분자량물질인대사체 (metabolite) 를그대상으로하는대사체학중하나로서, 약물에의해변화된많은대사체를동시에관찰하고, 대사체들간의관계를설명하는학문이라할수있다. 동시에관찰되는많은대사체의변화는통계적기법을이용하여해석할수있으며, 변화된대사체를통하여약물기전연구에중용한정보를제공하고, 판별분석모델링을통하여질병상태의예측또는약물반응의예후예측에대한가능성을제시하고있다. 이에본연구에서는, arachidonic acid에의하여유도되는혈소판응집작용에대한진세노사이드 Rk 1 의작용기전연구를약물대사체학을통해수행하고자하였다. 혈소판내의 metabolites profiling은 UPLC/Q-TOF를이용하여실시하였으며, 수집된데이터는 Profile analysis software에의해버켓팅 (Bucketing) 되었다. 주성분분석 (Principal component analysis PCA) 및판별분석 (Partial least square discriminant analysis, PLS-DA) 은 SIMCA-P (v 12.0) 를이용하여수행되었으며, 판별분석을통해그룹간의차이를규명할수있는대사체를효과적으로판별할수있었다. 혈소판이응집된샘플과진세노사이 i

4 드 Rk 1 이처리된샘플군에서 12(S)-Hydroxyeicosatetradecaenoic acid (12(S)- HETE) 가유의성있게감소된것을확인할수있었으며, 이는 positive control로사용했던아스피린처리군과도구별되는대사체임을확인하였다. 반면, Thromboxane B 2 의경우아스피린처리군과구별되지않음을확인할수있었다. 이에, 진세노사이드 Rk 1 의항혈소판작용은 TXB 2 의생성을억제하는아스피린의작용기전에도관여하며, 12(S)-HETE의생성기전이항혈소판응집작용에기여하는것으로확인하였다. 또한, 본실험에서는진세노사이드 Rk 1 이세포내칼슘유리및외부로부터의칼슘유입을효과적으로억제하는것을확인하였다. 이에, 칼슘농도변화와 12-LOX의연계성을확인하였고, 12-HETE의생성은칼슘의농도변화가 12-LOX의 translocation에영향을미침으로써나타내는현상으로판단하였다. 주요어 : 인삼 (Panax ginseng), 진세노사이드 (Ginsenoside), 항혈소판응집 (Antiplatelet aggregation), 약물대사체학 (Pharmacometabolomics) Arachidonic acid, 12- Hydroxyeicosatetradecaenoic acid 학번 : ii

5 목 차 국문초록 i 목차 iii List of Figures vi List of Tables viii List of Abbreviations ix Ⅰ. 서론 1 1. 약물대사체학 약물대사체학의의의및목적 주성분분석및판별분석 3 2. 혈소판의생리및활성화 5 3. Ginsenoside Rk 1 의항혈소판작용 8 Ⅱ. 실험방법 실험재료, 시약및기기 재료및시약 실험동물 11 iii

6 1-3. 실험기기 Ginsenoside Rk 1 의분리 가공인삼 (Sun ginseng, SG) 의제조 Ginsenoside Rk 1 의분리 Ginsenoside Rk 1 의혈소판응집억제활성 Washed platelets (WP) 의제조 혈소판응집억제활성 UPLC/Q-TOF MS 분석방법의검증 분석조건 분석법검증 (Method validation) 혈소판내의대사체분석 분석조건 분석샘플제조 데이터수집및처리 다변량분석 Cyclooxygenase (COX) 활성 COX 활성의검정 통계적처리법 23 iv

7 9. 혈소판내 calcium 변화측정 Western blot analysis 25 Ⅲ. 결과및고찰 Ginsenoside Rk 1 의항혈소판응집작용 시스템불순물분석및제거 UPLC/Q-TOFMS 분석법검증 UPLC/Q-TOFMS 를이용한대사체분석 주성분분석및판별분석 Eicosanoids 및 TXB 2 생성억제효과 Cyclooxygenase (COX) 저해활성 혈소판내 calcium 농도측정 Lipoxygenase (12-LOX) 의 translocation 53 Ⅵ. 결론 54 Ⅴ. 참고문헌 57 Abstract 62 v

8 List of Figures Figure 1: Workflow of pharmacometabolomics 4 Figure 2: Structural modification of dammarane glycosides by heat processing 6 Figure 3: Schematic procedure for sample preparation for washed platelets and metabolomics 16 Figure 4: Anti-platelet aggregation activity of ginsenoside Rk 1 and aspirin (ASA) 27 Figure 5: Identification of system impurities 29 Figure 6: Validation of the LC method; Precision test for mass to charge 31 Figure 7: Validation of the LC method; Precision test for retention time 32 Figure 8: Validation of the LC method; Linearity 33 Figure 9: The confirmation of carry-over effect in analysis 34 Figure 10: Base peak chromatograms of platelets samples using UPLC/micrOTOF MS 35 Figure 11: Calibration using calibrant injected before every sample run. 36 Figure 12: Correlations of m/z signals and the dilution factor 37 Figure 13: The calibration effect of measured m/z on database search 38 Figure 14: Multivariate analysis using metabolomes data 43 Figure 15: Validation plot of the statistical PLS-DA model 44 Figure 16: OPLS-DA score plots 45 Figure 17: Alteration of (S)-hydroxyeicosatetradecaenoic acids (HETEs) and thromboxane B 2 (TXB 2 ) 47 vi

9 Figure 18: Metabolite pathway of arachidonic acid 49 Figure 19: Cyclooxygenase (COX) inhibitory activities 50 Figure 20: Effects of ginsenoside Rk 1 on the arachidonic acid (AA) -induced changes in platelet cytoplasmic calcium concentration. 52 Figure 21: Effects of ginsenoside Rk 1 on 12-lipoxygenase translocation from cytosol to membrane. 53 Figure 22: Biological behavior of aspirin and ginsenoside Rk 1 on anti-platelet aggregatory activity. 56 vii

10 List of Tables Table 1. The list of metabolites observed in washed rat platelet, analyzed by UPLC/micrOTOF MS in negative mode Table 2. The list of metabolites observed in washed rat platelet, analyzed by UPLC/micrOTOF MS in positive mode. viii

11 List of Abbreviation AA ADP ANOVA ASA camp BPC COX C.V. DMSO EGTA GP GPCRs HETE HpETE LOD LOX PCA PLS-DA PG RT PRP : arachidonic acid : adenosine diphosphate : analysis of variance : acetylsalicylic acid : cyclic adenosine monophosphate : Base peak chromatography : cyclooxygenase : coefficient of variation : dimethyl sulfoxide : Ethylene glycol-bis(b-aminoethylether)-n -N -N -N -tetraactic acid : glycoprotein : G protein-coupled receptors :hydroxyeicosatetradecaenoic acid :hydroperoxyeicosatetradecaenoic acid : limit of detection : lipoxygenase : principal component analysis : partial least square-discriminant analysis : prostaglandin : retention time : platelet-rich plasma PGH 2 : prostaglandin H 2 Q-TOF : quadruple time of flight ix

12 RG RGUG : red ginseng : red ginseng unique ginsenosides RP-HPLC : reversed phase-high performance liquid chromatography S.D. SG : standard deviation : Sun ginseng TXA 2 : thromboxane A 2 TXB 2 : thromboxane B 2 U46619 UPLC vwf WP WG : thromboxane A 2 mimetic drug : ultra performance liquid chromatography : von Willebrand factor : washed platelet : white ginseng x

13 I. 서론 1. 약물대사체학 (Pharmacometabolomics) 1.1. 약물대사체학의의의및목적 최근진행되고있는 Genomics, Proteomics, Metabolomics 등의 `-omics' 는생명체에서합성하는유전자, 단백질, 대사물질의발현및조절, 변화로부터얻어지는다양한정보를획득하고처리하는학문으로써, 생명체에대한총체적이해를기반으로한다. 유전체학 (Genomics), 단백질체학 (Proteomics) 등과더불어 -omic 의한분야인대사체학 (metabolomics) 은세포, 조직, 혹은생체액속에포함되어있는저분자량물질인대사체 (metabolite) 를그대상으로하며, GC-MS, LC-MS 및 NMR 등과같은고감도분석기기를통해얻어진데이터를해석 (metabolites profiling) 함으로써, 1 식물또는동물의질병및생물학적메카니 즘의이해를돕는것을비롯하여질병에대한하나의진단마커를제시하는 학문이라고할수있다. 2-5 최근의분석기술의발전과더불어 metabolomics가지닌생물정보상의장점들을바탕으로, metabolomics가결국생명현상을이해하는데가장포괄적이며강력한수단이될것으로기대되고있으며, 한생명체가먹고, 마시고, 소화하고, 배설하는모든대사과정에서만들어지는대사체 ( 메타볼라이트 ) 는, 1

14 현재, 오줌, 혈액, 혈장, 혹은침등의시료로부터아미노산, 탄수화물, 당류, 지방같은저분자물질을분석하거나혹은대사체전체의패턴을분석함으 로써, 질병이나약물대사같은과정에서실마리를찾고있다. 대사체학중약물에의한대사체의변화를관찰하고, 이를설명하는학문인약물대사체학 (Pharmacometabolomics) 은개발된약물의기전연구및질병상태의예측등에대해많은정보를제공해줄수있으며, Figure 1과같은과정을통해연구를수행한다. 또한, 약물대사체학은통계적기법을통한판별분석모델링에의한새로운후보물질의발굴에도가능성을제시하고있다. 2

15 1.2. 주성분분석및판별분석 오믹스를기반으로하는학문은기존의분석방법에비해상당히크고복잡한양의데이터정보를수집하여분석을수행하고있다. 이때문에변수들간의인과관계및상관관계를통하여축약하고개체를분류하는다변량분석이필수적이라고할수있다. 본연구에서는다변량분석중변수를축약하는방법으로사용되는변수유도기법 (variable directed technique) 중오믹스연구에서주로사용하고있는주성분분석 (Principal component analysis, PCA) 를수행하고자한다. 주성분분석은다변량자료에존재하는비정규성 (abnormality) 이나이상치 (outlier) 를발견하기위하여공분산혹은상관행렬을이용하여상관관계가존재하지않는새로운변수를 ( 주성분 ) 을구하는방법으로주로, p개의변수들로부터서로독립인 k개의주성분을구해원변수의차원을줄이기위해사용된다. 오믹스연구에서는 PCA를통해 outlier를발견하고, biological diversity 및 toxicity trends하려는목적으로주로사용된다. 또한본연구에서는 partial least square-discriminant analysis (PLS-DA) 및 orthogonal partial least square-discriminant analysis (OPLS-DA) 분석법을이용하여판별분석을수행하였는데, 이는 2개이상으로나누어진개체들에대해분류에영향을미칠것같은특성을측정하고이를이용하여판별식을구해새로운개체를분류하는방법인판별분석의특성을이용, 약물처리그룹과처리하지않은그룹사이에서차이가나는대사체를규명하기위함이었다. 3

16 Figure 1. Workflow of Pharmacometabolomics 4

17 2. 혈소판의생리및활성화 혈액의한구성성분인혈소판은골수의거대세포 (megakaryocyte) 가수명을 다해세포질이혈액속으로떨어져나갈때생성되며, 약 7-10 일간혈액을 따라순환한다. 6 혈소판은혈관손상및출혈이생기는경우활성화되어지혈작용에중요한 역할을하는세포이다. 7, 8 혈소판의활성은혈관손상혹은여러자극에의 해수용성활성화물질들이증가함으로서유발되며, subendothelium ( 내피세포 ) 에 collagen이노출되면, von Willerbrand factor(vwf) 를매개로한내피세포로의부착 (adhesion) 및확장 (spreading) 이이루어짐으로서 1차응집반응이유도된다. 혈소판이 1차응집반응을할때, GP Ia/IIa 및 GP Ib/IX/V 등이주요한수용체로작용하게되며, 이러한특이적 GP 결합반응은세포내신호전달체계를활성화시키게된다. 신호전달체계의촉발은세포내칼슘의유입을증가시키고, 이에따라 cytoskeleton들의재조합이유발된다. Cytoskeleton의작용에의해혈소판의 shape change 및 spreading이일어나게 되며, 이는일차적인혈소판의활성을유발하게된다 이로부터 granule 에 함유되어있던혈소판활성물질들 (ADP, TXA 2 등 ) 이분비되고, 견고한혈소 판응집체를형성하게되는이차적인혈소판활성화를유발하게된다 (Figure 2)

18 Figure 2. Platelet thrombus formation (Ref. Barrett N.E. et al., Future innovations in anti-platelet therapies, British Journal of Pharmacology, 154, , 2008) 6

19 지혈작용은손상된부위에혈괴 (clot) 를생성함으로써혈액의손실을최소화하고혈액의정상적인순환을유지하기위한방어기전이라할수있으나, 과도한지혈작용또는비정상적인혈소판응집은혈전 (thrombus) 을초래하여혈액순환장애및심근경색증, 뇌졸중등다양한질환을유발한다 선진화및국민생활수준이높아짐에따라, 식습관이나생활방식에의한뇌혈관질환및혈행등의심혈관질환발병율이증가하고있으며, 그로인한사망률이높아지고있는추세로혈소판의응고기전을억제시키는약물의개발은심혈관계질환치료제로매우유용하다고할수있다. 또한개발된약물의기전연구는그약물이주는부작용의예방및다른약물과의상호작용의연구에중요하게연계될것으로여겨진다. 7

20 3. Ginsenoside Rk 1 의혈소판응집억제활성 인삼은오가피나무과 (Araliaceae) 에속하는다년생초본인 Panax ginseng C.A. MEYER의뿌리를이용한생약으로강장, 진통, 진정, 건위, 항종양등의약리작용을가진다고알려져있으며, 현재까지도인삼에함유된다양한성분들에대한연구가지속되고있다. 인삼의대표적인물질인진세노사이드 (ginsenoside) 는인삼유래배당체사포닌으로서, 가공하는조건에따라성분의화학적변화가나타나게된다. 이에, 수증기로쪄서말린홍삼 (red ginseng, RG) 에는 RG 특이진세노사이드 (red ginseng unique ginsenoside, RGUG) 의분리가보고되었으며, 이는물리화학적 가공과정에서, 열과압력에의해 deglycosylation 이일어나게되면, 극성이 적은진세노사이드가생성되는것으로알려져있다. 19 이렇게생성된진세노 20, 21 사이드에대한강한생리활성은많은연구를통해보고되었다. 또한, 이 성분들에대한혈소판응집억제활성에관한연구도끊임없이이루어져왔 다 한편, 본연구실에서 RGUG의함량을극대화하는방법을연구하던중개발된 선삼 (Sun ginseng, SG) 에는 RG에서발견되지않은새로운진세노사이드가존재하고있음을알게되었고, 이물질들에대한활성탐구노력역시이루어져왔다. 8

21 혈소판응집억제활성을가지는화합물의탐색결과로는, 진세노사이드 Rg 3 및 Rg 2 에대한항혈소판작용이보고된바있다. 25, 26 또한, 선행연구를통해, 진세노사이드중 Rk 1 이 arachidonic acid 에의하여유도되는혈소판응집작용을억제함을알수있었으며, 이는 ASA 를대조약물로사용하여실험하였을때, 기존의알려진항혈소판작용진세노사이드및 ASA 에비해, 강력한항혈소판응집작용을가지는것으로확인되었다. 27, 28 하지만, 이들진세노사이드의작용기전에대한추가적인연구는아직수행되지 않았다. 이에, 본연구에서는 ginsenoside Rk 1 을이용하여약물대사체학을 수행함으로서, 이를통해진세노사이드 Rk 1 의항혈소판작용기전이해및 고찰을실시하였다. 9

22 Figure 2. Structural modification of dammarane glycosides by heat processing 10

23 II. 실험방법 1. 실험재료, 시약및기기 1-1. 재료및시약 Acetylsalicylic acid (aspirin, ASA), arachidonic acid (AA), collagen, apyrase 및 formic acid (85%) 는 Sigma Aldrich (St. Louis, MO, USA) 로부터구입하였다. HPLC-grade methanol은 Duksan (Kyungki-Do, South Korea) 으로부터, Fluo-3 acetoxymethyl (AM) 은 Invitrogen (Molecular Probes, Eugene, USA) 으로부터구입하였으며, 5(S)-HETE, 8(S)-HETE, 11(S)-HETE, 12(S)-HETE, and 15(S)-HETE, 및 water는 Cayman Chemical (Ann Arbor, MI, USA) 로부터구입하였다 실험동물 Sprague-Dawley (SD) 계열웅성 (male) 흰쥐 (OrientBio, Seoul, Korea) 를실험동물로사용하였으며, 구입후 23 ± 0.5, 60% 상대습도가유지되는동물실에서 48시간이상순화과정을거친뒤실험에사용하였다. 사료는소동물용고형사료 ( 삼양유지, Seoul, Korea) 를사용하였으며, 실험동물의취급은서울대학교동물실험자원관리원의실험동물자원관리규정에따라시행되었다. 11

24 1-3. 실험기기 - Ultra performance liquid chromatograph (Waters, Milford, MA, USA) - Hystar chromatography software (Bruker Daltonik) - ESI-micrOTOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) - Reverse-phase C18 column (Acquity BEH 50 mm x 2.1 mm i.d.1.7μm, Waters) - FP-777 fluorometer (Jasco, Tokyo, Japan) R, 5810 R Centrifuge (Eppendorf AG, Hamburg, Germany) - Optima TLX bench-top ultracentrifuge (Beckman Coulter, Brea, CA) 12

25 2. Ginsenoside Rk 1 의분리 2-1. 가공인삼 (Sun ginseng, SG) 의제조 White ginseng (WG) 을 120 에서 3 시간 autoclave 안에서감압증숙한후, 건조기에서 24 시간이상건조시켜, SG 를제조하였다 Ginsenoside Rk 1 의분리 SG 2 kg을 MeOH 3 L로 70 에서 2 시간동안 3회 reflux 추출한뒤, 추출액을하나로합쳐감압농축하여 SG 추출물 380 g을얻었다. 농축된 SG 추출물에 H 2 O 1.5 L를가하여현탁한뒤, n-buoh 1.5 L로 3회용매추출하여 n-buoh 가용분획약 85 g을얻었다. n-buoh 가용분획중 30 g을취하여 600 g의 silica-gel을사용하여 silica-gel column chromatography를시행하였다. CHCl 3 : MeOH 으로구성된이동상을사용하였으며, 극성순차적인 stepwise gradient (40:1 à 30:1 à 20:1 à 10:1 à 5:1) 를시행하여총 10개의세부분획을 (Fr. 1 ~ Fr. 10) 얻었다. 얻어진세부분획중 Fr. 7을 10 mg/ml의농도로 MeOH에녹인뒤, semi-preparative RP-HPLC에주입하였다. 55% ACN aqueous solution ( 유속 : 5.0 ml/min) 을이동상으로하고 UV 203 nm에서검출하였을때, 약 13분에검출되는피크를대상으로하여해당시간대의용리액을반복적으로받았다. 얻어진물질의순도는기울기용리한 55% aqueous ACN을이동상으로하여 HPLC/ELSD를통하여확인하였으며, 이미분리되었던해당표준품 (authentic sample) 과의 ESI-MS spectral data 및머무름시간을비교하여물질을동정하였다. 13

26 3. Ginsenoside Rk 1 의혈소판응집억제활성 3-1. Washed platelets 의조제 Male SD rat (250 ± 20 g) 을디에칠에테르로흡입마취시킨후, 복강을절개하여심장으로부터전혈 (whole blood) 을채취하였다. 2.2% sodium citrate를항응고제로사용하였으며, 항응고제와전혈을 1:6의비율로하여전혈을채취하였다. 채혈액을 200 g에서 10 분간원심분리하여혈소판풍부혈장 (platelet-rich plasma, PRP) 을얻었으며, 얻어진 PRP는 washed platelet을조제하기위하여 200 unit/ml apyrase를첨가한후, 1900 rpm에서 10 분간원심분리하였다. Apyrase를첨가하는과정은총세번에걸쳐반복되었으며, 원심분리후얻어진 platelet pellet은 tyrode buffer로현탁시킨후, 혈소판측정기 (platelet analyzer) 로혈소판의개수를확인하였다. 실험에사용될 washed platelets은 600 ~ 개 /ml로맞추어제조되었다 혈소판응집억제활성 Ginsenoside Rk 1 의혈소판응집억제활성은 turbidimetric method 에기초한 방법으로 optical aggregometer 를이용하여검정하였다. WP 500 μl 를 37 에서 3 분간배양한후, 1100 rpm 으로교반하면서 DMSO 에용해시킨 14

27 ginsenoside Rk 1 solution 5 μl을넣고 30 초간추가배양하였다. 여기에 collagen ( 최종농도 1.0 μg/ml) AA ( 최종농도 3 μm) 을각각 5 μl씩넣고 37 에서 1100 rpm으로교반배양하였다. optical transmittance의변화를관찰하여혈소판의응집도에따라변화되는혼탁도 (turbidity) 로혈소판응집정도를판단하였다. 본실험에서는혈소판의 1차응집에따른모양의변화 (shape change) 를유도할수있도록 AA를가하기전에 Collagen을넣어주었으며, 같은농도의 Ginsenoside Rk 1 과 ASA를혈소판에처리하여, 두물질의응집억제활성도를비교하였다. 15

28 Figure 3. Schematic procedure for sample preparation for washed platelets (left) and metabolomics (right) 16

29 4. UPLC/Q-TOFMS 분석방법의검증 혈소판내대사체를분석하기에앞서, global metabolites profiling에적합한분석법을결정하고판단하기위하여 15개의 amino acids (alanine, arginine, aspartic acid, methionine, phenylalanine, glutamic acid, asparagine, glutamine, histidine, leucine, tryptophan, proline, serine, tyrosine, and valine) 및 TXB 2 를이용하여정밀성 (Precision), 직선성 (Linearity), 검출한계 (Limit of detection) 의항목을이용해분석방법을검증하였다 분석조건 Ultra Performance Liquid Chromatography (UPLC) - Gradient elution programs; 0 30% B (10 min), 80% B (25 min), 100% B (27 min). Solvent A: Water (0.1% formic acid), Solvent B: Acetonitrile (0.1% formic acid) - Flow rate: 0.2 ml/min Quadruple Time of Flight Mass Spectrometry (Q-TOF) - Capillary voltage: -4.5 kv (positive ion mode), 4.5 kv (negative ion mode) - Temperature dry gas (He, 99.9%): 200 C (4 L/min) - Calibrant: Lithium solution (Isopropanol, methanol and water) - Mass scan range: m/z 17

30 4.2. 분석법검증 (Method validation) 정밀성 (Precision) 분석방법의정밀성 (Precision) 은검출된 15개의아미노산과 TXB 2 의머무름시간및 mass to charge ratio (m/z) 을이용하여검증하였다. Intra precision은 60 μg/ml의농도에서하루동안세번시스템에반복주입함으로서실시하였으며, Inter precision은 μg/ml의농도범위안에서삼일동안시스템에주입하여분석함으로서수행되었다. 결과는 mean ± S.D. 로표시하였다 직선성 (Linearity) 분석방법의직선성 (Linearity) 은 15개의아미노산및 TXB 2 의 intensity를이용하여검증하였다. 검체농도의직선성은 y-절편, 회귀직선의기울기로부터 positive ion 및 negative ion mode에서각각평가되었다 검출한계 (Limit of detection) 분석대상물질을확실히검출할수있는최저의농도를확인하기위하여검출한계를평가하였으며, 검출한계 (LOD) 는표준편차와검량선의기울기에근거하는방법으로결정하였으며, 다음식에의해계산되었다. LOD = 3.3 * σ/s 여기서 σ는반응의표준편차를, S는검량선의기울기를말한다. 18

31 5. 혈소판내의대사체분석 5.1. 분석조건 Ultra Performance Liquid Chromatography (UPLC) - Gradient elution programs; 0 30% B (10 min), 80% B (25 min), 100% B (27 min). Solvent A: Water (0.1% formic acid), Solvent B: Acetonitrile (0.1% formic acid) - Flow rate: 0.2 ml/min Quadruple Time of Flight Mass Spectrometry (Q-TOF) - Capillary voltage: -4.5 kv (positive ion mode), 4.5 kv (negative ion mode) - Temperature dry gas (He, 99.9%): 200 C (4 L/min) - Calibrant: Lithium solution (Isopropanol, methanol and water) - Mass scan range: m/z 5.2. 분석샘플제조 혈소판응집샘플및물질을처리한샘플들은반응이끝난후, 샘플 200 μl 를취해새로운 eppendorf tube 에옮긴후, 바로 % MeOH 800 μl 를가하였다. 약 10 분간 voltexing 시킨후, 14,000 rpm 에서 20 분간원심분리하여상징액 500 μl 을취한후, 얻어진샘플을질소기류하에용매를건조하였다. 19

32 건조된샘플에 200 μl 의 70% MeOH 를넣어 3 분간 voltexing 한후, 0.2 μm PTFE syringe membrane filter 를이용해 filtration 하여 LC/MS 로주입하여 대사체분석을실시하였다. (Figure 3) 20

33 6. 데이터수집및처리 (Date processing) UPLC/Q-TOF으로부터얻어진데이터는 Profile Analysis software를이용하여데이터버켓팅 (Bucketing) 을수행하였다. UPLC/MS의데이터는 mass to charge ratio, retention time 및 intensity등으로이루어져있어, 통계학적분석을위해 2차원적인데이터로변환해야할필요성을가지고있다. 이에본연구에서는버켓팅이라는기능을이용하여데이터를 3차원에서 2차원으로만들어주었다. 버케팅은두가지방법으로접근할수있는데, 첫번째방법은얻어진데이터를일정한시간간격으로나눈뒤, 이를버켓으로지정하고지정된버켓안에검출된 m/z를일정한간격으로다시나눈후, 이에대한 intensity를측정, 2차원테이블화하는방식이다. 두번째방법은일정한시간간격으로나눈버켓에서검출된 m/z을대상으로 peak cluster 방법을이용, compound로지정되는 m/z만의 intensity를이용하여데이터를수집하는방식이다. Peak cluster 방법은 related isotope intensity 및 charge states를이용하여 compound를지정하는방법으로 find all compounds이라는기능을이용하여수행이가능하다. 본연구에서는두번째방법을사용하여테이블을얻었으며, find all compounds 기능으로 ΔRT 0.1 min, Δm/z 0.1 da 조건하에실시되었다. 버켓팅과정을통하여얻어진각 compounds에대하여 breakdown and a one-way analysis of variance (Tukey s HSD test p-values < 0.05) 를실시하여그룹간의차이를보고자하였다. 21

34 7. 다변량분석 (Multivariate analysis) 버켓팅을통하여얻어진피크테이블은 SIMCA-P+ (v12.0) 로추출된후, Principal component analysis (PCA) 및 partial least-squares discriminant analysis (PLS-DA) 분석을수행하였다. PLS-DA는 500-purmutations 방법에의하여 cross-validation을수행하였으며, 모델의적합성과예측성을확인하기위해서 R 2 및 Q 2 을계산하였다. 모델수행시의 missing data 값은 60% 로설정하였 으며, 버켓테이블내에있는모든변수들은 "Pareto Variance" 에의해 scaling 되었다. 그룹간의 outlier 를평가하기위해서, Student s t-distribution 의 multivariate generalization 을한 Hotelling s T 2 방법을사용하였다. 또한, 본연 구에서는 PLS-DA 분석수행후얻어진 variables important in the projection (VIP) 를이용하여그룹간의차이를규명하여주는대사체를선택하였다. - 22

35 8. Cyclooxygenase (COX) 활성 8-1. COX 활성의검정 AA에의한혈소판응집을강하게저해하는 ginsenoside Rk 1 의 TXA 2 생성억제정도를평가하기위하여 colorimetric COX inhibitor screening assay kit를이용하여 COX-1 및 COX-2에대한억제효과를검정하였다. 또한 ASA를 positive control로사용함으로써, 두약물간의 COX 억제정도를하고자하였다. COX 활성실험은, Kit에첨부된실험절차에따라실시하였으며, 96- well plate에 assay buffer 160 μl 및 heme 용액 10 μl을넣고, COX-1 시약 ( 또는 COX-2 시약 ) 10 μl 및시료 10 μl를가한후, 25 에서 5 분간 preincubation 하였다. 여기에 colorimetric reagent 20 μl와기질용액을 20 μl를가한뒤 25 에서 5분간 incubation 하여 590 nm에서흡광도를읽었다. COX-1 ( 또는 COX-2) 가들어가지않은 background와 DMSO만첨가한 vehicle control을측정하여각각의흡광도를 100% inhibition, 0% inhibition으로하여시료의활성억제도 (%) 를계산하였다 통계적처리법 모든실험은 3회반복실시되었으며, 결과는각시료의처리농도에대한저해활성도 (inhibition %) 의 mean ± S.D. 로표시하였다. 또한, 실험군의통계적유의성은 Duncan s multiple-range test를이용한 one-way ANOVA (Analysis of Variance) 를통해검정하였다. 23

36 9. 혈소판내 calcium 변화측정 Fluorescent Ca 2+ indicator 를세포안으로 loading 하기위하여준비된 washed platelets ( /ml) 에 5 mm Fluo-3 AM 및 2% pluonic-f127 을첨가한후, 60 분간실온에서반응시켰다. 이현탁액을 500 g 에서 10 min 간원심분리 시켜, platelet pellet 을얻은후, 생리식염수 (physiological saline) 로재현탁시켜 /ml 의혈소판을얻었다. Fluorometer cuvette 에 2 ml 의 WP 혈소판을 넣고 37 에서교반시키면 1 분간 pre-incubation 시키고, FP-777 fluorometer (Jasco, Tokyo, Japan) 에서 excitation at 488 nm, emission at 535 nm 조건에서형광 을측정하였다. Rk 1 또는용매 (DMSO) 를넣고 30 초간반응시킨후, collagen 0.5 mm 및 arachidonic acid 50 μm 을첨가하여증가하는 fluorescence intensity 를 측정하여세포내저장소에서유리된칼슘농도로환산하였다. 1 분후, CaCl 2 1 mm 을첨가하여증가한 fluorescence intensity 를측정하여세포 외부에서유입된칼슘농도로환산하였다. 칼슘의농도는다음식에의하여 계산하였다. [Ca 2+ ] = K d 24

37 10. Western blot analysis PRP로부터얻어진 washed platelet pellet은 HEPES tyrode buffer (ph =7.4) 로현탁시킨후, 혈소판측정기 (platelet analyzer) 로혈소판의개수를확인하였다. 실험에사용될 washed platelets은 개 /ml로맞추어제조되었다. WP 500 μl를 37 에서 3 분간배양한후, 1100 rpm으로교반하면서 DMSO에용해시킨 ginsenoside Rk 1 solution 5μL 및 vehicle을넣고 2 분간추가배양하였다. 여기에 collagen (1.0 μg/ml) 및 AA (3 μm) 을각각 5 μl씩넣고 37 에서 1100 rpm으로 5분간교반배양하였다. Cuvette을 ice-bath에넣음으로써반응을종결시켰고, 샘플들은 liquid nitrogen을이용 freeze-thawed를세번반복함으로써, homogenation 시켰다. 4, 100,000g에서 60분간원심분리한후, supernatant는 cytosolic, pellet은 membrane 분획으로각각사용되었다. 각분획은 50 mm Tris-Cl (ph 7.5), 150 ml NaCl, 1% Triton X-100, 1% Sodium deoxycholate, 0.1% SDS, 2mM EDTA, sterile solution 및 protease inhibitors (Gendepot, Barker, TX, USA) 를이용하여 lysis한후, 얻어진 protein lysates는 Precast 4% ~12% polyacrylamide gradient gel (Invitrogen, Carlsbad, CA, USA) 위에 loading 되었다. SDS-PAGE 를이용하여단백질을분리하였으며, polyvinylidene difluoride membrane (Pall, Pensacola, FL, USA) 로옮긴후, membrane을 3% nonfat milk로 blocking하였다. Anti-12 LO (1:1000, Abcam, Cambridge, MA, USA) 및 β-actin (1:1000, Santa Cruz, CA, USA) 을이용하여탐침하였으며, incubation 후, membranes은 TBS + 0.1% Tween 20을이용하여세척하였다. 다시 peroxidaseconjugated secondary antibody (1:5000, Santa Cruz, CA, USA) 와함께, 60분간실 25

38 온에서유지시킨후, Immunoactive proteins 는 WEST chemiluminescence substrate (Pierce Biotechnology Inc., Rockford, IL, USA) 에의해검출하였으며, Chemi Doc XRS (Bio-Rad Laboratories. Hercules. CA) 에의해영상화되었다. 26

39 III. 결과및고찰 1. Ginsenoside Rk 1 의항혈소판응집작용 Arachidonic acid (AA) 로유도된혈소판응집작용에대하여진세노사이드 Rk 1 은용량의존적인억제효과를보였으며 (Figure 4(a)), 항혈소판응집작용 을가지는아스피린을대조약물로하여실험하여본결과, 20 μm 에서 Rk 1 이아스피린에비해강력한응집억제효과를나타냈다. (Figure 4(b)) Figure 4. Anti-platelet aggregation activity of ginsenoside Rk 1 and aspirin (ASA) (a)the anti-platelet aggregatory activity by ginsenosides Rk 1 was dose-dependent manner on arachidonic acid -induced platelet aggregation. (b) The inhibitory activity of Rk 1 and aspirin were compared at the same concentration (50 μm) and the Rk 1 strongly inhibited the platelet aggregation. 27

40 2. 시스템불순물분석및제거 오믹스를기반으로하는분석을수행할시에는많은양의데이터를이용하여분석을실시하기때문에효과적인불순물제거는필수적이라고할수있다. 이에본실험에서는 UPLC/MS 분석에수행하기전에, 시스템으로부터나오는불순물을확인하고, 이를효과적으로제거하였다. 우선 syringe membrane filtration으로부터생성된두개의불순물을발견하였으며 (Figure 5(a)), 이두물질은각각 palmitic amide (Peak #1) 및 oleamide (Peak #2) 로확인되었다 (Figure 5(c)). 이들은각각 plastic syringe의 slip agent 로서 syringe의원활한 up-down을위해 syringe 제조시에묻혀나오는물질임을알수있었다. 이는 glass syringe의사용또는 MeOH washing에의하여효과적으로제거할수있었다 (Figure 5(b)). 28

41 Figure 5. Identification of system impurities. Base peak chromatograms of methanol filtered using (a) a plastic syringe, (b) the washed plastic syringe; (c) Mass spectrum of impurity produced by syringe filtering ; The peaks were identified as palmitic amide (peak #1, [M+H] + = ) and oleamide (peak #2, [M+H] + = ).The measured error were < 1 mda 29

42 3. UPLC/Q-TOFMS 분석법검증 본연구에서사용된분석방법은정밀성, 직선성및검출한계등의항목을통하여검증되었으며, 각항목은 15개의아미노산및 TXB 2 를이용하여평가되었다. 정밀성은 m/z 및머무름시간을이용하여평가되었으며, (Figure 6 및 Figure 7) mass to charge ratio의 tolerance는 ± 4 mda 내에서정밀성을, 머무름시간의 coefficient of variation (C.V.) 는 4% 이내에서정밀성을나타냈다. 직선성의경우 10 μg/ml ~ 80 μg/ml 의범위에서수행되었다. 직선성및검출한계의결과로서, chemical 성질에따라 positive 및 negative mode에서의결과가다르게나옴을확인할수있었다. (Figure 8) 예를들어, 트립토판의경우검출한계가 positive, negative ion mode에서각각 0.12 μg/ platelets 및 0.36 μg/ platelets 이였으며, ion mode에따라약 3배정도의검출한계가차이나는것을알수있었다. 또한, TXB 2 의검출한계는 positive, negative ion mode에서각각 6 ng/ platelets 및 30 ng/ platelets 로써, 낮은농도의 TXB 2 를분석하기에는 negative ion mode 가더적합함을제시해주었다. 30

43 Figure 6. Validation of the LC method Precision test for mass to charge (a) intra-precision of m/z in positive mode, (b) intra-precision of m/z in negative mode, (c) inter-precision of m/z in positive mode, (d) inter-precision of m/z in negative mode 31

44 Figure 7. Validation of the LC method; Precision test for retention time (a) intra-precision of m/z in positive mode, (b) intra-precision of m/z in negative mode, (c) inter-precision of m/z in positive mode, (d) inter-precision of m/z in negative mode 32

45 Figure 8. Validation of the LC method; Linearity The calibration curves were obtained using thromboxane B 2 (TXB 2 ) and 15 amino acids; the good linearity shown depends on their molecular characters in positive and negative ion mode, respectively, 1) positive mode, 2) negative mode, 3) both ion modes, 4) r 2 <0.9 33

46 4. UPLC/Q-TOF MS 를이용한대사체분석 샘플분석사이에영향을미칠수있는샘플 carry over 효과를고려하기위하여, 본연구에서는샘플의주입순서는 random sequence를이용하여결정하였으며, 매 8번의샘플의주입후 MeOH 주입을수행하였다. Random sequence는 randomsequnces.org를통해생성하였다. 8번의샘플주입후잔존하는불순물들은 negative ion mode에서는발견되지않았으며 (Figure 9 (d)), positive ion mode에서는약간잔존하는것으로확인되었다 (Figure 9 (b)). 하지만잔존하는피크들에대한규명은이루어지지못하였으며, 이에 meta-analysis 수행시, 이들피크들을제외하여분석을수행하였다. Figure 9. The confirmation of carry-over effect in analysis. Base peak chromatograms of (a) seventh injected sample, (b) post-blank run applied at eight measurements in positive mode; In negative mode, (c) 7 th injected, (d) 8 th postblank run. 34

47 혈소판내의대사체분석은 UPLC/Q-TOF MS 을이용하여수행되었으며, 대 조군, 혈소판응집, Rk 1, 아스피린샘플에대하여각각 positive ion mode 및 negative ion mode 에서 base peak chromatogram 을얻었다 (Figure 10). Figure 10. Base peak chromatograms of platelets samples Positive ion mode (a) 대조군 (b) 혈소판응집 (c) Ginsenoside Rk 1 (d) 아스피린, Negative ion mode (e) 대조군 (f) 혈소판응집 (g) Ginsenoside Rk 1 (h) 아스피린 35

48 UPLC/Q-TOF MS로부터얻어진데이터는 profile analysis software로추출된후, 버켓팅을실시하여, 버켓테이블을얻었다. 결과적으로, find all compounds에의해 positive 및 negative ion mode에서각각 10,717개및 12,087개의피크로이루어진버켓테이블을얻을수있었다. 본연구에서는 meta-analysis에방해요소로작용할수있는 chromatographic traces 및 peaks을제거하기위하여, dilution factor 를이용한 correlation test 를수행하였다. 30 Correlation 값이 0.5 이 하인변수를제거한후, 버켓테이블에나열된 m/z들은 UPLC/Q-TOF 분석시사용된 calibrant를이용하여 calibration을실시하였다 (Figure 11). 15개의아미노산혼합용액을이용하여 dilution factor를이용한 correlation test 를수행한결과는 Figure 12와같으며, 약 65% 변수가노이즈로간주되었다. Figure 11. Calibration using calibrant injected before every sample run. 36

49 Figure 12. Correlations of m/z signals and the dilution factor The standard mixture including 15 amino acids were diluted as 5 points and the total number of signals was obtained from each peak list generated by Bruker Profile Analysis Software. The correlations were calculated statistically and the signals did not correlated (correlation values <0.5) with the dilution factor were about 65%. Calibration된 m/z는 Human metabolome database ( 등의데이터베이스를기반으로대사체를확인하였다 (Figure 13, Table 1 및 2). Calibration 수행전에얻어진 m/z으로대사체를이용하여대사체를확인한결과, 정확한대사체를찾을수없었으며 (Figure 13 (a)), calibration후에얻어진 m/z를이용하여검색한결과, 정확하게찾을수있음을확인할수있었다 (Figure 13 (b)). 이는정확한대사체의검색을위하여 calibration이반드시필요함을제시하여주었다. 37

50 Figure 13. The calibration effect of measured m/z on database search. The m/z of thromboxane B 2 (TXB 2 ) was measured by UPLC/micrOTOF MS using authentic standard. It was calibrated by a calibrant prepared with lithium formate. The measured value ( m/z) was searched using human database and (a) identification could not performed properly, (b) the accurate identification was performed by calibrated m/z. 38

51 TABLE 1. The list of metabolites observed in washed rat platelet, analyzed by UPLC/micrOTOF MS in negative mode Retention m/z a Metabolite ID b Chemical Adduct time formula d forms cycle adenosine monophosphate c C 10 H 12 N 5 O 6 P M-H Adenosine triphosphate c C 10 H 16 N 5 O 13 P 3 M-H Serotonin c C 10 H 12 N 2 O M-H Taurohyocholate C 26 H 45 NO 7 S M-H c Thromboxane B 2 C 20 H 34 O 6 M-H Taurochenodesoxycholic acid C 26 H 45 NO 6 S M-H Hyocholic acid C 24 H 40 O 5 M-H Biocytin C 16 H 28 N 4 O 4 S M-H Glycoursodeoxycholic acid C 26 H 43 NO 5 M-H LysoPE e (16:0/0:0) g C 21 H 44 NO 7 P M-H LysoPE(20:3(8Z,11Z,14Z)/0:0) g C 25 H 46 NO 7 P M-H LysoPE(18:1(9Z)/0:0) g C 23 H 46 NO 7 P M-H LysoPC f (14:0) g C 22 H 46 NO 7 P M-H LysoPE(18:2(9Z,12Z)/0:0) g C 23 H 44 NO 7 P M-H LysoPE(20:4(8Z,11Z,14Z,17Z)/0:0) g C 25 H 44 NO 7 P M-H LysoPE(20:2(11Z,14Z)/0:0) g C 25 H 48 NO 7 P M-H LysoPC(16:1(9Z)) g C 24 H 48 NO 7 P M-H (S)-hydroxyeicosatetraenoic acid c C 20 H 32 O 3 M-H LysoPE(16:0/0:0) g C 21 H 44 NO 7 P M-H LysoPC(15:0) g C 23 H 48 NO 7 P M-H (S)- hydroxyeicosatetraenoic acid c C 20 H 32 O 3 M-H LysoPE(18:1(9Z)/0:0) g C 23 H 46 NO 7 P M-H PE(P-16:0e/0:0) g C 21 H 44 NO 6 P M-H (S)- hydroxyeicosatetraenoic acid c C 20 H 32 O 3 M-H LysoPE(22:4(7Z,10Z,13Z,16Z)/0:0) g C 27 H 48 NO 7 P M-H LysoPC(16:0) g C 24 H 50 NO 7 P M-H LysoPE(20:1(11Z)/0:0) g C 25 H 50 NO 7 P M-H LysoPE(22:2(13Z,16Z)/0:0) g C 27 H 52 NO 7 P M-H LysoPE(20:0/0:0) g C 25 H 52 NO 7 P M-H LysoPE(18:0/0:0) g C 23 H 48 NO 7 P M-H LysoPE(20:0/0:0) g C 25 H 52 NO 7 P M-H a Calibrated m/z by calibrant. b Observed metabolites were annotated by metabolomic public databases c Identification was confirmed based on retention time and m/z of authentic standards d Molecular formula of [M-H] e LysoPE is lysophosphatidyletanolamine f LysoPC; Lysophosphatidylcholine g Identification was performed using their fragments detected by LC/MS 39

52 TABLE 2. Metabolites list observed in washed rat platelet, analyzed by UPLC/micrOTOF MS in positive mode. Retention m/z a Metabolite ID b Chemical Adduct time formula d forms Cinnavalininate C 14 H 8 N 2 O 6 M+H ,17-Epiestriol C 18 H 24 O 3 M+H Carnosic acid C 20 H 28 O 4 M+H Salsoline-1-carboxylate C 12 H 14 NO 4 M+H Glutamylalanine C 8 H 14 N 2 O 5 M+H Hydroxy-2-methylpyridine-4,5- dicarboxylate C8H7NO5 M+H LysoPC e (20:4(8Z,11Z,14Z,17Z)) g C 28 H 50 NO 7 P M+H LysoPC(18:2(9Z,12Z)) g C 26 H 50 NO 7 P M+H L-Palmitoylcarnitine C 23 H 45 NO 4 M+H LysoPC(16:0) g C 24 H 50 NO 7 P M+H Oleoylcarnitine C 25 H 47 NO 4 M+H LysoPC(20:3(8Z,11Z,14Z)) g C 28 H 52 NO 7 P M+H LysoPC(18:3(9Z,12Z,15Z)) g C 26 H 48 NO 7 P M+H Sulfodeoxycholic acid C 23 H 38 O 7 S M+H CPA(18:1(9Z)/0:0) C 21 H 39 O 6 P M+H LysoPC(18:1(9Z)) g C 26 H 52 NO 7 P M+H LysoPC(P-18:1(9Z)) g C 26 H 52 NO 6 P M+H LysoPC(18:0) g C 26 H 54 NO 7 P M+H LysoPC(20:3(8Z,11Z,14Z)) g C 28 H 52 NO 7 P M+H SM(d18:1/12:0) C 35 H 71 N 2 O 6 P M+H Arachidonic acid c C 20 H 32 O 2 M+H Norcholestanehexol C 26 H 46 O 6 M+H alpha-carboxy-5alpha-cholesta-8-en- 3beta-ol C 28 H 46 O 3 M+H MG(20:3(8Z,11Z,14Z)/0:0/0:0) C 23 H 40 O 4 M+H Nor-5b-cholestane- 3a,7a,12a,24,25-pentol C 26 H 46 O 5 M+H a Calibrated m/z by calibrant b Observed metabolites were annotated by metabolomic public databases c Identification was confirmed based on retention time and m/z of authentic standards d Molecular formula of [M] e LysoPC; Lysophosphatidylcholine g Identification was performed using their fragments detected by LC/MS with databases of lipidmaps ( 40

53 5. 주성분분석및판별분석 Profile analysis software를이용하여버켓팅수행후얻어진버켓테이블을이용하여주성분분석 (Principal componenet analysis, PCA) 를실시하였다. Positive 및 negative로부터얻어진 PCA 결과는 Figure 14 (a), (b) 에각각나타냈다. Rk 1 및아스피린샘플은다른그룹군과차이가있음을보였으나, 혈소판이응집된샘플과대조군은크게차이를나타내지못하였다. 이에, 판별분석인 partial least-squares discriminant analysis (PLS-DA) 을실시하였고, 서로다른네개의그룹이주성분 1과 3에의해구별될수있었다 (Figure 14(c), (d)). 성립된 PLS-DA models로부터 R 2 및 Q 2 값을계산할수있으며, R 2 값은모델에의해데이터변수가얼마만큼설명되고있는지를알려주는지표이며, Q 2 의값은새로운변수를모델에적용시켰을때, 모델의예측도를알려주 는지표이다. 본실험에서얻어진 PLS-DA models 의타당성은 R 2 및 Q 2 의 값에의하여결정하였으며, positive 및 negative ion mode 에서 R 2 값은각각 0.98 and 0.99을가졌으며, Q 2 값은각각 0.82 and 0.83 값을가졌다. 이결과는성립된 PLS-DA가현재모델링에사용된변수들을높은비율로잘설명해주고, 새로운변수에대해서도높은예측도를가질수있음을의미한다. 얻어진 PLS-DA 결과는 500 permutations 방법에의해 cross validation 을실시하였고, Figure 15에보여진바와같이왼쪽에표시된값들이오른쪽에표시된 original point값보다낮고, Q 2 값의 y-intercept값이 0보다낮음으로써, positive ion mode(figure 15(a)) 및 negative ion mode (Figure 15(b)) 에서수행된 PLS-DA 모델들의 validation을확인할수있었다. 41

54 본연구에서는각그룹간의분리효과를극대화하기위해 ortho-partial leastsquares discriminant analysis (OPLS-DA) 를실시하였고 (Figure 16), 주성분 1 과 3 에의해 PLS-DA 결과에비해분리의극대화를확인할수있었다. 42

55 Figure 14. Multivariate analysis using metabolomes data Negative ion mode Principal component analysis (PCA) score plots were plotted by PC1 (1 st component) and PC2 (2 nd component) in (a) positive and (b) negative ion mode. The control, full aggregation, ginsenoside Rk 1 and aspirin groups were colored as black, red, blue and green, respectively. The score plots of partial least squares - discriminant analysis (PLS-DA) were plotted by PC1 (1 st component) and PC3 (3 rd component) in (c) positive ion mode and (d) negative ion mode. 43

56 Figure 15. Validation plot of statistical PLS-DA model. The PLS-DA were established from meta-data files analyzed by LC/MS and the validity of the PLS-DA models was evaluated by 500 permutation tests in (a) positive and (b) negative ion modes, respectively. The horizontal axis represents correlation between the permutated- and original y-vector and the criteria for validity were satisfied in both ion modes, an indication the PLS-DA models were valid. 44

57 Figure 16. OPLS-DA score plots The ortho-partial least-squares discriminant analysis was performed to maximize the segregation between groups and the clearer discrimination was observed in (a) positive mode and (b) negative ion mode. The y-axis represents the scores that summarizes the X variation orthogonal to Y. 45

58 6. Eicosanoids 및 TXB 2 생성억제효과 PLS-DA 판별분석을통해얻어진 variable importance in the projection (VIP) 로부터각그룹을나누는 m/z 가선택되었으며, 이들 m/z 은 breakdown and a one-way analysis of variance (Tukey s HSD test p-values < 0.05) 를수행하여유의성있는대사체를확인하였다. Eicosanoids 중에서는 15(S)-Hydroeicosatetradecaenoic acid (15(S)-HETE), 및 12(S)-HETE 가확인되었으며, 또한혈소판응집에중요한역할을하는 Thromboxane A 2 의생성지표인 Thromboxane B 2 (TXB 2 ) 를검출할수있었다. TXB 2 는 Rk 1 및아스피린처리모두에의해서유의성있게감소하는것 을확인할수있었으며, 100 μm ASA 및 10 μm Rk 1 에의해감소된 TXB 2 level 은각각 66% 및 77% 이였다. 12(S)-HETE 의경우, Rk 1 에의해항혈소판응집작용을나타낸샘플에서유의성있게감소하는한편, 아스피린이처리된샘플에서는오히려증가하는것을확인할수있었다 (Figure 17). 이와같은결과로서, Rk 1 에의한항혈소판응집작용은 TXA 2 및 12(S)-HETE 생성기전에영향을미치고있음을알수있었다. 아스피린의항혈소판응집작용은 cyclooxygenase 억제작용으로인한 TXA 2 생성억제로알려져있다. 이에, 본연구에서는 Rk 1 이 TXA 2 생성작용기전뿐만아니라, 12(S)-HETE 생성기전도효과적으로억제함으로서아스피린보다 arachidonic acid 에의한혈소판응집작용에강력한억제효과를나타낸것으로생각되었다. 46

59 Figure 17. Alteration of (S)-hydroxyeicosatetradecaenoic acids (HETEs) and thromboxane B 2 (TXB 2 ) (a) Thromboxane B 2, (b) 12(S)-HETE and (c) 15(S)-HETE; The (S)-HETEs and TXB 2 were determined using UPLC/Q-TOF system. Thromboxane B 2 decreased both Rk 1 and ASA. The increase in (S)-HETEs was observed in AA-induced platelet aggregation. In particular, 12(S)-HETE was significantly decreased by Rk 1 treatment, whereas it increased when aspirin was treated. Data were expressed as mean ± standard deviation and the y- axis of plot expressed as intensity of mass to charge ratio. Statistical analysis was performed by one-way anova. (* p<0.05) 47

60 7. Cyclooxygenase(COX) 저해활성 약물대사체학을통해, 그룹간의차이를규명하는대사체로 TXB 2 로확인하였으며, 아스피린및 ginsenoside Rk 1 에의해 TXB 2 생성을억제하는작용을분자수준에서규명하기위하여 COX에대한직접적길항효과를검정하였다. COX는 AA의대사과정에작용하는효소로서, 여러염증성인자및혈소판 응집에중요한역할을담당하고있는 TXA 2 생산을담당하고있다. 31 AA 는 COX 에의해 prostaglandin H 2 로대사된후, thromboxane synthase 에의해 TXA 2 로대사되고, 이는더안정한대사체인 TXB 2 를생성하게된다. 32 그렇기때 문에 TXB 2 의생성억제는 COX에대한저해효과를간접적으로예측할수있도록한다. COX는두가지의 isoform인 COX-1과 COX-2로존재하며, ASA 의경우, COX-1에대한억제효과가 COX-2에비해뛰어나 TXB 2 생성을억제하는것으로알려져있다. 33 본실험에서는 COX의활성에대한억제도를 COX의 peroxidase 효과에의한 oxidized N,N,N,N -tetramethyl-p-phenylenediamine (TMPD) 의생성으로, 확인하였으며, 생성된 TMPD는 590 nm에서검출하였다실험결과, ASA는알려진대로 COX-2에비해 COX-1에대하여억제효과가 강함을알수있었고, 농도의존적으로길항함을확인하였다. Ginsenoside Rk 1 의경우, COX-1 및 COX-2 에대해모두저해효과를나타냈다. COX-1 에대 한저해효과가 COX-2 에비해강한것을확인할수있었지만, ASA 에비하 48

61 여낮음을확인할수있었다. 이결과로미루어볼때, ginsenoside Rk 1 의 AA 의해유도된혈소판응집억제활성은부분적으로 COX 를억제하여나타 나는것임을알수있었다. Figure 18. Metabolic pathway of arachidonic acid 49

62 Figure 19: Cyclooxygenase (COX) inhibitory activities The inhibitory effects of ginsenoside Rk 1 and aspirin on (a) COX-1 and (b) COX-2 were measured by colorimetric inhibitor screening assay. Data were expressed as mean ± standard deviation. Statistical analysis was performed by one-way anova. (* p<0.005) 50

63 8. 혈소판내 [Ca 2+ ] 농도측정 약물대사체학을기반으로하여얻어진대사체의변화를살펴본결과, 진세노사이드 Rk 1 과아스피린처리샘플을구분짓는대사체로서 12(S)-HETE가확인되었다. 12-HETE는 AA 대사체중하나로서, AA가 12-LOX에의하여 12- HpETE로대사되고, 이는곧안정한대사체인 12-HETE로대사된다 (Figure 18). 12(S)-HETE가혈소판응집작용에미치는작용에대하여 1960년부터연 구가진행되어왔으며, 최근연구에서는천연물로부터분리된성분들중 항혈소판작용을나타내는물질들이 12(S)-HETE 생성기전에도영향을미치 는것으로보고되었다. 본연구에서는세포내저장소로부터 Ca 2+ 유리및유입이 12(S)-HETE 생성 에영향을미칠수있는가능성에초첨을두고 37 Rk 1 에의한세포내저장소 로부터 Ca 2+ 유리및외부로부터의 Ca 2+ 유입을측정하였다. 10 μm Rk 1 에의 하여세포내저장소로부터 Ca 2+ 유리가 54.8 ± 2.5% 감소하는것을확인할 수있었으며, 1 mm CaCl 2 의첨가후, 외부로부터 Ca 2+ 유입또한 48.8 ± 7.9% 억제하는것을확인하였다. (Figure 20) 51

64 Figure 20. Effects of ginsenoside Rk 1 on arachidonic acid (AA) -induced changes in platelet cytoplasmic calcium concentration. Fluorescence was measured in fluo-3 AM loaded washed platelets. The y- axis expressed as fluorescence intensity. Compared to (a) vehicle, the inhibition of AAinduced calcium influx and release by (b) Rk 1 could be observed. The effect of extracellular calcium was measured by addition of 1mM CaCl 2. 52

65 9. 12-Lipoxygenase(12-LOX) 의 translocation 세포내저장소로부터 Ca 2+ 유리및유입의결과로부터 12-HETE 생성이 Ca 2+ 유리및유입과밀접한관계를나타냄을확인할수있었다. 이에 12- HETE 를생성하는데중요한역할을하는 12-LOX 와 Ca 2+ 농도와의관계를 연구하였다. 38 본연구에서는, western blot analysis 을통해혈소판응집및항 혈소판응집작용이일어날때, 12-LOX가 cytosol에서 membrane으로 translocation되는것을확인해보았다. (Figure 21) 실험의결과, AA에의해혈소판응집이유도될때, 12-LOX가 cytosol에서 membrane으로 translocation되는것을확인할수있었고, ginsenoside Rk 1 의처리시, translocation이억제되는것을확인할수있었다. 결과적으로, ginsenoside Rk 1 의처리시 12-LOX이 membrane으로 translocation이억제됨으로써, 12-HETE의생성에영향을미치는것으로확인할수있었다. Figure 21. Effects of ginsenoside Rk 1 on 12-lipoxygenase translocation from cytosol to membrane. 53

66 IV. 결론 본연구는약물대사체학을이용하여 arachidonic acid에의한혈소판응집작용에대한진세노사이드 Rk 1 의항혈소판작용의기전연구를수행하였으며, 아스피린을대조군으로아스피린과의항혈소판작용기전의차이를규명하고자하였다. UPLC/Q-TOF 분석으로부터얻어진데이터는버켓팅을통하여패턴분석및판별분석에이용되었으며, 약물대사체학연구를통해, 아스피린과진세노사이드 Rk 1 의처리에의해 TXB 2 이모두감소하는것을확인할수있었다. 또한, 진세노사이드 Rk 1 가처리된그룹에서 12(S)-HETE가현저히감소하는것을확인할수있었다. 그룹간의차이를규명해주는 TXB 2 및 12(S)-HETE 모두 AA의대사체로서각각 COX 및 LOX에의해대사되어생성될수있으며, TXB 2 는혈소판응집작용에중요한역할을하는대사체로알려져있다. 이에, TXB 2 의감소에관련된작용기전을살펴보고자하였으며, TXB 2 생성기전중 COX의활성기전에초점을맞춰, 아스피린및진세노사이드 Rk 1 의 COX의활성억제도를평가하였다. 이미알려진대로아스피린은 COX-1에대한강한억제도를가지고있음을확인할수있었으며, 진세노사이드 Rk 1 에의한 COX-1에대한억제도또한확인할수있었다. 또다른대사체인 12(S)-HETE 역시, AA대사체로서 12-LOX에의해대사되어생성되며, 본연구에서는 12(S)-HETE의감소작용기전과관련해서 calcium 54

67 농도로부터기인된 12-LOX 의 translocation 에초점을맞추어실험을진행하 였다. 38 진세노사이드 Rk 1 은세포내저장소로부터 Ca 2+ 유리및외부로부터 의 Ca 2+ 유입을억제하는효과를나타내었으며, 진세노사이드 Rk 1 에의한 12(S)-HETE 의생성감소는이러한 Ca 2+ 농도변화에의한 12-lipoxygenase 의 translocation에의한 12-LOX의억제작용으로부터기인하는것으로확인되었다. 이로부터진세노사이드 Rk 1 가 COX 및 LOX의작용을효과적으로억제함으로서 AA에의해유도된혈소판응집작용에대해항혈소판응집작용을나타내는것을확인할수있었다. 이러한결과들을기반으로, 본연구는항혈소판작용을나타내는후보약물선정및항혈소판작용의기전연구에대한약물대사체학의접근가능성을제시하였다고할수있다. 55

68 Figure 22. Biological behavior of aspirin and ginsenoside Rk 1 on anti-platelet aggregatory activity 56

69 V. 참고문헌 1. Kell, D. B., Metabolomics and systems biology: making sense of the soup. Curr Opin Microbiol 2004, 7, (3), Sabatine, M. S.; Liu, E.; Morrow, D. A.; Heller, E.; McCarroll, R.; Wiegand, R.; Berriz, G. F.; Roth, F. P.; Gerszten, R. E., Metabolomic identification of novel biomarkers of myocardial ischemia. Circulation 2005, 112, (25), Lewis, G. D.; Wei, R.; Liu, E.; Yang, E.; Shi, X.; Martinovic, M.; Farrell, L.; Asnani, A.; Cyrille, M.; Ramanathan, A.; Shaham, O.; Berriz, G.; Lowry, P. A.; Palacios, I. F.; Tasan, M.; Roth, F. P.; Min, J.; Baumgartner, C.; Keshishian, H.; Addona, T.; Mootha, V. K.; Rosenzweig, A.; Carr, S. A.; Fifer, M. A.; Sabatine, M. S.; Gerszten, R. E., Metabolite profiling of blood from individuals undergoing planned myocardial infarction reveals early markers of myocardial injury. J Clin Invest 2008, 118, (10), Sreekumar, A.; Poisson, L. M.; Rajendiran, T. M.; Khan, A. P.; Cao, Q.; Yu, J.; Laxman, B.; Mehra, R.; Lonigro, R. J.; Li, Y.; Nyati, M. K.; Ahsan, A.; Kalyana-Sundaram, S.; Han, B.; Cao, X.; Byun, J.; Omenn, G. S.; Ghosh, D.; Pennathur, S.; Alexander, D. C.; Berger, A.; Shuster, J. R.; Wei, J. T.; Varambally, S.; Beecher, C.; Chinnaiyan, A. M., Metabolomic profiles delineate potential role for sarcosine in prostate cancer progression. Nature 2009, 457, (7231), Shaham, O.; Slate, N. G.; Goldberger, O.; Xu, Q.; Ramanathan, A.; Souza, A. L.; Clish, C. B.; Sims, K. B.; Mootha, V. K., A plasma signature of human mitochondrial disease revealed through metabolic profiling of spent media from cultured muscle cells. Proc Natl Acad Sci U S A 2010, 107, (4), Hartwig, J.; Italiano, J., Jr., The birth of the platelet. J Thromb Haemost 2003, 1, (7), Andrews, R. K.; Berndt, M. C., Platelet physiology and thrombosis. Thromb 57

70 Res 2004, 114, (5-6), Jurk, K.; Kehrel, B. E., Platelets: physiology and biochemistry. Semin Thromb Hemost 2005, 31, (4), Yip, J.; Shen, Y.; Berndt, M. C.; Andrews, R. K., Primary platelet adhesion receptors. Iubmb Life 2005, 57, (2), Clemetson, K. J., Platelet activation: signal transduction via membrane receptors. Thromb Haemost 1995, 74, (1), Ikeda, Y.; Handa, M.; Kamata, T.; Kawano, K.; Kawai, Y.; Watanabe, K.; Kawakami, K.; Sakai, K.; Fukuyama, M.; Itagaki, I.; et al., Transmembrane calcium influx associated with von Willebrand factor binding to GP Ib in the initiation of shear-induced platelet aggregation. Thromb Haemost 1993, 69, (5), Murugappan, S.; Shankar, H.; Kunapuli, S. P., Platelet receptors for adenine nucleotides and thromboxane A2. Semin Thromb Hemost 2004, 30, (4), Kroll, M. H.; Schafer, A. I., Biochemical mechanisms of platelet activation. Blood 1989, 74, (4), Barrett, N. E.; Holbrook, L.; Jones, S.; Kaiser, W. J.; Moraes, L. A.; Rana, R.; Sage, T.; Stanley, R. G.; Tucker, K. L.; Wright, B.; Gibbins, J. M., Future innovations in anti-platelet therapies. Br J Pharmacol 2008, 154, (5), Ruggeri, Z. M., Platelets in atherothrombosis. Nat Med 2002, 8, (11), Gawaz, M., Role of platelets in coronary thrombosis and reperfusion of ischemic myocardium. Cardiovasc Res 2004, 61, (3),

71 17. Abumiya, T.; Fitridge, R.; Mazur, C.; Copeland, B. R.; Koziol, J. A.; Tschopp, J. F.; Pierschbacher, M. D.; del Zoppo, G. J., Integrin alpha(iib)beta(3) inhibitor preserves microvascular patency in experimental acute focal cerebral ischemia. Stroke 2000, 31, (6), ; discussion Vivekananthan, D. P.; Patel, V. B.; Moliterno, D. J., Glycoprotein IIb/IIIa antagonism and fibrinolytic therapy for acute myocardial infarction. J Interv Cardiol 2002, 15, (2), Park, I. H.; Kim, N. Y.; Han, S. B.; Kim, J. M.; Kwon, S. W.; Kim, H. J.; Park, M. K.; Park, J. H., Three new dammarane glycosides from heat processed ginseng. Arch Pharm Res 2002, 25, (4), Kim, W. Y.; Kim, J. M.; Han, S. B.; Lee, S. K.; Kim, N. D.; Park, M. K.; Kim, C. K.; Park, J. H., Steaming of ginseng at high temperature enhances biological activity. J Nat Prod 2000, 63, (12), Park, I. H.; Piao, L. Z.; Kwon, S. W.; Lee, Y. J.; Cho, S. Y.; Park, M. K.; Park, J. H., Cytotoxic dammarane glycosides from processed ginseng. Chem Pharm Bull (Tokyo) 2002, 50, (4), Jung, K. Y.; Kim, D. S.; Oh, S. R.; Lee, I. S.; Lee, J. J.; Park, J. D.; Kim, S. I.; Lee, H. K., Platelet activating factor antagonist activity of ginsenosides. Biol Pharm Bull 1998, 21, (1), Teng, C. M.; Kuo, S. C.; Ko, F. N.; Lee, J. C.; Lee, L. G.; Chen, S. C.; Huang, T. F., Antiplatelet actions of panaxynol and ginsenosides isolated from ginseng. Biochim Biophys Acta 1989, 990, (3), Yu, J. Y.; Jin, Y. R.; Lee, J. J.; Chung, J. H.; Noh, J. Y.; You, S. H.; Kim, K. N.; Im, J. H.; Lee, J. H.; Seo, J. M.; Han, H. J.; Lim, Y.; Park, E. S.; Kim, T. J.; Shin, K. S.; Wee, J. J.; Park, J. D.; Yun, Y. P., Antiplatelet and antithrombotic activities of Korean Red Ginseng. Arch Pharm Res 2006, 29, (10),

72 25. Lee, W. M.; Kim, S. D.; Park, M. H.; Cho, J. Y.; Park, H. J.; Seo, G. S.; Rhee, M. H., Inhibitory mechanisms of dihydroginsenoside Rg3 in platelet aggregation: critical roles of ERK2 and camp. J Pharm Pharmacol 2008, 60, (11), Kimura, Y.; Okuda, H.; Arichi, S., Effects of various ginseng saponins on 5- hydroxytryptamine release and aggregation in human platelets. J Pharm Pharmacol 1988, 40, (12), Lee, J. G.; Lee, Y. Y.; Kim, S. Y.; Pyo, J. S.; Yun-Choi, H. S.; Park, J. H., Platelet antiaggregating activity of ginsenosides isolated from processed ginseng. Pharmazie 2009, 64, (9), Lee, J. G.; Lee, Y. Y.; Wu, B.; Kim, S. Y.; Lee, Y. J.; Yun-Choi, H. S.; Park, J. H., Inhibitory activity of ginsenosides isolated from processed ginseng on platelet aggregation. Pharmazie 2010, 65, (7), Kwon, S. W.; Han, S. B.; Park, I. H.; Kim, J. M.; Park, M. K.; Park, J. H., Liquid chromatographic determination of less polar ginsenosides in processed ginseng. J Chromatogr A 2001, 921, (2), Croixmarie, V.; Umbdenstock, T.; Cloarec, O.; Moreau, A. l.; Pascussi, J.- M.; Boursier-Neyret, C.; Walther, B., Integrated Comparison of Drug-Related and Drug-Induced Ultra Performance Liquid Chromatography/Mass Spectrometry Metabonomic Profiles Using Human Hepatocyte Cultures. Analytical Chemistry 2009, 81, (15), Hamberg, M.; Svensson, J.; Samuelsson, B., Thromboxanes: a new group of biologically active compounds derived from prostaglandin endoperoxides. Proc Natl Acad Sci U S A 1975, 72, (8), Needleman, P.; Turk, J.; Jakschik, B. A.; Morrison, A. R.; Lefkowith, J. B., Arachidonic acid metabolism. Annu Rev Biochem 1986, 55,

73 33. Kulmacz, R. J.; Lands, W. E., Requirements for hydroperoxide by the cyclooxygenase and peroxidase activities of prostaglandin H synthase. Prostaglandins 1983, 25, (4), Sekiya, F.; Takagi, J.; Usui, T.; Kawajiri, K.; Kobayashi, Y.; Sato, F.; Saito, Y., 12S-hydroxyeicosatetraenoic acid plays a central role in the regulation of platelet activation. Biochem Biophys Res Commun 1991, 179, (1), Coffey, M. J.; Jarvis, G. E.; Gibbins, J. M.; Coles, B.; Barrett, N. E.; Wylie, O. R.; O'Donnell, V. B., Platelet 12-lipoxygenase activation via glycoprotein VI: involvement of multiple signaling pathways in agonist control of H(P)ETE synthesis. Circ Res 2004, 94, (12), Chang, W. C.; Nakao, J.; Orimo, H.; Murota, S., Effects of reduced glutathione on the 12-lipoxygenase pathways in rat platelets. Biochem J 1982, 202, (3), Nyby, M. D.; Sasaki, M.; Ideguchi, Y.; Wynne, H. E.; Hori, M. T.; Berger, M. E.; Golub, M. S.; Brickman, A. S.; Tuck, M. L., Platelet lipoxygenase inhibitors attenuate thrombin- and thromboxane mimetic-induced intracellular calcium mobilization and platelet aggregation. J Pharmacol Exp Ther 1996, 278, (2), Baba, A.; Sakuma, S.; Okamoto, H.; Inoue, T.; Iwata, H., Calcium induces membrane translocation of 12-lipoxygenase in rat platelets. J Biol Chem 1989, 264, (27),

74 Abstract Analysis of the metabolome in a living organism or cell lines is important for understanding of the biological system. In the past, only individual preselected substances were measured and there was no ability to handle large amounts of data. Recently, advanced biotechnology with information technology has generated the omics and the dynamic behavior of metabolism can be evaluated. Ginsenosides isolated from white- or red ginseng have various biological activities and their effects on anti-platelets aggregation have been evaluated over the years. According to our previous reports, ginsenosides isolated from processed white ginseng also exhibited anti-platelet aggregatory activity on ADP, collagen, arachidonic acid (AA) and U induced aggregation. In particular, Rk 1 exhibited strong inhibitory activity in a dose-dependent manner against AA-induced aggregation and the activity was 8 to 22 fold higher than acetylsalicylic acid (ASA) which was used as positive control. ASA is a well-known inhibitor for the production of prostaglandin H 2 (PGH 2 ) by acetylation of cyclooxygenase; this is followed by decreased levels of thromboxane A 2 and results in anti-platelet aggregatory activity. However, the action of Rk 1 in platelets has not been studied yet. Thus, the aim of this study was to evaluate metabolome of washed-platelets induced by the ginsenoside Rk 1 to further understand Rk 1 behavior on AA-induced platelets aggregation. A solvent extraction method was used for metabolomics study and the extracts were subjected to UPLC/Q-TOFMS. Data files were processed with peak clustering and were evaluated by partial least squares and orthogonal projections to latent structures discriminant analyses. As the multivariate results, meaningful endogenous metabolites 62

75 eicosanoids and thromboxane B 2 (TBX 2 ) were identified. The TBX 2 acting as the key element in platelet aggregation was decreased in both aspirin and Rk 1 groups and the decreased level of TBX 2 was demonstrated as inhibition of cyclooxygenase activity based on the COX inhibition results. Meanwhile, the 12(S)-hydroxy-5, 8, 10, 14- eicosatetraenoic acid (12(S)-HETE) decreased significantly in Rk 1 - treated platelets compared to those in the AA-induced group, but an increase was observed in aspirin group. To understand the biological action of anti-platelet aggregation related to 12(S)- HETE, we focused on calcium-dependent 12-LOX translocation from cytosol to membranes and it was determined by western blot analysis. Therefore, this metabolomics study elucidates that the anti-platelet aggregation of ginsenoside Rk 1 resulted from two key metabolites, TXB 2 and 12-HETE, through their biological behaviors. This study shows that metabolomic analysis of metabolome in platelets is a possible approach to understand anti-platelet aggregation action and we expect that this metabolomics study will provide crucial information that can be used to discover other novel drugs by measuring metabolites and it may be useful to monitor the metabolic activation or inactivation that is triggered by the drug. Keywords: Ginsenosides Rk 1, Anti-platelet aggregation activity, Metabolomics, 12(S)- hydroxy-5, 8, 10, 14-eicosatetraenoic acid, arachidonic acid, Thromboxane B 2 Student Number: