Five New Triterpenoid Saponins from the Rhizomes of Panacis majoris and Their Antiplatelet Aggregation Activity.

Five new triterpenoid saponins (1-5) and four known triterpenoid saponins, ginsenoside Re5 (6), majonoside R1 (7), 24(R)-majonoside R1 (8), and ginsenoside Rf (9), were isolated from the rhizomes of Panacis majoris. The structures of new compounds were elucidated as (20S,24S,25R*)-6-O-[ß-D-glucopyranosyl-(1 → 2)-ß-D-glucopyranosyl]-dammar-20,24-epoxy-3ß,6α,12ß,25,26-pentaol (1), (20S,24R,25R)-6-O-[ß-D-glucopyranosyl-(1 → 2)-ß-D-glucopyranosyl]-dammar-20,24-epoxy-3ß,6α,12ß,25,26-pentaol (2), (20S)-6-O-[ß-D-glucopyranosyl-(1 → 2)-ß-D-glucopyranosyl]-dammar-20,25-epoxy-3ß,6α,12ß,24α-tetraol (3), 6-O-[ß-D-glucopyranosyl-(1 → 2)-ß-D-glucopyranosyl]-dammar-3ß,6α,12ß,20S,24R,25-hexaol (4), and 6-O-[ß-D-glucop-yranosyl-(1 → 2)-ß-D-glucopyranosyl]-dammar-25(26)-ene-3ß,6α,12ß,20S,24R-pentaol (5) on the basis of extensive spectral analysis and chemical methods. Ginsenoside Re5 was isolated from the plant for the first time. The similarities of the nine compounds lie in the fact that their aglycones are conjoined with the same glucopyranose moieties, the same linkage of the glycosyl chains, and the same glycosylation sites, while they have a varied C-17 side chain. Compounds 3 and 5 exhibited moderate antiplatelet aggregation activities induced by adenosine diphosphate with IC50 values of 23.24 and 18.43 µM, respectively. Compound 5 displayed moderate inhibition of arachidonic acid-induced platelet aggregation with an IC50 value of 30.11 µM.

Ionic liquid and aqueous two-phase extraction based on salting-out coupled with high-performance liquid chromatography for the determination of seven rare ginsenosides in Xue-Sai-Tong injection.

A method of ionic liquid salt aqueous two-phase extraction coupled with high-performance liquid chromatography has been developed for the analysis of seven rare ginsenosides including Rg6 , F4 , 20(S)-Rg3 , 20(R)-Rg3 , Rk3 , Rk1 , and Rg5 in Xue-Sai-Tong injection. The injection was mixed with ionic liquid 1-butyl-3-methylimidazolium bromide aqueous solution, and a mixture was obtained. With the addition of sodium dodecyl sulfate and dipotassium phosphate into the mixture, the aqueous two-phase mixture was formed after ultrasonic treatment and centrifuged. Rare ginsenosides were extracted into the upper phase. To obtain a high extraction factors, various influences were considered systematically, such as the volume of ionic liquid, the category and amount of salts, the amount of sodium dodecyl sulfate, the pH value of system, and the time of ultrasonic treatment. Under the optimal condition, rare ginsenosides in Xue-Sai-Tong injection were enriched and detected, the recoveries of seven rare ginsenosides ranged from 90.05 to 112.55%, while relative standard deviations were lower than 2.50%. The developed method was reliable, rapid and sensitive for the determination of seven rare ginsenosides in the injections.

Hypolipidemic effects of kaempferide-7-O-(4''-O-acetylrhamnosyl)-3-O-rutinoside in hyperlipidemic rats induced by a high-fat diet.

Kaempferide-7-O-(4''-O-acetylrhamnosyl)-3-O-rutinoside (A-F-B) is a novel flavonoid which is extracted from the leaves of Actinidia kolomikta. The aim of this study was to investigate the hypolipidemic effects of A-F-B in hyperlipidemic rats induced by a high-fat diet. Male Wistar rats were randomly divided into six groups: normal diet group, high-fat diet group, lovastatin (2.5 mg/kg) group and A-F-B (12.5, 25 or 50 mg/kg) groups. To evaluate the lipid-lowering effects of A-F-B, total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), atherogenic index (AI) and coronary risk index (CRI) were investigated. The activities of phosphatidate phosphohydrolase (PAP) and hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase in hepatic tissue were evaluated. Treatment with A-F-B to hyperlipidemic rats resulted in a significant decline in TC, TG, LDL-C, AI and CRI, with an increase in HDL-C level. The results also showed that A-F-B significantly decreased the activities of PAP and HMG-CoA reductase in hepatic tissue. These findings suggest that A-F-B improves lipid profiles. The mechanisms of A-F-B were associated with regulating the activities of PAP and HMG-CoA reductase in hepatic tissue.

Three new dammarane-type triterpene saponins from the leaves of Panax ginseng C.A. Meyer.

Three new dammarane-type triterpene ginsenosides, together with six known ginsenosides, were isolated from the leaves of Panax ginseng C.A. Meyer. The new saponins were named as ginsenoside Rh11, ginsenoside Rh12, and ginsenoside Rh13. Their structures were elucidated as (20S)-3ß,6α,12ß,20-tetrahydroxydammara-25-ene-24-one 20-O-ß-d-glucopyranoside (1), (20S)-3ß,12ß,20,24,25-pentahydroxydammarane 20-O-ß-d-glucopyranoside (2), and (20S,23E)-3ß,12ß,20,25-tetrahydroxydammara-23-ene 20-O-ß-d-glucopyranoside (3) on the basis of 1D and 2D NMR experiments and mass spectra. The known ginsenosides were identified as ginsenoside M(7cd), ginsenoside Rg6, ginsenoside Rb3, gypenoside XVII, gypenoside IX, and 20-(E)-ginsenoside F4.

Antiplatelet activity of beta-carboline alkaloids from Perganum harmala: a possible mechanism through inhibiting PLCgamma2 phosphorylation.

Beta-carboline alkaloids including harmalol, harmaline, norharmane, harmol, harmine and harmane are important constituents of the medicinal plant, Perganum harmala L. (Zygophylaceae), which has been used in traditional medicine. In the present study, the antiplatelet activities of six beta-carboline alkaloid compounds were investigated in vitro. At a concentration of 200 microM, these compounds have no effect on arachidonic acid (AA)-, thrombin- and U46619 (a thromboxane A2 mimic)-stimulated platelet aggregation. On the contrary, it was revealed that collagen-induced platelet aggregation could be inhibited by these compounds with different potencies (harmane and harmine were most potent, harmol had medium potency, and harmol, norharmane, harmalol and harmaline had a weak, non significant effect), indicating a selective inhibition on collagen-mediated platelet activation. Consistently, further study revealed that collagen-mediated phospholipase (PL) Cgamma2 and protein tyrosine phosphorylation, cytosolic calcium mobilization and arachidonic acid liberation were completely inhibited by harmane and harmine in a concentration-dependent manner, while the other compounds were only partially or not effective at all. Taken together, these results indicate that three of these six beta-carboline alkaloids can selectively affect collagen-induced platelet aggregation with different potencies; in particular, harmane and harmine were most potent, and their antiplatelet activities may be mediated by inhibiting PLCgamma2 and protein tyrosine phosphorylation with sequential suppression of cytosolic calcium mobilization and arachidonic acid liberation, indicating that harmane and harmine have a potential to be developed as a novel agent for atherothrombotic diseases.

Antiplatelet activity of epigallocatechin gallate is mediated by the inhibition of PLCgamma2 phosphorylation, elevation of PGD2 production, and maintaining calcium-ATPase activity.

We have previously reported that green tea catechins displayed a potent antithrombotic effect by inhibition of platelet aggregation. In the present study, the antiplatelet and antithrombotic activities of epigallocatechin gallate (EGCG), the major catechin derived from green tea, were extensively investigated. EGCG inhibited arterial thrombus formation and U46619-, collagen-, and arachidonic acid (AA)-induced washed rabbit platelet aggregation in a concentration-dependent manner, with IC50 values of 61 +/- 3, 85 +/- 4, and 99 +/- 4 microM, respectively. In line with the inhibition of collagen-induced platelet aggregation, EGCG revealed blocking of the collagen-mediated phospholipase (PL) Cgamma2 and protein tyrosine phosphorylation, and it caused concentration-dependent decreases of cytosolic calcium mobilization, AA liberation, and serotonin secretion. In addition, the platelet aggregation, intracellular Ca2+ mobilization, and protein tyrosine phosphorylation induced by thapsigargin, a Ca2(+)-ATPase pump inhibitor, were completely blocked by EGCG. Contrary to the inhibition of AA-induced platelet aggregation, EGCG failed to inhibit cyclooxygenase and thromboxane (TX) A2 synthase activities, but it concentration-dependently elevated AA-mediated PGD2 formation. In contrast, epigallocatechin (EGC), a structural analogue of EGCG lacking a galloyl group in the 3' position, slightly inhibited collagen-stimulated cytosolic calcium mobilization, but failed to affect other signal transductions as did EGCG in activated platelets and arterial thrombus formation. These results suggest that antiplatelet activity of EGCG may be attributable to its modulation of multiple cellular targets, such as inhibitions of PLCgamma2, protein tyrosine phosphorylation and AA liberation, and elevation of cellular PGD2 levels, as well as maintaining Ca2(+)-ATPase activity, which may underlie its beneficial effect on the atherothrombotic diseases.

[Study on variation of actinoside C in leaves of Actinidia kolomikta with different growth periods by RP-HPLC].

OBJECTIVE: To determine actinoside C in the leaves of Actinidia kolomikta with different growth periods. METHOD: The separation was performed at 25 degrees C on ZORBAX Extend C18 column (4.6 mm x 250 mm, 5 microm), using amixture of methanol and water (51:49) as a mobile phase. The flow rate was 1.2 mL x min(-1), and the wavelength for measurement was 267 nm. RESULT: The results showed that the contents of actinoside C in the leaves of A. kolomikta were variety in different growth periods. Actinoside C could reach its highest content in the middle ten days of June, then the content would decrease in the middle ten days of July slightly, it could reach their lowest content in the middle ten days of August. CONCLUSION: The optimal collective date for A. kolomikta are in the middle ten days of June.

Cudraflavanone A, a flavonoid isolated from the root bark of Cudrania tricuspidata, inhibits vascular smooth muscle cell growth via an Akt-dependent pathway.

In previous studies of the root bark of Cudrania tricuspidata, various isoprenylated xanthones and flavonoids were isolated, some of which have anticancer, hepatoprotective, and antiperoxidative activities. Cytokines and growth factors are involved in the regulation of vascular smooth muscle cells (VSMCs) in atherosclerotic plaques. To assess whether cudraflavanone A isolated from the root bark of C. tricuspidata may be useful in the prevention of atherosclerosis or restenosis after angioplasty, we investigated the ability of cudraflavanone A to inhibit VSMCs growth under 25 ng/mL platelet-derived growth factor BB (PDGF-BB)-stimulated conditions. Cudraflavanone A (0.1-1 microM) significantly inhibited PDGF-BB-induced cell numbers in a concentration-dependent manner. The antigrowth effects of cudraflavanone A on VSMCs were also examined in [3H]-thymidine incorporation and cell cycle assays. Consistent with the inhibitory effect on cell number, PDGF-BB-stimulated [3H]-thymidine incorporation and cell cycle progression in VSMCs was also concentration-dependently reduced by cudraflavanone A. Furthermore, PDGF-BB markedly activated PDGF-beta receptor (PDGF-Rbeta) tyrosine kinase activity, leading to activation of intracellular signals required for VSMC growth. However, PDGF-BB-induced this kinase activity was not affected by cudraflavanone A. PDGF-BB also increased the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), Akt, and phospholipase C gamma (PLCgamma)1, which are important signaling molecules in cell growth. Cudraflavanone A (0.1-1 microM) suppressed PDGF-BB-stimulated Akt activation, which is involved in cell survival, but had no effect on the activation of ERK1/2 and PLCgamma1. Selective modification of Akt activation by cudraflavanone A in VSMCs may suppress intimal thickening after angioplasty and plaque formation in atherosclerosis. These results suggest that cudraflavanone A from C. tricuspidata inhibits PDGF-BB-induced rat aortic VSMC growth via an Akt-dependent pathway.

Antiplatelet activity of carnosic acid, a phenolic diterpene from Rosmarinus officinalis.

Carnosic acid is a major phenolic diterpene derived from Rosmarinus officinalis and has been reported to have antioxidant, antibacterial, anticancer, antiobese and photoprotective activities. This study investigated the antiplatelet activity of carnosic acid. carnosic acid significantly inhibited collagen-, arachidonic acid-, U46619- and thrombin-induced washed rabbit platelet aggregation in a concentration-dependent manner, with IC50 values of 39+/-0.3, 34+/-1.8, 29+/-0.8 and 48+/-2.9 microM, respectively, while it failed to inhibit PMA-(a direct PKC activator) and ADP-induced platelet aggregation. In agreement with its antiplatelet activity, carnosic acid blocked collagen-, arachidonic acid-, U46619- and thrombin-mediated cytosolic calcium mobilization. accordingly, serotonin secretion and arachidonic acid liberation were also inhibited in a similar concentration-dependent manner. However, in contrast to the inhibition of arachidonic acid-induced platelet aggregation, carnosic acid had no effect on the formation of arachidonic acid-mediated thromboxane A2 and prostaglandin D2, thus indicating that carnosic acid has no effect on the cyclooxygenase and thromboxane A2 synthase activity. Overall, these results suggest that the antiplatelet activity of carnosic acid is mediated by the inhibition of cytosolic calcium mobilization and that carnosic acid has the potential of being developed as a novel antiplatelet agent.

Antithrombotic and antiplatelet activities of Korean red ginseng extract.

The antithrombotic and antiplatelet activities of Korean red ginseng extract (KRGE) were examined on rat carotid artery thrombosis in vivo and platelet aggregation in vitro and ex vivo. The KRGE significantly prevented rat carotid arterial thrombosis in vivo in a dose-dependent manner. Administration of the KRGE to rats significantly inhibited adenosine diphosphate (ADP)- and collagen-induced platelet aggregation ex vivo, although it failed to prolong coagulation times such as activated partial thromboplastin and prothrombin time indicating that the antithrombotic effect of the red ginseng may be due to its antiplatelet aggregation rather than anticoagulation effect. In line with the above observations, the red ginseng inhibited the U46619-, arachidonic acid-, collagen- and thrombin-induced rabbit platelet aggregations in vitro in a concentration-dependent manner, with IC(50) values of 390 +/- 15, 485 +/- 19, 387 +/- 11 and 335 +/- 15 microg/ml, respectively. Consistently, serotonin secretion was also inhibited by ginseng in the same pattern. These results suggest that the red ginseng has a potent antithrombotic effect in vivo, which may be due to the antiplatelet rather than the anticoagulation activity, and the red ginseng intake may be beneficial for individuals with high risks of thrombotic and cardiovascular diseases.

Antiplatelet and antithrombotic activities of Korean Red Ginseng.

The antiplatelet and antithrombotic activities of Korean Red Ginseng (KRG) were examined on rat carotid artery thrombosis in vivo, and platelet aggregation in vitro and ex vivo. Administration of KRG to rats not only prevented carotid artery thrombosis in vivo in a dose-dependent manner, but also significantly inhibited ADP- and collagen-induced platelet aggregation ex vivo, while failed to prolong coagulation times such as activated partial thromboplastin time (APTT) and prothrombin time (PT), indicating the antithrombotic effect of KRG might be due to its antiplatelet aggregation rather than anticoagulation effect. In line with the above observations, KRG inhibited U46619-, arachidonic acid-, collagen- and thrombin-induced rabbit platelet aggregation in vitro in a concentration-dependent manner, with IC50 values of 620 +/- 12, 823 +/- 22, 722 + 21 and 650 +/- 14 microg/mL, respectively. Accordingly, KRG also inhibited various agonists-induced platelet serotonin secretions as it suppressed platelet aggregation. These results suggest that KRG has a potent antithrombotic effect in vivo, which may be due to antiplatelet rather than anticoagulation activity, and KRG intake may be beneficial to the individuals with high risks of thrombotic and cardiovascular diseases.

[Determination of 20 (S)-ginsengnoside Rh2 in the alkali-hydrolysis product of saponins from leaves of Panax qinquefolium by RP-HPLC].

OBJECTIVE: To determine 20(S)-ginsengnoside Rh2 in the hydrolysis product of saponins from leaves of Panax qinquefolium. METHOD: The separation was performed on ZORBAX EXEND C18 column (4.6 mm x 250 mm, 5 microm), eluted with methanol and water (85:15) as mobile phase with the rate of 1.2 mL x min(-1) at 25 degrees C, the wavelength for measurement was 203 nm. RESULT: The calibration curve was linear in the range of 0.5-25 microg for 20(S)-ginsengnoside Rh2(r = 0.9999, n = 7). The average recovery was 99.7% (RSD= 1.0%). CONCLUSION: This method is simple, accurate, reliable and reproducible. The result shows that the transform ratio of 20(S)-ginsengnoside Rh2 is high by this hydrolysis method.

Inhibition of PDGF beta-receptor tyrosine phosphorylation and its downstream intracellular signal transduction in rat aortic vascular smooth muscle cells by kaempferol.

Kaempferol, a flavonoid present in human diet and plants, has been known to show cardiovascular protection via its anti-oxidant activity. In this study, we have investigated the effect of kaempferol on the proliferation of primary cultured rat aortic vascular smooth muscle cells (VSMCs). Kaempferol significantly inhibited 50 ng/mL platelet-derived growth factor (PDGF)-BB-induced proliferation and [3H]-thymidine incorporation into DNA at concentrations of 5, 20 and 50 microM without any cytotoxicity. Kaempferol also inhibited the c-fos mRNA expression induced by PDGF-BB concentration-dependently. In addition, consistent with the inhibition of cell proliferation and c-fos mRNA expression, kaempferol inhibited the PDGF beta-receptor (Rbeta) phosphorylation in a concentration-dependent manner. Accordingly, the downstream signal transductions of PDGF-Rbeta such as ERK1/2, Akt and PLC-gamma1 phosphorylation were also inhibited by kaempferol in the same pattern. These findings suggest that, in addition to its anti-oxidant activity, the cardiovascular protective effect of kaempferol may be mediated, at least in part, by the suppression of VSMC proliferation, which is due to the inhibition of PDGF-Rbeta tyrosine phosphorylation and its downstream intracellular signal transduction.

Treatment of pig serum-induced rat liver fibrosis with Boschniakia rossica, oxymatrine and interferon-alpha.

AIM: To investigate the effect of Boschniakia rossica (BR), oxymatrine (OM) and interferon-alpha (IFN-alpha) 1b on the therapy of rat liver fibrosis and its mechanism. METHODS: By establishing a rat model of pig serum-induced liver fibrosis, liver/weight index and serum alanine transaminase (ALT) were observed to investigate the therapeutic effect of BR,OM and IFN-alpha. Radioimmunoassay was utilized to measure procollagen type III (PCIII) and collagen type IV (CIV). RT-PCR was used to assay the expression of liver transforming growth factor-beta 1 (TGF-beta1) mRNA. Immunohistochemistry of alpha-smooth muscle actin (alpha-SMA) and pathologic changes of liver tissues were also under investigation. RESULTS: Serum PCIII and CIV in BR, OM and IFN-alpha groups were significantly declined compared with those in model group, and their RT-PCR revealed that TGF-beta1 mRNA expression was also reduced more than that in model group. Immunohistochemistry demonstrated that alpha-SMA also declined more than that in model group. Serum ALT in IFN-alpha, control and model groups was within normal level. Serum ALT in BR group had no significant difference from those of IFN-alpha, control and model groups. Serum ALT in OM group was significantly higher than those in BR, IFN-alpha, model, and control groups. CONCLUSION: BR, OM and IFN-alpha can prevent pig serum-induced liver rat fibrosis by inhibiting the activation of hepatic stellate cells and synthesizing collagen. OM has hepatotoxicity to rat liver fibrosis induced by pig serum.

Antiplatelet effect of green tea catechins: a possible mechanism through arachidonic acid pathway.

We have previously reported that green tea catechins (GTC) showed an antithrombotic activity, which might be due to antiplatelet effect rather than anticoagulation. The present study was performed to investigate the effect of GTC on the arachidonic acid (AA) metabolism in order to elucidate a possible antiplatelet mechanism. GTC inhibited the collagen-, AA- and U46619-induced rabbit platelet aggregation in vitro in a concentration-dependent manner, with IC50 values of 61.0+/-2.5, 105.0+/-4.9 and 67.0+/-3.2 microg/ml, respectively. Moreover, GTC administered orally into rats inhibited the AA-induced platelet aggregation ex vivo by 46.9+/-6.1% and 95.4+/-2.2% at the doses of 25 and 50 mg/kg, respectively. [3H]AA liberation induced by collagen in [3H]AA incorporated rabbit platelets was significantly suppressed by GTC compared to the control. GTC also significantly inhibited the thromboxane A2 (TXA2) and prostaglandin D2 (PGD2) generations induced by addition of AA in intact rabbit platelets. GTC significantly inhibited TXA2 synthase activity in a concentration-dependent manner. Moreover, adenosine triphosphate (ATP) release from dense granule was inhibited by GTC in washed platelets. These results suggest that the antiplatelet activity of GTC may be due to the inhibition of TXA2 formation through the inhibition of AA liberation and TXA2 synthase.

Excisanin H, a novel cytotoxic 14,20-epoxy-ent-kaurene diterpenoid, and three new ent-kaurene diterpenoids from Rabdosia excisa.

A novel 14,20-epoxy-ent-kaurene diterpenoid, excisanin H (1), and three new ent-kaurene diterpenoids, 2-4, were isolated from aerial parts of Rabdosia excisa along with eight known ent-kaurene diterpenoids, 5-12. The structural elucidations were made using spectral (HREIMS, IR, (1)H, (13)C, and 2D NMR) methods. The absolute configuration of 1 was determined by demonstrating that oxidation of kamebakaurin (8) produced excisanin H (1). These ent-kaurene diterpenoids all showed significant cytotoxic activity against P388 murine leukemia cells.