Free Radical Inhibition Using a Water-Soluble Curcumin Complex, NDS27: Mechanism Study Using EPR, Chemiluminescence, and Docking.

There is a growing interest in the use of natural compounds to tackle inflammatory diseases and cancers. However, most of them face the bioavailability and solubility challenges to reaching cellular compartments and exert their potential biological effects. Polyphenols belong to that class of molecules, and numerous efforts have been made to improve and overcome these problems. Curcumin is widely studied for its antioxidant and anti-inflammatory properties as well as its use as an anticancer agent. However, its poor solubility and bioavailability are often a source of concern with disappointing or unexpected results in cellular models or in vivo, which limits the clinical use of curcumin as such. Beside nanoparticles and liposomes, cyclodextrins are one of the best candidates to improve the solubility of these molecules. We have used lysine and cyclodextrin to form a water-soluble curcumin complex, named NDS27, in which potential anti-inflammatory effects were demonstrated in cellular and in vivo models. Herein, we investigated for the first time its direct free radicals scavenging activity on DPPH/ABTS assays as well as on hydroxyl, superoxide anion, and peroxyl radical species. The ability of NDS27 to quench singlet oxygen, produced by rose bengal photosensitization, was studied, as was the inhibiting effect on the enzyme-catalyzed oxidation of the co-substrate, luminol analog (L012), using horseradish peroxidase (HRP)/hydrogen peroxide (H2O2) system. Finally, docking was performed to study the behavior of NDS27 in the active site of the peroxidase enzyme.

Targeting Myeloperoxidase Activity and Neutrophil ROS Production to Modulate Redox Process: Effect of Ellagic Acid and Analogues.

Malaria is an infectious disease caused by a Plasmodium genus parasite that remains the most widespread parasitosis. The spread of Plasmodium clones that are increasingly resistant to antimalarial molecules is a serious public health problem for underdeveloped countries. Therefore, the search for new therapeutic approaches is necessary. For example, one strategy could consist of studying the redox process involved in the development of the parasite. Regarding potential drug candidates, ellagic acid is widely studied due to its antioxidant and parasite-inhibiting properties. However, its low oral bioavailability remains a concern and has led to pharmacomodulation and the synthesis of new polyphenolic compounds to improve antimalarial activity. This work aimed at investigating the modulatory effect of ellagic acid and its analogues on the redox activity of neutrophils and myeloperoxidase involved in malaria. Overall, the compounds show an inhibitory effect on free radicals as well as on the enzyme horseradish peroxidase- and myeloperoxidase (HRP/MPO)-catalyzed oxidation of substrates (L-012 and Amplex Red). Similar results are obtained with reactive oxygen species (ROS) produced by phorbol 12-mystate acetate (PMA)-activated neutrophils. The efficiency of ellagic acid analogues will be discussed in terms of structure-activity relationships.

Muscle Derived Mesenchymal Stem Cells Inhibit the Activity of the Free and the Neutrophil Extracellular Trap (NET)-Bond Myeloperoxidase.

Mesenchymal stem cells (MSCs) are known to migrate to tissue injury sites to participate in immune modulation, tissue remodelling and wound healing, reducing tissue damage. Upon neutrophil activation, there is a release of myeloperoxidase (MPO), an oxidant enzyme. But little is known about the direct role of MSCs on MPO activity. The aim of this study was to investigate the effect of equine mesenchymal stem cells derived from muscle microinvasive biopsy (mdMSC) on the oxidant response of neutrophils and particularly on the activity of the myeloperoxidase released by stimulated equine neutrophils. After specific treatment (trypsin and washings in phosphate buffer saline), the mdMSCs were exposed to isolated neutrophils. The effect of the suspended mdMSCs was studied on the ROS production and the release of total and active MPO by stimulated neutrophils and specifically on the activity of MPO in a neutrophil-free model. Additionally, we developed a model combining adherent mdMSCs with neutrophils to study total and active MPO from the neutrophil extracellular trap (NET). Our results show that mdMSCs inhibited the ROS production, the activity of MPO released by stimulated neutrophils and the activity of MPO bound to the NET. Moreover, the co-incubation of mdMSCs directly with MPO results in a strong inhibition of the peroxidase activity of MPO, probably by affecting the active site of the enzyme. We confirm the strong potential of mdMSCs to lower the oxidant response of neutrophils. The novelty of our study is an evident inhibition of the activity of MPO by MSCs. The results indicated a new potential therapeutic approach of mdMSCs in the inhibition of MPO, which is considered as a pro-oxidant actor in numerous chronic and acute inflammatory pathologies.

Priming of mesenchymal stem cells with a hydrosoluble form of curcumin allows keeping their mesenchymal properties for cell-based therapy development.

Mesenchymal stem cells are increasingly studied for their use as drug-carrier in addition to their intrinsic potential for regenerative medicine. They could be used to transport molecules with a poor bioavailability such as curcumin in order to improve their clinical usage. This natural polyphenol, well-known for its antioxidant and anti-inflammatory properties, has a poor solubility that limits its clinical potential. For this purpose, the use of NDS27, a curcumin salt complexed with hydroxypropyl-beta-cyclodextrin (HPßCD), displaying an increased solubility in aqueous solution, is preferred. This study aims to evaluate the uptake of NDS27 into skeletal muscle-derived mesenchymal stem cells (mdMSCs) and the effects of such uptake onto their mesenchymal properties. It appeared that the uptake of NDS27 into mdMSCs is concentration-dependent and not time-dependent. The use of a concentration of 7 µmol/L which does not affect the viability and proliferation also allows preservation of their adhesion, invasion and T cell immunomodulatory abilities.

Modulation of mitochondrial respiration rate and calcium-induced swelling by new cromakalim analogues.

A cellular model of cardiomyocytes (H9c2 cell line) and mitochondria isolated from mouse liver were used to understand the drug action of BPDZ490 and BPDZ711, two benzopyran analogues of the reference potassium channel opener cromakalim, on mitochondrial respiratory parameters and swelling, by comparing their effects with those of the parent compound cromakalim. For these three compounds, the oxygen consumption rate (OCR) was determined by high-resolution respirometry (HRR) and their impact on adenosine triphosphate (ATP) production and calcium-induced mitochondrial swelling was investigated. Cromakalim did not modify neither the OCR of H9c2 cells and the ATP production nor the Ca-induced swelling. By contrast, the cromakalim analogue BPDZ490 (1) induced a strong increase of OCR, while the other benzopyran analogue BPDZ711 (2) caused a marked slowdown. For both compounds, 1 displayed a biphasic behavior while 2 still showed an inhibitory effect. Both compounds 1 and 2 were also found to decrease the ATP synthesis, with pronounced effect for 2, while cromakalim remained without effect. Overall, these results indicate that cromakalim, as parent molecule, does not induce per se any direct effect on mitochondrial respiratory function neither on whole cells nor on isolated mitochondria whereas both benzopyran analogues 1 and 2 display totally opposite behavior profiles, suggesting that compound 1, by increasing the maximal respiration capacity, might behave as a mild uncoupling agent and compound 2 is taken as an inhibitor of the mitochondrial electron-transfer chain.

Anti-inflammatory and antioxidant activities of Rungia congoensis, a traditional vegetable consumed by Yombe people from Kongo Central area (DR. Congo).

Rungia congoensis, a traditional vegetable from Kongo Central area (DR. Congo) was studied for establishing microscopic characters and characterised by chromatographic techniques and their in vitro biochemical activities against ROS production were evaluated in cellular models and on an enzyme, myeloperoxidase (MPO), involved in inflammation. Microscopically leaf can be characterised by non-glandular and glandular trichomes, sinuous anticlinal epidermal cells, diacytic stomata and helical vessels. Methanolic extract displayed high cellular antioxidant activity at the concentrations range of 0.1-10 µg mL-1 and 1-20 µg mL-1 using lucigenin on neutrophils and DCFH-DA on HL 60, respectively. This extract also showed, more efficient effects on extracellular and intracellular ROS production and MPO activity. Antioxidant and anti-inflammatory activities of R. congoensis were significantly higher, positively correlated with their phytochemical constituents such as flavonoids, iridoids and phenolic acids; and could justify their use as traditional vegetable and potent local nutraceutical resource.

Flavonoid composition, cellular antioxidant activity and (myelo)peroxidase inhibition of a Bryonia alba L. (Cucurbitaceae) leaves extract.

OBJECTIVES: The aim of the present study consisted in the isolation of flavonoids from the leaves of Bryonia alba L. and evaluation of their antioxidant activity and inhibition on peroxidase-catalysed reactions. METHODS: Flavonoids were isolated by preparative HPLC-DAD and their structures were elucidated by MS and NMR. Inhibitory effect was tested by the horseradish peroxidase and the myeloperoxidase assays. Cellular antioxidant assays consisted in testing the inhibitory activity on the reactive oxygen species released upon activation of neutrophils freshly isolated ex vivo from equine blood and of human monocytes-derived macrophages in vitro. Whole organism toxicity was assessed on zebrafish larvae. KEY FINDINGS: Four flavonoids (lutonarin, saponarin, isoorientin and isovitexin) were isolated. The performed assays showed significant antioxidant activity and inhibition for the peroxidase-catalysed reactions. Absence of cellular and zebrafish toxicity was confirmed. CONCLUSIONS: Bryonia alba L. leaves are particularly interesting for their flavonoids content and showed significant inhibitory effect on peroxidase-catalysed oxidation of substrates (Amplex Red and L012), as well as antioxidant/antiradical activity, proving that this species has a medicinal potential. Moreover, the present study highlights the absence of the toxicity of these leaves and offers though a novel perspective on the species, previously known as being toxic.

Sevoflurane modulates the release of reactive oxygen species, myeloperoxidase, and elastase in human whole blood: Effects of different stimuli on neutrophil response to volatile anesthetic in vitro.

Volatile anesthetics have been shown to modulate polymorphonuclear neutrophil (PMN) functions. The aim of this study was to examine the impact of clinically relevant concentrations of sevoflurane (SEVO), a volatile anesthetic, on the release of reactive oxygen species (ROS), myeloperoxidase (MPO), and elastase (EL) from human activated PMNs. For this purpose, samples of whole blood were collected from healthy volunteers and exposed in vitro to 2.3% or 4.6% SEVO in air. To assess for a stimulus-dependent effect of the volatile anesthetic, PMNs were activated using different validated protocols. Artificial stimulation of neutrophils involved either a combination of cytochalasin B (CB) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA). In addition, a combination of lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-α) was also tested as a natural activation mean of PMNs. The production of ROS by PMNs was assessed by L-012 chemiluminescence. Total MPO and EL released in supernatant were measured by enzyme-linked immunosorbent assay (ELISA). Furthermore, degranulation of the active fraction of MPO was also measured by specific immunological extraction followed by enzymatic detection (SIEFED). Overall, SEVO enhanced the release of ROS, MPO, and EL following artificial stimulation of PMNs but the volatile anesthetic inhibited the degranulation of active MPO and EL after neutrophil exposure to LPS and TNF-α. This study highlighted that the effect of SEVO on activated PMNs is dependent on the conditions of cell stimulation. These properties should be taken into consideration in future studies investigating immunomodulatory effects of volatile anesthetics.

Comparison of metabolic profiles and bioactivities of the leaves of three edible Congolese Hibiscus species.

Methanolic and dichloromethane extracts from the leaves of Congolese Hibiscus species were characterised by chromatographic and spectroscopic techniques and their in vitro biochemical activities against ROS production were evaluated in cellular models and on an enzyme, myeloperoxidase (MPO), involved in inflammation. Hibiscus acetosella has a chemical fingerprint different from Hibiscus cannabinus and Hibiscus sabdariffa both having similar fingerprints. Major compounds were polyphenols, represented mainly by caffeoyl-hydroxycitric acid for H. acetosella and neochlorogenic acid for the two other species. All extracts displayed high cellular antioxidant activity with IC50 values ranging from 0.5 to 3 µg mL-1 using lucigenin on neutrophils. Dichloromethane extracts showed more efficient effects on extracellular ROS production and MPO activity. Antioxidant and anti-inflammatory activities of caffeoyl-hydroxycitric acid were significantly higher than those of neochlorogenic acid. The bioactivities of Hibiscus species were positively correlated with their phytochemical content and could justify their use as local nutraceutical resources and medicines.

Assessment of systemic inflammation by time-trends of blood granulocyte count and plasma myeloperoxidase and elastase concentrations following colic surgery in horses.

OBJECTIVE: To determine changes in blood granulocyte counts and in plasma myeloperoxidase (MPO) and elastase (ELT) concentrations in surgical colic cases, and to determine the relationship between these changes and the surgical procedure performed, occurrence of postoperative ileus, and final outcome. DESIGN: Prospective clinical study conducted over a 12-month period. SETTING: University teaching hospital. ANIMALS: Fifty-three horses undergoing emergency laparotomy and surviving at least 12 hours postoperatively. INTERVENTIONS: Blood samples were taken before surgery, during surgery, at the recovery from anesthesia, and then serially until the 150th hour after the first blood sampling. Granulocyte counts were performed by an automated cell hematology analyzer. Specific ELISAs were performed for the MPO and ELT measurements. Mixed models were used to compare the time-trends of the 3 parameters. MEASUREMENTS AND MAIN RESULTS: Taking all horses together, the time-trends of MPO and ELT were not significantly different from each other, but they were significantly different from the granulocyte time-trend. The type of surgical procedure did not influence the time-trends of the 3 parameters. Significant changes in the granulocyte time-trends were associated with postoperative ileus and outcome. Significant changes in the MPO time-trends were associated with outcome. The ELT time-trends were not influenced by ileus or outcome. CONCLUSIONS: Granulocyte counts and MPO change over time and are related to the severity of the inflammatory reaction in surgical colic cases. These time-trends may allow evaluation of treatment efficacy in an effort to modulate excessive granulocyte activation and degranulation.

NDS27 combines the effect of curcumin lysinate and hydroxypropyl-ß-cyclodextrin to inhibit equine PKCδ and NADPH oxidase involved in the oxidative burst of neutrophils.

Polymorphonuclear neutrophils (PMNs) are involved in host defence against infections by the production of reactive oxygen species (ROS), but excessive PMN stimulation is associated with the development of inflammatory diseases. After appropriate stimuli, protein kinase C (PKC) triggers the assembly of NADPH oxidase (Nox2) which produces superoxide anion (O2 (•) (-)), from which ROS derive. The therapeutic use of polyphenols is proposed to lower ROS production by limiting Nox2 and PKC activities. The purpose of this study was to compare the antioxidant effect of NDS27 and NDS28, two water-soluble forms of curcumin lysinate respectively complexed with hydroxypropyl-ß-cyclodextrin (HPßCD) and γ-cyclodextrin (γ-CD), on the activity of Nox2 and PKCδ, involved in the Nox2 activation pathway. Our results, showed that NDS27 is the best inhibitor for Nox2 and PKCδ. This was illustrated by the combined effect of HPßCD and curcumin lysinate: HPßCD, but not γ-CD, improved the release of curcumin lysinate and its exchange against lipid or cholesterol as demonstrated by the lipid colouration with Oil Red O, the extraction of radical lipophilic probes recorded by ESR and the HPLC measurements of curcumin. HPßCD not only solubilised and transported curcumin, but also indirectly enhanced its action on both PKC and Nox2 activities. The modulatory effect of NDS27 on the Nox2 activation pathway of neutrophils may open therapeutic perspectives for the control of pathologies with excessive inflammatory reactions.

Training modifies innate immune responses in blood monocytes and in pulmonary alveolar macrophages.

In humans, strenuous exercise causes increased susceptibility to respiratory infections associated with down-regulated expression of Toll-like receptors (TLRs) and costimulatory and antigen-presenting molecules. Lower airway diseases are also a common problem in sport and racing horses. Because innate immunity plays an essential role in lung defense mechanisms, we assessed the effect of acute exercise and training on innate immune responses in two different compartments. Blood monocytes and pulmonary alveolar macrophages (PAMs) were collected from horses in untrained, moderately trained, intensively trained, and deconditioned states before and after a strenuous exercise test. The cells were analyzed for TLR messenger ribonucleic acid (mRNA) expression by real-time PCR in vitro, and cytokine production after in vitro stimulation with TLR ligands was measured by ELISA. Our results showed that training, but not acute exercise, modified the innate immune responses in both compartments. The mRNA expression of TLR3 was down-regulated by training in both cell types, whereas the expression of TLR4 was up-regulated in monocytes. Monocytes treated with LPS and a synthetic diacylated lipoprotein showed increased cytokine secretion in trained and deconditioned subjects, indicating the activation of cells at the systemic level. The production of TNF-α and IFN-ß in nonstimulated and stimulated PAMs was decreased in trained and deconditioned horses and might therefore explain the increased susceptibility to respiratory infections. Our study reports a dissociation between the systemic and the lung response to training that is probably implicated in the systemic inflammation and in the pulmonary susceptibility to infection.

Effect of different kinds of anoxia/reoxygenation on the mitochondrial function and the free radicals production of cultured primary equine skeletal myoblasts.

Horses are outstanding athletes, performing in many different disciplines involving different kinds of efforts and metabolic responses. Depending on exercise intensity, their skeletal muscle oxygenation decreases, and the reperfusion at cessation of the exercise can cause excessive production of free radicals. This study on cultured primary equine myoblasts investigated the effect of different kinds of anoxia/reoxygenation (A/R) on routine respiration, mitochondrial complex I specific activity and free radicals production. Our data revealed that short cycles of A/R caused a decrease of all the parameters, opposite to what a single long period of anoxia did. A preconditioning-like effect could explain our first pattern of results whereas mild uncoupling could be more appropriate for the second one. Anyway, it seems that mitochondrial complex I could play a major role in the regulation of the balance between metabolic and antioxidant protection of the muscular function of athletic horses.

Intra- and extracellular antioxidant capacities of the new water-soluble form of curcumin (NDS27) on stimulated neutrophils and HL-60 cells.

Phagocytic cells, especially neutrophils (PMNs) are specialized in the production of reactive oxygen species (ROS) to kill pathogenic agents, but an excessive ROS production is associated with tissue damages and inflammatory diseases. Phagocytes are thus prime therapeutic targets to control inflammatory events associated to ROS production. Nowadays, there is a growing interest for the use of polyphenols to modulate the inflammatory response. The aim of this work was to study the antioxidant effect of NDS27, a highly water-soluble form of the polyphenolic molecule curcumin, on in vitro stimulated equine PMNs and human promyelocytic leukemia cells (HL-60). NDS27 was either pre-incubated with cells and eliminated before their activation (intracellular effect) or let in the medium (extracellular effect). Our results indicate that NDS27 significantly and dose-dependently (10(-6) M-10(-4) M) inhibited the ROS production in both cell types without affecting their viability. NDS27 was able to cross and interact with cell membrane, especially for HL-60 cells, while we observed a better intracellular antioxidant effect with PMNs. The activity of myeloperoxidase (MPO) released by PMNs and HL-60 cells, was decreased by NDS27, but more efficiently for PMNs. These results suggested that the greater efficiency of NDS27 in PMNs is due to an inhibitory effect on cells which are more mature for ROS production, probably by targeting the enzymes implied in respiratory burst like MPO. The modulatory effect of NDS27 on the oxidant activity of cells involved in immune and inflammatory responses opens perspectives for a therapeutic control of pathologies with excessive inflammatory reactions.

An in vitro whole blood model to test the effects of different stimuli conditions on the release of myeloperoxidase and elastase by equine neutrophils.

Horses are particularly sensitive and exposed to excessive inflammatory responses evolving toward an important stimulation of polymorphonuclear neutrophils (PMNs). The aim of this work was to stimulate equine neutrophils in whole blood and to evaluate their response by measuring the release of total and active myeloperoxidase (MPO) and total elastase, considered as markers of neutrophil stimulation and degranulation. Because of the critical importance of the concomitant presence of LPS and TNF-α in equine pathological situations, we combined these two natural mediators to stimulate PMN and compared the response with those obtained after the PMN stimulation with each mediator used alone and well-known artificial stimulation systems such as 12-phorbol 13-myristate acetate (PMA) and the combination of cytochalasin B (CB) and N-formyl-methionyl-leucyl-phenylalanine (fMLP). All the activation systems, PMA, CB/fMLP, TNF-α, LPS and LPS/TNF-α, induced a significant release of total MPO in whole blood but only the combinations CB/fMLP and LPS/TNF-α significantly favored the release of active MPO. Regarding the total elastase, we did not observe a significant release in all the stimulated conditions except with PMA. It appears clearly that the choice of the neutrophil stimulation model is fundamental for the selection of potentially active pharmacological agents, especially on MPO activity.

Polyphenol content and modulatory activities of some tropical dietary plant extracts on the oxidant activities of neutrophils and myeloperoxidase.

Young leaves of Manihot esculenta Crantz (Euphorbiaceae), Abelmoschus esculentus (Malvaceae), Hibiscus acetosella (Malvaceae) and Pteridium aquilinum (Dennstaedtiaceae) are currently consumed as green vegetables by peoples in sub-Saharan Africa, Latin America, Asia and their migrants living in Western Europe. Sub-Saharan peoples use Manihot, Abelmoschus and Hibiscus also in the folk medicine to alleviate fever and pain, in the treatment of conjunctivitis, rheumatism, hemorrhoid, abscesses, ... The present study investigates the effects of aqueous extracts of those plants on the production of reactive oxygen species (ROS) and the release of myeloperoxidase (MPO) by equine neutrophils activated with phorbol 12-myristate 13-acetate (PMA). The ROS production was measured by lucigenin-enhanced chemiluminescence (CL), and the release of total MPO by an ELISA method. The study also investigates the effect of the extracts on the activity of MPO by studying its nitration activity on tyrosine and by using a new technique called SIEFED (Specific Immunological Extraction Followed by Enzymatic Detection) that allows studying the direct interaction of compounds with the enzyme. In all experiments, the aqueous extracts of the plants developed concentration-dependent inhibitory effects. A moderate heat treatment did not significantly modify the inhibitory capacity of the extracts in comparison to not heated ones. Total polyphenol and flavonoid contents were determined with an HPLC-UV/DAD analysis and a spectroscopic method using Folin-Ciocalteu reagent. Some polyphenols with well-known antioxidant activities (caffeic acid, chlorogenic acid, hyperoside, rosmarinic acid and rutin) were found in the extracts and may partly explain the inhibitory activities observed. The role of those dietary and medicinal plants in the treatment of ROS-dependent inflammatory diseases could have new considerations for health.

Antioxidant and antiradical activities of Manihot esculenta Crantz (Euphorbiaceae) leaves and other selected tropical green vegetables investigated on lipoperoxidation and phorbol-12-myristate-13-acetate (PMA) activated monocytes.

Abelmoschus esculentus (Malvaceae), Hibiscus acetosella (Malvaceae), Manihot esculenta Crantz (Euphorbiaceae) and Pteridium aquilinum (Dennstaedtiaceae) leaves are currently consumed as vegetables by migrants from sub-Saharan Africa living in Western Europe and by the people in the origin countries, where these plants are also used in the folk medicine. Manihot leaves are also eaten in Latin America and some Asian countries. This work investigated the capacity of aqueous extracts prepared from those vegetables to inhibit the peroxidation of a linoleic acid emulsion. Short chain, volatile C-compounds as markers of advanced lipid peroxidation were measured by gas chromatography by following the ethylene production. The generation of lipid hydroperoxides, was monitored by spectroscopy using N-N'-dimethyl-p-phenylene-diamine (DMPD). The formation of intermediate peroxyl, and other free radicals, at the initiation of the lipid peroxidation was investigated by electron spin resonance, using α-(4-pyridyl-1-oxide)-N-tert-butylnitrone as spin trap agent. The ability of the extracts to decrease the cellular production of reactive oxygen species (ROS) in "inflammation like" conditions was studied by fluorescence technique using 2',7'-dichlorofluorescine-diacetate as fluorogenic probe, in a cell model of human monocytes (HL-60 cells) activated with phorbol ester. Overall the extracts displayed efficient concentration-dependent inhibitory effects. Their total polyphenol and flavonoid content was determined by classic colorimetric methods. An HPLC-UV/DAD analysis has clearly identified the presence of some polyphenolic compounds, which explains at least partially the inhibitions observed in our models. The role of these plants in the folk medicine by sub-Saharan peoples as well as in the prevention of oxidative stress and ROS related diseases requires further consideration.

Evaluation of the antioxidant activity of passion fruit (Passiflora edulis and Passiflora alata) extracts on stimulated neutrophils and myeloperoxidase activity assays.

The antioxidant activity of methanol extracts from Passiflora edulis and Passiflora alata pulp, and P. edulis rinds, healthy or infected with the passion fruit woodiness virus (PWV), was investigated using the oxidant activities of the neutrophil and the neutrophil granule enzyme myeloperoxidase (MPO), both playing key roles in inflammation. The reactive oxygen species produced by stimulated neutrophils were evaluated by lucigenin-enhanced chemiluminescence (CL) and the activity of purified MPO was measured by SIEFED (Specific Immunological Extraction Followed by Enzymatic Detection), a technique for studying the direct interaction of a compound with the enzyme. The rind extracts of P. edulis possessed higher and dose-dependent inhibitory effects on CL response and on the peroxidase activity of MPO than total pulp extracts from both passion fruit species. The quantification of isoorientin in the extracts showed a correlation with their antioxidant activity, suggesting the potential of P. edulis rinds as functional food or as a possible source of natural flavonoids.

Resveratrol inhibits the activity of equine neutrophil myeloperoxidase by a direct interaction with the enzyme.

Resveratrol is a polyphenolic antioxidant present in beverage and food known for its multiple protective effects. We report the inhibitory effects of resveratrol on equine myeloperoxidase (MPO), a hemic peroxidase present in the granules of the neutrophils involved in the inflammatory response. Resveratrol inhibited the production of reactive oxygen species (ROS) by stimulated equine neutrophils by acting as a direct scavenger of the ROS released by the cells but did not modify the degranulation of the stimulated neutrophils as the amounts of released MPO were unchanged. Resveratrol strongly inhibited the chlorination, oxidation, and nitration activities of MPO in a dose-dependent manner. By an original technique of specific immunological extraction followed by enzymatic detection (SIEFED), we demonstrated that resveratrol inhibited the peroxidasic activity of the MPO measured by a direct interaction such as the fixation of resveratrol on the enzyme. The observation of a decrease of the accumulation of compound II suggested that resveratrol acts as an electron donor for MPO reduction.

A specific method for measurement of equine active myeloperoxidase in biological samples and in in vitro tests.

An original method called SIEFED (specific immunological extraction followed by enzymatic detection) was developed for the specific detection of the activity of equine myeloperoxidase (MPO). The method consists of the extraction of MPO from aqueous solutions by immobilized anti-MPO antibodies followed by washing (to eliminate proteins and interfering molecules) and measurement of MPO activity using a detection system containing a fluorogenic substrate, hydrogen peroxide, and nitrite as reaction enhancer. The SIEFED technique was applied to study active MPO in horse biological fluids and the effects of 2 polyphenolic molecules, curcumin and resveratrol, on MPO activity. The detection limit of the SIEFED was 0.23 mU/ml. The SIEFED exhibited good precision with intra-assay and interassay coefficients of variation below 10% and 20%, respectively, for MPO activities ranging from 0.25 to 6.4 mU/ml. The activity of MPO was generally higher than 1 mU/ml in the fluids collected from horses with inflammatory diseases. Curcumin and resveratrol exerted a dose-dependent inhibition on MPO activity and, as they were removed before the enzymatic detection of MPO, the results suggest a direct drug-nzyme interaction or an enzyme structure modification by the drug. The SIEFED is a new tool that would be useful for specific detection of active MPO in complex media and for selection of MPO activity modulators.