臺灣博碩士論文加值系統

黃芩(Scutellariae Radix) 為Labiatae屬Scutellaria baicalensis Georgi之根除去周皮之生藥。在「神農本草經」上列為草部中品，並記載有清熱燥濕、降火解毒及安胎等功用。近來黃芩也被發現有抗發炎和抗氧化的作用，因此本研究探討黃芩水抽取物及其4種主要flavonids成分─ baicalin、baicalein、oroxylin A、wogonin對於人類血小板凝集作用之抑制活性測定結果如下：（1）黃芩水抽取物對collagen (10 μg/ml)、U46619 (2 μM) 所引起血小板凝集之反應具有劑量依存性(dose-dependent)，其IC50分別為73.5、73.8 μg/ml。（2）Baicalin、baicalein對collagen (10 μg/ml)、U46619 (2 μM)、ADP (10 μM)所引起血小板凝集之反應均具有劑量依存性(dose-dependent)，其IC50分別為74.5、35.2、61.5 μg/ml及34.5、37.7、65.8 μg/ml；但對於collagen引起的TxB2生成卻無抑制作用；另外對血小板懸浮液也無提昇cAMP、cGMP和NO的生成。由上述結果顯示：baicalin和baicalein是藉由抑制common pathway (如Ca2+ mobilization或PI turnover )而產生抗血小板凝集作用。（3）Oroxylin A對U46619 (2 μM) 所引起的血小板凝集反應之抑制亦具有濃度依存性，其IC50為45 μg/ml，同時對由collagen引起的TxB2生成(4.9 ± 0.6 pg，control 21.1 ± 0.5 pg)亦有抑制。此外oroxylin A也可以提高NO的生成(8.5 ± 0.4 μM，control 2.6 ± 0.1 μM)。但是對於cAMP、cGMP則沒有作用。因此推測oroxylin A抑制血小板凝集之機制是經由抑制TxA2的生合成及誘生NO而來抑制血小板的凝集。（4）Wogonin對collagen (10 μg/ml)、U46619 (2 μM) 所引起的血小板凝集反應中呈現劑量依存性，其IC50為59.1、45 μg/ml，並抑制由collagen所引起的TxB2之生成(6.8 ± 0.5 pg，control 21.1 ± 0.5 pg)。此外wogonin也提升NO的生成(15.5 ±1.2 μM，control 2.6 ± 0.1 μM)。但是對於cAMP、cGMP皆沒有提高生成的作用。另外在Schild analysis和competitive binding sites測定結果中，wogonin於一定濃度時U46619有隨著濃度的增加而有更大的反應(maximum response)，且隨著wogonin濃度增加，U46619的劑量有向右移動(shift to the right)的現象。進一步以Schild plot計算pA2和pA10 values值分別為5.95及4.50，顯示wogonin與U46619作用在相同的部位，因此會與作用劑(U46619)競爭相同的部位。從以上結果顯示，wogonin對血小板的抑制作用可能是是經由與TxA2競爭receptors及誘生NO之產生而抑制血小板的凝集。
綜合以上結果顯示，黃芩水抽取物具有對人類血小板凝集之抑制作用，而baicalin、baicalein、oroxylin A、wogonin為其活性主成分。

Scutellariae Radix, the roots of Scutellaria baicalensis Georgi (Labiatae) has been demonstrated that the therapeutic effects on treatment of bloodless in Chinese medicinal books. However, the action mechanism of Scutellariae Radix on blood function is undefined. Therefore, study the functions of Scutellariae Radix on platelet aggregation was performed in this project.
Baicalin、baicalein、oroxylin A and wogonin are the major compounds of Scuteiilaire Radix and several biological activities have been demonstrated in these compounds including antioxidant activity and anti-inflammation. In order to study the activities from Scutellariae Radix on platelet aggregation, these four compounds isolated from Scutellariae Radix were used to investigate their inhibitory effects on platelet aggregation induced by collagen, U46619 or ADP in human platelet-rich plasma. The results showed that these four polyphenolic compounds showed the significant inhibition on collagen-, U46619- or ADP-induced aggregation. The IC50 values of baicalin, baicalein and wogonin are 74.5, 34.5, >100, 59.1 μg/ml in the collagen-induced aggregation, and 35.2, 37.7, 45, 45 μg/ml in the U46619-induced aggregation, respectively. As the same part of experiment, both baicalin and baicalein also showed the obvious dose-dependent inhibition of ADP-induced platelet aggregation, and the IC50 values are 61.5 and 65.8 μg/ml, respectively. These data provided evidences that Scutellariae radix inhibits stimulators induced platelet aggregation.
Nitric oxide (NO) and TxB2 have been implicated to play as modulators in the process of platelet aggregation. Upon oroxylin A and wogonin treated platelet, NO production was significantly increased to 8.5 ± 0.4 and 15.5 ± 1.2 μM, respectively, as compared with the controlled group (2.6 ± 0.1 μM).
A binding site for TxA2 antagonist wogonin to PRP was studied. Wogonin competitively antagonized aggregation of PRP by the TxA2 mimetic U46619. A Schild analysis of the pharmacology study revealed a pA2 of 3.90 and pA10 of 5.01. It suggests that this high-affinity binding sites is the site responsible for inhibition of aggregation induced by U46619.
These data provided evidences that Scutellariae Radix inhibits stimulators induced platelet aggregation.

考試委員名錄
致謝
目錄 ………………………………………………………… i
縮寫 ………………………………………………………… iii
中文摘要 …………………………………………………… 1
英文摘要 …………………………………………………… 3
前言 ………………………………………………………… 5
實驗部分 …………………………………………………… 10
I 實驗材料 ………………………………………………… 10
1藥材 ……………………………………………………… 10
2實驗試劑 ………………………………………………… 11
II實驗儀器 ………………………………………………… 12
III實驗方法 …………………………………………………12
1試劑製備 ……………………………………………………12
2測試藥品之調製 ……………………………………………13
3富含血小板血漿的製備 ……………………………………13
4人類血小板懸浮液的製備 …………………………………13
5血小板凝集反應的測定 ……………………………………14
6血小板內TxB2的測定 ………………………………………15
7統計方法 ……………………………………………………16
結果 …………………………………………………………17
I黃芩水萃取物對人類血小板凝集作用 ……………………17
II黃芩中flavonoids對人類血小板凝集作用 …………… 17
III黃芩中flavonoids對血小板內TxB2含量的影響 ………18
IV黃芩中flavonoids對血小板內cAMP含量的影響 ………18
V黃芩中flavonoids對血小板內NO含量的影響 ……………19
VI Wogonin對TxA2 receptors的影響………………………19
討論 …………………………………………………………21
圖表 …………………………………………………………24
參考文獻 ……………………………………………………42

1.Holmsen H. Significance of testing platelet functions in vitro. Eur J Clin Invest 1994; 24: 3-8.
2. Berridge MJ, Irvine RF. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature 1984; 312: 315-21.
3.Berridge MJ, Irvine RF. Inositol phosphates and cell signalling. Nature 1989; 341: 197-205.
4.Bishop WR, Ganong BR, Bell RM. Attenuation of 1,2-diacylglycerol second messengers by diacylglycerol kinase. Inhibition by diacylglycerol analogs in vitro and in human platelets. J Biol Chem 1986; 261: 6993-7000.
5.Bishop WR, Bell RM. Attenuation of sn-1,2-diacylglycerol second messengers. Metabolism of exogenous diacylglycerols by human platelets. J Biol Chem 1986; 261: 12513-9.
6.Siess W, Lapetina EG. Ca2+ mobilization primes protein kinase C in human platelets. Ca2+ and phorbol esters stimulate platelet aggregation and secretion synergistically through protein kinase C. Biochem J 1988; 255: 309-18.
7.Rink TJ, Smith SW, Tsien RY. Cytoplasmic free Ca2+ in human platelets: Ca2+ thresholds and Ca-independent activation for shape-change and secretion. FEBS Lett 1982; 148: 21-6.
8.Adunyah SE, Dean WL. Inositol triphosphate-induced Ca2+ release from human platelet membranes. Biochem Bioph Res Commun 1985; 128: 1274-80.
9.Wilson DB, Connolly TM, Bross TE, Majerus PW, Sherman WR, Tyler AN, Rubin LJ, Brown JE. Isolation and characterization of the inositol cyclic phosphate products of polyphosphoinositide cleavage by phospholipase C. Physiological effects in permeabilized platelets and Limulus photoreceptor cells. J Biol Chem 1985; 260: 13496-501.
10.Lapetina EG, Reep B, Ganong BR, Bell RM. Exogenous sn-1,2-diacylglycerols containing saturated fatty acids function as bioregulators of protein kinase C in human platelets. J Biol Chem 1985; 260: 1358-61.
11.Maruyama I, Majerus PW. The turnover of thrombin-thrombomodulin complex in cultured human umbilical vein endothelial cells and A549 lung cancer cells. Endocytosis and degradation of thrombin. J Biol Chem 1985; 260: 15432-8.
12.Nishizuka Y. Studies and perspectives of protein kinase C. Science 1986; 233: 305-12.
13.Bell RL, Kennerly DA, Stanford N, Majerus PW. Diglyceride lipase: a pathway for arachidonate release from human platelets. Proc Natl Acad Sci USA 1979; 76: 3238-41.
14.Cook HW, Lands WE. Mechanism for suppression of cellular biosynthesis of prostaglandins. Nature 1976; 260: 630-2.
15.Calzada C, Vericel E, Mitel B, Lagarde M. Stimulation of platelet aggregation in response to arachidonic acid hydroperoxide via phospholipase activation. Lipids 1999; 34: S295.
16.Hamberg M, Svensson J, Samuelsson B. Thromboxanes: a new group of biologically active compounds derived from prostaglandin endoperoxides. Proc Natl Acad Sci USA 1975; 72: 2994-8.
17.Sims PJ, Ginsberg MH, Plow EF, Shattil SJ. Effect of platelet activation on the conformation of the plasma membrane glycoprotein IIb-IIIa complex. J Biol Chem 1991; 266: 7345-52.
18.Wu H, Peng L, Li J, Wu W, Hou Q, Liu H, Wang M. Reversible exposure of human platelet fibrinogen receptors by antiplatelet tetraspanin monoclonal antibodies via induction of a conformational change in membrane glycoprotein IIb/IIIa complex. Throm Res 1999; 95: 245-53.
19.Mais DE, Burch RM, Saussy DL Jr, Kochel PJ, Halushka PV. Binding of a thromboxane A2/prostaglandin H2 receptor antagonist to washed human platelets. J Pharmacol Exp Ther 1985; 235: 729-34.
20.Moro MA, Russel RJ, Cellek S, Lizasoain I, Su Y. Darley-Usmar VM. Radomski MW. Moncada S. cGMP mediates the vascular and platelet actions of nitric oxide: confirmation using an inhibitor of the soluble guanylyl cyclase. Proc Natl Acad Sci USA 1996; 93: 1480-5.
21.Ignarro LJ. Heme-dependent activation of guanylate cyclase by nitric oxide: a novel signal transduction mechanism. Blood Vessels 1991; 28(1-3): 67-73.
22.Moncada S, Higgs A. The L-arginine-nitric oxide pathway. New Eng J Med 1993; 329: 2002-12.
23.Nathan C, Xie QW. Regulation of biosynthesis of nitric oxide. J Biol Chem 1994; 269: 13725-8.
24.Radomski MW, Palmer RM, Moncada S. An L-arginine/nitric oxide pathway present in human platelets regulates aggregation. Proc Natl Acad Sci USA 1990; 87: 5193-7.
25.Knowles RG. Nitric oxide synthases. Biochem Soc T 1996; 24: 875-8.
26.Furchgott RF, Vanhoutte PM. Endothelium-derived relaxing and contracting factors. FASEB J 1989; 3: 2007-18.
27.Radomski MW, Palmer RM, Moncada S. Characterization of the L-arginine:nitric oxide pathway in human platelets. Brit J Pharmacol 1990; 101: 325-8.
28.Malinski T, Radomski MW, Taha Z, Moncada S. Direct electrochemical measurement of nitric oxide released from human platelets. Biochem Bioph Res Commun 1993; 194: 960-5.
29.Radomski MW, Palmer RM, Moncada S. An L-arginine/nitric oxide pathway present in human platelets regulates aggregation. Proc Natl Acad Sci USA 1990; 87: 5193-7.
30.Swier P, Mohammad SF, Olsen DB, Kolff WJ. A comparison of the antiplatelet effect of aspirin on human and bovine platelets. ASAIO Trans 1989; 35: 205-8.
31.Geiger J, Brich J, Honig-Liedl P, Eigenthaler M, Schanzenbacher P, Herbert JM, Walter U. Specific impairment of human platelet P2Y(AC) ADP receptor-mediated signaling by the antiplatelet drug clopidogrel. Arterioscl Throm Vas 1999; 19: 2007-11.
32.Rivey MP, Alexander MR, Taylor JW. Dipyridamole: a critical evaluation. Drug Intell Clin Pharm 1984; 18: 869-80.
33.Donayre CE, Ouriel K, Rhee RY, Shortell CK. Future alternatives to heparin: low-molecular-weight heparin and hirudin. J Vasc Surg 1992; 15: 675-82.
34.Ghaffari S, Kereiakes DJ, Lincoff AM, Kelly TA, Timmis GC, Kleiman NS, Ferguson JJ. Miller DP. Califf RA. Topol EJ. Platelet glycoprotein IIb/IIIa receptor blockade with abciximab reduces ischemic complications in patients undergoing directional coronary atherectomy. EPILOG Investigators. Evaluation of PTCA to Improve Long-term Outcome by c7E3 GP IIb/IIIa Receptor Blockade. Am J Cardiol 1998; 82: 7-12.
35.Taher A, Ammash Z, Dabajah B, Nasrallah A, Mourad FH. Ticlopidine-induced aplastic anemia and quick recovery with G-CSF: case report and literature review. Am J Hematol 2000; 63: 90-3.
36.Keltz TN, Innerfield M, Gitler B, Cooper JA. Dipyridamole-induced myocardial ischemia. JAMA 1987; 257: 1515-6.
37.Peter K, Straub A, Kohler B, Volkmann M, Schwarz M, Kubler W, Bode C. Platelet activation as a potential mechanism of GP IIb/IIIa inhibitor-induced thrombocytopenia. Am J Cardiol 1999; 84: 519-24.
38.Sheu JR, Kan YC, Hung WC, Ko WC, Yen MH: Mechanisms involved in the antiplatelet activity of tetramethylpyrazine in human platelets. Thrombo Res 1997; 88: 259-70.
39.Lee CM, Jiang LM, Shang HS, Hon PM, He Y, Wong HN: Prehispanolone , a novel platelet activating factor receptor antagonist from Leonurus heterophyllus. Bri J Pharm 1991 103: 1719-24.
40.Sheu JR, Lee CR, Hsiao G, Hung WC, Lee YM, Chen YC, Yen MH. Comparison of the relative activities of alpha-tocopherol and PMC on platelet aggregation and antioxidative activity. Life Sci 65:197-206, 1999.
41.Sheu JR, Lee CR, Lin CC, Kan YC, Lin CH, Hung WC, Lee YM, Yen MH. The antiplatelet activity of PMC, a potent alpha-tocopherol analogue, is mediated through inhibition of cyclo-oxygenase. Bri J Pharm 1999; 127, 1206-12.
42.Landolfi R. Mower RL. Steiner M. Modification of platelet function and arachidonic acid metabolism by bioflavonoids. Structure-activity relations. Biochem Pharm 1984; 33:1525-30.
43.Tomimori T, Miyaichi Y, Kizu H. On the flavonoid constituents from the roots of Scutellaria baicalensis Georgi. I. Yakugaku Zasshi - J Pharm Soci Japan 1982; 102: 388-91.
44.Gao Z, Huang K, Yang X, Xu H. Free radical scavenging and antioxidant activities of flavonoids extracted from the radix of Scutellaria baicalensis Georgi. Biochim Biophys Acta 1999; 1472: 643-50.
45.Ng TB, Liu F, Wang ZT. Antioxidative activity of natural products from plants. Life Sci 2000; 66: 709-23.
46.Gao D, Sakurai K, Chen J, Ogiso T. Protection by baicalein against ascorbic acid-induced lipid peroxidation of rat liver microsomes. Res Commun Mol Path Pharm 1995; 90: 103-14.
47.Butenko IG. Gladtchenko SV. Galushko SV. Anti-inflammatory properties and inhibition of leukotriene C4 biosynthesis in vitro by flavonoid baicalein from Scutellaria baicalensis Georgy roots. Agents Actions 39 :C49-51, 1993.
48.Nagai T, Moriguchi R, Suzuki Y, Tomimori T, Yamada H. Mode of action of the anti-influenza virus activity of plant flavonoid, 5,7,4''-trihydroxy- 8-methoxyflavone, from the roots of Scutellaria baicalensis. Antivir Res 1995; 26: 11-25.
49.Nagai T, Suzuki Y, Tomimori T, Yamada H. Antiviral activity of plant flavonoid, 5,7,4''-trihydroxy-8-methoxyflavone, from the roots of Scutellaria baicalensis against influenza A (H3N2) and B viruses. Biol Pharm Bull 1995; 18: 295-9.
50.Kim HM, Moon EJ, Li E, Kim KM, Nam SY, Chung CK. The nitric oxide-producing activities of Scutellaria baicalensis. Toxicology 1999; 135: 109-15.
51.Kyo R, Nakahata N, Sakakibara I, Kubo M, Ohizumi Y. Baicalin and baicalein, constituents of an important medicinal plant, inhibit intracellular Ca2+ elevation by reducing phospholipase C activity in C6 rat glioma cells. J Pharm Pharmacol 1998; 50: 1179-82.
52.Mustard JF, Kinlough-Rathbone RL, Packham MA. Isolation of human platelets from plasma by centrifugation and washing. Methods Enzymol 1989; 169, 3-11.
53.Mustard JF, Perry DW, Ardlie NG, Packham MA: Preparation of suspensions of washed platelets from humans. Brit J Haematol 1972; 22: 193-204.
54.Kornecki E, Niewiarowski S, Morinelli TA, Kloczewiak M: Effect of chymotrypsin and adenosine diphosphate on the exposure of fibrinogen receptors on normal human and Glanzmann’s thrombasthenic platelets. J Biol Chem 1981; 256: 5696-5701.
55.O’Brien JR: Platelet aggregation II. Some results from a new method of study. J Clin Path 1962; 15:452-455.
56.Born GVR, Cross MJ: The aggregation of blood platelet. J Physiol 1963; 168: 178-195.
57.McNicol A. Platelet preparation and estimation of functional responses. In: Watson SP, Authi KS, editors. Platelets: A practical approach. Oxford: IRL Rress; 1-26, 1996.
58.Karniguian A, Legrand YJ, Caen JP: Prostaglandins: specific inhibition of platelet adhesion to collagen and relationship with camp level. Prostaglandins 1982; 23: 437-457.
59.Grynkiewicz G, Poenie M, Timmons S: Prostacylin inhibits mobilization of fibrinogen-binding sites on human ADP- and thrombin- treated platelets. Nature 1980; 283: 195-197.
60.Yoshimoto H, Suehiro A, Kakishita E. Exogenous nitric oxide inhibits platelet activation in whole blood. J Cardiovasc Pharm 1999; 33: 109-15.
61.Malinski T, Radomski MW, Taha Z, Moncada S. Direct electrochemical measurement of nitric oxide released from human platelets. Biochem Bioph Res Commun 1993; 194: 960-5.
62.Tadeusz M, Marek WR, Ziad T, Salvador M. Direct electrochemical measurement of nitric oxide released from human platelets. Biochem Biophys Res Commun 1993; 194: 960-965.
63.Schulz K, Kerber S, Kelm M. Reevaluation of the Griess method for determining NO/NO2- in aqueous and protein-containing samples. Nitric Oxide 1999; 3: 225-34.
64.Wolfe SM, Shulman NR. Inhibition of platelet energy production and release reaction by PGE1, theophylline and cAMP. Biochem Biophy Res Commun 1970; 41: 128-34.
65.Feinstein MB, Egan JJ, Sha''afi RI, White J. The cytoplasmic concentration of free calcium in platelets is controlled by stimulators of cyclic AMP production (PGD2, PGE1, forskolin). Biochem Biophy Res Commun 1983; 113: 598-604.
66.Feinstein MB, Egan JJ, Opas EE. Reversal of thrombin-induced myosin phosphorylation and the assembly of cytoskeletal structures in platelets by the adenylate cyclase stimulants prostaglandin D2 and forskolin. J Biol Chem 1983; 258: 1260-7.
67.Herman F, Hadhazy P, Magyar K. In vivo measurement of the dis-aggregatory action of prostacyclin. A methodological study. Thromb Res 1986; 44: 575-85.
68.Mellion BT, Ignarro LJ, Ohlstein EH, Pontecorvo EG, Hyman AL, Kadowitz PJ. Evidence for the inhibitory role of guanosine 3'', 5''-mono- phosphate in ADP-induced human platelet aggregation in the presence of nitric oxide and related vasodilators. Blood 1981; 57: 946-55.
69.Kubo M. Matsuda H, Tanaka M, Kimura Y, Okuda H, Higashino M, Tani T, Namba K, Arichi S. Studies on Scutellariae radix. VII. Anti-arthritic and anti-inflammatory actions of methanolic extract and flavonoid components from Scutellariae radix. Chem Pharm Bull 1984; 32: 2724-9.
70.Kyo R, Nakahata N, Sakakibara I, Kubo M, Ohizumi Y. Baicalin and baicalein, constituents of an important medicinal plant, inhibit intracellular Ca2+ elevation by reducing phospholipase C activity in C6 rat glioma cells. J Pharm Pharmacol 1998; 50: 1179-82.
71.Sekiya K, Okuda H. Selective inhibition of platelet lipoxygenase by baicalein. Biochem Biophys Res Commun 1982,105: 1090-5.
72.Chen YC, Yang LL, Lee TJF. Oroxylin A inhibition of lipopolysaccharide-induced iNOS and COX-2 gene expression via suppression of nuclear factor-κB activation. Biochem pharmacol 2000: 59, 1445-57.
73.You KM, Jong HG, Kim HP. Inhibition of cyclooxygenase/lipoxygenase from human platelets by polyhydroxylated/methoxylated flavonoids isolated from medicinal plants. Arch Pharm Res 1999; 22: 18-24.
74.Liu XF, Liu ML, Iyanagi T, Legesse K, Lee TD, Chen SA. Inhibition of rat liver NAD(P)H:quinone acceptor oxidoreductase (DT-diaphorase) by flavonoids isolated from the Chinese herb scutellariae radix (Huang Qin). Mol Pharmaco 1990; 37:911-5.
75.Wakabayashi I. Inhibitory effects of baicalein and wogonin on lipopolysaccharide-induced nitric oxide production in macrophages. Pharmacol Toxiol 1999; 84: 288-91.
76.Lin CN, Kuo SH, Chung MI, Ko FN, Teng CM. A new flavone C-glycoside and antiplatelet and vasorelaxing flavones from Gentiana arisanensis. J Nat Prod 60: 851-3, 1997.
77.Tsai WJ, Hsin WC, Chen CC. Antiplatelet flavonoids from seeds of Psoralea corylifolia. J Nat Prod 1996; 59: 671-2.
78.Goker H, Tuncbilek M, Leoncini G, Buzzi E, Mazzei M, Rolland Y, Ertan R. Synthesis and inhibitory activities on platelet aggregation of some flavonoid analogues. Arzneimittel-Forschung. 45: 150-5, 1995.
79.Bourdillat B. Delautier D. Labat C. Benveniste J. Potier P. Brink C. Hispidulin, a natural flavone, inhibits human platelet aggregation by increasing cAMP levels. Eur J Pharmacol 1998; 147:1-6.