Effects of Korean red ginseng and its mixed prescription on the high molecular weight dextran-induced blood stasis in rats and human platelet aggregation.

This study was undertaken to evaluate the antithrombotic effects of Korean Red Ginseng (KRG) and its new prescription (KRGP) consisting of five herbs such as Korean red ginseng, Ganoderma, Cinnamomi Cortex, Glycyrrhizae Radix and Laminaria. In rats with blood stasis induced by high molecular weight dextran, KRG and KRGP significantly restored not only the number of platelets and fibrinogen, but also suppressed the fibrin degradation products (FDP) to normal range. In platelet aggregation assay with human platelet rich plasma (PRP), KRG and KRGP significantly inhibited thrombin and collagen-induced platelet aggregation. The IC(50) values of KRG and KRGP were >2 and 0.23+/-0.01 mg/ml for thrombin, 0.32+/-0.01 and 0.17+/-0.02 mg/ml for collagen and 0.72+/-0.25 and >2 mg/ml for ADP, respectively. In coagulation assay, KRG and KRGP significantly prolonged activated partial prothrombin time (APPT) and prothrombin time (PT) as compared with control data. KRGP was found to be more effective than KRG alone on antithrombotic activity. These results suggest that KRGP may exert its antithrombotic activity due to inhibition of platelet aggregation and coagulation activity more than KRG.

Effect of genistein analogs on oxygen radical production in leukocytes stimulated by unopsonized zymosan.

Effects of genistein analogs on oxygen radical production have been analyzed in human neutrophils, human monocytes or murine macrophages Raw264.7 stimulated with unopsonized zymosan by lucigenin- and luminol-enhanced chemiluminescence assays. Genistein exhibited IC50 values of 10.7-11.5 microM on the oxygen radical production in human neutrophils, 10.9-11.0 microM in human monocytes, and 14.8-27.3 microM in Raw264.7 cells. Orobol, a genistein analog with an additional hydroxy group at the 3' position, exhibited IC50 values of 3.0-3.3 microM on the oxygen radical production in human neutrophils, 2.8-3.1 microM in human monocytes, and 1.5-3.9 microM in Raw264.7 cells. Genistin and sophoricoside are genistein glycosides with a glucose moiety at 7 or 4' position, respectively. The genistein glycosides exhibited 23-37% inhibitory effects at 100 microM on the oxygen radical production.

Protective effect of green tea extract on ischemia/reperfusion-induced brain injury in Mongolian gerbils.

Free radical-induced oxidative damages of macromolecules and cell death are important factors in the pathogenesis of ischemia/reperfusion brain injury. In the present study, an investigation as to whether green tea extract reduces ischemia/reperfusion-induced brain injury in Mongolian gerbils was conducted. The effect of green tea on the ischemia/reperfusion-induced production of hydrogen peroxide, lipid peroxidation and oxidative DNA damage (formation of 8-hydroxydeoxyguanosine), and cell death in addition to locomotor activity was studied. Two doses (0.5 or 2%) of green tea extract were added into the drinking water and to be accessed by animals ad libitum for 3 weeks prior to the induction of ischemia. A global ischemia was induced by the bilateral occlusion of the common carotid arteries for 5 min. Reperfusion was achieved by releasing the occlusion and restoring blood circulation for 48 h. The infarction volumes were 112+/-31 mm(3) and 76+/-11 mm(3) in the 0.5 and 2% green tea pretreated animals compared to 189+/-12 mm(3) in the ischemia/reperfusion animals. Green tea extract also reduced the levels of ischemia/reperfusion-induced hydrogen peroxide (from 1470+/-170 to 1034+/-46 and 555+/-30 nmole/mg protein), lipid peroxidation products (from 1410+/-210 to 930+/-40 and 330+/-20 nmole/mg protein) and 8-oxodG (from 3.9+/-0.1 to 2.8+/-0.3 and1.9+/-0.3 ng/microg DNA, x10(-2)) by pretreatment of 0.5 or 2% green tea for 3 weeks, respectively. Moreover, green tea also reduced the number of ischemia/reperfusion-induced apoptotic cells (from 59+/-12 to 37+/-8, 15+/-11 apoptotic cells/high power field in the striatum region) and locomotor activity (from 15140+/-2940 to 3900+/-600 and 4100+/-1200). This study therefore suggests that green tea may be a useful agent for the prevention of cerebral ischemia damage.

Neuroprotective effect of green tea extract in experimental ischemia-reperfusion brain injury.

Eicosanoids accumulation and formation of oxygen free radicals have been implicated in the pathogenesis of ischemia/reperfusion brain injury. In the present study, we examined whether green tea extract protects against ischemia/reperfusion-induced brain injury by minimizing eicosanoid accumulation and oxygen radical-induced oxidative damage in the brain. Green tea extract (0.5%) was orally administered to Wistar rats for 3 weeks before induction of ischemia. Ischemia was induced by the occlusion of middle cerebral arteries for 60 min and reperfusion was achieved for 24 h. Infarction volume in the ipsilateral hemisphere of ischemia/reperfusion animals was 114 +/- 16 mm(3) in the 0.5% green tea pretreated animals compared to 180 +/- 54 mm(3) in left hemisphere of nontreated animals. Green tea extract (0.5%) also reduced ischemia/reperfusion-induced eicosanoid concentration: Leukotriene C(4) (from 245 +/- 51 to186 +/- 22), prostoglandin E(2) (from 306 +/- 71 to 212 +/- 43) and thromboxane A(2) (327 +/- 69 to 251 +/- 87 ng/mg protein). Ischemia/reperfusion-induced increases of hydrogen peroxide level (from 688 +/- 76 to 501 +/- 99 nmole/mg protein), lipid peroxidation products (from 1010 +/- 110 to 820 +/- 70 nmole/mg protein) and 8-oxodG formation (from 1.3 +/- 0.3 to 0.8 +/- 0.2 ng/microg DNA, x10(-2)) were also reduced. Moreover, 0.5% green tea extract also reduced the apoptotic cell number (from 44 +/- 11 to 29 +/- 1 in the striatum, and from 72 +/- 11 to 42 +/- 5 apoptotic cells/high power field in the cortex region). Green tea extract pretreatment also promoted recovery from the ischemia/reperfusion-induced inhibition of active avoidance. The present study shows that the minimizing effect of green tea extract on the eicosanoid accumulation and oxidative damage in addition to the reduction of neuronal cell death could eventually result in protective effect on the ischemia/reperfusion-induced brain injury and behavior deficit.

Antithrombotic activities of green tea catechins and (-)-epigallocatechin gallate.

The antithrombotic activities and mode of action of green tea catechins (GTC) and (-)-epigallocatechin gallate (EGCG), a major compound of GTC, were investigated. Effects of GTC and EGCG on the murine pulmonary thrombosis in vivo, human platelet aggregation in vitro, and ex vivo, and coagulation parameters were examined. GTC and EGCG prevented death caused by pulmonary thrombosis in mice in vivo in a dose-dependent manner. They significantly prolonged the mouse tail bleeding time of conscious mice. They inhibited adenosine diphosphate- and collagen-induced rat platelet aggregation ex vivo in a dose-dependent manner. GTC and EGCG inhibited ADP-, collagen-, epinephrine-, and calcium ionophore A23187-induced human platelet aggregation in vitro dose dependently. However, they did not change the coagulation parameters such as activated partial thromboplastin time, prothrombin time, and thrombin time using human citrated plasma. These results suggest that GTC and EGCG have the antithrombotic activities and the modes of antithrombotic action may be due to the antiplatelet activities, but not to anticoagulation activities.

Effects of tetrandrine and fangchinoline on human platelet aggregation and thromboxane B2 formation.

Tetrandrine (TET) and fangchinoline (FAN) are two major components of the radix of Stephania tetrandra. The effects of TET and FAN on human platelet aggregation and formation of thromboxane (TX) B2, a stable metabolite of TXA2, were examined in the aspect of platelet aggregation. TET and FAN inhibited platelet-activating factor (PAF)-induced human platelet aggregation. IC50 values for TET and FAN were 28.6+/-3.24 microM and 21.7+/-2.61 microM, respectively. In the PAF-receptor binding assay, neither TET nor FAN showed any inhibitory effects on the specific bindings of PAF to its receptor. TET and FAN also inhibited PAF-, thrombin- and arachidonic acid-induced thromboxane B2 formation in human washed platelet. These results indicate that TET and FAN inhibit the platelet aggregation by interfering with the intracellular messengers system, but not by inhibiting the binding of PAF to PAF-receptor on the platelet membrane directly, and the suppression of TXA2 formation by TET and FAN may be responsible for their inhibitory activities on the platelet aggregation and further on the thrombosis.

Epigallocathechin-3 gallate selectively inhibits the PDGF-BB-induced intracellular signaling transduction pathway in vascular smooth muscle cells and inhibits transformation of sis-transfected NIH 3T3 fibroblasts and human glioblastoma cells (A172).

Enhanced activity of receptor tyrosine kinases such as the PDGF beta-receptor and EGF receptor has been implicated as a contributing factor in the development of malignant and nonmalignant proliferative diseases such as cancer and atherosclerosis. Several epidemiological studies suggest that green tea may prevent the development of cancer and atherosclerosis. One of the major constituents of green tea is the polyphenol epigallocathechin-3 gallate (EGCG). In an attempt to offer a possible explanation for the anti-cancer and anti-atherosclerotic activity of EGCG, we examined the effect of EGCG on the PDGF-BB-, EGF-, angiotensin II-, and FCS-induced activation of the 44 kDa and 42 kDa mitogen-activated protein (MAP) kinase isoforms (p44(mapk)/p42(mapk)) in cultured vascular smooth muscle cells (VSMCs) from rat aorta. VSMCs were treated with EGCG (1-100 microM) for 24 h and stimulated with the above mentioned agonists for different time periods. Stimulation of the p44(mapk)/p42(mapk) was detected by the enhanced Western blotting method using phospho-specific MAP kinase antibodies that recognized the Tyr204-phosphorylated (active) isoforms. Treatment of VSMCs with 10 and 50 microM EGCG resulted in an 80% and a complete inhibition of the PDGF-BB-induced activation of MAP kinase isoforms, respectively. In striking contrast, EGCG (1-100 microM) did not influence MAP kinase activation by EGF, angiotensin II, and FCS. Similarly, the maximal effect of PDGF-BB on the c-fos and egr-1 mRNA expression as well as on intracellular free Ca2+ concentration was completely inhibited in EGCG-treated VSMCs, whereas the effect of EGF was not affected. Quantification of the immunoprecipitated tyrosine-phosphorylated PDGF-Rbeta, phosphatidylinositol 3'-kinase, and phospholipase C-gamma1 by the enhanced Western blotting method revealed that EGCG treatment effectively inhibits tyrosine phosphorylation of these kinases in VSMCs. Furthermore, we show that spheroid formation of human glioblastoma cells (A172) and colony formation of sis-transfected NIH 3T3 cells in semisolid agar are completely inhibited by 20-50 microM EGCG. Our findings demonstrate that EGCG is a selective inhibitor of the tyrosine phosphorylation of PDGF-Rbeta and its downstream signaling pathway. The present findings may partly explain the anti-cancer and anti-atherosclerotic activity of green tea.

Effects of tetrandrine and fangchinoline on experimental thrombosis in mice and human platelet aggregation.

Tetrandrine (TET) and fangchinoline (FAN) are two naturally occurring analogues with a bisbenzylisoquinoline structure. The present study was undertaken to investigate the effects of TET and FAN on the experimental thrombosis induced by collagen plus epinephrine (EP) in mice, and platelet aggregation and blood coagulation in vitro. In the in vivo study, the administration (50 mg/kg, i.p.) of TET and FAN in mice showed the inhibition of thrombosis by 55% and 35%, respectively, while acetylsalicylic acid (ASA, 50 mg/kg, i.p.), a positive control, showed only 30% inhibition. In the vitro human platelet aggregations induced by the agonists used in tests, TET and FAN showed the inhibitions dose dependently. In addition, neither TET nor FAN showed any anticoagulation activities in the measurement of the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) using human-citrated plasma. These results suggest that antithrombosis of TET and FAN in mice may be mainly related to the antiplatelet aggregation activities.

Inhibition of lens-aldose reductase activity by brazilin and haematoxylin.

Brazilin and haematoxylin, plant pigments, were examined for their effects on the Bovine-Lens aldose reductase (LAR)-activity. About 50% inhibition was observed in a concentration of 10 (-4) M-brazilin and 10 (-4) M-haematoxylin, and above 95% inhibition was observed in a concentration of 10 (-3) M-brazilin and 10 (-3)M-haematoxylin. In order to determine the type of inhibition, kinetic studies were also conducted with brazilin and haematoxylin, in which both were found to be noncompetitive inhibitors.