Metabolic fingerprinting for discrimination of DNA-authenticated Atractylodes plants using 1H NMR spectroscopy.

Identifying different species of the genus Atractylodes which are commonly used in Chinese and Japanese traditional medicine, using chromatographic approaches can be difficult. 1H NMR metabolic profiling of DNA-authenticated, archived rhizomes of the genus Atractylodes was performed for genetic and chemical evaluation. The ITS region of the nuclear rDNA was sequenced for five species, A. japonica, A. macrocephala, A. lancea, A. chinensis, and A. koreana. Our samples had nucleotide sequences as previously reported, except that part of the A. lancea cultivated in Japan had a type 5, hybrid DNA sequence. Principal component analysis (PCA) using 1H NMR spectra of extracts with two solvent systems (CD3OD, CDCl3) was performed. When CDCl3 extracts were utilized, the chemometric analysis enabled the identification and classification of Atractylodes species according to their composition of major sesquiterpene compounds. The 1H NMR spectra using CD3OD contained confounding sugar peaks. PCA removal of these peaks gave the same result as that obtained using CDCl3 and allowed species distinction. Such chemometric methods with multivariate analysis of NMR spectra will be useful for the discrimination of plant species, without specifying the index components and quantitative analysis on multi-components.

Enhanced secretion of tissue plasminogen activator by simultaneous use of retinoic acid and ascorbic acid from tissue cultured gastroepiploic artery.

Tissue-type plasminogen activator (tPA) is a key enzyme in the fibrinolysis system and the regulation of its expression has been extensively studied in cultured vascular endothelial cells. Many kinds of supplements including growth factors are needed, however, to keep endothelial cells viable, which leads the culture condition far from the physiological milieu. Using a new device of amorphous calcium phosphate coated culture plate, we succeeded in culturing ring-cut gastroepiploic artery in a basic medium of RPMI 1640 containing 10% fetal calf serum. The overall normal vessel architecture and the antigenicity of von Willebrand factor, tPA and plasminogen activator inhibitor type 1 (PAI-1) were retained for at least 9 days. tPA was constantly secreted into the conditioned medium at least up to day 12. Employing this organ culture technique, we analyzed the effects of two well-known profibrinolytic vitamins of retinoic acid (Vit. A) and ascorbic acid (Vit. C) on the release of tPA and PAI-1. The cultured artery responded well and the tPA secretion was enhanced by factors of 1.5 fold by Vit. A, 1.7 fold by Vit C and 3.2 fold by their combination, whereas none of these stimuli increased PAI-1 secretion. These results suggested that the cultured ring-cut artery retained functional endothelial cells for at least 9 days and was suitable in analyzing the regulatory mechanism of protein synthesis and secretion from the vascular wall. Using this method, vitamins A and C were shown to lead the intravascular condition to a profibrinolytic state.