Improving endothelial health with food-derived H2S donors: an in vitro study with S-allyl cysteine and with a black-garlic extract enriched in sulfur-containing compounds.

A healthy vascular endothelium plays an essential role in modulating vascular tone by producing and releasing vasoactive factors such as nitric oxide (NO). Endothelial dysfunction (ED), the loss of the endothelium physiological functions, results in the inability to properly regulate vascular tone, leading to hypertension and other cardiovascular risk factors. Alongside NO, the gasotransmitter hydrogen sulfide (H2S) has emerged as a key molecule with vasodilatory and antioxidant activities. Since a reduction in H2S bioavailability is related to ED pathogenesis, natural H2S donors are very attractive. In particular, we focused on the sulfur-containing amino acid S-allyl cysteine (SAC), a bioactive metabolite, of which black garlic is particularly rich, with antioxidant activity and, among others, anti-diabetic and anti-hypertensive properties. In this study, we analyzed the protective effect of SAC against ED by evaluating reactive oxygen species level, H2S release, eNOS phosphorylation, and NO production (by fluorescence imaging and western blot analysis) in Bovine Aortic Endothelial cells (BAE-1). Furthermore, we chemically characterized a Black Garlic Extract (BGE) for its content in SAC and other sulfur-containing amino acids. BGE was used to carry out an analysis on H2S release on BAE-1 cells. Our results show that both SAC and BGE significantly increase H2S release. Moreover, SAC reduces ROS production and enhances eNOS phosphorylation and the consequent NO release in our cellular model. In this scenario, a natural extract enriched in SAC could represent a novel therapeutic approach to prevent the onset of ED-related diseases.

Bioactive Triterpenes of Protium heptaphyllum Gum Resin Extract Display Cholesterol-Lowering Potential.

Hypercholesterolemia is one of the major causes of cardiovascular disease, the risk of which is further increased if other forms of dyslipidemia occur. Current therapeutic strategies include changes in lifestyle coupled with drug administration. Statins represent the most common therapeutic approach, but they may be insufficient due to the onset of resistance mechanisms and side effects. Consequently, patients with mild hypercholesterolemia prefer the use of food supplements since these are perceived to be safer. Here, we investigate the phytochemical profile and cholesterol-lowering potential of Protium heptaphyllum gum resin extract (PHE). Chemical characterization via HPLC-APCI-HRMS2 and GC-FID/MS identified 13 compounds mainly belonging to ursane, oleanane, and tirucallane groups. Studies on human hepatocytes have revealed how PHE is able to reduce cholesterol production and regulate the expression of proteins involved in its metabolism. (HMGCR, PCSK9, LDLR, FXR, IDOL, and PPAR). Moreover, measuring the inhibitory activity of PHE against HMGR, moderate inhibition was recorded. Finally, molecular docking studies identified acidic tetra- and pentacyclic triterpenoids as the main compounds responsible for this action. In conclusion, our study demonstrates how PHE may be a useful alternative to contrast hypercholesterolemia, highlighting its potential as a sustainable multitarget natural extract for the nutraceutical industry that is rapidly gaining acceptance as a source of health-promoting compounds.

Quali-Quantitative Characterization of Volatile and Non-Volatile Compounds in Protium heptaphyllum (Aubl.) Marchand Resin by GC-MS Validated Method, GC-FID and HPLC-HRMS2.

Protium heptaphyllum (Aubl.) Marchand (PH) trees are endemic to the tropical region of South America, mostly Brazil. Antibacterial, antinociceptive, anti-inflammatory, anxiolytic, antidepressant and anti-hyperlipidemic/anti-hypercholesterolemic effects were reported for its resinous exudate Protiumheptaphyllum resin (PHR). This work aims to provide a qualitative and quantitative consistent chemical profiling of the major constituents of this resin and two extracts enriched in acid (acidic triterpene concentrated extract, ATCE) and neutral triterpenes (α and ß-amyrin concentrated extract, AMCE). GC-MS/GC-FID was used for volatile terpene fraction, a validated GC-MS method was developed for quantification of neutral α and ß-amyrin and HPLC-APCI HRMS2 was used for acidic triterpenes analysis. The chemical investigation reported 29 molecules, including 14 volatile terpenes, 6 neutral triterpenes and 11 acid triterpenes. The most abundant compounds were α-amyrin (251.28 g kg-1, 123.98 g kg-1 and 556.82 g kg-1 in PHR, ATCE and AMCE, respectively), ß-amyrin (172.66 g kg-1, 95.39 g kg-1 and 385.58 g kg-1 in PHR, ATCE and AMCE, respectively), 3-oxo-tirucalla-7,24-dien-21-oic acid (80.64 g kg-1, 157.10 g kg-1 and 15.31 g kg-1 in PHR, ATCE and AMCE, respectively) and 3α-hydroxy-tirucalla-8,24-dien-21-oic acid (77.71 g kg-1, 130.40 g kg-1 and 11.64 g kg-1 in PHR, ATCE and AMCE, respectively). Results showed specific enrichment of acidic and neutral triterpenoids in the two respective extracts.

Formation of by-products during chemical interesterification of lipids. Detection and characterization of dialkyl ketones by non-aqueous reversed-phase liquid chromatography-high resolution mass spectrometry and gas chromatography-mass spectrometry.

A new class of foreign substances present in the unsaponifiable fraction of vegetable oils undergone to chemical interesterification was systematically investigated. Their chemical structure, corresponding to dialkyl ketones (DAK) molecules, was elucidated both by gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-high resolution mass spectrometry (LC-HRMS). An analytical protocol aimed to qualitative and quantitative detection of DAK molecules in vegetable oils of confectionery industry interest was developed. Being the range of concentration levels to be evaluated dependent on the technological task of interesterification process, the quantitation step was thoroughly examined. All the validation parameters were satisfactory and particularly the concentration determinations were made more reliable by the contemporary use of several quantitation standards. GC-MS and LC-HRMS analytical techniques exhibited comparable performances even if the second one shown better detection sensitivity.

Qualitative characterization of Desmodium adscendens constituents by high-performance liquid chromatography-diode array ultraviolet-electrospray ionization multistage mass spectrometry.

The many effects of the African medicinal herb Desmodium adscendens were studied in the 1980s and 1990s. In spite of this, a comprehensive analytical protocol for the quality control of its constituents (soyasaponins, alkaloids and flavonoids) has not yet been formulated and reported. This study deals with the optimization of extraction conditions from the plant and qualitative identification of the constituents by HPLC-diode array UV and multistage mass spectrometry. Plant constituents were extracted from leaves by liquid-liquid and solid matrix dispersion extraction. Separation was achieved via RP-C18 liquid chromatographywith UV and MS(n) detection and mass spectrometry analysis was conducted by electrospray ionization ion trap or orbitrap mass spectrometry. High resolution mass spectrometry (HRMS) was used for structural identification of active molecules relating to soyasaponins and alkaloids. The flavonoid fragmentations were preliminarily studied by HRMS in order to accurately characterize the more common neutral losses. However, the high number of isomeric species induced us to make recourse to a more extended chromatographic separation in order to enable useful tandem mass spectrometry and ultraviolet spectral interpretation to propose a reasonable chemical classification of these polyphenols. 35 compounds of this class were identified herein with respect to the five reported in literature in this way we made up a comprehensive protocol for the qualitative analysis of the high complexity content of this plant. This result paves the way for both reliable quality control of potential phytochemical medicaments and possible future systematic clinical studies.

Study of the photocatalytic transformation of synephrine: a biogenic amine relevant in anti-doping analysis.

The occurrence of some cases of positive results in anti-doping analysis of octopamine requires clarification as to whether its methylated derivative synephrine could be a metabolic precursor of octopamine itself. Synephrine is a natural phenylethylamine derivative present in some food supplements containing Citrus aurantium, permitted in sport regulations. A simulative laboratory study had been done using a photocatalytic process, to identify all possible main and secondary transformation products, in a clean matrix; these were then sought in biological samples obtained from three human volunteers and four rats treated with synephrine; the parent compound and its new potential metabolic products were investigated in human urine and rat plasma samples. The transformation of synephrine and octopamine and the formation of intermediate products were evaluated, adopting titanium dioxide as photocatalyst. Several products were formed and characterized using the HPLC-HRMS(n) technique. The main intermediates identified in these experimental conditions were compared with the major synephrine metabolites found in in vivo studies on rats and humans. Some more oxidized species, already formed in the photocatalytic process, were also found in urine and plasma samples of treated animals. These new findings could be of interest in further metabolism studies. The main photocatalytic pathway involving synephrine appears to be N-demethylation to give octopamine. On the contrary, we demonstrate the inconsistency of this reaction in both rat and human in vivo determinations, resulting in forensic importance.