Bioassay-coupled LC-QTOF MS/MS to Characterize Constituents Inhibiting Nitric Oxide Production of Thuja orientalis

The ethyl acetate fractions prepared from the leaves of Thuja orientalis significantly inhibited nitric oxide (NO) production in lipopolysaccharide-stimulated BV2 microglial cells. According to bioassay-coupled LC-QTOF MS/MS, the components near 22 and 25 mins in the mass chromatogram highly inhibited NO production and were expected to be labdane diterpenes, and the active components were characterized via further isolation. The results of the NO production inhibitory assay of the isolated compounds correlated well with the results of bioassay-coupled LC-QTOF MS/MS. Among the identified constituents, NO production inhibitory activities of 16-hydroxy-labda-8(17),13-diene-15,19-dioic acid butenolide (2) and 15-hydroxypinusolidic acid (3) were newly reported. Taken together, these results demonstrated that LC-QTOF MS/MS coupled with NO production inhibition assay was a powerful tool for accurately predicting new anti-inflammatory constituents in the extracts from natural products. Moreover, it provided a potential basis for broadening the application of bioassay-coupled LC-QTOF MS/MS in natural product research.

Asiatic Acid Induces Apoptosis and Autophagy and Reduces MiR-17 and MiR-21 Expression in Pancreatic Cancer Cell Lines

This study investigated the cytotoxic effects and mechanism of action of asiatic acid in pancreatic cancer cell lines. First, we confirmed the cell viability of MIA PaCa-2 and PANC-1 cells after asiatic acid administration for 48 and 72 h. The viability of MIA PaCa-2 and PANC-1 cells decreased in a dose-dependent manner following asiatic acid administration. To investigate the underlying mechanism, we performed a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, annexin V assay, and western blotting. Asiatic acid induced apoptosis and autophagy through activation of AMP-activated protein kinase (AMPK) and inhibition of mammalian target of rapamycin (mTOR) in MIA PaCa-2 cells. Finally, the expression of miR-17 and miR-21, known as oncogenes in pancreatic cancer, was decreased by asiatic acid. These results indicate that asiatic acid has potential as a new therapeutic agent against pancreatic cancer.

Oocyte maturation under a biophoton generator improves preimplantation development of pig embryos derived by parthenogenesis and somatic cell nuclear transfer

This study was conducted to determine the effects of biophoton treatment during in vitro maturation (IVM) and/or in vitro culture (IVC) on oocyte maturation and embryonic development in pigs. An apparatus capable of generating homogeneous biophoton energy emissions was placed in an incubator. Initially, immature pig oocytes were matured in the biophoton-equipped incubator in medium 199 supplemented with cysteine, epidermal growth factor, insulin, and gonadotrophic hormones for 22 h, after which they were matured in hormone-free medium for an additional 22 hr. Next, IVM oocytes were induced for parthenogenesis (PA) or provided as cytoplasts for somatic cell nuclear transfer (SCNT). Treatment of oocytes with biophoton energy during IVM did not improve cumulus cell expansion, nuclear maturation, intraoocyte glutathione content, or mitochondrial distribution of oocytes. However, biophoton-treated oocytes showed higher (p < 0.05) blastocyst formation after PA than that in untreated oocytes (50.7% vs. 42.7%). In an additional experiment, SCNT embryos produced from biophoton-treated oocytes showed a greater (p < 0.05) number of cells in blastocysts (52.6 vs. 43.9) than that in untreated oocytes. Taken together, our results demonstrate that biophoton treatment during IVM improves developmental competence of PA- and SCNT-derived embryos.

Identification of Antioxidative Constituents from Polygonum aviculare using LC-MS Coupled with DPPH Assay

A method for simultaneously identifying antioxidative compounds was developed using time-based LC-MS coupled with DPPH assay regardless of the time consuming process. The methanolic extract of Polygonum aviculare (Polygonaceae) showed significant DPPH radical scavenging activity. Time-based DPPH assay for simultaneous identification of active compounds from the extracts of P. aviculare was used. Major peaks of ethyl acetate fraction of P. aviculare showed high DPPH radical scavenging activity. A simple phenolic compound (1) and six flavonoids (2-7) were isolated from the ethyl acetate fraction of P. aviculare by silica gel and sephadex LH-20 column chromatography. The structures of seven compounds were determined to be protocatechuic acid (1), catechin (2), myricitrin (3), epicatechin-3-O-gallate (4), avicularin (5), quercitrin (6), and juglanin (7) based on the analysis of the 1H-NMR, 13C-NMR and ESI-MS data. All compounds exhibited significant antioxidant activity on DPPH assay and active compounds were well correlated with predicted one.