In neutrophils and other defense cells, the RNOS production starts with the generation of superoxide anion (O₂ ^•−) by NADPH oxidase, whose activation is mediated by protein kinase C (PKC) [39]. However, an excessive RNOS production induces negative side effects for cell and health [2]. Several studies show that the inhibition of the oxidant response of neutrophils becomes a therapeutic challenge to control or modulate an excessive inflammatory response. Beside both steroidal and non-steroidal anti-inflammatory drugs (NSAIDs), there is an increasing interest to find naturally occurring agents with very few side effects for therapeutic substitution [26,40,41].

Thus, we were interested to investigate on isolated neutrophils the anti-inflammatory capacities of aqueous extracts made from some tropical dietary plants which are also used in the folk medicine to alleviate fever and pain and in the treatment of some inflammatory diseases. Here we compared the effect of the different extracts on the oxidant activity of external RNOS and on total MPO release by stimulated equine neutrophils, as well as on the nitration and peroxidasic activity of MPO.

To investigate the effect on the RNOS amount produced by neutrophils, we designed a lucigenin-enhanced chemiluminescence assay (CL) because lucigenin is a good luminescent probe to measure the O₂ ^•− amount produced by activated neutrophils (or other inflammatory cells). In fact, lucigenin reacts specifically with O₂ ^•− in the extra-cellular medium [42] and is divided into two symmetric luminescent adducts [43]. All extracts exerted a dose dependent inhibitory effect on RNOS amount produced by neutrophils stimulated with PMA. The inhibition effect was more powerful for Pteridium and Hibiscus. Interestingly, these two extracts contained the highest total polyphenol, all phenolic acids and flavonoids screened during the HPLC-UV/DAD analysis. The higher content of polyphenols in these extracts in comparison to Manihot and Abelmoschus can explain the more efficient inhibitory effect on ROS production.

Since the extracts were always present during the neutrophil stimulation, polyphenols can act on several ways, either by scavenging and neutralizing the RNOS produced by the cells on a direct stoichiometric relationship, or by interfering with molecules that act on the enzymes responsible for the RNOS production. Some polyphenols have been reported to interfere with the signal transduction involved in the NOx activation [17,20,21,44]. Da Cunha et al. (2004) demonstrated that caffeic acid, present in high concentration in Pteridium, exerts in vitro and in vivo anti-inflammatory actions, at least by scavenging ^•NO and modulating the gene expression for iNOs and probably other inflammatory mediators [45]. Recently, Kim et al. (2011) demonstrated that hyperoside, found in high concentration in Hibiscus, might decrease inflammation by suppressing the activation of nuclear factor-κB in mouse peritoneal macrophages [46]. However, it appears evident that the protective effects of the extracts against the oxidant response of neutrophils could not be attributed to only one or few polyphenols but rather to a synergic effect of all polyphenols present in the extract.

Our results are in agreement with findings reported about the antioxidant activities developed by extracts of the same plants in other assays such as DPPH, ABTS, hydroxyl radical scavenging, and metal chelating [24–32]. Recently we demonstrated the potential of the extracts to decrease the oxidative response of human monocytes (HL-60 cells) activated with PMA and to inhibit lipid peroxidation [35].

Except RNOS production, neutrophils are also specialized in the release of MPO, elastase and other proteolytic enzymes outside their granules and the cell [47]. MPO is a very oxidant enzyme considered as an important marker of inflammation and cardiovascular risk [48]. Our results demonstrate that all extracts slightly decrease the MPO amount released by the neutrophils stimulated with PMA. The mechanisms involved in the degranulation of neutrophils depend on several factors such as the ligation of selectin and integrin, the phosphorylation and activation of proteins like phospholipase C and D, the intracellular Ca²⁺ uptake, the actin cytoskeleton and the activation of protein kinase C (PKC) [1,39,49,50]. Many of these factors are intracellular; it could be possible that the aqueous extracts that we tested contained not enough lipophilic compounds that could interact with the cell membrane or reach the cytosol. This moderate effect of the extracts could also be explained by the use of the stimulating agent. PMA acts directly on PKC activity and bypasses all previous events involved in the stimulation cascade of neutrophils and where some polyphenols could play a role. Interestingly, rutin was found in all extracts. Sheloum et al. (2003) [51] demonstrated that rutin developed anti-inflammatory activities: it interfered into the chemotaxis of neutrophils, inhibited partially the exocytosis of elastase and probably modulated the activation pathways through an interaction with PI3-kinase and mitogen-activated protein kinase (MAPK). Vauzour et al. (2010) [20] illustrated that flavonoids interact with MAP-kinase and PI3-kinase mediated cellular signaling and thereby induce the activation of transcription factors involved in gene expression.

Although the extracts are not very effective on the intracellular mechanisms involved in neutrophil degranulation, they could act efficiently on the activity of MPO released in the extracellular milieu. Evidence has emerged that oxidant compounds derived from the activity of MPO are necessary for their killer function toward bacterial and other pathogen agents, but also provoke tissues damages, initiate and propagate acute and chronic inflammatory diseases [8–11]. Thus, there is a growing interest to modulate the activity of oxidant enzymes with new reversible inhibitors including natural compounds [12–14,26,40,41]. We tested the effects of the extracts on the nitration and peroxidase activitiy of MPO with two techniques. All extracts exert a dose dependent inhibitory effect on both activities as measured by tyrosine nitration assay and SIEFED technique, suggesting that polyphenols interfere on the enzymatic activity of MPO. Whatever the technique used, the best effects were observed for Pteridium which contains the highest amount of total polyphenols and tannins, and Manihot, which has a high content of flavonoids. Through their chemical structure, polyphenols are efficient electron donors [15,16]. Thus, the inhibition observed on the nitration activity of MPO can be due to a competitive effect between the polyphenols of the plant extracts and NO₂ ⁻ as electron donors for the reduction of Cp I - MPO into Cp II - MPO. The oxidant potential of Cp I - MPO is higher than that of Cp II - MPO. By favoring the reduction of Cp I and the accumulation of Cp II, some inhibitors have the ability to shift MPO from the chlorination cycle to the peroxidase cycle [52]. Additionally, polyphenols can also interact with NO₂ ⁻, making it less available as substrate for the activity of Cp I. With SIEFED technique, we clearly demonstrate that the polyphenols present in the extracts interact directly with the enzyme [38]. Indeed, after incubation of purified MPO with the extracts, the SIEFED technique allows to capture MPO by antibodies and to wash the excess of the extracts before the enzymatic measurement of its nitration–peroxidasic activity. Any inhibition of the enzyme activity, persisting after the elimination of the extract, evidences that some molecules of the extract directly interact with the enzyme, modify its structure, hinder its active site and thus limit or block the access of substrates to this site. Several studies demonstrated the ability of purified polyphenols (resveratrol, curcumin, gallic acid, catechin) to interfere in MPO activity, to modulate [53] or to inhibit it [6,53–57].

Each plant that we have studied contains at least one polyphenol with expected AOX, anti-radical and anti-inflammatory activities resulting from its chemical structure. As seen above, the hierarchy of content was different depending on the phytochemical subclass that was considered. The inhibitory effects developed by each extract are probably the effect of a synergy resulting from all its water-soluble antioxidant compounds present in the sample, since the experiments were done with crude aqueous extracts. The HPLC-UV/DAD analysis identified clearly the presence of phenolic acids (caffeic acid, chlorogenic acid and rosmarinic acid) and flavonoids (rutin and hyperoside). The hydroxyl groups around the aromatic rings at the positions 5, 7, 3′, 4′ for hyperoside and 5, 7, 4′, 5′ for rutin, the C2-C3 double bond in the central C ring and the oxo-function on C4 are classic criteria for a powerful AOX activity by flavonoids [15,16]. Thus, these structures can sufficiently explain the inhibitory effects that we observed. However, some eluted compounds remained still unidentified and need further analysis to achieve their identification.

Usually, the plant parts that we have tested are previously chopped, immersed in water, roasted for 10 min, crushed and then cooked in water for 30 to 45 min before their consumption as green vegetables. On this way, cyanosids and ptaquilosids, well-known toxic compounds of Manihot and Pteridium, are eliminated or decrease under a not-toxic level. Consequently, the consumption provokes neither digestive problems nor allergic symptoms [58,59]. In our conditions, the not-heated samples and the boiled ones had no toxic effect on neutrophils, because either the toxic compounds were completely eliminated during the sample preparation, or the low final concentrations applied on the cells could develop no effect on cell viability. The results obtained with our model of cell viability are in agreement with this traditional use and literature data [58,59].

Moreover, the moderate heat treatment of the samples did not modify their AOX and anti-inflammatory capacities. The effects of heat treatment on the AOX potential of polyphenols found in vegetal foodstuffs depend on the matrix of the foodstuff, the kind of the bonds between the AOX compounds and other components within the matrix, as well as on the level and duration of the heat treatment. The literature reports controversial effects of heat treatment on the AOX capacity depending on the plant that was analyzed and the heat treatment that was applied [60–62]. The findings issued from our model encourage cooking green vegetables with moderate heat treatment, because such processing might preserve the AOX potential of polyphenols contained in those plants. At the same concentration, the little differences of AOX activity among the plants that we tested might be explained by the profile of polyphenols contained in each plant, and the synergy of their chemical action [15,16].

Analytical-grad methanol and ethanol, CaCl₂, KCl, NaCl, H₂O₂ 30%, NaOH and Tween 20 were supplied by Merck (VWRI, Leuven, Belgium). NaNO₂, paranitrophenyl phosphate, bovine serum albumin (BSA), ethylene diamine tetra-acetic acid (EDTA) disodium salt, bis-N-methylacridinium nitrate (lucigenin), phorbol 12-myristate 13-acetate (PMA), Percoll, Folin-Ciocalteu reagent, sheepskin powder, dimethylsulfoxide (DMSO), hyperoside and pyrogallol were purchased from Sigma (Bornem, Belgium). Trypan blue was from ICN Biomedicals, Inc (Ohio, USA), all aqueous solutions were prepared with water previously purified in a Milli-Q water system (Millipore, Bedford, MA, USA).

Abelmoschus, Hibiscus, Manihot young leaves and Pteridium crosses and leaves were gathered in Kisantu, province Bas-Congo, Democratic Republic Congo (DRC) in March 2007. The National Institute for Research in Agronomics (INRA, University of Kinshasa, DRC) authenticated the crude vegetal material and provided voucher specimens. The plant parts were dried in the dark, ground and sieved at 180 μm particle size. The recovered powder was loaded into hermetic and opaque flasks and stored at room temperature out of light until further preparations.

For this preparation, we applied the procedure presented in our previous report [35]. Hundred g powder were stirred at 300 rpm for 6 h at room temperature in 500 mL ultra pure water until filtration and lyophilization (24 h). The lyophilized extracts were weighed and kept in hermetic and opaque flasks at −22 °C. The extraction yields increased respectively in the following order: Abelmoschus 6.9% < Hibiscus 7.3% < Manihot 7.4% < Pteridium 7.9%. For each extract, not heated and boiled samples were prepared to the final concentrations required for the assays. All preparations were carried out in darkness.

Neutrophils were isolated from horse blood using EDTA disodium salt (1.6 mg/mL) as anticoagulant. The blood was drawn from the jugular vein of healthy horses bred and fed under identical conditions and submitted to no medical treatment (Faculty of Veterinary Medicine, University of Liège, Belgium). The neutrophils were isolated at room temperature (18–22 °C) by centrifugation (400 g; 45 min; 20 °C) on a discontinuous Percoll density gradient according to the method described by Pycock et al. (1987) [63]. The cells were gently collected and washed with two volumes of physiological saline solution. The cell pellets were resuspended in 20 mM phosphate buffered saline (PBS) at pH 7.4 with 137 mM NaCl and 2.7 mM KCl. Each batch of neutrophils was prepared with 90 mL of blood from one horse. The cells were used within 4 h after isolation. For extracts, each concentration was tested in triplicate; each assay was repeated at last three times with different batches of cells collected from different horses.

PMA was dissolved in DMSO and aliquots were kept at −20 °C. Before use, ultra pure water was added to the aliquots to obtain a solution of 16 μM PMA concentration with 1% DMSO. Parallel assays were carried out with cell suspensions in PBS alone or PBS in 0.05% DMSO, i.e., without activator of the cells. The plant extracts were solubilized in ultra pure water to the final concentrations designed respectively for each assay.

Within an experiment, each measure was repeated three times, and each assay was done at least three times (n ≥ 9). The statistical analysis was performed with GraphPad Instat 3.05 (GraphPad Software, San Diego CA, USA). A One-way Analysis of variance (ANOVA) and a “Student-Newman- Keuls Multiple Comparisons” test were carried out. The IC₅₀ and R² values were calculated with GraphPad Prism 5.0 after converting the concentrations into their decimal logarithm and applying the function “log (inhibitor) vs. normalized response-variable slope”. All results are expressed as mean ± standard deviation (SD) in percentage vs. controls, which were set as 100%. * p-value < 0.05 was considered as significant.