Benzylidene Barbituric Acid Derivatives Shown Anticonvulsant Activity on Pentylenetetrazole-Induced Seizures in Mice: Involvement of Nitric Oxide Pathway

ABSTRACT Background: Barbituric acid derivatives have long been used as central nervous system (CNS) suppressants, such as sedatives, hypnotics and anticonvulsants. In addition, previous studies have implicated the involvement of nitric oxide (NO) in the anticonvulsive effects of barbiturates in CNS. Therefore, the purpose of this study was to figure out the effects of a novel class of barbituric acid derivatives on pentylenetetrazole (PTZ)-induced seizures in male mice. Methods: Thirteen synthesized barbituric acid derivatives (a-m) and phenobarbital were administered intraperitoneally (i.p.) 30 min before induction of seizures by PTZ administration. The mechanisms of PTZ-induced seizures in the mice was evaluated using a non-selective nitric oxide synthase (NOS) inhibitor, selective inducible NOS (iNOS) inhibitor, a selective neuronal NOS (nNOS) inhibitor, and NO substrate. Results: Administration of most of the above mentioned derivatives significantly increased the seizures threshold (P<0.001). The most potent derivative (compound a), was chosen in order to investigate the mechanism of action involving in anticonvulsant activity. Administration of a non-selective NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) and a selective nNOS inhibitor, 7-nitroindazole (7-NI) reversed anticonvulsant activity of compound a. However, injection of the nitric oxide precursor, L-arginine (L-Arg) and a selective iNOS inhibitor, aminoguanidine (AG), did not change anticonvulsant activity of the mentioned compound. Conclusion: These results indicated that the NO system, specifically nNOS may contribute to the anticonvulsant activity of benzylidene barbituric acid derivative a. Therefore, this compound is a good candidate in order to designing new anticonvulsant medication

Naphtoyl-Glycyl-Glycyl-Glycine: A New Substrate for Angiotensin Converting Enzyme (ACE) Assay Using HPLC

Background: Several in vitro assays are used to determine Angiotensin Converting Enzyme (ACE) activity. The purpose of the present investigation, was developing a practical and extraction-free chromatographic method to determine ACE activity. Methods: The method relies on UV-detection of Naphthoyl-glycine (NG), which is resulted from enzymatic hydrolysis of the synthetic substrate, Naphthoyl-glycyl-glycyl-glycine (NGGG), applying a reverse phase chromatographic separation. In this regard, experimental conditions for the assay such as Enzyme/Substrate (E/S) ratio and incubation time were optimized. Chromatographic separation was performed on a reverse phase C18 column (250 × 4.6 mm), using a mobile phase consisting of acetonitrile/water (20:80, v/v), pH = 5.0 with a flow rate of 2.0 mL/min and a detection wavelength of 280 nm. Results: The optimized Enzyme/Substrate (E/S) ratio and incubation time were 10 mU/nmol and 35 min respectively. The results indicated that the calibration curve was linear (r2 = 0.994) and the average recovery (n = 6) of NG was 99.5 ± 1.3% (mean ± RSD). Conclusion: In this study, we introduced a method which is an efficient approach to determine ACE activity due to its sensitive, accurate, and reliable performance with great repeatability

Probing Angiotensin Converting Enzyme (ACE) Domain-Dependent Inhibition of Onopordia, Isolated from Onopordon acanthium L., Using a Continuous Fluorescent Assay

Background: Somatic ACE is a two-domain protein, C and N which are resulted from gene duplication. Presence of two active sites with particular properties, demonstrates functional significance of each domain. Increased levels of circulating N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), could be the result of ACE N-domain selective inhibition. Moreover, ACE C-domain specific inhibitors are able to inactivate bradykinin and inhibit the conversion of angiotensin I to angiotensin II in order to regulate blood pressure as well as reduced side effect profiles. Methods: The present study was designed to determine ACE domain specificity of the novel ACE inhibitor, onopordia which was recently isolated from Onopordon acanthium L. The ACE inhibition activity was determined using Abz-SDK (Dnp)P-OH and Abz-LFK(Dnp)-OH as ACE domain selective substrates. IC50 values of onopordia determined and compared with those of captopril as the standard. Results: IC50 values of onopordia for ACE N and C- domains were 180 µM and 244 µM respectively which demonstrated approximately similar affinity of the mentioned compound to ACE C and N-domains. A pharmacophore model was further generated based on onopordia interactions with the relevant ACE domain active sites. Conclusion: According to onopordia interactions in the ACE C and N-domain active sits, it is a potential to generate more potent and also specific inhibitor based on this new scaffold by doing accurate adjustments. Therefore, this study provides the molecular basis for further designing ACE inhibitors, which are new therapeutics in combating tissue fibrosis diseases