Cytotoxic and antimigratory effects of Cratoxy formosum extract against HepG2 liver cancer cells.

The aim of the present study was to investigate the molecular mechanisms underlying Cratoxylum formosum (CF) Dyer-induced cancer cell death and antimigratory effects in HepG2 liver cancer cells. The cytotoxic, antiproliferative and antimigratory effects of CF leaf extract on human liver cancer HepG2 cell lines were evaluated using sulforhodamine B, colony formation, and wound healing assays. In addition, apoptosis induction mechanisms were investigated via reactive oxygen species (ROS) formation, caspase 3 activities, and mitochondrial membrane potential (ΔΨm) disruption. Gene expression and apoptosis-associated protein levels were measured by reverse transcription-quantitative polymerase chain reaction and western blotting. CF induced HepG2 cell death in a time- and dose-dependent manner with half maximal inhibitory concentration values of 219.03±9.96 and 124.90±6.86 µg/ml at 24 and 48 h, respectively. Treatment with CF caused a significant and dose-dependent decrease in colony forming ability and cell migration. Furthermore, the present study demonstrated that CF induced ROS formation, increased caspase 3 activities, decreased the ΔΨm, and caused HepG2 apoptosis. CF marginally decreased the expression level of the cell cycle regulatory protein, ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1) and the downstream protein, cyclin dependent kinase 6. Additionally, CF significantly enhanced p21 levels, reduced cyclin D1 protein levels and triggered cancer cell death. CF leaf extracts induced cell death, stimulated apoptosis and inhibited migration in HepG2 cells. Thus, CF may be useful for developing an anticancer drug candidate for the treatment of liver cancer.

Antioxidant, antibacterial and DNA protective activities of protein extracts from Ganoderma lucidum.

Crude proteins of cultured mycelia and fruiting bodies of Ganoderma lucidum were investigated for antioxidant, antibacterial and DNA protective activities. It was found that the half maximal inhibitory concentration (IC50) of the mycelia protein and fruiting bodies protein extracts against 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) radical (ABTS(•+)) were 2.47 ± 0.01 and 2.77 ± 0.11 µg protein/ml and against 2,2-diphenylpicrylhydrazyl radical (DPPH(•)) were 2.5 ± 0.01 and 3.42 ± 0.01 µg protein/ml, respectively. The ferric reducing-antioxidant power (FRAP) values of those samples were 1.73 ± 0.01 and 2.62 ± 0.01 µmole trolox/µg protein respectively. Protein hydrolysates prepared by pronase exhibited a weaker antioxidant activity. Both crude proteins showed antibacterial activity, whereas only the mycelia protein extract could protect DNA damage by hydroxyl ((•)OH) radicals. This protein extract was partial purified by Diethyl amino ethyl (DEAE)-Sepharose column and Sulfopropyl (SP)-Sepharose column, obtained major protein with molecular weight about 45 kilo Dalton (kDa). In conclusion, G. lucidum protein extracts have promise potential for applications as antioxidant and antibacterial agents.

Acetic acid fermentation of acetobacter pasteurianus: relationship between acetic acid resistance and pellicle polysaccharide formation.

Acetobacter pasteurianus strains IFO3283, SKU1108, and MSU10 were grown under acetic acid fermentation conditions, and their growth behavior was examined together with their capacity for acetic acid resistance and pellicle formation. In the fermentation process, the cells became aggregated and covered by amorphous materials in the late-log and stationary phases, but dispersed again in the second growth phase (due to overoxidation). The morphological change in the cells was accompanied by changes in sugar contents, which might be related to pellicle polysaccharide formation. To determine the relationship between pellicle formation and acetic acid resistance, a pellicle-forming R strain and a non-forming S strain were isolated, and their fermentation ability and acetic acid diffusion activity were compared. The results suggest that pellicle formation is directly related to acetic acid resistance ability, and thus is important to acetic acid fermentation in these A. pasteurianus strains.

Characterization of thermotolerant Acetobacter pasteurianus strains and their quinoprotein alcohol dehydrogenases.

We isolated several thermotolerant Acetobacter species of which MSU10 strain, identified as Acetobacter pasteurianus, could grow well on agar plates at 41 degrees C, tolerate to 1.5% acetic acid or 4% ethanol at 39 degrees C, similarly seen with A. pasteurianus SKU1108 previously isolated. The MSU10 strain showed higher acetic acid productivity in a medium containing 6% ethanol at 37 degrees C than SKU1108 while SKU1108 strain could accumulate more acetic acid in a medium supplemented with 4-5% ethanol at the same temperature. The fermentation ability at 37 degrees C of these thermotolerant strains was superior to that of mesophilic A. pasteurianus IFO3191 strain having weak growth and very delayed acetic acid production at 37 degrees C even at 4% ethanol. Alcohol dehydrogenases (ADHs) were purified from MSU10, SKU1108, and IFO3191 strains, and their properties were compared related to the thermotolerance. ADH of the thermotolerant strains had a little higher optimal temperature and heat stability than that of mesophilic IFO3191. More critically, ADHs from MSU10 and SKU1108 strains exhibited a higher resistance to ethanol and acetic acid than IFO3191 enzyme at elevated temperature. Furthermore, in this study, the ADH genes were cloned, and the amino acid sequences of ADH subunit I, subunit II, and subunit III were compared. The difference in the amino acid residues could be seen, seemingly related to the thermotolerance, between MSU10 or SKU1108 ADH and IFO 3191 ADH.