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Prostaglandins (PG) derived from COX-1 are essential for the maintenance of mucosal integrity but COX-2 isoform synthesizes PG at a site of inflammation. Recently, COX-2 mRNA expression was demonstrated at the ulcer edge during healing of chronic gastric ulcers but the role for expression of COX-2 and its products such as PGE(2) and cytokines including interleukin (IL-1beta) and tumor necrosis factor alpha (TNFalpha) in ulcer healing remains unknown. In this study, Wistar rats with gastric ulcers produced by serosal application of acetic acid (ulcer area 28 mm(2)) received daily treatment either with: (1) vehicle (saline); (2) NS-398 (10 mg/kg-d i.g.) and Vioxx (5 mg/kg-d i.g.), both, highly specific COX-2 inhibitors; (3) meloxicam (5 mg/kg-d i.g.), a preferential inhibitor of COX-2; (4) resveratrol (10 mg/kg-d i.g.), a specific COX-1 inhibitor; (5) indomethacin (5 mg/kg-d i.g); and (6) aspirin (ASA; 50 mg/kg-d i.g.), non-selective inhibitors of both COX-1 and COX-2. At day 3, 7, and 14 after ulcer induction, the animals were sacrificed and the area of gastric ulcers was determined by planimetry and histology, gastric blood flow (GBF) at ulcer base and margin was measured by H(2) clearance technique, and blood was withdrawn for measurement of plasma IL-1beta and TNFalpha levels. The mucosal biopsy samples were taken for the determination of PGE(2) generation by RIA and expression of COX-1, COX-2, IL-1beta, and TNFalpha mRNA by RT-PCR. In vehicle-treated rats, gastric ulcers healed progressively and at day 14 the healing was completed, accompanied by a significant rise in the GBF at ulcer margin. The IL-1beta, TNFalpha, and COX-1 mRNA were detected in intact and ulcerated gastric mucosa, whereas COX-2 mRNA were upregulated only in ulcerated mucosa with peak observed at day 3 after ulcer induction. The plasma IL-1beta level was significantly increased at day 3 and 7 but then declined at day 14 to that measured in vehicle-controls. Indomethacin and ASA, which suppressed PGE(2) generation both in the non-ulcerated and ulcerated gastric mucosa, significantly delayed the rate of ulcer healing and this was accompanied by the fall in GBF at ulcer margin and further elevation of plasma IL-1beta and TNFalpha levels, which was sustained up to the end of the study. Treatment with NS-398 and Vioxx, which caused only a moderate decrease in the PGE(2) generation in the non-ulcerated gastric mucosa, delayed ulcer healing and attenuated significantly the GBF at ulcer margin and PGE(2) generation in the ulcerated tissue, while raising the plasma IL-1beta and TNFalpha similarly to those observed in indomethacin- and ASA-treated rats. Resveratrol, which suppressed the PGE(2) generation in both non-ulcerated and ulcerated gastric mucosa, prolonged ulcer healing and this was accompanied by the fall in the GBF at the ulcer margin and a significant increase in plasma IL-1beta and TNFalpha levels. We conclude that (1) classic NSAID delay ulcer healing due to suppression of endogenous PG, impairment in GBF at ulcer area, and excessive cyt...

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Melatonin, a major hormone of pineal gland, was recently shown to attenuate acute gastric lesions induced by strong irritants because of the scavenging of free radicals but its role in ulcer healing has been little investigated. In this study we compared the effects of intragastric (i.g.) administration of melatonin and its precursor, L-tryptophan, with or without concurrent treatment with luzindole, a selective antagonist of melatonin MT2 receptors, on healing of chronic gastric ulcers induced by serosal application of acetic acid (ulcer area 28 mm2). The involvement of endogenous prostaglandins (PG), nitric oxide (NO) and sensory nerves in ulcer healing action of melatonin and L-tryptophan was studied in rats treated with indomethacin and NG-nitro-L-arginine (L-NNA) to suppress, respectively, cyclo-oxygenases (COX) and NO synthases or in those with functionally deactivated sensory nerves with capsaicin. The influence of melatonin on gastric secretion during ulcer healing was tested in separate group of rats with gastric ulcer equipped with gastric fistulas (GF). At day 8 and 15 upon the ulcer induction, the area of gastric ulcers was measured by planimetry, the mucosal blood flow (GBF) was determined by H2-gas clearance technique and gastric luminal NO2-/NO3- levels was assessed by Griess reaction. Plasma melatonin and gastrin levels were measured by specific radioimmunoassay (RIA). Biopsy mucosal samples were taken for expression of constitutive NO-synthase (cNOS) and inducible NOS (iNOS) by reverse transcriptase-polymerase chain reaction (RT-PCR). Melatonin (2.5-20 mg/kg-d i.g.) and L-tryptophan (25-100 mg/kg-d i.g.) dose-dependently accelerated ulcer healing, the dose inhibiting by 50% (ED50) of ulcer area being 10 and 115 mg/kg, respectively. This inhibitory effect of melatonin (10 mg/kg-d i.g.) and L-tryptophan (100 mg/kg-d i.g.) on ulcer healing was accompanied by a significant rise in the GBF at ulcer margin and an increase of plasma melatonin. luminal NO2-/NO3- and plasma gastrin levels. Gastric acid and pepsin outputs were significantly inhibited during the ulcer healing in melatonin-treated gastric mucosa as compared with those in vehicle-treated animals. Luzindole abolished completely the healing effects of melatonin and L-tryptophan and attenuated significantly the rise in plasma gastrin evoked by the hormone and its precursor. Indomethacin (5 mg/kg-d i.p). that blocked PG biosynthesis by 90% or L-NAME (20 mg/kg i.v), inhibitor of NOS. that suppressed luminal NO release, attenuated significantly melatonin and L-tryptophan-induced acceleration of ulcer healing and accompanying rise in GBF at ulcer margin and luminal NO release. The melatonin-induced acceleration of ulcer healing, hyperemia at ulcer margin and increase in the release of NO were enhanced when L-arginine but not D-arginine was added to L-NAME. The ulcer healing and the GBF effects of melatonin and L-tryptophan were significantly impaired in rats with capsaicin-induced denervation of sensory nerves and both, ulcer healing and the hyperem...

HPLC coupled to a mass spectrometer (MS) was used for the analysis of galanthamine and lycorine in natural extracts of Leucojum aestivum and in their in vitro cultures grown with a precursor (ACC), inhibitors (AgNO(3), STS), or an absorber (KMnO(4)) of ethylene. The maximum galanthamine (0.002%) and lycorine (0.02%) concentrations in tissue cultures were obtained in the presence of KMnO(4). GCMS was used to investigate underivatized alkaloid mixtures from L. aestivum. Seven alkaloids were identified in in vivo bulbs. KMnO(4) led to the highest diversity of alkaloids in tissue culture extracts.

Cytokinins are growth regulators that stimulate cell division and control morphogenesis in plants, however their role in regulating secondary metabolism is not well studied. The influence of various cytokinins (benzyladenine, zeatin, kinetin, meta‐topolin, thidiazuron) and culture systems (solid and temporary immersion RITA® system) on the quality Leucojum aestivum plant regenerated from somatic embryos was investigated. The largest number of regenerated plants (181.6 and 168.8) was obtained from the embryos cultivated on media enriched with meta‐topolin and benzyladenine. Thidiazuron and meta‐topolin led to the highest number of normally developed plants (94.8 and 90.6). The random amplified polymorphic DNA analysis of in vitro and in vivo plants showed four clusters of similarity. The highest biomass (growth index: 2.49) was obtained with the temporary immersion RITA® system. Alkaloid extracts were analyzed by LC‐MS, leading to the quantification of galanthamine and lycorine both in plant materials and in liquid media. The highest contents of galanthamine (0.05% dry weight) were observed in plants cultivated in the presence of thidiazuron in bioreactor system. Galanthamine was accumulated (highest content 0.05% dry weight) in plants cultivated in the presence of thidiazuron in bioreactor system whereas lycorine was synthetized mainly in plants cultivated on solid media.

The influence of ethylene on in vitro morphogenesis of Leucojum aestivum and galanthamine accumulation was studied. Calli were cultivated on Murashige and Skoog (MS) medium supplemented with 25 lM 4-amino-3,5,6-trichloropicolinic acid (picloram) and 0.5 lM benzyladenine (BA). During incubation under these conditions, callus cultures produced ethylene (9.5 nL/g fresh weight: F.W.) whereas no ethylene was found in somatic embryos cultivated on medium supplemented with 0.5 lM a-naphthalene acetic acid (NAA) and 5 lM zeatin. Application of the precursor of ethylene 1-aminocyclopropane-1-carboxylic acid (ACC) increased ethylene production in both cultures, and decreased callus growth by a factor of 1.2, whereas callus growth was enhanced by a factor of 1.1 in the presence of an inhibitor of ethylene silver nitrate (AgNO 3 ) or by a factor of 1.2 with an absorbent potassium permanganate (KMnO 4 ). ACC enhanced the induction of somatic embryos and the development of globular embryos. Removal of ethylene by KMnO 4 during somatic embryogenesis led to the development of plants with greater length. Silver thiosulphate (STS) induced galanthamine production in callus cultures (0.1% dry weight), whereas ACC induced galanthamine production in somatic embryo cultures (2% dry weight).

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