The Components of Flemingia macrophylla Attenuate Amyloid ß-Protein Accumulation by Regulating Amyloid ß-Protein Metabolic Pathway.

Flemingia macrophylla (Leguminosae) is a popular traditional remedy used in Taiwan as anti-inflammatory, promoting blood circulation and antidiabetes agent. Recent study also suggested its neuroprotective activity against Alzheimer's disease. Therefore, the effects of F. macrophylla on Aß production and degradation were studied. The effect of F. macrophylla on Aß metabolism was detected using the cultured mouse neuroblastoma cells N2a transfected with human Swedish mutant APP (swAPP-N2a cells). The effects on Aß degradation were evaluated on a cell-free system. An ELISA assay was applied to detect the level of Aß1-40 and Aß1-42. Western blots assay was employed to measure the levels of soluble amyloid precursor protein and insulin degrading enzyme (IDE). Three fractions of F. macrophylla modified Aß accumulation by both inhibiting ß-secretase and activating IDE. Three flavonoids modified Aß accumulation by activating IDE. The activated IDE pool by the flavonoids was distinctly regulated by bacitracin (an IDE inhibitor). Furthermore, flavonoid 94-18-13 also modulates Aß accumulation by enhancing IDE expression. In conclusion, the components of F. macrophylla possess the potential for developing new therapeutic drugs for Alzheimer's disease.

Tournefolic acid B attenuates amyloid beta protein-mediated toxicity by abrogating the calcium overload in mitochondria and retarding the caspase 8-truncated Bid-cytochrome c pathway in rat cortical neurons.

The effect of tournefolic acid B (TAB) on amyloid beta protein-mediated neurotoxicity and the underlying mechanisms were investigated. Amyloid beta protein 25-35 elicited neuronal death as determined by calcein/ethidium homodimer-1 staining. 10 microM amyloid beta protein 25-35 caused cell death at a level of 41.5+/-3.8% by MTT reduction. 50 microM TAB attenuated the amyloid beta protein 25-35-induced cell death by 49.7+/-11.1%. TAB also abrogated amyloid beta protein-induced activation of caspases 8 and 9 by about 50-60%. Furthermore, TAB significantly diminished the amyloid beta protein 25-35-induced elevation of calcium level in mitochondria, whereas it did not affect the calcium level in cytosol or endoplasmic reticulum. TAB markedly retarded the amyloid beta protein-mediated release of cytochrome c from mitochondria. Amyloid beta protein 25-35 elevated mitochondrial truncated BH3 interacting domain death agonist (tBid) and decreased the level of B-cell leukemia/lymphoma-2alpha (Bcl-2alpha) in mitochondria. Moreover, amyloid beta protein induced a slight up-regulation of Bcl-2 agonist killer 1 (Bak) in cytosol. 50 microM TAB decreased the amyloid beta protein-induced elevation of mitochondrial tBid and the level of Bak, whereas it did not affect the amyloid beta protein-mediated decrease in mitochondrial Bcl-2alpha. Caspase 8 inhibitor significantly inhibited the amyloid beta protein-mediated increase in mitochondrial tBid and the release of cytochrome c. Therefore, TAB blocked the overload of calcium in mitochondria and impaired the amyloid beta protein-mediated activation of the caspase 8-tBid-cytochrome c pathway, thereby conferring its neuroprotective effects on amyloid beta protein-mediated neurotoxicity.

Ester derivatives of tournefolic acid B attenuate N-methyl-D-aspartate-mediated excitotoxicity in rat cortical neurons.

The effects of tournefolic acid B (TAB) and two ester derivatives, TAB methyl ester (TABM) and TAB ethyl ester (TABE), on N-methyl-D-aspartate (NMDA)-mediated excitotoxicity and the underlying mechanisms were investigated. Treatment with 50 microM NMDA elicited neuronal death by 48.7 +/- 5.1%, coinciding with the appearance of injured morphology. TABM (50 microM) attenuated the NMDA-induced cell death by 60.9 +/- 19.7%, and to a lesser extent by TABE. The NMDA-mediated activation of calpain was not affected by TABM and TABE, as determined by the cleavage of alpha-spectrin. NMDA increased the activity of caspases 2, 3, 6, 8, and 9 and reached the maximum after 8-h treatment. TABM and TABE abrogated NMDA-induced activation of caspases 2, 3, 6, and 8 by approximately 80 to 90% and 50 to 60%, respectively, and to a higher extent for caspase 9. TABM and TABE also blocked the NMDA-mediated activation of caspase 12. Furthermore, TABM and TABE eliminated the NMDA-induced accumulation of superoxide anion (O2-*). NMDA evoked significant depolarization of mitochondria, whereas TABM elicited a mild decrease of mitochondrial membrane potential as determined by tetramethylrhodamine methyl ester perchlorate. NMDA treatment induced elevation of Ca2+ levels in cytosol, endoplasmic reticulum (ER), and mitochondria. TABM (50 microM) significantly diminished the NMDA-induced elevation of Ca2+ levels in mitochondria and ER but not cytosol. Therefore, TABM decreased mitochondrial membrane potential and attenuated the NMDA-mediated Ca2+-loading in ER and mitochondria. These events subsequently eliminated the accumulation of O2-* and blocked the activation of caspase cascade, thereby conferring their neuroprotective effects on NMDA-mediated excitotoxicity.

In vitro protective effects of salvianolic acid B on primary hepatocytes and hepatic stellate cells.

The production of reactive oxygen species (ROS) is believed to be involved in liver injury and hepatic fibrosis. Activation of hepatic stellate cells (HSCs) is a key feature of liver fibrosis. Salvia miltiorrhiza is a traditional Chinese herb used in the treatment of cardiovascular and liver diseases to resolve stasis. The effects of salvianolic acid B (Sal B), a major component of Salvia miltiorrhiza, on oxidative damage include free radical DPPH scavenging, malondialdehyde (MDA) formation and ROS generation in primary rat hepatocytes and HSCs, and on alpha-SMA, and collagen expression in transforming growth factor-beta1 (TGF-beta1)-stimulated HSCs were examined. Results indicated that Sal B scavenged DPPH potently with an IC50 2.2+/-0.2 microg/ml (3.06+/-0.3 microM), inhibited lipid peroxidation and eliminated ROS accumulation in a concentration-dependent manner on primary rat hepatocytes and HSCs. Sal B also reduced alpha-SMA and collagen synthesis and deposition in HSCs, and had no direct cytotoxicity on both hepatocytes and HSCs. Our results suggest that Sal B ameliorated oxidative damage and eliminated ROS accumulation in hepatocytes, and attenuated HSC activation, potentially conferring hepatoprotective and anti-fibrogenic effects.

Neuroprotective flavonoids from Flemingia macrophylla.

Using an Abeta-induced neurotoxicity blocking assay to direct fractionation, three new flavonoids, fleminginin (1), flemingichromone (2), and flemingichalcone (3), and twenty known compounds were isolated from the active fractions of the aerial parts of Flemingia macrophylla. The structures of 1 - 3 were elucidated on the basis of spectroscopic data. When tested for neuroprotective activity, compound 2, osajin ( 4), 5,7,4'-trihydroxy-6,8-diprenylisoflavone (5), 5,7,4'-trihydroxy-6,3'-diprenylisoflavone (6), and aureole (7) protected neuronal cells from Abeta-induced damage with EC50 values of 31.43 +/- 3.16, 5.01 +/- 1.28, 11.25 +/-1.51, 4.47 +/- 0.65, 12.09 +/- 2.55 microM, respectively.

Falcarindiol impairs the expression of inducible nitric oxide synthase by abrogating the activation of IKK and JAK in rat primary astrocytes.

The effects of falcarindiol on the expression of inducible nitric oxide synthase (iNOS) induced by lipopolysaccharide/interferon-gamma (LPS/IFN-gamma) in rat primary astrocytes were investigated. The molecular mechanisms underlying falcarindiol that confers its effect on iNOS expression were also elucidated. Falcarindiol abrogated the LPS/IFN-gamma-mediated induction of iNOS by about 80%. Falcarindiol attenuated the induction of iNOS in a concentration-dependent manner. The inhibitory effect of falcarindiol on iNOS induction was attributable to decrease in the protein content and the mRNA level of iNOS. Treatment with 50 microM of falcarindiol for 30 min decreased LPS/IFN-gamma-induced nuclear factor-kappaB (NF-kappaB) activation by 32%. Treatment with 50 microM of falcarindiol for 60 min diminished the LPS/IFN-gamma-mediated activation of IkappaB kinase-alpha (IKK-alpha) and IKK-beta by 28.2 and 29.7%, respectively. Falcarindiol modulated the nuclear translocation of signal transducer and activator of transcription 1 (Stat1) in a time-dependent manner. Falcarindiol (50 microM) decreased the tyrosine phosphorylation of janus kinase 1 (JAK1) by 84.8% at 5 min. Falcarindiol also abrogated the tyrosine phoshorylation of JAK2 by 82.3% at 10 min.The present study demonstrates that falcarindiol attenuated the activation of IKK and JAK contributing to the blockade of activation of NF-kappaB and Stat1, thereby leading to the suppression of iNOS expression.

Tournefolic acid B methyl ester attenuates glutamate-induced toxicity by blockade of ROS accumulation and abrogating the activation of caspases and JNK in rat cortical neurons.

The effects of nine polyphenolic compounds on glutamate-mediated toxicity were investigated. The underlying mechanisms by which a polyphenolic compound confers its effect were also elucidated. Treatment of cortical neurons with 50 microm glutamate for 24 h decreased cell viability by 45.8 +/- 7.9%, and 50 microm of tournefolic acid B methyl ester attenuated glutamate-induced cell death by 46.8 +/- 17.8%. Glutamate increased the activity of caspase 35.2-fold, and to a similar extent for caspase 2, 6, 8 and 9. Tournefolic acid B methyl ester abrogated glutamate-induced activation of caspase 2, 3, 6 and 9 by about 70%, and to a lesser extent for caspase 8. Treatment with glutamate for 1 h elevated reactive oxygen species (ROS) by 208.3 +/- 21.3%. Tournefolic acid B methyl ester eliminated the glutamate-induced accumulation of ROS. Glutamate increased the phosphorylation of p54-c-jun N-terminal kinase (JNK) concomitantly with activation of the endogenous antioxidant defense system. Tournefolic acid B methyl ester at 50 microm diminished the activity of p54-JNK in control and glutamate-treated cells, coinciding with the abolishment of the glutamate-triggered antioxidant defense system. Therefore, tournefolic acid B methyl ester blocked the activation of the caspase cascade, eliminated ROS accumulation and abrogated the activation of JNK, thereby conferring a neuroprotective effect on glutamate-mediated neurotoxicity.