Phosphodiesterase 5 and Arginase Inhibitory Activities of the Extracts from Some Members of Nelumbonaceae and Nymphaeaceae Families.

The objectives of this study were (1) to investigate the effect of extracts from some plants in the families Nelumbonaceae and Nymphaeaceae on phosphodiesterase 5 (PDE5) and arginase, which have been used in erectile dysfunction treatment, and (2) to isolate and identify the compounds responsible for such activities. The characterization and quantitative analysis of flavonoid constituents in the active extracts were performed by HPLC. Thirty-seven ethanolic extracts from different parts of plants in the genus Nymphaea and Victoria of Nymphaeaceae and genus Nelumbo of Nelumbonaceae were screened for PDE5 and arginase inhibitory activities. The ethanolic extracts of the receptacles and pollens of Nelumbo nucifera Gaertn., petals of Nymphaea cyanea Roxb. ex G.Don, Nymphaea stellata Willd., and Victoria amazonica (Poepp.) Sowerby and the petals and receptacles of Nymphaea pubescens Willd. showed IC50 values on PDE5 of less than 25 µg/mL while none of the extracts showed effects on arginase. The most active extract, N. pubescens petal extract, was fractionated to isolate and identify the PDE5 inhibitors. The results showed that six flavonoid constituents including quercetin 3'-O-ß-xylopyranoside (1), quercetin 3-methyl ether 3'-O-ß-xylopyranoside (2), quercetin (3), 3-O-methylquercetin (4), kaempferol (5) and 3-O-methylkaempferol (6) inhibited PDE5 with IC50 values at the micromolar level.

Mammalian Arginase Inhibitory Activity of Methanolic Extracts and Isolated Compounds from Cyperus Species.

Polyphenolic enriched extracts from two species of Cyperus, Cyperus glomeratus and Cyperus thunbergii, possess mammalian arginase inhibitory capacities, with the percentage inhibition ranging from 80% to 95% at 100 µg/mL and 40% to 64% at 10 µg/mL. Phytochemical investigation of these species led to the isolation and identification of two new natural stilbene oligomers named thunbergin A-B (1-2), together with three other stilbenes, trans-resveratrol (3), trans-scirpusin A (4), trans-cyperusphenol A (6), and two flavonoids, aureusidin (5) and luteolin (7), which were isolated for the first time from C.thunbergii and C. glomeratus. Structures were established on the basis of the spectroscopic data from MS and NMR experiments. The arginase inhibitory activity of compounds 1-7 was evaluated through an in vitro arginase inhibitory assay using purified liver bovine arginase. As a result, five compounds (1, 4-7) showed significant inhibition of arginase, with IC50 values between 17.6 and 60.6 µM, in the range of those of the natural arginase inhibitor piceatannol (12.6 µM). In addition, methanolic extract from Cyperus thunbergii exhibited an endothelium and NO-dependent vasorelaxant effect on thoracic aortic rings from rats and improved endothelial dysfunction in an adjuvant-induced arthritis rat model.

Arginase inhibitory properties of flavonoid compounds from the leaves of Mulberry (Morus alba, Moraceae).

OBJECTIVES: We aimed to isolate and identify bioactive molecules from Morus alba (Moraceae) leaves having arginase inhibitory activity towards the combat of clinical outcomes related to endothelial dysfunction. METHOD: Extraction and isolation were carried out by successive macerations, prepurification by using a Solid Phase Extraction (SPE) and separation using preparative PLC. The structures of the isolated components were established and confirmed by spectroscopic analyses, including the ESI-HRMS and NMR spectroscopic investigations. Biological evaluation was performed by using an in vitro assay with liver bovine purified arginase and by an ex vivo aortic ring study. KEY FINDINGS: We demonstrated that a phenolic extract from the leaves of M. alba possesses mammalian arginase inhibitory capacities. Investigation of the chemical constituents of its leaves results in the isolation and identification of ten compounds investigated in vitro for their arginase inhibitory capacities. Four compounds showed significant inhibition of arginase, with percentage inhibition ranging from 54% to 83% at 100 µm. In isolated rat aortic rings incubated with NO synthase inhibitor, Luteolin-7-diglucoside compound (2) was able to increase acetylcholine-induced relaxation. CONCLUSIONS: These results demonstrated the attractive ability of M. alba to be a potential source for the discovery of new active products on vascular system.

Vasorelaxant effects of Crataegus pentagyna: Links with arginase inhibition and phenolic profile.

ETHNOPHARMACOLOGICAL RELEVANCE: Crataegus leaves, flowers and fruits have been traditionally used to improve blood circulation, numerous preclinical and clinical studies supporting the cardiovascular benefits of Crataegus preparations. In this respect, there is very limited data on Crataegus pentagyna; in addition, the chemical profile of this species is still incompletely elucidated. AIM OF THE STUDY: The objective of this study was to examine the cardiovascular benefits of Crataegus pentagyna Waldst. et Kit. ex Willd. (small-flowered black hawthorn, Rosaceae) extracts (leaf, flower and fruit ethyl acetate extracts) and the underlying mechanisms. We hypothesized that C. pentagyna extracts might exert vasodilatory effects and inhibit arginase activity due, in large part, to their polyphenolic constituents. MATERIALS AND METHODS: C. pentagyna extracts induced-relaxation and the mechanisms involved were studied ex vivo in isolated aortic rings from Sprague-Dawley rats. The inhibitory effects on bovine liver arginase I were assessed by an in vitro assay. Metabolite profiling of C. pentagyna extracts was performed and the most endothelium- and nitric oxide synthase-dependent; flower extract additionally reduced Ca2+ entry and, to a lesser extent, Ca2+ release from the sarcoplasmic reticulum. C. pentagyna proved to be an important source of arginase inhibitors with potential benefits in endothelial dysfunction that remains to be explored.

Vascular Arginase Is a Relevant Target to Improve Cerebrovascular Endothelial Dysfunction in Rheumatoid Arthritis: Evidence from the Model of Adjuvant-Induced Arthritis.

Emerging data revealed that rheumatoid arthritis (RA) is associated with higher risk of cerebrovascular diseases. Whereas cerebral endothelial dysfunction is acknowledged as a critical aspect of cerebrovascular diseases, its presence in RA and the mechanisms involved are currently unknown. By using the model of rat adjuvant-induced arthritis (AIA), the present study investigated cerebrovascular reactivity in pressurized middle cerebral arteries (MCA) on day 33 post-immunization. The results revealed that arthritis induced a dramatic decrease in the vasodilatory response to acetylcholine (ACh), ADP, and bradykinin (n = 7-9 arteries, p < 0.0001). By using nor-NOHA, L-NAME, BH4, and Tempol, the results showed that the reduced response to ACh relied on arginase overactivation (n = 8), low NOS activity (n = 8), BH4 deficiency (n = 9), and excessive superoxide production (n = 9). Immunohistological analysis revealed an endothelial upregulation of arginase 2 (p < 0.05, n = 5-6) and NADPH oxidase (p < 0.05, n = 5-7) while eNOS expression was unchanged in AIA (n = 6). To assess whether arginase inhibition may be a relevant therapeutic, AIA rats were treated with an arginase inhibitor (nor-NOHA, 40 mg/kg/day, i.p., n = 20 rats) daily from day 10 to day 33 post-immunization. The treatment alleviated the impaired response of MCA to endothelium-dependent agonists, through an increase in NOS signaling and a suppression of BH4 deficiency and superoxide overproduction. By contrast, it did not change the course of arthritis. In conclusion, arthritis induced a cerebrovascular endothelial dysfunction involving an imbalance in the arginase/NOS pathway. Arginase inhibition appears as a promising therapy beyond anti-rheumatic drugs for reducing the risk of cerebrovascular diseases in RA.

Metabolite profiling, arginase inhibition and vasorelaxant activity of Cornus mas, Sorbus aucuparia and Viburnum opulus fruit extracts.

The present study investigated the effects of Cornus mas, Sorbus aucuparia and Viburnum opulus fruit extracts on arginase activity and arterial vasodilation. V. opulus fruit extract exerted the highest vasorelaxant activity in phenylephrine precontracted rat aortic rings (EC50 = 6.31 ±â€¯1.61 µg/mL) and a significant inhibition of arginase (IC50 = 71.02 ±â€¯3.06 µg/mL). By contrast, S. aucuparia and C. mas fruit extracts showed no important anti-arginase activity and a significantly weaker activity in the rat aortic rings relaxation assay (EC50 = 100.9 ±â€¯11.63 and 78.52 ±â€¯8.59 µg/mL, respectively). For all extracts, the main mechanism of vasodilation was proven to be endothelium-dependent. HPLC-ESI-Q-TOF-MS/MS studies revealed a very complex metabolite profiling in all three extracts with chlorogenic acid accounting for 30.89, 0.72 and 2.03 mg/g in V. opulus, C. mas and S. aucuparia fruit extracts, respectively. All extracts were declared non-toxic in the brine shrimp acute toxicity test. Our study highlights potential benefits of V. opulus fruit extract in diseases associated with endothelial dysfunction and impaired vasodilation.

Methotrexate did not improve endothelial function in rheumatoid arthritis: a study in rats with adjuvant-induced arthritis.

OBJECTIVES: Rheumatoid arthritis is associated with an increased cardiovascular risk, secondary to endothelial dysfunction. There is accumulating evidence that methotrexate reduces cardiovascular risk in rheumatoid arthritis, but the mechanisms involved are still unknown. In this study, we aimed to determine the effect of methotrexate on endothelial function and traditional cardiovascular risk factors in the adjuvant-induced arthritis (AIA) rat model. METHODS: On the first signs of arthritis, methotrexate (1 mg/kg/week, s.c.) or saline (Vehicle) was administered to AIA for 3 weeks. Endothelial function was studied in aortic rings relaxed with acetylcholine in the presence or not of inhibitors of nitric oxide synthase, cyclooxygenase-2, arginase, EDHF and superoxide anions production. Arthritis and radiological scores, blood pressure and blood levels of cytokines, triglycerides, cholesterol, homocysteine and BMP-4 were measured. RESULTS: Although methotrexate significantly reduced the arthritis score, it had no effect on Ach-induced relaxation. As regards mechanisms, methotrexate increased nitric oxide synthase activity and reduced the superoxide anions production but did not change arginase, cyclooxygenase-2 and EDHF pathways. Methotrexate did not change the radiological score or blood pressure, lipid, glucose and homocysteine levels. By contrast, methotrexate significantly reduced plasma IL-1ß and TNF-α levels and increased serum BMP-4 level. CONCLUSIONS: Despite a reduction of clinical and biological inflammation, methotrexate did not improve endothelial function in AIA rats. Overall data suggest that mechanisms other than the ED reduction are likely involved, and remain to be elucidated to better understand the cardiovascular benefits of methotrexate in rheumatoid arthritis.

Eulophia macrobulbon extract relaxes rat isolated pulmonary artery and protects against monocrotaline-induced pulmonary arterial hypertension.

BACKGROUND: Extract of the wild orchid, Eulophia macrobulbon (EM) inhibits phosphodiesterase5 (PDE5) suggesting it could preferentially dilate the pulmonary vasculature. PURPOSE AND STUDY DESIGN: To pharmacologically characterize the vascular actions of EM ethanolic extract and its active compound, 1-(4'-hydroxybenzyl)-4,8-dimethoxyphenanthrene-2,7-diol using isolated pulmonary arteries (PA) from rats having pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT). PA were fixed and prepared for histology. RESULTS: EM extract relaxed PA (EC50 = 0.17  mg/ml, Emax ∼ 94%) but less so for aorta (EC50 = 0.51 mg/ml, Emax ∼ 62%), suggesting some selectivity towards the pulmonary circulation. PA vasorelaxation was reduced by endothelial removal or NG-nitro-L-arginine methyl ester, but unaffected by indomethacin, apamin +charybdotoxin, 4-aminopyridine, glibenclamide, iberiotoxin, or 1H - [1,2,4]oxadiazolo[4,3-a]quinoxalin -1- one. Sodium nitroprusside-induced relaxation was enhanced by EM extract, probably via PDE5 inhibition. EM extract reduced contractions evoked by extracellular Ca2+application, and inhibited intracellular Ca2+release activated by phenylephrine. The phenanthrene relaxed PA independently of the endothelium. MCT thickened walls and decreased lumens of PA, and hypertrophied right ventricular myocytes, effects ameliorated by 3 weeks of oral sildenafil (20  mg/kg) or EM extract (15, 450 or 1000  mg/kg). CONCLUSION: PAH is improved by EM extract acting through PA relaxation mediated through endothelial NO, reduced Ca2+-mobilization, and reduced PA wall thickness and right ventricular hypertrophy.

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).

Diclofenac but not celecoxib improves endothelial function in rheumatoid arthritis: A study in adjuvant-induced arthritis.

BACKGROUND AND AIMS: We aimed at investigating the effect of celecoxib (COX-2 selective inhibitor) and diclofenac (non-selective COX inhibitor) on endothelial function, and at identifying the underlying mechanisms in adjuvant-induced arthritis (AIA). METHODS: At the first signs of AIA, diclofenac (5 mg/kg twice a day, i.p), celecoxib (3 mg/kg/day, i.p) or saline (Vehicle) was administered for 3 weeks. Endothelial function was studied in aortic rings relaxed with acetylcholine (Ach) with or without inhibitors of NOS, arginase, EDHF and superoxide anions (O2-°) production. Aortic expression of eNOS, Ser1177-phospho-eNOS, COX-2, arginase-2, p22phox and p47phox was evaluated by Western blotting analysis. Arthritis scores, blood pressure, glycaemia and serum ADMA levels were measured. RESULTS: Diclofenac and celecoxib significantly reduced arthritis score to the same extent (p<0.05). As compared to vehicle-treated AIA, celecoxib did not change whereas diclofenac improved endothelial function (p<0.05) through increased EDHF production, decreased arginase activity and expression, decreased superoxide anions production and expression of p22phox and p47phox. Diclofenac but not celecoxib significantly enhanced blood pressure and serum ADMA levels. Glycaemia was unchanged by both treatments. CONCLUSIONS: Our study reveals that the effect of NSAIDs on endothelial function cannot be extrapolated from their impact on arthritis severity and suggest that changes in blood pressure and plasma ADMA levels may not be useful to predict CV risk of NSAIDs in RA.

Etanercept improves endothelial function via pleiotropic effects in rat adjuvant-induced arthritis.

OBJECTIVES: To determine the effect of etanercept on endothelial dysfunction and on traditional cardiovascular (CV) risk factors in the adjuvant-induced arthritis (AIA) rat model. METHODS: At the first signs of arthritis, etanercept (10 mg/kg/3 days, s.c.) or saline was administered for 3 weeks in AIA rats. Body weights and arthritis scores were monitored daily. Endothelial function was studied in aortic rings relaxed with acetylcholine (Ach) with or without inhibitors of nitric oxide synthase (NOS), cyclo-oxygenase (COX-2), arginase, endothelium-derived hyperpolarizing factor and superoxide anions (O2 (-)°) production. Aortic expression of endothelial nitic oxide synthase (eNOS), Ser1177-phospho-eNOS, COX-2, arginase-2, p22(phox) and p47(phox) was evaluated by western blotting analysis. Blood pressure, heart rate and blood levels of triglycerides, cholesterol and glucose were measured. RESULTS: Etanercept significantly reduced arthritis score (P < 0.001). It improved Ach-induced relaxation (P < 0.05) as a result of increased NOS activity, decreased COX-2/arginase activities and decreased O2 (-)° production. These functional effects relied on increased eNOS expression and phosphorylation, and decreased COX-2, arginase-2 and p22(phox) expressions. No correlation was found between arthritis score and Ach-induced relaxation. The treatment did not change triglycerides, cholesterol and glucose levels, but significantly increased systolic blood pressure and heart rate (P < 0.05). CONCLUSION: Our data demonstrated that efficient dosage of etanercept on inflammatory symptoms improved endothelial function in AIA. This beneficial effect on endothelial function is disconnected from its impact on CV risk factors and relates to pleiotropic effects of etanercept on endothelial pathways. These results suggest that etanercept could be a good choice for patients with rheumatoid arthritis at high risk of CV events.

Endothelial Dysfunction in Rheumatoid Arthritis: Mechanistic Insights and Correlation with Circulating Markers of Systemic Inflammation.

OBJECTIVES: To determine mechanisms involved in endothelial dysfunction (ED) during the course of arthritis and to investigate the link between cytokines, chemokines and osteoprotegerin. APPROACH AND RESULTS: Experiments were conducted on aortic rings at day 4 (preclinical), day 11 (onset of disease), day 33 (acute disease) and day 90 (chronic disease) after adjuvant-induced arthritis (AIA) in Lewis rats. At day 4, the unique vascular abnormality was a reduced norepinephrine-induced constriction. At day 11, endothelial function assessed by the relaxation to acetylcholine was normal despite increased cyclo-oxygenase-2 activity (COX-2) and overproduction of superoxide anions that was compensated by increased nitric oxide synthase (NOS) activity. At day 33, ED apparition coincides with the normalization of NOS activity. At day 90, ED was only observed in rats with a persisting imbalance between endothelial NOS and COX-2 pathways and higher plasma levels of IL-1ß and TNFα. Plasma levels of IL-1ß, TNFα and MIP-1α negatively correlated with Ach-induced relaxation throughout the course of AIA. CONCLUSIONS: Our data identified increased endothelial NOS activity as an important compensatory response that opposes the ED in the early arthritis. Thereafter, a cross-talk between endothelial COX-2/NOS pathways appears as an important element for the occurrence of ED. Our results encourage determining the clinical value of IL-1ß, TNFα and MIP-1α as biomarkers of ED in RA.