Purification and characterization of a novel extracellular tripeptidyl peptidase from Rhizopus oligosporus

A novel extracellular tripeptidyl peptidase (TPP) was homogenously purified from the culture supernatant of Rhizopus oligosporus by sequential fast protein liquid chromatography. The purified enzyme was a 136.5 kDa dimer composed of identical subunits. The effects of inhibitors and metal ions indicated that TPP is a metallo- and serine protease. TPP was activated by divalent cations, such as Co(2+) and Mn(2+), and completely inhibited by Cu(2+). Enzyme activity was optimal at pH 7.0 and 45 °C with a specific activity of 281.9 units/mg for the substrate Ala-Ala-Phe-pNA. The purified enzyme catalyzed cleavage of various synthetic tripeptides but not when proline occupied the P1 position. Purified TPP cleaved the pentapeptide Ala-Ala-Phe-Tyr-Tyr and tripeptide Ala-Ala-Phe, confirming the TPP activity of the enzyme.     

Metabolic dependence of green tea on plucking positions revisited: a metabolomic study

The dependence of global green tea metabolome on plucking positions was investigated through (1)H nuclear magnetic resonance (NMR) analysis coupled with multivariate statistical data set. Pattern recognition methods, such as principal component analysis (PCA) and orthogonal projection on latent structure-discriminant analysis (OPLS-DA), were employed for a finding metabolic discrimination among fresh green tea leaves plucked at different positions from young to old leaves. In addition to clear metabolic discrimination among green tea leaves, elevations in theanine, caffeine, and gallic acid levels but reductions in catechins, such as epicatechin (EC), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epigallocatechin-3-gallate (EGCG), glucose, and sucrose levels were observed, as the green tea plant grows up. On the other hand, the younger the green tea leaf is, the more theanine, caffeine, and gallic acid but the lesser catechins accumlated in the green tea leaf, revealing a reverse assocation between theanine and catechins levels due to incorporaton of theanine into catechins with growing up green tea plant. Moreover, as compared to the tea leaf, the observation of marked high levels of theanine and low levels of catechins in green tea stems exhibited a distinct tea plant metabolism between the tea leaf and the stem. This metabolomic approach highlights taking insight to global metabolic dependence of green tea leaf on plucking position, thereby providing distinct information on green tea production with specific tea quality.     

Simultaneous determination of orysastrobin and its isomers in rice using HPLC-UV and LC-MS/MS

Orysastrobin is a new strobilurin-type fungicide to control leaf and panicle blast and sheath blight in rice. An analytical method was developed to determine the residues of orysastrobin and its two isomers, the main metabolite F001 and the major impurity F033, in hulled rice by the use of high-performance liquid chromatography with ultraviolet photometry (HPLC-UV) and liquid chromatography with tandem mass spectrometry (LC-MS/MS). All compounds were extracted with acetone from hulled rice samples. The extract was diluted with saline water, and an extraction step using dichloromethane/n-hexane partition was used to recover analytes from the aqueous phase. An n-hexane/acetonitrile partition and Florisil column chromatography were employed to further remove interfering coextractives prior to instrumental analysis. An octadecylsilyl column was successfully applied to identify orysastrobin and its isomers in sample extracts. Net recovery rates of orysastrobin, F001, and F033 from fortified samples ranged from 80.6 to 114.8% using HPLC-UV and LC-MS/MS. Relative standard deviations for the analytical methods were all <20%, and the quantification limits of the method were in the 0.002-0.02 mg/kg range. The proposed methods were reproducible and sufficiently accurate to evaluate the terminal residue of orysastrobin and its isomers in rice.     

Osthole enhances glucose uptake through activation of AMP-activated protein kinase in skeletal muscle cells

AMP-activated protein kinase (AMPK) is an energy sensor that regulates cellular metabolism. Activation of AMPK in skeletal muscles, the liver, and adipose tissues results in a favorable metabolic milieu for preventing and treating type 2 diabetes, i.e., decreased levels of circulating glucose, plasma lipids, and ectopic fat accumulation and enhanced insulin sensitivity. Osthole was extracted from a Chinese herbal medicine, and we found that it had glucose lowering activity in our previous study. However, the detailed glucose lowering mechanisms of osthole are still unclear. In this study, we used skeletal muscle cells to examine the underlying molecular mechanisms of osthole's glucose lowering activity. A Western blot analysis revealed that osthole significantly induced phosphorylation of AMPK and acetyl-CoA carboxylase (ACC). Next, we found that osthole significantly increased the level of translocation of glucose transporter 4 (GLUT4) to plasma membranes and glucose uptake in a dose-dependent manner. Osthole-induced glucose uptake was reversed by treatment with Compound C, an AMPK inhibitor, suggesting that osthole-induced glucose uptake was mediated in an AMPK-dependent manner. The increase in the AMP:ATP ratio was involved in osthole's activation of AMPK. Finally, we found that osthole counteracted hyperglycemia in mice with streptozotocin-induced diabetes. These results suggest that the increase in the AMP:ATP ratio by osthole triggered activation of the AMPK signaling pathway and led to increases in plasma membrane GLUT4 content and glucose uptake level. Therefore, osthole might have potential as an antidiabetic agent for treating diabetes.     

Combined genomic-metabolomic approach for the differentiation of geographical origins of natural products: deer antlers as an example

The correct identification of the geographical origin of deer antlers is essential to quality control, as its positive physiological effects correlate with chemical components. In this study, we applied both genomics and metabolomics to the origin-identification of 101 samples from Canada, New Zealand, and Korea. The genomics identified deer species in each country but failed to categorize all the samples, due to the presence of identical species in different countries. For identical species, NMR-based metabolomics gave clean separations, compounds specific to each country were identified, and the validity was confirmed by prediction analysis. As the genomics provided unambiguous read-outs for different species, and the metabolomics cleanly distinguished among identical species from different countries, their combined use could be a robust method for origin-identification even in difficult cases. We believe the method to be generally applicable to many herbal medicinal products for which various species are grown internationally.     

Comparison of the α-amylase inhibitor-1 from common bean (Phaseolus vulgaris) varieties and transgenic expression in other legumes--post-translational modifications and immunogenicity

The seeds of peas (Pisum sativum) and chickpeas (Cicer arietinum) expressing a gene for α-amylase inhibitor-1 (αAI) from the common bean (Phaseolus vulgaris) are protected from damage by old world bruchids (pea and cowpea weevils). Here, we used electrospray ionization time-of-flight mass spectrometry to compare the post-translational modifications of αAI from transgenic sources with the processed forms of the protein from several bean varieties. All sources showed microheterogeneity with differences in the relative abundance of particular variants due to differences in the frequency of addition of glycans, variable processing of glycans, and differences of C-terminal exopeptidase activity. The structural variation among the transgenics was generally within the range of the bean varieties. Previously, mice showed allergic reactions following ingestion of transgenic pea αAI but not bean αAI. Here, only minor differences were observed following intraperitoneal sensitization. Both of the transgenic pea and bean forms of αAI elicited Th1 and Th2 antibody isotype responses, suggesting that both proteins are immunogenic and could potentially be allergenic.     

Antioxidant activities of Korean rice wine concentrates

The antioxidant activities of six Korean rice wine (KRW) concentrates were measured by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and lipid/malonaldehyde (MA) assays. In the DPPH assay, the antioxidant activities of the KRW concentrates, including Maesilju (MSJ), Kookhwaju-1 (KHJ-1), Kookhwaju-2 (KHJ-2), Gugijaju (GGJ), Sasamju (SSJ), and Sogokju (SGJ), were 40%, 66%, 64%, 35%, 35%, and 63%, respectively. Furthermore, the concentrates inhibited the formation of MA from cod liver oil by 49%, 83%, 75%, 82%, 89%, and 90%, respectively, according to the lipid/MA assay. The sample wines were also analyzed for pH, titratable acidity, soluble solids (°Bx), and reducing sugars. The antioxidant activities of volatile extracts of the KRWs extracted by a solvent assisted flavor evaporation (SAFE) apparatus were evaluated by aldehyde/carboxylic acid assay. The volatile extracts of MSJ, KHJ-1, KHJ-2, GGJ, SSJ, and SGJ inhibited the oxidation of hexanal by 97%, 99%, 90%, 90%, 50%, and 51%, respectively. Among the nonvolatile extracts of KRWs, KHJ-2 showed the highest inhibitory effect on MA formation.     

Influenza virus neuraminidase inhibitory activity of phlorotannins from the edible brown alga Ecklonia cava

Influenza A virus infections continue to pose a major threat to humans and several animal species. Neuraminidase (NA) is one of the most promising targets for the development of drugs against influenza viruses because of its critical role in the viral life cycle. During the course of a search for NA inhibitors from edible natural sources, we found that the ethyl acetate layer of ethanol extracts of Ecklonia cava showed extremely high NA-inhibitory activity (72.1% inhibition at 30 μg/mL). Bioactivity-guided fractionation of the ethyl acetate layer yielded five phlorotannins, identified as phloroglucinol (1), eckol (2), 7-phloroeckol (3), phlorofucofuroeckol (4), and dieckol (5). The inhibitory activities of these compounds (1-5) against NAs from group-1 (A/Bervig_Mission/1/18 [H1N1], A/PR/8/34 [H1N1]) and group-2 (A/Hong Kong/8/68 [H3N2], A/Chicken/Korea/MS96/96 [H9N2]) influenza A were evaluated to determine potencies and kinetic behavior. Analyses using various in vitro influenza A virus NA assays showed that all five phlorotannin derivatives were selective NA inhibitors. Of the phlorotannins, phlorofucofuroeckol (4) exhibited the most potent inhibitory activities toward group-1 NAs (IC?? values, 4.5 and 14.7 μM), whereas dieckol (5) potently inhibited group-2 NAs. Kinetic analyses indicated that compounds 1-5 were all noncompetitive. Notably, these noncompetitive inhibitors synergized with oseltamivir to enhance the NA-inhibitory effects of oseltamivir.