Yun-Chin Chung

Mutagenicity and safety evaluation of water extract of fermented Toona sinensis Roemor leaves.

The purpose of this study was to evaluate the mutagenicity and safety of water extract of fermented Toona sinensis Roemor leaves (WFTS). The WFTS was prepared by fermenting Toona sinensis Roemor leaves anaerobically for 14 d, and then extracting with boiling water. The mutagenic effects of WFTS were investigated using Ames test. No mutagenicity was found toward all tester strains (Salmonella typhimurium TA98, TA100, TA102, TA1535). In the acute oral toxicity study, a single limit dose of 2.5 or 5 g/kg body weight (bw) WFTS was given to male Sprague-Dawley (SD) rats, then the rats were observed for 14 d. No acute lethal effect at a maximal dose of 5 g/kg bw WFTS was observed in rats. In the subacute study, the male rats were administered daily by gavage at a dose of 0.5 or 1 g/kg bw/d of WFTS for 28 d. The results indicated that no significant toxic effect was found in the parameters of body and organ weight, as well as hematological, biochemical, urinary, and pathological parameters between control and the WFTS-treated rats. The level of no observed adverse effect level (NOAEL) of WFTS in male rats was 1 g/kg bw for subacute toxicity study.

Purification and characterization of two chitosanase isoforms from the sheaths of bamboo shoots.

Two thermally stable chitosanase isoforms were purified from the sheaths of chitosan-treated bamboo shoots. Isoforms A and B had molecular masses of 24.5 and 16.4 kDa and isoelectric points of 4.30 and 9.22, respectively. Using chitosan as the substrate, both isoforms functioned optimally between pH 3 and 4, and the optimum temperatures for the activities of isoforms A and B were 70 and 60 °C, respectively. The kinetic parameters K(m) and V(max) for isoform A were 0.539 mg/mL and 0.262 μmol/min/mg, respectively, and for isoform B were 0.183 mg/mL and 0.092 μmol/min/mg, respectively. Chitosans were susceptible to degradation by both enzymes and could be converted to low molecular weight chitosans between 28.2 and 11.7 kDa. Furthermore, the most susceptible chitosan substrates were 50-70 and 40-80% deacetylated for isoforms A and B, respectively. Both enzymes could also degrade chitin substrates with lower efficacy. N-Bromosuccinimide and Woodwards reagent K strongly inhibited both enzymes.

Characterization of an Acidic Chitinase from Seeds of Black Soybean (Glycine max (L) Merr Tainan No. 3)

Using 4-methylumbelliferyl-β-D-N,N′,N″-triacetylchitotrioside (4-MU-GlcNAc3) as a substrate, an acidic chitinase was purified from seeds of black soybean (Glycine max Tainan no. 3) by ammonium sulfate fractionation and three successive steps of column chromatography. The purified chitinase was a monomeric enzyme with molecular mass of 20.1 kDa and isoelectric point of 4.34. The enzyme catalyzed the hydrolysis of synthetic substrates p-nitrophenyl N-acetyl chitooligosaccharides with chain length from 3 to 5 (GlcNAcn, n = 3-5), and pNp-GlcNAc4 was the most degradable substrate. Using pNp-GlcNAc4 as a substrate, the optimal pH for the enzyme reaction was 4.0; kinetic parameters K m and kcat were 245 µM and 10.31 min−1, respectively. This enzyme also showed activity toward CM-chitin-RBV, a polymer form of chitin, and N-acetyl chitooligosaccharides, an oligomer form of chitin. The smallest oligomer substrate was an N-acetylglucosamine tetramer. These results suggested that this enzyme was an endo-splitting chitinase with short substrate cleavage activity and useful for biotechnological applications, in particular for the production of N-acetyl chitooligosaccharides.

In vitro and in vivo safety evaluation of low molecular weight chitosans prepared by hydrolyzing crab shell chitosans with bamboo shoots chitosanase.

Our previous study demonstrated that the oral administration of low molecular weight chitosans (LMWC), prepared by hydrolyzing crab shell chitosans with bamboo shoots chitosanase in an appropriate dose, reduced aristolochic acid-induced renal lesions in mice. The objectives of this study were to evaluate the safety of LMWC using genetic and animal toxicity assays. Two assays for genotoxicity were performed: the chromosomal aberration of Chinese hamster ovary cells (CHO-K1 cells) (in vitro) and micronucleus assays in mice (in vivo). Acute oral toxicity and 28-day repeated feeding toxicity tests were performed via the oral gavage method in Sprague-Dawley (SD) rats. LMWC did not induce an increase in micronucleus ratios in vivo, and the chromosome aberration assay indicated that the LMWC was safe in terms of clastogenicity in doses up to 5.0 mg/ml. No acute lethal effect at a maximum tested dose of 5.0 g LMWC/kg body weight (bw) was observed in rats. The results of the 28-day study revealed no adverse effects on the body weight, feed consumption, hematology, blood biochemical parameters, organ weights or pathology. The no observed adverse effect level (NOAEL) of LMWC in rats was 1.0 g/kg bw for the subacute toxicity study.

Improving antioxidant status in aged mice by 50% ethanol extract from red bean fermented by Bacillus subtilis.

BACKGROUND The purpose of this study was to investigate the effects of 50% ethanol extracts from red bean non-fermented (RBE) and fermented by Bacillus subtilis (RBNE) on the antioxidant status of aged ICR mouse. RESULTS Compared to 2-month-old ICR mouse, the plasma total antioxidant status (TAS) in 12-month-old ICR mouse decreased about 57%, while malondialdehyde (MDA) levels in the liver and brain of 12-month-old ICR mouse increased 56% and 30%, respectively. Orally administration of RBE or RBNE could completely recover the changes of MDA and plasma TAS levels due to the aging process. Vitamin E contents declined 88% in the liver and 74% in the brain of aged ICR mouse. At a level of 0.3 or 0.6 g kg(-1) body weight, RBNE raised vitamin E content in the liver and brain; however, RBE showed no significant influence. All antioxidant enzymes activities in the liver and brain of aged ICR mouse decreased compared to those activities in 2-month-old ICR mouse. RBNE could significantly enhance the superoxide dismutase activity in the brain of aged ICR mouse. CONCLUSION Oral administration of RBE or RBNE could improve antioxidant status in aged ICR mouse. Fermentation by Bacillus subtilis could enhance the antioxidant properties of red bean.

Antimutagenic Potential of Graptopetalum paraguayense E. Walther Extracts

According to the archaic Chinese prescription, Graptopetalum paraguayense E. Walther is used for medication purpose; however, no literature on its biophysical functions is presently available. This study was aimed to evaluate the antimutagenicity activities of water (GWE), 50% ethanolic (GE50), and 95% ethanolic (GE95) extracts of Graptopetalum paraguayense. The mutagenic and antimutagenic properties of the three extracts were investigated using Ames test. The results showed that all the extracts had no-mutagenicity effect toward all tester strains (Salmonella typhimurium TA97, TA98, TA100, TA102, and TA1535). All tested extracts had marked 100% inhibition effect against the mutagenicity of the diagnostic mutagens, 2-aminofluorene (2-AF) and 2-aminoanthracene (2-AA), in all tester strains with the S9 mix system.

Red Bean Extract Inhibits Lipopolysaccharide-Induced Inflammation and H2O2-Induced Oxidative Stress in RAW 264.7 Macrophages

Red bean (Phaseolus radiatus L. var. Aurea) is a leguminous seed and mainly used as one of the popular ingredients in oriental desserts. The objective of this study was to evaluate the anti-inflammatory activity of 50 g/kg ethanolic extract of red bean (RBE) by measuring lipopolysaccharide (LPS)-induced expressions of nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in RAW 264.7 macrophages. On the other hand, the antioxidant activity of RBE was determined by thiobarbituric acid reactive substances method and comet assay using H2O2-induced macrophages. The results showed that RBE at the concentrations of 50-200 μg/mL can significantly suppress the inflammatory responses in LPS-stimulated macrophages through the reduction of cellular NO and downregulation of the gene expressions of iNOS, COX-2, TNF-α, and IL-6 in a dose-dependent manner. Furthermore, RBE can diminish H2O2-induced oxidative damage in RAW 264.7 macrophage. Phenolic compounds and cyanidin-3-O-glucoside from BRE may have efficacy as overall in vitro anti-inflammatory and antioxidant agents. Red bean exerts an anti-inflammatory response and has potential as a health-promoting ingredient.

In vitro and in vivo safety of aqueous extracts of Graptopetalum paraguayense E. Walther

ETHNOPHARMACOLOGICAL RELEVANCE Graptopetalum paraguayense E. Walther, a widely consumed vegetable in Taiwan, has many biological effects and has been used in folk medicine to alleviate hepatic disorders, exert diuretic effects, and relieve pain and infections. However, little data exist regarding its safety. MATERIALS AND METHODS Two genotoxicity assays were performed: chromosomal aberration of Chinese hamster ovary (CHO-K1 cells) (in vitro) and micronucleus assay in mice (in vivo). Acute oral toxicity and 28-day repeated feeding toxicity tests were performed by oral gavage in Sprague-Dawley (SD) rats. RESULTS GWE did not increase micronucleus ratios in vivo, and by chromosome aberration assay, GWE was safe up to 1.2mg/ml with regard to clastogenicity. Chromatid breakage was observed at high concentrations (2.5 and 5.0mg/ml) of GWE. GWE had no acute lethal effect at the maximum dose (5g/kg bw) in rats. In the 28-day study, there were no adverse effects on body weight, feed consumption, hematology, blood biochemical parameters, organ weight, or pathology. CONCLUSION The acute toxicity study showed that the LD(50) of GWE was greater than the tested dose (up to 1g/kg bw) in SD rats. In the subacute toxicity study, the no observed adverse effect level (NOAEL) of GWE in rats was 1g/kg bw. The in vivo study of mammalian erythrocyte micronuclei confirmed the Ames test results, demonstrating that GWE has no mutagenicity. High doses of GWE require further examination due to its clastogenic potential.

Characterization of a Chitosanase from Jelly Fig (Ficus awkeotsang Makino) Latex and Its Application in the Production of Water-Soluble Low Molecular Weight Chitosans

A chitosanase was purified from jelly fig latex by ammonium sulfate fractionation (50–80% saturation) and three successive column chromatography steps. The purified enzyme was almost homogeneous, as determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and gel activity staining. The molecular mass of the enzyme was 20.5 kDa. The isoelectric point (pI) was <3.5, as estimated by isoelectric focusing electrophoresis on PhastGel IEF 3-9. Using chitosan as the substrate, the optimal pH for the enzyme reaction was 4.5; the kinetic parameters Km and Vmax were 0.089 mg mL-1 and 0.69 μmol min-1 mg-1, respectively. The enzyme showed activity toward chitosan polymers which exhibited various degrees of deacetylation (21–94%). The enzyme hydrolyzed 70–84% deacetylated chitosan polymers most effectively. Substrate specificity analysis indicated that the enzyme catalyzed the hydrolysis of chitin and chitosan polymers and their derivatives. The products of the hydrolysis of chitosan polymer derivatives, ethylene glycol (EG) chitosan, carboxymethyl (CM) chitosan and aminoethyl (AE) chitosan, were low molecular weight chitosans (LMWCs); these products were referred to as EG-LMWC, CM-LMWC and AE-LMWC, respectively. The average molecular weights of EG-LMWC, CM-LMWC and AE-LMWC were 11.2, 11.2 and 8.89 kDa, respectively. All of the LMWC products exhibited free radical scavenging activities toward ABTS•+, superoxide and peroxyl radicals.

Engineering Saccharomyces cerevisiae for improvement in ethanol tolerance by accumulation of trehalose.

ABSTRACT A genetic recombinant Saccharomyces cerevisiae starter with high ethanol tolerance capacities was constructed. In this study, the gene of trehalose-6-phosphate synthase (encoded by tps1), which catalyzes the first step in trehalose synthesis, was cloned and overexpressed in S. cerevisiae. Moreover, the gene of neutral trehalase (encoded by nth1, trehalose degrading enzyme) was deleted by using a disruption cassette, which contained long flanking homology regions of nth1 gene (the upstream 0.26 kb and downstream 0.4 kb). The engineered strain increased its tolerance against ethanol and glucose stress. The growth of the wild strain was inhibited when the medium contained 6 % or more ethanol, whereas growth of the engineered strain was affected when the medium contained 10 % or more ethanol. There was no significant difference in the ethanol yield between the wild strain and the engineered strain when the fermentation broth contained 10 % glucose (p > 0.05). The engineered strain showed greater ethanol yield than the wild type strain when the medium contained more than 15 % glucose (p < 0.05). Higher intracellular trehalose accumulation by overexpression of tps1 and deletion of nth1 might provide the ability for yeast to protect against environmental stress.