ABSTRACT A liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous determination of six bioactive constituents including vitexin, orientin, isoorientin, 2"-O-β-D-xylopyranosyl isoorientin, 2"-O-β-D-xylopyranosyl isovitexin, and 6-C-L-α-arabipyranosyl vitexin in rats’ various tissues using isoquercitrin as the internal standard. Biological samples were pretreated by protein precipitation with acetonitrile. Chromatographic separation was carried out on a C18 column with a gradient mobile phase consisting of acetonitrile and 0.1% aqueous formic acid. All analytes and internal standard were quantitated through electrospray ionization in negative ion selected reaction monitoring mode. The mass transitions were as follows: m/z 431 → 311 for vitexin, m/z 447 → 327 for orientin or isoorientin, m/z 579 → 459 for 2"-O-β-D-xylopyranosyl isoorientin, m/z 563 → 293 for 2"-O-β-D-xylopyranosyl isovitexin, m/z 563 → 353 for 6-C-L-α-arabipyranosyl vitexin, and m/z 463 → 300 for the internal standard, respectively. The lower limits of quantification for the six analytes in different tissue homogenates were 0.8–2.2 ng/ml. The validated assay was successfully applied to a tissue distribution study of the six components in rats after intravenous administration of total flavonoids of Scorzonera austriaca Willd; Asteraceae. The results of the tissue distribution study showed that the high concentrations of six components were mainly in the kidney.

In this study, we used microarray analysis to investigate the biogenesis and progression of intervertebral disc degeneration. The gene expression profiles of 37 disc tissue samples obtained from patients with herniated discs and degenerative disc disease collected by the National Cancer Institute Cooperative Tissue Network were analyzed. Differentially expressed genes between more and less degenerated discs were identified by significant analysis of microarray. A total of 555 genes were significantly overexpressed in more degenerated discs with a false discovery rate of < 3%. Functional annotation showed that these genes were significantly associated with membrane-bound vesicles, calcium ion binding and extracellular matrix. Protein-protein interaction analysis showed that these genes, including previously reported genes such as fibronectin, COL2A1 and f-catenin, may play key roles in disc degeneration. Unsupervised clustering indicated that the widely used morphology-based Thompson grading system was only marginally associated with the molecular classification of intervertebral disc degeneration. These findings indicate that detailed, systematic gene analysis may be a useful way of studying the biology of intervertebral disc degeneration.