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.

5-HT4 receptors in the hippocampus modulate rat locomotor activity.

In the present study, the ability of 5-hydroxytryptamine-4 (5-HT4) receptors in the hippocampus to enhance locomotor activity in rats was investigated by local infusion via microdialysis probes. The local infusion of 5-HT bilaterally into the striatum did not alter rat motor activity. The local infusion of 1.0 mM 5-HT into the bilateral hippocampus, but not lower doses, significantly increased motor activity as compared with the baseline values or the control rats. During the day hours (0700-1900, light on), the local infusion of either 5-HT4 agonist, 5-MeOT (100 microM) or mosapride (10 microM), but not in their lower concentrations, into the bilateral hippocampus significantly increased motor activity as compared with the baseline values or the control rats. Almost all increased motor activity was normal forward locomotion. This 5-MeOT-induced hyperlocomotion was completely reversed by the combined infusion of a 5-HT4 antagonist, either GR125487D (100 microM), SB204070 (100 microM) or RS23597-190 (100 microM). During the night hours (1900-0700, light off), the local infusion of either SB204070 (100 microM) or RS23597-190 (100 microM), but not in their lower concentrations, into the bilateral hippocampus significantly decreased rat motor activity and inhibited rat nocturnal hyperactivity. These hypoactivities during the night hours induced by 5-HT4 antagonist were reversed by the combined infusion of a 5-HT4 agonist, 5-MeOT (100 microM). The present study demonstrates that the serotonergic neurons projecting to the hippocampus, but not to the striatum, modulate rat locomotor activity by stimulating 5-HT4 receptors in the hippocampus.

L-158,809 and (D-Ala(7))-angiotensin I/II (1-7) decrease PAI-1 release from human umbilical vein endothelial cells.

The endothelium is a major source of plasminogen activator inhibitor-1 (PAI-1), which plays a critical role in the regulation of fibrinolysis. There are many reports on the increase in the expression of PAI-1 by angiotensin II (Ang II). In the present study, we investigated the effects of angiotensin-related substances on the release of PAI-1 from human umbilical vein endothelial cells (HUVECs). Ang II increased PAI-1 and tissue plasminogen activator (t-PA) release, while its metabolite angiotensin-(1-7) (Ang-(1-7)) amino acid fragment decreased them. Angiotensin Type 1 (AT1) receptor antagonist, L-158,809 (L-1), and Ang-(1-7) receptor antagonist, (D-Ala(7))-angiotensin I/II (1-7) (D-Ala), decreased PAI-1 and t-PA release; angiotensin Type 2 (AT2) antagonist, PD123,319 (PD), however, did not have any effects on the release of PAI-1 and t-PA. The addition of the equal concentration or 10-times-higher concentration of L-1 to Ang II did not change PAI-1 release compared to that by Ang II. Although Ang-(1-7) and L-1 decreased PAI-1 release, there were no additional effects on the decrease of the amounts of PAI-1 by the mixture of Ang-(1-7) and the equal concentration or 10-times-higher concentration of L-1 compared to those by Ang-(1-7). The equal concentration of D-Ala to Ang II did not change the amounts of PAI-1, but the addition of the 10-times-higher concentration of D-Ala to Ang II resulted in significant decrease of the amounts of PAI-1 compared to those by Ang II. The addition of equal concentration or 10-times-higher concentration of D-Ala to Ang-(1-7) showed the significant decrease of the amounts of PAI-1 compared to those by Ang-(1-7). In conclusion, L-158,809 and (D-Ala(7))-angiotensin I/III (1-7) may be used as profibrinolytic drugs.

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.