Preface to the Special Issue "Glutathione: Chemistry and Biochemistry".

This year we celebrate the 135th anniversary of the discovery of glutathione (L-γ-glutamyl-L-cysteinyl-glycine) [...].

(E)-2-Benzylidenecyclanones: Part XVIII Study the Possible Link between Glutathione Reactivity and Cancer Cell Cytotoxic Effects of Some Cyclic Chalcone Analogs A Comparison of the Reactivity of the Open-Chain and the Seven-Membered Homologs.

Non-enzymatic thiol addition into the α,ß-unsaturated carbonyl system is associated with several biological effects. In vivo, the reactions can form small-molecule thiol (e.g., glutathione) or protein thiol adducts. The reaction of two synthetic (4'-methyl- and 4'-methoxy substituted) cyclic chalcone analogs with reduced glutathione (GSH) and N-acetylcysteine (NAC) was studied by (high-pressure liquid chromatography-ultraviolet spectroscopy) HPLC-UV method. The selected compounds displayed in vitro cancer cell cytotoxicity (IC50) of different orders of magnitude. The structure of the formed adducts was confirmed by (high-pressure liquid chromatography-mass spectrometry) HPLC-MS. The incubations were performed under three different pH conditions (pH 3.2/3.7, 6.3/6.8, and 8.0/7.4). The chalcones intrinsically reacted with both thiols under all incubation conditions. The initial rates and compositions of the final mixtures depended on the substitution and the pH. The frontier molecular orbitals and the Fukui function were carried out to investigate the effects on open-chain and seven-membered cyclic analogs. Furthermore, machine learning protocols were used to provide more insights into physicochemical properties and to support the different thiol-reactivity. HPLC analysis indicated diastereoselectivity of the reactions. The observed reactivities do not directly relate to the different in vitro cancer cell cytotoxicity of the compounds.

Glutathione-Related Enzymes and Proteins: A Review.

The tripeptide glutathione is found in all eukaryotic cells, and due to the compartmentalization of biochemical processes, its synthesis takes place exclusively in the cytosol. At the same time, its functions depend on its transport to/from organelles and interorgan transport, in which the liver plays a central role. Glutathione is determined as a marker of the redox state in many diseases, aging processes, and cell death resulting from its properties and reactivity. It also uses other enzymes and proteins, which enables it to engage and regulate various cell functions. This paper approximates the role of these systems in redox and detoxification reactions such as conjugation reactions of glutathione-S-transferases, glyoxylases, reduction of peroxides through thiol peroxidases (glutathione peroxidases, peroxiredoxins) and thiol-disulfide exchange reactions catalyzed by glutaredoxins.

New Insights on Glutathione's Supramolecular Arrangement and Its In Silico Analysis as an Angiotensin-Converting Enzyme Inhibitor.

Angiotensin-converting enzyme (ACE) inhibitors are one of the most active classes for cardiovascular diseases and hypertension treatment. In this regard, developing active and non-toxic ACE inhibitors is still a continuous challenge. Furthermore, the literature survey shows that oxidative stress plays a significant role in the development of hypertension. Herein, glutathione's molecular structure and supramolecular arrangements are evaluated as a potential ACE inhibitor. The tripeptide molecular modeling by density functional theory, the electronic structure by the frontier molecular orbitals, and the molecular electrostatic potential map to understand the biochemical processes inside the cell were analyzed. The supramolecular arrangements were studied by Hirshfeld surfaces, quantum theory of atoms in molecules, and natural bond orbital analyses. They showed distinct patterns of intermolecular interactions in each polymorph, as well as distinct stabilizations of these. Additionally, the molecular docking study presented the interactions between the active site residues of the ACE and glutathione via seven hydrogen bonds. The pharmacophore design indicated that the hydrogen bond acceptors are necessary for the interaction of this ligand with the binding site. The results provide useful information for the development of GSH analogs with higher ACE inhibitor activity.

The Intestinal and Biliary Metabolites of Ibuprofen in the Rat with Experimental Hyperglycemia.

Hyperglycemia is reported to be associated with oxidative stress. It can result in changes in the activities of drug-metabolizing enzymes and membrane-integrated transporters, which can modify the fate of drugs and other xenobiotics; furthermore, it can result in the formation of non-enzyme catalyzed oxidative metabolites. The present work aimed to investigate how experimental hyperglycemia affects the intestinal and biliary appearance of the oxidative and Phase II metabolites of ibuprofen in rats. In vivo studies were performed by luminal perfusion of 250 µM racemic ibuprofen solution in control and streptozotocin-treated (hyperglycemic) rats. Analysis of the collected intestinal perfusate and bile samples was performed by HPLC-UV and HPLC-MS. No oxidative metabolites could be detected in the perfusate samples. The biliary appearance of ibuprofen, 2-hydroxyibuprofen, ibuprofen glucuronide, hydroxylated ibuprofen glucuronide, and ibuprofen taurate was depressed in the hyperglycemic animals. However, no specific non-enzymatic (hydroxyl radical initiated) hydroxylation product could be detected. Instead, the depression of biliary excretion of ibuprofen and ibuprofen metabolites turned out to be the indicative marker of hyperglycemia. The observed changes impact the pharmacokinetics of drugs administered in hyperglycemic individuals.

A new quinolinone-chalcone hybrid with potential antibacterial and herbicidal properties using in silico approaches.

Quinolinone-chalcones are hybrid compounds consisting of chalcone and quinolone moieties with biological activity related to their hybrid structure. This work seeks to describe the structural and theoretical parameters related to the physicochemical properties and biological activity of a new quinolinone-chalcone. The synthesis, structural characterization by X-ray diffraction, molecular topology by Hirshfeld surfaces and QTAIM, molecular electronic calculations, and pharmacophore analysis were described. The weak interactions C-H…O, C-H…π, and C-H…Br were responsible for crystal growth and stabilized the crystalline state. The DFT analysis shows that the sulfonamide group region is susceptible to observed interactions, and the frontier molecular orbitals indicate high kinetic stability. Also, pharmacophore analysis revealed potential antibacterial and herbicidal activity; by docking within the active site of TtgR, a transcription regulator for the efflux pump TtgABC from the highly resistant Pseudomonas putida (P. putida) strain DOT-TIE, we showed that the activation of TtgR relies upon the binding of aromatic-harboring compounds, which plays a crucial role in bacterial evasion. In this context, a new quinolinone-chalcone has a higher binding affinity than tetracycline, which suggests it might be a better effector for TtgR.

Reaction of Chalcones with Cellular Thiols. The Effect of the 4-Substitution of Chalcones and Protonation State of the Thiols on the Addition Process. Diastereoselective Thiol Addition.

Several biological effects of chalcones have been reported to be associated with their thiol reactivity. In vivo, the reactions can result in the formation of small-molecule or protein thiol adducts. Both types of reactions can play a role in the biological effects of this class of compounds. Progress of the reaction of 4-methyl- and 4-methoxychalcone with glutathione and N-acetylcysteine was studied by the HPLC-UV-VIS method. The reactions were conducted under three different pH conditions. HPLC-MS measurements confirmed the structure of the formed adducts. The chalcones reacted with both thiols under all incubation conditions. The initial rate and composition of the equilibrium mixtures depended on the ratio of the deprotonated form of the thiols. In the reaction of 4-methoxychalcone with N-acetylcysteine under strongly basic conditions, transformation of the kinetic adduct into the thermodynamically more stable one was observed. Addition of S-protonated N-acetylcysteine onto the polar double bonds of the chalcones showed different degrees of diastereoselectivity. Both chalcones showed a Michael-type addition reaction with the ionized and non-ionized forms of the investigated thiols. The initial reactivity of the chalcones and the equilibrium composition of the incubates showed a positive correlation with the degree of ionization of the thiols. Conversions showed systematic differences under each set of conditions. The observed differences can hint at the difference in reported biological actions of 4-methyl- and 4-methoxy-substituted chalcones.

Insights on a new sulfonamide chalcone with potential antineoplastic application.

Chalcones (E)-1,3-diphenyl-2-propene-1-ones, a class of biosynthetic precursor molecules of flavonoids, have a wide variety of biological applications. Besides the natural products, many synthetic derivatives and analogs became an object of continued interest in academia and industry. In this work, a synthesis and an extensive structural study were performed on a sulfonamide chalcone 1-Benzenesulfonyl-3-(4-bromobenzylidene)-2-(2-chlorophenyl)-2,3-dihydro-1H-quinolin-4-one with potential antineoplastic application. In addition, in silico experiments have shown that the sulfonamide chalcone fits well in the ligand-binding site of EGFR with seven µ-alkyl binding energy interactions on the ligand-binding site. Finally, the kinetic stability and the pharmacophoric analysis for EGFR indicated the necessary spatial characteristics for potential activity of sulfonamide chalcone as an antagonist.

Toxicokinetic Study of a Gastroprotective Dose of Capsaicin by HPLC-FLD Method.

BACKGROUND: A low dose of capsaicin and its natural homologs and analogs (capsaicinoids) have shown to prevent development of gastric mucosal damage of alcohol and non-steroid anti-inflammatory drugs. Based on this experimental observation, a drug development program has been initiated to develop per os applicable capsaicin containing drugs to eliminate gastrointestinal damage caused by non-steroid anti-inflammatory drugs. METHODS: As a part of this program, a sensitive and selective reverse-phase high-performance liquid chromatography-based method with fluorescence detection has been developed for quantification of capsaicin and dihydrocapsaicin in experimental dog's plasma. RESULTS: The method was evaluated for a number of validation characteristics (selectivity, repeatability, and intermediate precision, LOD, LOQ, and calibration range). The limit of detection (LOD) was 2 ng/mL and the limit of quantification (LOQ) was 10 ng/mL for both capsaicin and dihydrocapsaicin. The method was used for analysis of capsaicin and dihydrocapsaicin in the plasma samples obtained after per os administration of low doses (0.1, 0.3, and 0.9 mg/kg bw) of Capsaicin Natural (USP 29) to the experimental animals. CONCLUSIONS: The obtained results indicated that the administered capsaicinoids did not reach the general circulation.

Investigation of intestinal elimination and biliary excretion of ibuprofen in hyperglycemic rats.

An in vivo intestinal perfusion model was used to investigate how experimental hyperglycemia affects intestinal elimination and biliary excretion in the rat. Experimental diabetes was induced by administration of streptozotocin (65 mg/kg, i.v.). The intestinal perfusion medium contained 250 µM (±)-ibuprofen. An isocratic high-performance liquid chromatography method with UV-visible detection was developed to quantitate ibuprofen in the intestinal perfusate, while a gradient method was applied to quantitate ibuprofen and ibuprofen-ß-d-glucuronide in the bile. The limit of quantitation of ibuprofen was found to be 0.51 µM in the perfusate of the small intestine. In the bile, the limit of quantitation of ibuprofen and ibuprofen-ß-d-glucuronide was 4.42 and 10.3 µM, respectively. Unconjugated ibuprofen and ibuprofen-ß-d-glucuronide were detected in the bile; however, no ß-d-glucuronide of ibuprofen could be detected in the intestinal perfusate. The results indicate that experimental diabetes can cause a decrease in the disappearance of ibuprofen from the small intestine. Excretion of both ibuprofen and ibuprofen-ß-d-glucuronide decreased to the bile in experimental diabetes. The results can be explained by the results of molecular biological studies indicating streptozotocin-initiated alterations in the intestinal and hepatic transport processes.

HPLC study on Fenton-reaction initiated oxidation of salicylic acid. Biological relevance of the reaction in intestinal biotransformation of salicylic acid.

Fenton-reaction initiated in vitro oxidation and in vivo oxidative biotransformation of salicylic acid was investigated by HPLC-UV-Vis method. By means of the developed high performance liquid chromatography (HPLC) method salicylic acid, catechol, and all the possible monohydroxylated derivatives of salicylic acid can be separated. Fenton oxidations were performed in acidic medium (pH 3.0) with two reagent molar ratios: (1) salicylic acid: iron: hydrogen peroxide 1:3:1 and (2) 1:0.3:1. The incubation samples were analysed at different time points of the reactions. The biological effect of elevated reactive oxygen species concentration on the intestinal metabolism of salicylic acid was investigated by an experimental diabetic rat model. HPLC-MS analysis of the in vitro samples revealed presence of 2,3- and 2,5-dihydroxybenzoic acids. The results give evidence for nonenzyme catalysed intestinal hydroxylation of xenobiotics.

Reagent-induced asymmetric induction in addition reaction of reduced glutathione onto bis-Mannich chalcones.

The stereochemistry of non-enzyme catalyzed nucleophilic addition of GSH to 4'-hydroxychalcone 1 and its bis-Mannich derivative 2 was investigated at different pH values (pH 3.2, 6.1, 7.4, and 8.0). The stereochemical outcome of the reactions was evaluated by HPLC-UV-Vis method. Under strongly acidic conditions (pH 3.2), an unexpected diastereoselective addition of GSH onto the bis-Mannich derivative 2 was observed. Such a selectivity could not be observed in the similar reaction of 2 with N-acetylcysteine. The observed stereoselectivity can be rationalized by ion-pair formation between the protonated Mannich nitrogens and the deprotonated GSH(glutamate)-carboxylate. To the best of our knowledge, this is the first example of reagent-induced asymmetric induction in Michael-type additions of thiols.

Oxidation of Hydroxy- and Dihydroxybenzoic Acids Under the Udenfriend's Conditions. An HPLC Study.

BACKGROUND: Non-enzymatic hydroxylation of aromatic compounds to the respective phenolic derivatives is a possible metabolic pathway of xenobiotics. The formed metabolites can undergo consecutive oxidative reactions with free radicals to form potential toxic molecules. OBJECTIVE: Development of HPLC methods to separate, identify and quantitate the main products formed from salicylic acid, 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid under in vitro hydroxylation conditions (Udenfriend's system). METHOD: An RP-HPLC-UV-Vis method was developed to separate salicylic acid and isomeric dihydroxybenzoic acids formed in the Udenfriend's system. Confirmation of structures of the oxidized products of salicylic acid, 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid was performed by HPLC-ESI-MS/MS method. RESULTS: The HPLC-UV-Vis method was evaluated for a number of validation characteristics (selectivity, repeatability and intermediate precision, LOD, LOQ and calibration range). It was found that oxidation of salicylic acid resulted in the formation of 2,3- and 2,5-dihydroxybenzoic acids. Furthermore, the hydroxylated metabolites can be further metabolized under the Udenfriend's conditions. CONCLUSION: The results give evidence for possible involvement of the oxidized metabolites of salicylic acid in the development of biological action of salicylates at the site of inflammation, where high hydroxyl radical level can be detected.

Changes in hepatic metabolic enzyme activities and biliary excretion of 4-nitrophenol in streptozotocin induced diabetic rats

Abstract Activity of hepatic metabolic enzymes of glucuronidation and sulfation of 4-nitrophenol (PNP) and biliary excretion of its glucuronide (PNP-G) and sulfate (PNP-S) conjugates have been investigated in control and streptozotocin (STZ)-induced diabetic rats. 500 µM PNP solution was luminally perfused in a cannulated jejunal loop for 90 minutes. It was found that biliary excretion of PNP-G was significantly decreased in the diabetic rats. This effect of STZ could be completely reversed by administration of rapid-acting insulin. Activity of hepatic UDP-glucuronyltransferase and ß-glucuronidase was also depressed by the STZ pretreatment. Administration of insulin antagonized the inhibitory action of STZ on UDP-glucuronyltransferase, but the reduced activity of ß-glucuronidase was not reversed. Biliary excretion of PNP-S was also depressed in the diabetic rats. Whereas, different effects of insulin administration were observed. Namely, the lower biliary excretion rate of PNP-S was not changed after administration of insulin. Activity of the sulfotransferase and the arylsulfatase enzymes was not altered either by STZ pretreatment or by insulin administration. Biliary excretion of PNP was also significantly depressed by STZ and this depression was not changed after insulin administration. The results call attention to hepatobiliary circulation of low molecular weight xenobiotics and their glucuronide and sulfate conjugates

HPLC analysis of in vivo intestinal absorption and oxidative metabolism of salicylic acid in the rat.

In vivo absorption and oxidative metabolism of salicylic acid in rat small intestine was studied by luminal perfusion experiment. Perfusion through the lumen of proximal jejunum with isotonic medium containing 250 µm sodium salicylate was carried out. Absorption of salicylate was measured by a validated HPLC-DAD method which was evaluated for a number of validation characteristics (specificity, repeatability and intermediate precision, limit of detection, limit of quantification, linearity and accuracy). The method was linear over the concentration range 0.5-50 µg/mL. After liquid-liquid extraction of the perfusion samples oxidative biotransformation of salicylate was also investigated by HPLC-MS. The method was linear over the concentration range 0.25-5.0 µg/mL. Two hydroxylated metabolites of salicylic acid (2,5-dihydroxybenzoic acid and 2,3-dihydroxybenzoic acid) were detected and identified. The mean recovery of extraction was 72.4% for 2,3-DHB, 72.5% for 2,5-DHB and 50.1% for salicylic acid, respectively. The methods were successfully applied to investigate jejunal absorption and oxidative metabolism of sodium salicylate in experimental animals. The methods provide analytical background for further metabolic studies of salycilates under modified physiological conditions.

Chalcone derivatives cause accumulation of colon cancer cells in the G2/M phase and induce apoptosis.

AIMS: Chalcones, naturally occurring open-chain polyphenols abundant in plants, have demonstrated antiproliferative activity in several cancer cell lines. In the present study, the potential anticancer activity of two synthetic analogues named Ch1 and Ch2 in colon cancer cell line was investigated. MAIN METHODS: Antiproliferative activities of both synthetic analogues were assessed by Growth Inhibition Assay (MTT) and xCELLigence cell analysis. Apoptosis was assessed by annexin V/PI staining (early stage) or by DNA fragmentation (final stage). To study the cell death mechanism induced by tested substances, we assessed a series of assays including measurements of the caspase 3 activity, membrane mitochondrial potential (MMP) changes, reactive oxygen species (ROS) production by flow cytometry and expression of important apoptosis-related genes by realtime PCR. KEY FINDINGS: We found concentration and time-dependent cytotoxicity, inhibition of proliferation of Caco-2 cells after Ch1 and Ch2 treatment in parallel with G2/M phase cell cycle arrest and increased cell proportion in subG0/G1 population with annexin V positivity. We demonstrated that both Ch1 and Ch2 induced caspase-dependent cell death associated with increased ROS production, suppressed Bcl-2 and Bcl-xL and enhanced Bax expression. Treatment of Ch1 also suppressed α-, α1- and ß5-tubulins, on the other hand Ch2 only suppressed α-tubulin expression. SIGNIFICANCE: Presented chalcones induce apoptosis by intrinsic pathways, and therefore may be an interesting strategy for cancer therapy.

Effect of experimental diabetes and insulin replacement on intestinal metabolism and excretion of 4-nitrophenol in rats.

Luminal appearance of 4-nitrophenol (PNP) metabolites (4-nitrophenol-ß-glucuronide (PNP-G) and 4-nitrophenol-sulfate (PNP-S)) and activity of the related metabolic enzymes have been investigated in control and experimental diabetic rats. Experimental diabetes was induced by administration of streptozotocin (65 mg/kg i.v.). PNP (500 µmol/L) was luminally perfused in the small intestine and the metabolites were determined in the perfusion solution. Effect of insulin replacement was also investigated in the diabetic rats. It was found that experimental diabetes increased the luminal appearance of PNP-G, which could be completely compensated by rapid-acting insulin administration (1 U/kg i.v.). Activities of the enzymes involved in PNP-G production (UDP-glucuronyltransferase and ß-glucuronidase) were also elevated; however, these changes were only partially compensated by insulin. Luminal appearance of PNP-S was not significantly changed by administration of streptozotocin and insulin. Activities of the enzymes of PNP-S production (sulfotransferases and arylsulfatases) did not change in the diabetic rats. The results indicate that experimental diabetes can provoke changes in intestinal drug metabolism. It increased intestinal glucuronidation of PNP but did not influence sulfate conjugation. No direct correlation was found between the changes of metabolic enzyme activities and the luminal appearance of the metabolites.

A validated HPLC-FLD method for analysis of intestinal absorption and metabolism of capsaicin and dihydrocapsaicin in the rat.

A sensitive and selective reverse-phase high performance liquid chromatographic method with fluorescence detection has been developed for determination of capsaicin (8-methyl-N-vanillyl-(trans)-6-nonenamid) and dihydrocapsaicin (8-methyl-N-vanillylnonanamide) in samples generated in rat small intestine luminal perfusion experiments. The experiments were designed to study the biotransformation of capsaicinoids in the small intestine in the rat. The chromatographic separation was performed at room temperature on a ZORBAX Eclipse(®) XDB-C8 column using isocratic elution with a mobile phase consisting 0.05M orthophosphoric acid solution and acetonitrile (60:40, v/v; pH 3.0) with a flow rate of 1.5mL/min. Fluorescence detection was performed at excitation and emission wavelengths of 230 and 323nm, respectively. The method was evaluated for a number of validation characteristics (accuracy, repeatability and intermediate precision, limit of detection, limit of quantification and calibration range). The limit of detection (LOD) was 50ng/mL and the limit of quantification (LOQ) was 100ng/mL for both capsaicin and dihydrocapsaicin reference standards dissolved in blank perfusate. The method was successfully applied for investigation of intestinal absorption of capsaicin and dihydrocapsaicin while 30µg/mL standardized Capsicum extract - containing capsaicin and dihydrocapsaicin - was luminally perfused for a 90min period. The structure of the glucuronide metabolites of capsaicin and dihydrocapsaicin appeared in the perfusate was identified by mass spectrometry.

Development and validation of an HPLC-DAD analysis for pharmacopoeial qualification of industrial Capsicum extracts.

A reverse-phase high-performance liquid chromatography method was developed to quantify capsaicin (trans-8-methyl-N-vanillyl-6-nonenamid), dihydrocapsaicin (8-methyl-N-vanillylnonanamide) and the main capsaicinoid contents of Capsicum extracts. The chromatographic separation was carried out on a C8 column using isocratic mobile phase consisting of 40% (v/v) acetonitrile and 60% (v/v) orthophosphoric acid solution with flow rate of 1.5 mL/min. The concentration of the eluting compounds was monitored by a diode-array detector at wavelength of 281 nm. The method was evaluated for number of validation characteristics (selectivity, accuracy (confidence intervals <1%), repeatability and intermediate precision (RSD% < 2.5%), limit of detection (LOD), limit of quantification (LOQ) and calibration range). The LOD was 0.25 µg/mL and the LOQ was 0.5 µg/mL. Using methanolic solutions of United States Pharmacopoeia (USP) Capsaicin and Dihydrocapsaicin Reference Standards, the method was linear over the concentration range 0.0005-0.5000 mg/mL for both capsaicinoids. The method was applied to qualify capsaicinoid content of two industrial capsicum extracts according to the USP 29.

3-benzylidene-4-chromanones: a novel cluster of anti-tubercular agents.

In a quest for developing novel anti-tubercular agents, a series of 3-benzylidene-4-chromanones 1a-l were evaluated for growth inhibition of Mycobacterium tuberculosis H37Rv. Three promising compounds 1d, g, j emerged as the lead compounds with the IC50 and IC90 values of less than 1 µg/mL. Evaluation of the potent compounds 1d, g, j and k against Vero monkey kidney cells revealed that these compounds are far more toxic to M. tuberculosis than to Vero cells. Structure-activity relationships demonstrated that 3-benzylidene-4-chromanones are more potent against M. tuberculosis than the related 2-benzylidene cycloalkanones and the meta substituted chromanone derivatives are more active than their ortho- and para-counterparts. Some guidelines for amplifying the project are presented.