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Abstract: Arginase, which converts arginine into ornithine and urea, is a promising therapeutic target. Arginase is involved in cardiovascular diseases, parasitic infections and through a critical role in immunity, in some cancers. There is a need to develop effective arginase inhibitors and therefore efforts to identify and optimize new inhibitors are increasing. Several methods of evaluating arginase activity are available, but few directly measure the product. Radiometric assays need to separate urea and dying reactions require acidic conditions and sometimes heating. Hence, there are a variety of different approaches available, and each approach has its own limits and benefits. In this review, we provide an update on arginase inhibitors, followed by a discussion on available arginase assays and alternative methods, focusing on the intrinsic biases and parameters that are likely to impact results.

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.

International audience; Arginase represents a promising therapeutic target for various pathologies including inflammatory, cardiovascular, and parasitic diseases or cancers. In the current work, we report, for the first time, about the development of a thin‐layer chromatography‐based bioautography which can be used to rapidly detect arginase inhibitors in complex matrices such as plant extracts. The assay is based on the detection of urea produced by arginase using the coloring reagent α‐isonitrosopropiophenone, resulting in the formation of a pink background on thin‐layer chromatography plates. The assay conditions were optimized in order to provide sufficient contrast between the pink colored thin‐layer chromatography plate and the clearer zones generated by the presence of arginase inhibitors. Different parameters were tested, such as incubation time and temperature, atmospheric conditions, as well as substrate and enzyme concentrations. This technique makes it possible to detect 0.1 μg of a known arginase inhibitor, Nω‐hydroxy‐nor‐Arginine, after it has been spotted, either pure or mixed with a Myrtus communis methanolic fruit extract, and the plate has been developed in an appropriate solvent. The newly developed method was used to reveal the presence of an inhibitor in hempseed cakes (Cannabis sativa L.).

Essential oils or their components are increasingly used to fight bacterial infections. Cinnamaldehyde (CNA), the main constituent of cinnamon bark oil, has demonstrated interesting properties in vitro against various pathogens, including Pseudomonas aeruginosa. In the present study, we investigated the mechanisms and possible therapeutic consequences of P. aeruginosa adaptation to CNA. Exposure of P. aeruginosa PA14 to subinhibitory concentrations of CNA caused a strong albeit transient increase in the expression of operons that encode the efflux systems MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY/OprM. This multipump activation enhanced from 2- to 8-fold the resistance (MIC) of PA14 to various antipseudomonal antibiotics, including meropenem, ceftazidime, tobramycin, and ciprofloxacin. CNA-induced production of pump MexAB-OprM was found to play a major role in the adaption of P. aeruginosa to the electrophilic biocide, through the NalC regulatory pathway. CNA was progressively transformed by bacteria into the less toxic metabolite cinnamic alcohol (CN-OH), via yet undetermined detoxifying mechanisms. In conclusion, the use of cinnamon bark oil or cinnamaldehyde as adjunctive therapy to treat P. aeruginosa infections may potentially have antagonistic effects if combined with antibiotics because of Mex pump activation.

International audience; The inhibition of arginase is of substantial interest for the treatment of various diseases of public health interest including cardiovascular diseases. Using an ex vivo experiment on rat aortic rings and an in vitro assay with liver bovine purified arginase, it was demonstrated that several polyphenolic extracts from Cyperus and Carex species possess vasorelaxant properties and mammalian arginase inhibitory capacities. Phytochemical studies performed on these species led to the identification of eight compounds, including monomers, dimers, trimers, and tetramers of resveratrol. The potential of these stilbenes as inhibitors of mammalian arginase was assessed. Five compounds, scirpusin B (5), ε-viniferin (4), cyperusphenol B (6), carexinol A (7), and the new compound virgatanol (1), showed significant inhibition of arginase, with percentage inhibition ranging from 70% to 95% at 100 μg/mL and IC50 values between 12.2 and 182.1 μM, confirming that these stilbenes may be useful for the development of new pharmaceutical products.

In an effort to identify novel inhibitors of arginase, a phytochemical study was performed on hempseed cakes (Cannabis sativa L.). It led to the isolation of a new lignanamide, cannabisin I ( 1), together with seven known lignanamides, cannabisins A, B, C, F, M, 3,3′-demethylgrossamide, grossamide, and two phenylpropanoid amides, N-trans-caffeoyltyramine and N-trans-caffeoyloctopamine, among which was later identified for the first time from C. sativa. Their structures were elucidated by comprehensive analysis of NMR spectroscopy and mass spectrometry data. These compounds were evaluated on mammal arginase (purified liver bovine arginase), showing that N-trans-caffeoyltyramine exhibited the higher activity with an IC50 value of 20.9 µM, which remains, however, less active than the reference compound S-(2-boronoethyl)-l-cysteine (IC50 = 4.3 µM). Radical scavenging capacity of these compounds was determined by the ORAC-FL method. All tested cannabisins displayed antioxidant activity close to or better than the reference compounds. N-trans-Caffeoyltyramine has both arginase inhibitory property and antioxidant capacity.

Polyphenols are plant secondary metabolites which possess many positive effects on human health. Although these beneficial effects could be mediated through an increase in nitric oxide synthase activity, little is known regarding the inhibitory effect of polyphenols on mammal arginase, an enzyme which competes with nitric oxide synthase for their common substrate, L-arginine. The aim of the present study was to determine the potential of a series of polyphenols as mammalian arginase inhibitors and to identify some structure-activity relationships. For this purpose, we first developed a simple and cost-effective in vitro colorimetric microplate method using commercially-available mammal bovine liver arginase (b-ARG 1). Among the ten tested polyphenolic compounds [chlorogenic acid, piceatannol, resveratrol, (−)-epicatechin, taxifolin, quercetin, fisetin, caffeic acid, quinic acid, and kaempferol], cholorogenic acid and piceatannol exhibited the highest inhibitory activities (IC50 = 10.6 and 12.1 µM, respectively) but were however less active as (S)-(2-Boronoethyl)-L-cysteine (IC50 = 3.3 µM), used as reference compound. Enzyme kinetic studies showed that both chlorogenic acid and piceatannol are competitive arginase inhibitors. Structural data identified the importance of the caffeoyl (3,4-dihydroxycinnamoyl)-part and of the catechol function in the inhibitory activity of the tested compounds. These results identified chlorogenic acid and piceatannol as two potential core structures for the design of new arginase inhibitors.