Multi-target 1,4-dihydropyridines showing calcium channel blockade and antioxidant capacity for Alzheimer's disease therapy.

In this work we describe the synthesis, Ca+2 channel blockade capacity and antioxidant power of N3,N5-bis(2-(5-methoxy-1H-indol-3-yl)ethyl)-2,6-dimethyl-4-aryl-1,4-dihydropyridine-3,5-dicarboxamides 1-9, a number of multi-target small 1,4-dihydropyridines (DHP), designed by juxtaposition of melatonin and nimodipine. As a result, we have identified antioxidant DHP 7 (Ca2+ channel blockade: 55%, and 8.78 Trolox/Equivalents), the most balanced DHP analyzed here, for potential Alzheimer's disease therapy.

Vasorelaxant and Hypotensive Effects of an Ethanolic Extract of Eulophia macrobulbon and Its Main Compound 1-(4'-Hydroxybenzyl)-4,8-Dimethoxyphenanthrene-2,7-Diol.

Background: Ethnopharmacological studies demonstrated the potential for Eulophia species to treat inflammation, cancer, and cardio-metabolic diseases. The aim of the study was to investigate the vasorelaxant effect of ethanolic Eulophia macrobulbon (EM) extract and its main phenanthrene on rat isolated mesenteric artery and to investigate the hypotensive effect of EM. Methods: The vasorelaxant effects of EM extract or phenanthrene and the underlying mechanisms were evaluated on second-order mesenteric arteries from Sprague Dawley rats. In addition, the acute hypotensive effect was evaluated in anesthetized rats infused with cumulative concentrations of the EM extract. Results: Both EM extract (10-4-1 mg/ml) and phenanthrene (10-7-10-4 M) relaxed endothelium-intact arteries, an effect that was partly reduced by endothelium removal (p < 0.001). A significant decrease in the relaxant effect of the extract and the phenanthrene was observed with L-NAME and apamin/charybdotoxin in endothelium-intact vessels, and with iberiotoxin in denuded vessels. SNP (sodium nitroprusside)-induced relaxation was significantly enhanced by EM extract and phenanthrene. By contrast, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one), 4-aminopyridine and glibenclamide (endothelium-denuded vessels) and indomethacin (endothelium-intact vessels) had no effect. In calcium-free solution, both the EM extract and phenanthrene inhibited extracellular Ca2+-induced contraction in high KCl and phenylephrine (PE) pre-contracted rings. They also inhibited the intracellular Ca2+ release sensitive to PE. The acute infusion of EM extract (20 and 70 mg/kg) induced an immediate and transient dose-dependent hypotensive effect. Conclusion: The ethanolic extract of EM tubers and its main active compound, 1-(4'-hydroxybenzyl)-4,8-dimethoxyphenanthrene-2,7-diol (phenanthrene) induced vasorelaxant effects on rat resistance vessels, through pleiotropic effects including endothelium-dependent effects (NOS activation, enhanced EDH production) and endothelium-independent effects (opening of KCa channels, inhibition of Ca2+ channels, inhibition of intracellular Ca2+ release and PDE inhibition).

Magnesium deficiency affects HNF1ß expression in rat liver in vivo and in vitro.

Hepatocyte nuclear factor 1ß (HNF1ß) is a transcription factor that is involved in embryonic development and tissue-specific gene expression in several organs, including the kidney and the liver. HNF1ß mutations are associated with hypomagnesemia and renal magnesium wasting; however, to date, the exact molecular mechanism involved in this regulation is unclear. Furthermore, it is not known whether the Mg concentration could per se participate to this regulation by modifying HNF1ß expression. We have studied in rats the effects of a 6-week diet with deficient or supplemented Mg concentrations compared to a diet with a standard Mg concentration on HNF1ß protein expression. HNF1ß expression was increased in the Mg-deficient group as compared to the other groups in the liver but not in the kidney. No changes in tissue Mg level were obtained in both organs. By contrast, a significant correlation between plasma Mg concentration and HNF1ß level in the rat liver was evidenced. In rat hepatocyte cultures exposed for 72h to various extracellular Mg concentrations, HNF1ß expression was modified after 72h of treatment of the hepatocytes with the lowest Mg concentrations as compared to the other Mg conditions. Moreover, these changes were correlated with extracellular but not intracellular Mg concentrations. In conclusion, HNF1ß expression is modified by the extracellular Mg concentration in the liver, both in vivo and in vitro, suggesting regulations with membrane events in hepatocytes.

Effects of a chronic l-arginine supplementation on the arginase pathway in aged rats.

While ageing is frequently associated with l-arginine deficiency, clinical and experimental studies provided controversial data on the interest of a chronic l-arginine supplementation with beneficial, no or even deleterious effects. It was hypothesized that these discrepancies might relate to a deviation of l-arginine metabolism towards production of l-ornithine rather than nitric oxide as a result of age-induced increase in arginase activity. This study investigated the effect of ageing on arginase activity/expression in target tissues and determined whether l-arginine supplementation modulated the effect of ageing on arginase activity. Arginase activity and expression were measured in the heart, vessel, brain, lung, kidney and liver in young rats (3-months old) and aged Wistar rats (22-24-months-old) with or without l-arginine supplementation (2.25% in drinking water for 6weeks). Plasma levels of l-arginine and l-ornithine were quantified in order to calculate the plasma l-arginine/l-ornithine ratio, considered as a reflection of arginase activity. Cardiovascular parameters (blood pressure, heart rate) and aortic vascular reactivity were also studied. Ageing dramatically reduced plasma l-arginine and l-arginine/l-ornithine ratio, decreased liver and kidney arginase activities but did not change activities in other tissues. l-Arginine supplementation normalized plasma l-arginine and l-arginine/l-ornithine ratio, improved endothelial function and decreased systolic blood pressure. These effects were associated with decreased arginase activity in aorta along with no change in the other tissues except in the lung in which activity was increased. A strong mismatch was therefore observed between arginase activity and expression in analyzed tissues. The present study reveals that ageing selectively changes arginase activity in clearance tissues, but does not support a role of the arginase pathway in the potential deleterious effect of the l-arginine supplementation in aged patients. Moreover, our data argue against the use of the measurement of plasma l-arginine/l-ornithine ratio to estimate arginase activity in aged patients.

Treatment with an extract of Terminalia superba Engler & Diels decreases blood pressure and improves endothelial function in spontaneously hypertensive rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The stem bark of Terminalia superba (TS) is widely used as a decoction by Cameroonian folk medicine for the treatment of hypertension. The aim of the present study was to evaluate the effect of a chronic treatment with a TS extract on spontaneously hypertensive rats (SHR) with respect to efficacy, biochemical mechanisms and safety. MATERIALS AND METHODS: Eleven-week-old SHR and normotensive Wistar Kyoto rats (WKY) were daily treated by gavage with a methylene chloride extract of stem bark of Terminalia superba (TMSE, 150mg/kg) or with the vehicle for 5 weeks. Systolic blood pressure (SBP) was measured weekly using the tail-cuff method. At the end of the treatment period, vascular function was assessed on isolated thoracic rings, urinary 8-iso-PGF2α levels were measured and cytochrome P-450 3A (CYP 3A) activity was evaluated in liver microsomes. RESULTS: TMSE reduced SBP (P<0.001) in SHR but not in WKY rats. In SHR, the vasorelaxant response to acetylcholine was significantly improved by TMSE as a result of increased nitric oxide synthase (NO) activity and decreased superoxide anion production. In addition, TMSE reduced the vasoconstrictive effect of phenylephrine and improved the sensitivity of smooth muscle cells to NO. TMSE dramatically decreased 8-iso-PGF2α levels in SHR. By contrast, TMSE did not affect all these parameters in WKY rats. Neither diuresis nor the hepatic CYP 3A activity was modified in both animal groups. CONCLUSIONS: This study demonstrated that Terminalia superba has a potent antihypertensive activity in SHR which is partly due to endothelium-dependent and endothelium-independent effects as well as decreased oxidative stress. The data also provide evidence for the lack of herb-drug interaction through hepatic CYP 3A.

Dietary Mg2+ regulates the epithelial Mg2+ channel TRPM6 in rat mammary tissue.

The epithelial Mg(2+) channel TRPM6 is considered a pivotal component in active Mg(2+)absorption and re-absorption in the intestine and kidney, but its expression and function in other tissues are largely unknown. We have previously demonstrated that extracellular Mg(2+) availability modulates TRPM6, but not the ubiquitous TRPM7, in cultured mammary epithelial cells; in addition, TRPM6 protein expression correlated to Mg(2+) influx capacities. Our results closely remind the modulation of TRPM6 described by others in murine kidney and colon following Mg(2+) dietary restriction. We sought to validate our observations by investigating whether TRPM6 modulation by extracellular Mg(2+)also occurs in vivo. To this aim, we exploited a model consisting of rats fed either with a Mg(2+)-deficient or a Mg(2+)-enriched diet, and studied TRPM6 expression in breast and kidney tissues. Immunohistochemical and western blot analyses confirmed that rat mammary tissues express TRPM6 protein levels similar to those found in the kidney, and that protein expression is modulated by dietary Mg(2+). In particular, Mg(2+) restriction upregulated TRPM6 expression, while Mg(2+) supplementation resulted in a significant decrease in protein levels. This work confirms and extends our previous results on TRPM6 modulation by Mg(2+) availability in mammary tissues. Further studies are required to clarify the functional significance of these findings, and the role of TRPM6 in tissue-specific magnesium homeostasis.

Effects of Andrographis paniculata extract and Andrographolide on hepatic cytochrome P450 mRNA expression and monooxygenase activities after in vivo administration to rats and in vitro in rat and human hepatocyte cultures.

The expression of cytochrome P450 (CYP) is regulated by both endogenous factors and foreign compounds including drugs and natural compounds such as herbs. When herbs are co-administrated with a given drug in modern medicine it can lead to drug-herb interaction that can be clinically significant. The ability of Andrographis paniculata extract (APE) and Andrographolide (AND), the most medicinally active phytochemical in the extract, to modulate hepatic CYP expression was examined in vivo in rats and in vitro in rat and human hepatocyte cultures. After in vivo administration, APE at dose levels of 0.5 g/kg/day (i.e. 5 mg/kg/day AND equivalents) and at 2.5 g/kg/day (i.e. 25 mg/kg/day AND equivalents) and AND at dose levels of 5 and 25 mg/kg/day significantly decreased CYP2C11 activity. In primary cultures of rat and human hepatocytes, treatment with AND 50 microM and APE-containing 50 microM AND also resulted in significant decreases in CYP2C expression and activity. In addition, in human hepatocytes, treatment with APE and AND 50 microM resulted in a decrease in CYP3A expression and activity. In conclusion, this study suggests that AND and APE could cause herb-drug interactions in humans through modulation of CYP2C9 and CYP3A4 expression and activities.

Effects of long-term dietary intake of magnesium on oxidative stress, apoptosis and ageing in rat liver.

In the present study, we investigated the effect of long-term dietary Mg intake on the rate of oxidative stress, apoptosis and ageing in rat livers. To address this issue, rats were fed diets containing either a moderately deficient (0.15 g Mg/kg diet), a standard (0.8 g Mg/kg diet) or a high (3.2 g Mg/kg diet) Mg dose for two years. It is noteworthy that a higher percentage of animal mortality was observed in the lowest Mg diet, as compared to the other groups. Oxidative stress and antioxidant status were evaluated by measuring different enzyme activities, among which glutathione peroxidase activity was significantly reduced when Mg content was decreased in the diet. Moreover, we obtained an activation of caspase-3 and a higher lipid peroxidation in the Mg-deficient group, as compared to the Mg standard group, while no changes in Mg-supplemented group were observed, in accordance with our previously published data in primary cultures of rat hepatocytes (Martin et al., J Nutr 2003). Telomere shortening was measured in rat livers, as a marker of ageing. We found that telomere length was decreased in old animals, as compared to young animals confirming that telomere shortening correlated well with ageing events. Moreover, in old animals, we obtained a decrease of telomere length in the Mg-deficient group, as compared to the other groups. Taken together, our results show that a long-term chronic Mg deficiency led to oxidative stress, apoptosis and an acceleration of ageing in rat livers.

Effect of chrysin and natural coumarins on UGT1A1 and 1A6 activities in rat and human hepatocytes in primary culture.

Flavonoids and coumarins are naturally occurring compounds that are widely distributed in vegetables and have a broad pharmacological activity. Inducibility of UDP-glucuronosyltransferases (UGTs) by xenobiotics is well documented and can be considered beneficial for health. In particular, UGT1A1-dependent bilirubin conjugation plays a critical role in the detoxification of neurotoxic bilirubin and phenobarbital-mediated UGT1A1 induction therapy is commonly used in the treatment of unconjugated hyperbilirubinemic diseases such as Crigler-Najjar type II disease. In the present study, the effects of the flavone chrysin and six natural coumarins isolated from various Rutaceous plants on UGT1A6-dependent P-nitrophenol and/or UGT1A1-dependent bilirubin glucuronoconjugation activities were evaluated in cultured rat and human hepatocytes and compared to those of the prototypical UGT1A inducers beta-naphthoflavone, phenobarbital and clofibric acid. After 3 days of treatment at a concentration of 25 microM, the pyranocoumarins avicennin and CIS-avicennol, and the furocoumarins bergapten and imperatorin, increased by 2-fold UGT1A1-dependent activity, equivalent to the increases obtained with chrysin at 25 microM, whereas in the presence of the simple coumarins such as coumarin or umbelliferone, UGT1A1-dependent activity was not modified. In terms of structural requirements for UGT1A1 induction, the present study suggests that the B-ring (phenyl) for chrysin and the furan or pyran rings for coumarins are essential for the biological activity.

Magnesium deficiency induces apoptosis in primary cultures of rat hepatocytes.

The effects of extracellular magnesium (Mg) concentration on the rate of apoptosis in rat hepatocytes in primary culture were examined. After overnight attachment, incubations were conducted for up to 72 h in serum-free media containing low (0-0.4 mmol/L), physiological (0.8 mmol/L) or high (2 and 5.6 mmol/L) Mg concentrations. At 72 h, we observed numerous rounded hepatocytes on top of a shrunken cell monolayer at extracellular Mg concentrations < 0.8 mmol/L. These morphological features were associated with Mg-dependent differences in the total protein levels. The various Mg concentrations did not affect DNA synthesis; however, at a concentration < 0.8 mmol/L, the susceptibility of cultured rat hepatocytes to oxidative stress was increased as shown by the reduced glutathione concentration (10.6 +/- 2.8 vs. 37.3 +/- 4.1 nmol/mg protein with 0 and 0.8 mmol/L, respectively; P < 0.05) and increased lipid peroxidation (0.36 +/- 0.03 vs. 0.21 +/- 0.01 nmol malondialdehyde/mg protein with 0 and 0.8 mmol/L, respectively; P < 0.05). Fluorescence microscopy after Hoechst dye staining revealed numerous apoptotic figures in Mg-free monolayers compared with 0.8 and 5.6 mmol/L Mg conditions. These observations were confirmed quantitatively by flow-cytometric analysis after propidium iodide staining. The proportion of subdiploid cells decreased with increasing Mg concentration; for example, it was greater at 72 h in Mg-free cultures (76%) than in cultures containing 0.8 mmol/L or 5.6 mmol/L Mg (28%; P < 0.05). Caspase-3 was highly activated in Mg-free cultures after 48 h of treatment compared with 0.8 and 5.6 mmol/L conditions (P < 0.05). Overall, these results show that extracellular Mg deficiency has a negative effect on the survival of cultured rat hepatocytes by inducing apoptosis; however, supplementation of extracellular Mg did not reduce the spontaneous apoptosis that occurred over time in rat hepatocyte cultures.