Riccardo Bertin

EC50 estimation of antioxidant activity in DPPH assay using several statistical programs

DPPH(·) assay is a reliable method to determine the antioxidant capacity of biological substrates. The DPPH(·) radical scavenging activity is generally quantified in terms of inhibition percentage of the pre-formed free radical by antioxidants, and the EC(50) (concentration required to obtain a 50% antioxidant effect) is a typically employed parameter to express the antioxidant capacity and to compare the activity of different compounds. In this study, the EC(50) estimation was performed using a comparative approach based on various regression models implemented in six specialized computer programs: GraphPad Prism® version 5.01, BLeSq, OriginPro® 8.5.1, SigmaPlot® 12, BioDataFit® 1.02, and IBM SPSS Statistics® Desktop 19.0. For this project, quercetin, catechin, ascorbic acid, caffeic acid, chlorogenic acid and acetylcysteine were screened as antioxidant standards with DPPH(·) assay to define the EC(50) parameters. All the statistical programs gave similar EC(50) values, but GraphPad Prism® five-parameter analysis pointed out a best performance, also showing a minor variance in relation to the actual EC(50).

Endothelium-independent vasorelaxation by ticlopidine and clopidogrel in rat caudal artery

Objectives  Thienopyridines are prodrugs currently used as anti‐aggregating agents. The aim of this study was to determine if these compounds might have vascular activity independent of hepatic bioactivation.

Activity of myricetin and other plant-derived polyhydroxyl compounds in human LDL and human vascular endothelial cells against oxidative stress

Studies indicate that oxidative modifications of endothelium and LDL play a preeminent role in atherogenesis; therefore, the preservation of the endothelial antioxidant capacity and the inhibition of LDL oxidation by use of plant-derived compounds are an appealing strategy against several vascular disorders. On this basis, baicalein, eupatorin, galangin, magnolol, myricetin, oleuropein, silibinin and bilobalide were studied against various oxidative conditions. The radical scavenging capacity was analysed using DPPH and ORAC assays. Furthermore, the LDL oxidation was detected by measuring the formation of thiobarbituric acid reactive substances (TBARS) and by monitoring the oxidation kinetics. Further, we used cultured HUVEC to investigate the activities of the polyhydroxyl compounds towards the oxidative stress induced by H2O2. The lowest levels of TBARS were observed in the presence of oleuropein and baicalein, while myricetin, magnolol and eupatorin inhibited these ones to a lesser extent. In addition, oleuropein and myricetin exhibited higher protection in copper-induced LDL oxidation kinetics. However, only myricetin and galangin showed significant protective effects against H2O2 oxidative injury in HUVEC cells. Taken all together the results indicate myricetin as the most active agent among the selected plant-derived polyhydroxyl compounds, with prominent capacities against ox-LDL and ROS production in HUVEC.

Vasorelaxation by extracts of Casimiroa spp. in rat resistance vessels and pharmacological study of cellular mechanisms

AIM OF THE STUDY Casimiroa spp. are Mexican plants traditionally used for treatment of hypertension. To study their antihypertensive action, we determined the arterial dilatation induced by extracts from leaves and seeds of Casimiroa calderoniae F. Chiang & Medrano, Casimiroa edulis Llave et Lex, and Casimiroa pubescens Ramirez. MATERIALS AND METHODS The vascular effects of Casimiroa spp. extracts were investigated on rat caudal and aortic arteries. In addition, the extracts were characterized by HPLC using heraclenol, isopimpinellin, heraclenin and phellopterin as external standards. The methanolic extract of Casimiroa pubescens seeds (Cp12) was also studied by H-NMR and LC-MS (ESI-TOF) for the determination of casimiroin and zapotin. RESULTS The hexanic and methanolic extracts of Casimiroa spp. (20 μg/ml) showed vasorelaxation in arterial tissues precontracted by phenylephrine (0.5 μM); the extracts from seeds always caused a greater relaxation in comparison to those from leaves. The most active were the methanolic seed extracts of Casimiroa edulis (Ce8) and Casimiroa pubescens (Cp12). To study the pharmacological mechanisms of vasodilatation we used various inhibitors selective to different receptor subtypes or intracellular enzymes. The vasorelaxant effect of Ce8 (20 μg/ml) remained unaffected by the pretreatment with pyrilamine (10 μM), an antagonist of histamine H(1) receptors, but was inhibited by atropine (0.1 μM), a muscarinic receptor antagonist. Therefore, to determine muscarinic receptor subtypes, we used pirenzepine (1 μM), a selective inhibitor of M(1) receptor, and 4-diphenylacetoxyl-N-methylpiperidine methiodide (DAMP, 0.01 μM), a selective inhibitor of M(3) receptor. Only the latter reduced the vasodilatation by Ce8 and Cp12. To investigate the role of the nitric oxide synthase (NOS), we used N(G)-nitro-l-arginine methyl ester (l-NAME, 10 μM), a selective NOS inhibitor, which decreased the dilatation induced by Ce8 and Cp12. Finally, we studied the action of (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) (ODQ, 3 μM), a selective guanylyl cyclase inhibitor, which inhibited the dilatation by Casimiroa extracts. CONCLUSION The data show that methanolic seed extracts of Casimiroa edulis (Ce8) and Casimiroa pubescens (Cp12) induce vasorelaxation by M(3) receptor through the activation of cGMP-dependent NO signaling. These results support the traditional use of Casimiroa decoctions for antihypertensive treatments in the Mexican ethnomedicine.

Age-dependent vasorelaxation of Casimiroa edulis and Casimiroa pubescens extracts in rat caudal artery in vitro

ETHNOPHARMACOLOGICAL RELEVANCE Leaf and seed decoctions of Casimiroa spp. are used in Mexican traditional medicine to treat high blood pressure. Previous researches showed as Casimiroa extracts are able to induce relaxation of rat aortic and caudal arteries. To study the influence of the aging, we determined the vascular effect induced by extracts of Casimiroa edulis and Casimiroa pubescens in arterial tissues from young and old rats. MATERIALS AND METHODS The activity of Casimiroa edulis extracts: hexanic-leaf (Ce5), methanolic-leaf (Ce6), hexanic-seed (Ce7) and methanolic-seed (Ce8), and Casimiroa pubescens: hexanic-leaf (Cp9), methanolic-leaf (Cp10), hexanic-seed (Cp11) and methanolic-seed (Cp12) were investigated in precontracted caudal arteries of young (4 months) and old (20 months) rats. RESULTS The Casimiroa extracts tested at 20 μg/ml induced vasorelaxation in phenylephrine-precontracted arterial tissues both in young and old arterial tissues. Methanolic seed extracts of Casimiroa edulis (Ce8) and Casimiroa pubescens (Cp12) caused a higher relaxation in young than in old arterial tissues. Nifedipine (0.01 μM) did not change the vasorelaxation induced by Casimiroa edulis extract either in young and old rat arterial tissues. CONCLUSIONS The vasorelaxation induced by Casimiroa edulis and Casimiroa pubescens extracts is decreased from aging since the effects were higher in young than in old rat arterial tissues. However, the methanolic-seed extracts of both plant species induced a relevant vasorelaxation also in old arterial tissues. Thus the results support the traditional use of Casimiroa decoctions as antihypertensive, also in elderly.