Quantification and characterisation of cyclo-oxygenase and lipid peroxidation inhibitory anthocyanins in fruits of Amelanchier.

The levels of bioactive anthocyanins in the fruits of Amelanchier alnifolia, A. arborea and A. canadensis have been determined by HPLC. Cyanidin 3-galactoside (1) was present in the fresh fruit of the three species at concentrations of 155, 390 and 165 mg/100 g, respectively. Cyanidin 3-glucoside (2) was present only in A. alnifolia and A. canadensis at concentrations of 54 and 48 mg/100 g, respectively. The anthocyanins were confirmed by LC-ESI/MS and NMR studies. At 100 ppm, anthocyanin mixtures from the three species inhibited cyclo-oxygenase (COX)-1 and -2 enzymes at 66 and 67%, 60 and 72%, and 51 and 76%, respectively. The positive controls used in the COX assays were aspirin, Celebrex and Vioxx at 180, 1.67 and 1.67 ppm, respectively, and showed 74 and 69%, 5 and 82% and 0 and 85% COX-1 and COX-2 inhibition, respectively. Anthocyanins 1 and 2 and cyanidin (3) inhibited COX-1 enzyme 50.5, 45.62 and 96.36%, respectively, at 100 ppm, whereas COX-2 inhibition was the highest for 3 at 75%. In the lipid peroxidation inhibitory assay, anthocyanin mixtures at 10 ppm from the three species showed activities of 72, 73 and 68%, respectively, compared with 89, 87 and 98% for commercial anti-oxidants butylated hydoxyanisole, butylated hydroxytoluene, and tert-butylhydroxyquinone at 1.67, 2.2 and 1.67 ppm, respectively. At 10 ppm, compounds 1-3 inhibited lipid peroxidation by 70, 75 and 78%, respectively.

Duodenal ulcer in South Africa: home-pounded versus milled maize.

BACKGROUND: In South Africa there is suggestive evidence that home-pounded maize protects against duodenal ulceration. Therefore the purpose of the present paper was to test, in an animal model, whether oil from home-pounded maize gives protection against ulceration and whether this effect is present in commercially prepared maize oil. METHODS: Gastric ulceration was induced in rats with topical ethanol 1 h after giving oil prepared either from fresh-pounded or from commercially treated maize. The lengths of the linear ulcers produced were measured with a planimeter and summed in each rat. Control observations were made using arachis oil (which is known not to be ulceroprotective) and horse gram lipid (which is known to be strongly ulceroprotective). Statistical comparisons were performed mainly with the Mann-Whitney U-test, but also with reference to the normal distribution. Thin-layer chromatography (TLC) was performed on the oil from fresh maize, and the fractions similarly investigated for ulceroprotective activity. RESULTS: Fresh maize oil was strongly ulceroprotective (P = 0.0039), commercial maize oil was not (P = 0.2864). The active ingredient in the fresh maize oil was located in the fraction near the solvent front. CONCLUSION: These findings support the hypothesis that home-pounded maize protects against duodenal ulceration.

A comprehensive study of Easter lily poisoning in cats.

This study was conducted with 3 objectives in mind: first, to identify the toxic fraction (aqueous or organic) in leaves and flowers; second, to identify diagnostic marker(s) of toxicosis in cats; and, third, to evaluate the morphologic effects of intoxication. The study was conducted in 2 phases. Phase 1 was to identify which extract, organic or aqueous, was nephrotoxic and also to determine the appropriate dose for use in the phase 2 studies. Results indicated that only the aqueous extracts of leaves and flowers were nephrotoxic and pancreotoxic. To identify the proximate toxic compound, cats in the phase 2 study were orally exposed to subfractions of the aqueous flower extract, 1 subfraction per cat. Results confirmed vomiting, depression, polyuria, polydipsia, azotemia, glucosuria, proteinuria, and isosthenuria as toxic effects of the Easter lily plant. Another significant finding in serum was elevated creatinine kinase. Significant histologic kidney changes included acute necrosis of proximal convoluted tubules and degeneration of pancreatic acinar cells. Renal ultrastructural changes included swollen mitochondria, megamitochondria, edema, and lipidosis. Subfraction IIa3 of the aqueous floral extract contained most of the toxic compound(s). These studies reproduced the clinical disease, identified the most toxic fraction of the Easter lily, and helped characterize the clinical pathology, histopathology, and ultrastructural pathology associated with the disease.

Constituents in Easter lily flowers with medicinal activity.

Easter lily (Lilium longiflorum) flowers have been used in traditional medicine for alleviating many ailments. However, the chemical basis of its bioactivity has not been investigated. We have determined bioactive components in Easter lily flowers using lipid peroxidation and cyclooxygenase enzyme inhibitory assays and found to be kaempferol (1), kaempferol glycosides (2, 3, 4, 8, 9 and 10), quercetin glycosides (5, 6 and 7), a regaloside (11), a chalcone (12) and a fatty acid fraction (13). The structures of compounds were determined by NMR, IR, UV/VIS and mass spectroscopic studies. Compound 1 showed the highest COX-1 inhibition (94.1%) followed by 3, 8 and 12 with 38.7, 30.8 and 32.4%, respectively. Only compound 1 inhibited COX-2 enzyme by 36.9% at 80 ppm. In lipid peroxidation inhibitory assay, kaempferol showed 37 and 100 % inhibitions at 1 and 10 ppm, respectively. At 10 ppm, more than 20% inhibition was observed for compounds 4, 7, 10, 11 and 12 and 53% for compound 3. The compounds reported in here are isolated for the first time from Easter lily flowers including novel compounds 10, 11 and 12. Our results suggest that kaempferol and quercetin flavonoids contributed to the anecdotal medicinal properties of Easter lily flowers.

Bioactive terpenoids and guggulusteroids from Commiphora mukul gum resin of potential anti-inflammatory interest.

Guggulu, the gum resin from Commiphora mukul, is one of the components of various formulations of traditional Ayurvedic medicine to treat inflammation, obesity, and lipid disorders. In most preparations of Ayurvedic medicine in India, guggulu is boiled prior to its use. Therefore, guggulu was boiled with H2O prior to extractions in our study. Bioassay-guided isolation of compounds from the hexane-soluble portion of the MeOH extract of guggulu yielded cembrenoids, 1-6, a bicyclic diterpene, 7, guggulusterone derivatives, 8-11, myrrhanone derivatives, 12, myrrhanol derivative, 13, and a lignan, 14. The structures of these compounds were confirmed by spectroscopic methods. Compounds 5, 6, 7, 10, and 12-14 are novel. These compounds were assayed for lipid peroxidation and cyclooxygenase (COX) enzyme inhibitory activities. At 100 ppm, compounds 3, 6, and 14 inhibited the lipid peroxidation by 79, 57, and 58%, respectively, and the rest of isolated compounds showed 20-40% inhibitory activity with respect to the controls. In COX-1 and COX-2 enzyme inhibitory assays, compound 3 showed 79 and 83%, and compound 8 gave 67 and 54% of inhibition, respectively, at 100 ppm. All fourteen compounds inhibited COX-1 enzyme at 100 ppm. The lipid peroxidation and COX enzyme inhibitory activities exhibited by compounds isolated from C. mukul may substantiate its use in traditional medicine.

Duodenal ulcer prevalence: research into the nature of possible protective dietary lipids.

The varying geographical prevalence of duodenal ulceration has suggested a relationship to staple diet. Previous experiments on animal peptic ulcer models showed that certain foods, particularly the lipid fraction, are ulceroprotective. This paper reports experiments on animal models further to investigate the nature of the protective substances in the most active lipid, that of horse gram. The free fatty acids and triglycerides, sterols, sterol esters and phospholipids from horse gram were extracted and tested for protective activity on rat peptic ulcer models: the pyloric ligation model which is chronic, involving 14 days pre-feeding, and two acute models using ethanol or cysteamine to induce ulceration. The results showed that sterol esters, but not sterols, were protective in the pyloric ligation model. Sterols were protective in the acute models using ethanol-induced and cysteamine-induced ulceration. Phospholipids were protective in both types of model. The free fatty acids and triglycerides gave no protection using the pyloric ligation model. The presence of sterols, sterol esters and phospholipids in the lipid fraction of foods in staple diets may account for the low prevalence of duodenal ulcer in certain geographical areas, despite a uniformly high prevalence of Helicobacter pylori infection.

Rat and rabbit plasma distribution of free and chylomicron-associated BIRT 377, a novel small molecule antagonist of LFA-1-mediated cell adhesion.

PURPOSE: The objectives of this study are to determine the plasma distribution of free and chylomicron-associated BIRT 377 within rats and rabbits. METHODS: For the rat studies free and chylomicron-associated BIRT 377 was incubated in plasma from CD 1 non-fasted rats for 60 minutes at 37 degrees C. Following incubation the plasma was separated into its lipoprotein and lipoprotein-deficient plasma (LPDP) fractions by three different methods and analyzed for BIRT 377 content by HPLC. For the rabbit studies New Zealand fasted white rabbits (3 kg; n=4) were administered an intravenous dose of free BIRT 377 (1 mg/kg). Following administration, serial blood samples were obtained and the plasma was analyzed for BIRT 377. The plasma conected at the 0.083-h time point was separated into each of its lipoprotein fractions and analyzed for BIRT 377. RESULTS: 37.8 +/- 1.2% of the original drug amount incubated in rat plasma was recovered within the lipoprotein-rich fraction. 41.5 +/- 0.4% of the original chylomicron-associated drug concentration incubated was recovered within the lipoprotein-rich fraction. The percentage of drug recovered within the TRL fraction was significantly greater following the incubation of chylomicron-associated BIRT 377 compared to free BIRT 377. In addition, BIRT 377 apparently follows a two-compartment pharmacokinetic model following single intravenous dose administration to rabbits. CONCLUSIONS: These findings suggest that plasma lipoprotein binding of BIRT 377 is evident and may be a factor in evaluating the pharmacological fate of this drug when administered to patients that exhibit changes in their plasma lipoprotein lipid.

Dietary factors in relation to the distribution of duodenal ulcer in India as assessed by studies in rats.

BACKGROUND: The prevalence of duodenal ulcer is less in the northern wheat-eating regions of India and China than in the southern rice-eating areas. METHODS AND RESULTS: Experiments were conducted on rat peptic ulcer models in which controls were fed on either known ulcerogenic rice or rice plus tapioca diets or on non-ulcerogenic stock diet. By using an ulcerogenic diet and pyloric ligation, unrefined wheat, wheat bran and their respective oils were protective against ulceration. Refined wheat, wheat germ and its oil were not protective. Freshly milled rice and unmilled rice were protective, but stored rice bran and its oil increased the ulceration. Fresh rice bran oil was not ulcerogenic, but on storage, it became ulcerogenic. By using stock diet and alcohol-induced ulceration, the findings with whole wheat oil, wheat bran and wheat germ oil were confirmed. Rats fed on the stock diet subjected to pyloric ligation developed ulcers following intragastric injection of stored rice bran oil. This ulcerogenicity was counteracted by whole wheat oil. CONCLUSION: These results suggest that the factor of diet may well explain the regional differences in the prevalence of duodenal ulceration between North and South India and China where other etiologic factors are similar.

Duodenal ulcer prevalence: experimental evidence for the possible role of dietary lipids.

BACKGROUND AND AIMS: Mapping the geographical distribution of duodenal ulcer in relation to staple diets, and experiments on animal peptic ulcer models suggested that the lipid fraction in certain foodstuffs had a protective effect which was most marked in the lipid obtained from Horse gram (Dolichos biflorus). Lipid obtained from stored polished rice or rice bran was ulcerogenic. Further animal experiments were designed to investigate the protective and healing effects of Horse gram lipid (HGL) against peptic ulceration. METHODS: Three effects were investigated in rats: (i) the protective effect of HGL on peptic ulceration produced by using pyloric ligation in combination with South Indian diet or rice bran oil, or by cysteamine, alcohol or aspirin; (ii) the effect of HGL on mast cell degranulation in response to pyloric ligation and rice bran oil; and (iii) the healing effect of HGL on acute gastric ulceration produced by alcohol, on chronic gastric ulceration produced by topical acetic acid or on chronic duodenal ulcer following cysteamine. RESULTS: Horse gram lipid was shown to be protective and to promote ulcer healing in all the models used. Mast cell degranulation was inhibited. CONCLUSION: The experiments confirm the presence of a lipid in certain staple foods that have protective and healing properties in experimental peptic ulcer animal models. The differences in the prevalence of duodenal ulceration between different regions in some developing countries with a high prevalence of Helicobacter pylori infection might be explained by the presence or absence of protective lipids or ulcerogenic factors in the staple diet.

Lipid-based delivery systems for improving the bioavailability and lymphatic transport of a poorly water-soluble LTB4 inhibitor.

Ontazolast is a potent inhibitor (IC50 = 1 nm) of calcium ionophore A23187-stimulated leukotriene B4 (LTB4) biosynthesis in human peripheral blood leukocytes. The compound is practically insoluble in water (0.14 microgram/mL) and previous studies in animals have demonstrated extensive presystemic drug clearance through hepatic first-pass metabolism. Bioavailability of a suspension formulation in rats was less than 1%, but increased to approximately 9% when administered as a 20% soybean oil-in-water emulsion. The emulsion formulation and three additional lipid-based formulations were administered by gavage to conscious, minimally restrained rats in a novel, double-cannulated model to determine the effects of formulation on systemic blood absorption and mesenteric lymph transport of ontazolast. The bioavailability of ontazolast was significantly and substantially enhanced by all of the lipid-based formulations. While these formulations also significantly increased the amount of ontazolast transported by the lymph, the total amounts transported were insufficient to account for the improvement in bioavailability, which may be due to the elimination or reduction of the barriers of poor aqueous solubility and slow dissolution to absorption of ontazolast from the gastrointestinal tract, or the effects of lipid on the gastrointestinal membrane permeability, transit time, or metabolism of ontazolast. Semisolid SEDDS formulations, composed of Peceol and Gelucire 44/14, produced bioavailability similar to the emulsion formulation. The total amount of ontazolast transported by the lymph varied directly with the amount of concurrent triglyceride transport and appeared to be favored by formulations that prolong gastric emptying time or promote rapid absorption of ontazolast from the gastrointestinal tract.

Duodenal mucosal histology and histochemistry in active, treated and healed duodenal ulcer: correlation with duodenal prostaglandin E2 production.

We investigated whether impaired duodenal mucosal prostaglandin E2 (PGE2) production previously observed in duodenal ulcer (DU) was a primary pathophysiological abnormality or secondary to mucosal architectural changes that accompany ulceration. One hundred patients were studied: at endoscopy, paired duodenal biopsies were taken in patients with normal endoscopies and from the ulcer edge or scar and background mucosa in active or healed DU. One of the pair of biopsies was used to estimate PGE2 synthesis ability, the other was processed for histology and histochemistry. The following features graded: goblet cell numbers and staining with Periodic acid-Schiff reagent (PAS), epithelial staining with PAS, villous atrophy, columnar cell height, inflammatory cell infiltrate and micro-erosions and gastric metaplasia taken as a whole. Patients were found to have normal endoscopy (n = 31), active untreated DU (n = 20) active DU on treatment with either cimetidine or ranitidine (n = 13), healed DU on maintenance treatment (n = 27) and healed DU off treatment (n = 9). Active duodenal ulceration was found to be associated with decreased numbers of goblet cells, loss and blunting of villi, increased columnar cell height, increased epithelial cell PAS staining and with gastric metaplasia. After healing, only villous blunting remained. These changes were present, but less marked, at sites removed from the ulcer and were not apparent in the patient groups with healed ulcers. A strong correlation between overall gastric metaplasia and epithelial cell PAS staining and the reduced ability to synthesize PGE2 (P < 0.001) was only apparent when biopsies from all patients were grouped together, but not within individual patient subgroups. There was no consistent correlation between PGE2 generation and individual parameters of pathological change in duodenum. We conclude that, although inflammatory and mucosal changes may contribute, the evidence suggests that the impaired PGE2 generation in DU disease is, to a large extent, independent of histological and histochemical features.

Mexiletine effects on theophylline disposition.

The effect of mexiletine administration on steady-state plasma theophylline concentrations was studied in eight normal healthy men in a prospective open label nonrandomized two-way crossover trial. Repeated doses of 300 mg of sustained-release theophylline were given every 12 hours for 9 days. Mexiletine hydrochloride, 200 mg every 8 hours, was given for five consecutive doses starting on the morning of day 6. Mexiletine increased theophylline levels in all subjects. Mean predose (trough) levels rose from 8.1 +/- 0.1 microgram.ml-1 to 13.4 +/- 0.6 micrograms.ml-1 and AUC(0-12) from 96.8 +/- 9.1 to 160.2 +/- 3.7 micrograms.ml-1.hr. Plasma clearance was reduced by mexiletine from 44.7 +/- 5.1 to 25.4 +/- 1.2 ml.hr-1. Both N-demethylated metabolites of theophylline were decreased by 60% by mexiletine, whose levels remained within its therapeutic range. Theophylline levels returned to pre-mexiletine values when this drug was discontinued. Mexiletine reduces theophylline clearance and increases its plasma concentration by inhibiting N-demethylation of theophylline. Plasma theophylline levels should be monitored when mexiletine is added.

Phase I trial of dipyridamole with 5-fluorouracil and folinic acid.

We have performed two Phase I trials of the combination of dipyridamole, 5-fluorouracil (5-FU), and folinic acid in patients with advanced refractory malignancy, based upon in vitro evidence that dipyridamole can modulate the cytotoxicity of 5-FU. In the first trial, patients were treated every 4 wk with dipyridamole (50 mg/m2) p.o. every 6 h on Days 0 to 6, beginning 24 h prior to the i.v. administration of folinic acid (200 mg/m2) and escalating doses of i.v. 5-FU on Days 1 to 5. The maximum tolerated daily dose of 5-FU that could be given with this combination was 375 mg/m2. Because dipyridamole is extensively bound to plasma proteins, it was hypothesized that the concentrations of free dipyridamole achieved with a dose of 50 mg/m2 were inadequate to modulate the cytotoxicity of 5-FU and folinic acid. Therefore, a second Phase I trial of escalating dose of p.o. dipyridamole was performed. Folinic acid (200 mg/m2) and 5-FU (375 mg/m2) were given i.v. on Days 1 to 5 every 4 wk, beginning 24 h after the start of therapy with dipyridamole; dipyridamole was administered p.o. on Days 0 to 6 at doses of 75, 100, 125, 150, 175, or 200 mg/m2/dose to successive cohorts of patients. Dose-limiting neutropenia, mucositis, and nausea were produced at a dose of 200 mg/m2/dose; the recommended dose of dipyridamole for use in Phase II studies is 175 mg/m2 p.o. every 6 h, or 700 mg/m2/day. At this dose, a mean peak plasma concentration of total dipyridamole of 16.32 mumol and a mean peak plasma concentration of free dipyridamole of 38.30 nmol were observed. Trough concentrations of free dipyridamole averaged 60% of the peak concentrations. Objective antitumor responses were seen in a number of tumor types; five of 13 patients with breast cancer treated with high-dose p.o. dipyridamole, 5-FU, and folinic acid responded. High-dose p.o. dipyridamole can produce plasma concentrations of free dipyridamole within the range shown to modulate the cytotoxicity of 5-FU and other agents. Phase II trials of this combination are justified.

Differences in mucosal appearances and in relapse rates in duodenal ulceration treated with sucralfate or cimetidine.

A total of 46 patients with duodenal ulcer were randomly assigned, without the knowledge of the investigators, to treatment with cimetidine 200 mg three times daily and 400 mg at night or sucralfate 1 g four times daily followed by one year of maintenance treatment with cimetidine 400 mg at night or sucralfate 1 g twice daily, respectively, in those patients with healed ulcers. The endoscopic healing rates and relapse rates during the maintenance period were similar, four relapses occurring in each group. All four relapses in the sucralfate group occurred at 12 months and only two were symptomatic. All the cimetidine relapses were symptomatic, two occurring at six months, one at nine months, and one at 12 months. Following the one year maintenance period, 13 cimetidine patients and 11 sucralfate patients were followed up for 36 months. During the first two years, nine of 13 (69 percent) cimetidine-treated and two of 11 (18 percent) sucralfate-treated patients had relapses. During the third year, three more sucralfate-treated patients and one more cimetidine-treated patient had relapses, making a total of 10 of 13 (77 percent) and five of 11 (45 percent) in the cimetidine and sucralfate groups, respectively. Duodenal biopsy specimens obtained before and after healing and after one year of maintenance were examined by light and electron microscopy. The sucralfate group showed greater improvement after one year of maintenance therapy than did the cimetidine group, although the appearances in either group were not predictive of subsequent relapse. The results show that relapses are less frequent and occur later after sucralfate therapy and also that the morphologic appearances are more normal after treatment with sucralfate than after treatment with cimetidine.