Anti-inflammatory, anti-angiogenic, and anti-nociceptive activities of the chloroform fraction of a methanol extract from Rosa davurica Pall. leaves in experimental animal models.

Rosa davurica Pall. has been traditionally used to treat inflammatory diseases and tumors. Its dried leaves were extracted with absolute methanol, and the methanol extract was successively fractionated into n-hexane, chloroform, ethyl acetate, n-butanol, and aqueous fractions. Anti-angiogenic and anti-nociceptive activities were determined using the chick embryo chorioallantoic membrane (CAM) assay and acetic acid-induced writhing response, respectively. Anti-inflammatory activity was evaluated using two in vivo mouse models, acetic acid-induced vascular permeability and carrageenan-induced inflammation in the air-pouch. The methanol extract gave rise to significant inhibition in the CAM angiogenesis, and showed marked 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. Among the fractions prepared from the methanol extract, the chloroform fraction exhibited highest inhibitory effect in the CAM angiogenesis. The chloroform fraction displayed anti-inflammatory activities in vascular permeability and air-pouch models. In the air-pouch model, it was able to diminish exudate volume, number of polymorphonulcear leukocytes, and nitrite content. It also showed anti-nociceptive activity in the writhing response model in mice. The leaves of R. davurica possess anti-angiogenic and related anti-inflammatory and anti-nociceptive activities, which would provide some therapeutic support on its traditional use.

Hepatoprotective and antioxidant effects of Morus bombycis Koidzumi on CCl4-induced liver damage.

The antioxidant activity and liver protective effect of Morus bombycis Koidzumi were investigated. Aqueous extracts of M. bombycis Koidzumi had higher superoxide radical scavenging activity than other types of extracts. The aqueous extract at a dose of 100 mg/kg showed significant hepatoprotective activity when compared with that of a standard agent. The biochemical results were confirmed by histological observations indicating that M. bombycis Koidzumi extract together with CCl(4) treatment decreased ballooning degeneration. The water extract recovered the CCl(4)-induced liver injury and showed antioxidant effects in assays of FeCl(2)-ascorbic acid-induced lipid peroxidation in rats. Based on these results, we suggest that the hepatoprotective effect of the M. bombycis Koidzumi extract is related to its antioxidative activity.

Anti-angiogenic and inhibitory activity on inducible nitric oxide production of the mushroom Ganoderma lucidum.

Fresh fruit bodies of Ganoderma lucidum were extracted with 70% ethanol at room temperature. The extract (GL) showed significant anti-angiogenic activity, which was detected using a chick embryo chorioallantoic membrane assay. GL significantly inhibited LPS-induced NO production in RAW 264.7 macrophages. These results support the anti-tumor effect of Ganoderma lucidum.

Anti-angiogenic, antioxidant and xanthine oxidase inhibition activities of the mushroom Phellinus linteus.

Fruiting bodies of Phellinus linteus were extracted with 70% ethanol at room temperature. The Phellinus linteus extract (PL) showed strong anti-angiogenic activity, which was detected using the chick embryo chorioallantoic membrane (CAM) assay. The in vitro antioxidant activities of PL were evaluated using two different bioassays. PL was comparable to Vitamin C in scavenging the stable free radical 1,1-diphenyl-2-picrylhyrazyl (DPPH). It also inhibited lipid peroxidation (LPO) in a concentration-dependent manner. These results suggest that antioxidant and anti-angiogenic activities of Phellinus linteus would be partly responsible for its anti-tumor effect.

Regulation of the gene encoding glutathione synthetase from the fission yeast.

The fission yeast cells that contained the cloned glutathione synthetase (GS) gene showed 1.4-fold higher glutathione (GSH) content and 1.9-fold higher GS activity than the cells without the cloned GS gene. Interestingly, gamma-glutamylcysteine synthetase activity increased 2.1-fold in the S. pombe cells that contained the cloned GS gene. The S. pombe cells that harbored the multicopy-number plasmid pRGS49 (containing the cloned GS gene) showed a higher level of survival on solid media with cadmium chloride (1 mM) or mercuric chloride (10 microM) than the cells that harbored the YEp357R vector. The 506 bp upstream sequence from the translational initiation point and N-terminal 8 amino acid-coding region were fused into the promoterless beta-galactosidase gene of the shuttle vector YEp367R to generate the fusion plasmid pUGS39. Synthesis of beta-galactosidase from the fusion plasmid pUGS39 was significantly enhanced by cadmium chloride and NO-generating S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SN). It was also induced by L-buthionine-(S,R)-sulfoximine, a specific inhibitor of gamma-glutamylcysteine synthetase (GCS). We also found that the expression of the S. pombe GS gene is regulated by the Atf1-Spc1-Wis1 signal pathway.

Activities of antioxidant and redox enzymes in human normal hepatic and hepatoma cell lines.

The cellular defense system (including glutathione, glutathione-related enzymes, antioxidant and redox enzymes) plays a crucial role in cell survival and growth in aerobic organisms. To understand its physiological role in tumor cells, the glutathione content and related enzyme activities in the human normal hepatic cell line, Chang and human hepatoma cell line, HepG2, were systematically measured and compared. Superoxide dismutase, catalase, and glutathione peroxidase activities are 2.8-, 4.3-, and 2.9-fold higher in HepG2 cells than in Chang cells. Total glutathione content is also about 1.4-fold higher in HepG2, which is supported by significant increases in gamma-glutamylcysteine synthetase and glutathione synthetase activities. Two other glutathione-related enzymes, glutathione reductase and gamma-glutamyltranspeptidase, are upregulated in HepG2 cells. However, thioredoxin reductase and glutathione S-transferase activities are significantly lower in HepG2 cells. These results propose that defense-related enzymes are largely modulated in tumor cells, which might be linked to their growth and maintenance.

Regulation of Schizosaccharomyces pombe gene encoding copper/zinc superoxide dismutase.

Copper/zinc superoxide dismutase (Cu/Zn SOD) is an abundant enzyme that scavenges superoxide radicals. To independently examine the regulation of the Cu/Zn SOD gene of the fission yeast Schizosaccharomyces pombe, the 882 bp upstream region of the Cu/Zn SOD gene was fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357R, which generated the fusion plasmid pSC601. Cupric chloride (4.5 microM), aluminum chloride (10 mM), cadmium chloride (30 microM, 50 microM), mercuric chloride (1 microM), zinc chloride (11 mM), and hydrogen peroxide (0.3 mM) enhanced the synthesis of beta-galactosidase from the fusion plasmid. These results indicate that the expression of the S. pombe Cu/Zn SOD gene is, therefore, regulated by various metal ions, however superoxide-generating menadione did not affect the expression of the S. pombe Cu/Zn SOD gene. The expression of the S. pombe Cu/Zn SOD gene is also regulated by the transcription factor Pap1.

Pap1-dependent regulation of the GSTII gene from the fission yeast.

The genomic DNA encoding a second glutathione S-transferase (GSTII) was previously isolated from the fission yeast Schizosaccharomyces pombe. Its expression was shown to be induced by menadione, mercuric chloride, o-dinitrobenzene, and NO-generating S-nitroso-N-acetylpenicillamine using the GSTII-lacZ fusion harboring the 910 bp upstream region from the translational initiation point. In this study, the additional fusion plasmids pGST50-590 and pGST50-6R-590 were constructed to carry the 590 bp upstream region in the vectors YEp357 and YEp367R, respectively. The synthesis of beta-galactosidase from the fusion plasmid pGST50-590 was about 3-fold higher than that from the fusion plasmid pGST50-F, indicating the presence of negatively activating sequence in the -910 to approximately -590 region. It was also enhanced by the same agents, which induced the synthesis of beta-galactosidase from the fusion plasmid pGST50-F. The synthesis of beta-galactosidase from both fusion plasmids pGST50-F and pGST50-590 was enhanced by the overexpressed Pap1 protein. The synthesis of beta-galactosidase from the two YEp367R derivatives pGST50-6R-F and pGST50-6R-590 was greatly decreased in the Pap1-negative strain TP108-3C. These results propose the Pap1-dependent regulation of the GSTII gene from the fission yeast.

The Pap1-independent induction by metal ions of a third gene encoding glutathione S-transferase gene from the fission yeast.

A third gene that encodes glutathione S-transferase (GSTIII) was previously cloned from the fission yeast Schizosaccharomyces pombe. Using the GSTIII-lacZ fusion plasmid pGDA-19, its expression was shown to be enhanced by various metal ions. In the present study, four additional fusion plasmids, pGDA-29, pGDA-39, PGDA-49, and pGDA-59, were designed to carry 998, 378, 276, and 115 bp upstream regions from the translational initiation point, respectively. The major activation region was located between -998 and -378 bp upstream of the GSTIII gene. Regulatory sequences that are responsible for the induction by metal ions reside between -998 and -378 bp and between -276 and -115 bp upstream of the gene. The overexpressed Pap1 exerts a repression effect on the GSTIII expression via -998 to approximately -378 bp region, whereas it exerts an activation effect on the GSTIII expression via -270 to approximately -115 bp region. However, the induction of the GSTIII gene by metal ions occurs independent of Pap1.