Carob pods (Ceratonia siliqua L.) inhibit human neutrophils myeloperoxidase and in vitro ROS-scavenging activity

Abstract

Natural antioxidants such as phenolic compounds protect cells against the damaging effects of reactive oxygen species (ROS). In this study, we investigated the effect of carob pods aqueous extract (CPAE) on the reactive oxygen species (ROS) production by human neutrophils, myeloperoxidase (MPO) activity and expression as well as lactoferrin and NADPH oxidase phosphorylation. Neutrophils were isolated from whole human blood using the ficoll–dextran method and ROS generation was measured by luminol-amplified chemiluminescence. Superoxide anion generation was detected by chemiluminescence using the lucigenin method. H₂O₂ was detected by the chemiluminescence assay. MPO activity was measured by the tetramethylbenzidine oxidation method. Western blotting analysis was used to determine the MPO and lactoferrin as well as P47phox–Ser-328 phosphorylation. The use of HPLC technique revealed the identification of many phenolic compounds in carob pods with pyrogallol as the main compound in the pulp and tannic acid in the seeds. We also found that CPAE inhibits luminol-amplified chemiluminescence in human neutrophils stimulated by PMA and that it is able to scavenge superoxide anion and hydrogen peroxide. Carob extract significantly reduces MPO activity and expression. More importantly, CPAE inhibits PMA-induced p47^phox phosphorylation on Ser328 as well as lactoferrin release by neutrophils in a concentration-dependent manner. The effects are generally more marked for the seeds compared to the pulp. In conclusion, we suggest in the present study that Carob pods (Ceratonia siliqua L.) inhibit human neutrophils myeloperoxidase and in vitro ROS production.

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1. Introduction

The immune system is especially vulnerable to oxidative damage because many immune cells, such as neutrophils, produce reactive oxygen and nitrogen species (ROS/RNS) as part of the body's defense mechanisms to destroy invading pathogens.¹ They release reactive oxygen species and cytokines outside the cells to kill extracellular bacteria and recruit additional leukocytes to the region of infection or inflammation.² Therefore, neutrophils represent the body's primary line of defense against invading pathogens and inflammation.³ Neutrophil granules are classified into three distinct subsets based on the presence of characteristic granule proteins: primary (azurophil) granules (myeloperoxidase (MPO)), secondary (specific) granules (lactoferrin), and tertiary (gelatinase) granules (gelatinase). Excessive neutrophil degranulation is a common feature of many inflammatory disorders such as severe asphyxic episodes of asthma, acute lung injury, rheumatoid arthritis, and septic shock.¹

The generation of microbicidal oxidants by neutrophils leads to the activation of a multiprotein enzyme complex known as the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which is responsible for transferring electrons from NADPH to O₂, resulting in the formation of a superoxide anion. This multi-component enzyme system is composed of cytosolic proteins (p47phox, p67phox, p40phox, and rac1/2) and membrane proteins (p22phox and gp91phox, which form cytochrome b558), which assemble at membrane sites upon cell activation. NADPH oxidase activation is tightly regulated because of potential damage to the surrounding tissues.^4–6 P47^phox phosphorylation on several serines plays a pivotal role in oxidase activation in intact cells.^7,8

The response of neutrophils to several biologically active substances, e.g. to arachidonic acid, phorbolmyristate acetate (PMA), N-formyl-methionylleucylphenylalanine (FMLP) or to calcium ionophore A₂₃₁₈₇, mimics the inflammation-induced burst of neutrophils as well as the production and release of superoxide anion radical (O₂˙⁻). Subsequently, O₂˙⁻ may initiate the formation of derived ROS: hydrogen peroxide (H₂O₂), singlet oxygen, hydroxyl radical (OH˙) and hypochlorous acid (HOCl).^9–11 Therefore, depending on the factors of concentration, duration, and localization of their production, ROS either play a beneficial role by participating in tissue homeostasis or when inappropriate or excessive production occurs, can directly damage the surrounding tissues and participate in inflammatory disorders.¹² In addition to the ROS production, the cytoplasmic granules of the neutrophils discharge hydrolytic and proteolytic enzymes and myeloperoxidase (MPO; EC 1.11.1.7) that is a hemic enzyme specific of azurophilic granules. MPO is unique because it has a dual activity of peroxidase and chlorination reaction.

In this perspective, the use of natural polyphenols is promising. These compounds have antioxidant, anti-inflammatory and antitumor activities.¹³ Antioxidant activities of plant polyphenols have been claimed to have beneficial health functions for retarding aging and preventing cancer and cardiovascular diseases.¹⁴ In vitro, Gismondi et al.¹⁵ recently showed some antioxidant capabilities of the African medicinal plant as well as the capabilities of cell cycle arrest and differentiation in B16F10 melanoma cells. With the same cell model, Forni et al.¹⁶ suggested an antineoplastic activity of strawberry (Fragaria × ananassa Duch.) crude extracts. For these reasons, the natural antioxidants and anti-inflammatory compounds have recently become a major area of research.

The carob tree (Ceratonia siliqua L., Leguminosae family) has been widely cultivated for years in Mediterranean countries, including Tunisia.¹⁷ The carob fruit, a brown pod 10–25 cm in length, contains two major components: pulp and the seeds.¹⁸ This plant is rich in bioactive molecules such as phenolic compounds.¹⁹ Recently, we have shown that the water extract of carob pods presents a good in vitro free-radical scavenging ability,²⁰ higher than many other foods rich in phenolic compounds such as blueberries and grapes.^21,22

The aim of the present study was to examine the effect of carob pods aqueous extract (CPAE) on ROS production by human neutrophils as well as its effect on MPO, lactoferrin expression, and P47phox–Ser-328 phosphorylation.

2. Materials and methods

2.1. Chemicals and reagents

Luminol, PMA, lucigenin, and HRPO were from Sigma-Aldrich (Saint-Quentin Fallavier, France). HBSS and HEPES were from Gibco. Ficoll and dextran T500 were from GE Healthcare. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and western blotting reagents were purchased from Bio-Rad (Richmond, CA, USA). Polyclonal rabbit anti-p47^phox and antibodies were previously described.²³ The rabbit polyclonal antibodies against anti-phospho-Ser328–p47^phox, lactoferrin and MPO have been produced using specific peptides made by PolyPeptide Laboratories (Strasbourg, France).²⁴ The different solutions were diluted in phosphate-buffered saline (PBS) immediately before use.

2.2. Ethics statement

The necessary permits for the field studies and collection of carob pod samples were obtained from the Ministry of Agriculture in Tunisia and identified by Mrs Mouhiba Ben-Naceur, professor of taxonomy in the Higher Institute of Biotechnology of Béja-Tunisia. The voucher specimens have been deposited with the herbarium of the Higher Institute of Biotechnology of Beja and also in the Department of Biological Sciences, Faculty of Science of Tunis.

The Clinical Research Committee at the Xavier Bichat hospital (Paris, France) approved all protocols, and we obtained written consent from each blood donor.

2.3. Preparation of carob extracts

The mature carob pods were collected from the region of Tabarka (North-West of Tunisia) during October 2013. Briefly, the seeds were rigorously separated from the pulp, dried in an incubator at 50 °C during 72 hours and powdered in an electric blender (Moulinex Ovatio 2, FR). Seeds and pulp powder were dissolved in double distilled water and filtered through a colander (0.5 mm mesh size). Finally, the lyophilized extracts of carob pulp and seeds were immediately used.

2.4. Hydrolysis and analysis of phenolic compounds using RP-HPLC

Dried samples from pulp and seeds were hydrolyzed according to the slightly modified method of Proestos et al.²⁵ The acidic hydrolysis was used to release the aglycones to simplify the identification process since the free forms of phenolic compounds are rarely present in plants and they occur as esters, glycosides, or bound to the cell wall.²⁶ Twenty milliliters of methanol were added to 0.5 g of a dried sample. Then, 10 mL of 1 M HCl was added. The mixture was stirred carefully and sonicated for 15 min and refluxed in a water bath at 90 °C for 2 h. The obtained mixture was injected into a HPLC. The separation was carried out on a 250 mm × 4.6 mm, 5 μm Hypersil ODS C18 reversed phase column at ambient temperature. The mobile phase consisted of acetonitrile (solvent A) and water with 0.2% formic acid (solvent B). The flow rate was maintained at 0.7 mL min⁻¹. The gradient program was as follows: 35% A/65% B, 0–6 min; 60% A/40% B, 6–9 min; 80% A/20% B, 9–14 min and 100% A, 14–25 min. The injection volume was 20 μL, and peaks were monitored at 280 nm. Samples were filtered through a 0.45 μm membrane filter before injection. Phenolic compounds were identified by congruent retention times compared with standards. Analyses were performed in triplicate.

2.5. Fractionation of human neutrophils

Venous blood was collected from healthy adult volunteers and neutrophils were isolated by dextran sedimentation and density gradient centrifugation as previously described.²⁷ Erythrocytes were removed by hypotonic lysis. After the isolation, the cells were resuspended in an appropriate medium such as Hank's balanced salt solution (HBSS). The cells were counted and their viability was determined with the trypan blue exclusion method.

2.6. Measurement of ROS production by chemiluminescence

Isolated cells were resuspended in HBSS at a concentration of 1 million per mL. Cell suspensions (5 × 10⁵) in 0.5 mL of HBSS containing 10 μM luminol in the presence or absence of CPAE were added after dilution using a micropipette, preheated at 37 °C in the thermostated chamber of a luminometer (Berthold-Biolumat LB937) and allowed to stabilize. After a baseline reading, cells were stimulated with PMA (100 ng mL⁻¹). Changes in chemiluminescence were measured over a 30 min period.²⁸

2.7. Preparation of azurophilic granules and measurement of MPO activity

Neutrophils were lysed by nitrogen cavitation and the granule fraction was purified by Percol gradient centrifugation.²⁹ The granules were sonicated in 0.2 CTAB and MPO activity was assessed using the H₂O₂-dependent tetramethylbenzidine (TMB) oxidation assay at 650 nm.³⁰ MPO content was also measured by western blotting analysis and ELISA.

2.8. H₂O₂ chemiluminescence assay

The chemiluminescence luminol-based method has been used for the determination of H₂O₂, which is based on a reaction with H₂O₂ catalyzed by horseradish peroxidase (HRP).³¹

2.9. Lucigenin-dependent chemiluminescence assay for O₂˙⁻

The accumulation of O₂˙⁻ is measured by the chemiluminescence of lucigenin.³²

2.10. Electrophoresis and immunoblotting

Immunoprecipitated proteins were separated on SDS-polyacrylamide gels using standard techniques.³³ Proteins were transferred to nitrocellulose, and the phosphorylation of p47-phox, P47phox–Ser-328, MPO and lactoferrin were monitored by immunoblotting using monoclonal or polyclonal antibodies.

2.11. Statistical analysis

The data were analyzed by one-way analysis of variance (ANOVA) and were expressed as means ± standard error of the mean (SEM). The data are representative of five independent experiments. All statistical tests were two-tailed, and a p value of 0.05 or less was considered significant.

3. Results

3.1. Identification and quantification of phenolic compounds by HPLC analysis

The use of HPLC technique (Fig. 1) revealed the identification of various phenolic compounds, as shown in Table 1. The principal compounds are as follows: pyrogallol (48.02% ± 3.55% and 9.12% ± 2.04%), catechin (19.10% ± 2.11% and 6.25% ± 1.10%) and tannic acid (9.01% ± 1.40% and 19.03% ± 2.13%), respectively, in pulp and seed compartments.

Fig. 1 Chromatographic profiles of aqueous extract of carob pods ((A) pulp and (B) seeds).

Table 1 Identification and quantification of phenolic compounds in carob pods by HPLC analysis

	% of phenolic compounds
	Pulp	Seeds
Gallic acid	3.10 ± 0.62	0.80 ± 0.30
Tannic acid	9.01 ± 1.40	19.03 ± 2.13
Caffeic acid	1.71 ± 0.41	00
Ferulic acid	3.10 ± 0.72	00
Vanillic acid	00	2.71 ± 0.64
Pyrogallol	48.02 ± 3.55	9.12 ± 2.04
Catechin	19.10 ± 2.11	6.25 ± 1.10

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3.2. CPAE inhibits luminol-amplified chemiluminescence in human neutrophils stimulated by PMA

To study the effect of carob extract on human neutrophil ROS production, cells were incubated with different CPAE concentrations and ROS were detected by luminol-amplified chemiluminescence. Results show that CPAE (pulp and seeds) inhibited luminol-amplified chemiluminescence on neutrophils stimulated by PMA in a concentration-dependent manner (Fig. 2). The CI₅₀ values calculated from the graph demonstrate that the inhibition of ROS production by neutrophils was more marked for the seeds (CI₅₀ = 3.4 μg mL⁻¹) compared to the pulp (CI₅₀ = 12.5 μg mL⁻¹).

Fig. 2 Effect of CPAE (pulp and seeds) on luminol-amplified chemiluminescence of human neutrophils. Human neutrophils (5 × 10⁵) were incubated in the presence or absence of CPAE and stimulated with PMA (100 ng mL⁻¹). Luminol-amplified chemiluminescence was measured during 30 min (data are presented as means ± SEM of five independent experiments, *p < 0.05).

3.3. The effect of CPAE on neutrophil superoxide anion production

To investigate the effect of aqueous extract of carob pods on NADPH oxidase activity, we studied its effect on superoxide anion production using a lucigenin-dependent chemiluminescence assay. As shown in Fig. 3, CPAE of pulp and seeds significantly inhibit PMA-induced superoxide anion production in a concentration-dependent manner. The CI₅₀ values calculated from the graph clearly show that the effect is more marked for the seeds (CI₅₀ = 5.3 μg mL⁻¹) compared to the pulp (CI₅₀ = 6.7 μg mL⁻¹).

Fig. 3 Effect of CPAE (pulp and seeds) on superoxide anion production by neutrophils in the lucigenin-dependent chemiluminescence assay. Human neutrophils (5 × 10⁵) were incubated in the presence or absence of CPAE and stimulated with PMA (100 ng mL⁻¹). Luminol-amplified chemiluminescence was measured during 30 min (data are presented as means ± SEM of five independent experiments, *p < 0.05).

3.4. Effect of CPAE on horseradish peroxidase-induced H₂O₂ production

We further examined the effect of aqueous extract of carob pods on horseradish peroxidase (HRP)-induced H₂O₂ production. Results show that CPAE (pulp and seeds) significantly and dose-dependently decreased the luminol-amplified chemiluminescence (Fig. 4). Contrary to all ROS and superoxide anion production, the effect on H₂O₂ is more marked for the pulp (CI₅₀ = 25 μg mL⁻¹) compared to the seeds (CI₅₀ = 87.4 μg mL⁻¹).

Fig. 4 Effect of CPAE (pulp and seeds) on luminol-amplified chemiluminescence of H₂O₂. Luminol-amplified chemiluminescence was measured during 30 min (data are presented as means ± SEM of five independent experiments, *p < 0.05).

3.5. Effect of CPAE on MPO activity and expression from azurophilic granules

We also tested the effect of CPAE on MPO activity and expression. As shown in Fig. 5, CPAE inhibited the MPO activity in a concentration-dependent manner. Moreover, the western blot analysis shows that PMA administration clearly stimulated the expression of MPO, whereas the CPAE co-treatment significantly abolished PMA-induced MPO expression in a dose-dependent manner (60% and 85%, respectively, for the pulp and the seeds with the high concentration of CPAE) (Fig. 6). In both cases, the effect is more marked for the seeds compared to the pulp.

Fig. 5 Human neutrophils (5 × 10⁵) from healthy donors were pretreated or not with CPAE (pulp and seeds) for 30 min and stimulated with PMA (100 ng mL⁻¹) for 3 min. Cells were centrifuged and MPO activity was determined in the supernatants using H₂O₂ and TMB (data are presented as means ± SEM of five independent experiments, *p < 0.05).

Fig. 6 Human neutrophils (5 × 10⁵) from healthy donors were pretreated or not with CPAE (pulp and seeds) for 30 min, stimulated with PMA (100 ng mL⁻¹) for 3 min. Cells were centrifuged and MPO activity was determined with western blotting analysis as described in Material and methods. Western blots from different experiments were scanned; MPO were quantified by densitometry (data are presented as means ± SEM of five independent experiments, *p < 0.05).

3.6. CPAE inhibits PMA-induced p47^phox phosphorylation on Ser328

Using a specific antibody directed against this phosphorylated site, we further analyzed the effect of CPAE on intracellular protein phosphorylation. Results showed that CPAE inhibited PMA-induced p47^phox phosphorylation on Ser328 in a dose-dependent manner (50% and 90%, respectively, for the pulp and the seeds with the high concentration of CPAE). Western blot analysis also showed the same amount of proteins for all tested conditions (Fig. 7).

Fig. 7 Effect of CPAE (pulp and seeds) on PMA-induced p47phox phosphorylation on Ser328. (A) Neutrophils (5 × 10⁵) were pretreated with increasing concentrations of CPAE (10 to 40 μg) for 30 minutes, then incubated with PMA (100 ng mL⁻¹) and immunoblotting with anti-phospho-Ser328 antibody (pSer328) or anti-p47^phox antibody (p47^phox). (B) Western blots from different experiments were scanned; phosphorylated and total p47^phox were quantified by densitometry, and the intensity of phosphorylated p47^phox was corrected for the amount of p47^phox (data are presented as means ± SEM of five independent experiments, *p < 0.05).

3.7. CPAE inhibits PMA-induced lactoferrin release by neutrophils

We further examined the effect of aqueous extract of carob pods (pulp and seeds) on the release of lactoferrin present in the secondary granules of neutrophil granulocytes. As shown in Fig. 8, the use of western blot analysis shows that CPAE co-treatment inhibited the PMA-induced lactoferrin release in a concentration-dependent manner, indicating an inhibition of neutrophil degranulation. The effect is more marked for the seeds (50%) compared to the pulp (40%) with the high carob extract concentration.

Fig. 8 Effect of CPAE (pulp and seeds) on PMA-induced lactoferrin release by neutrophils. Neutrophils (5 × 10⁵) were pretreated with increasing concentrations of CPAE (10 to 40 μg) for 30 minutes, then incubated with PMA (100 ng mL⁻¹) and immunoblotting with anti-lactoferrin antibody. Western blots from different experiments were scanned; lactoferrin was quantified by densitometry (data are presented as means ± SEM of five independent experiments, *p < 0.05).

4. Discussion

In the present study, we investigated the effect of carob pod (Ceratonia siliqua L.) aqueous extracts on human neutrophils myeloperoxidase and on in vitro ROS-scavenging activity.

Our phytochemical study firstly revealed that CPAE is rich in total polyphenols, flavonoids and condensed tannins. In addition, using the ABTS radical-scavenging assay, we found that CPAE of pulp and seeds exhibits a high scavenging capacity, albeit lesser than ascorbic acid, which was used as a reference molecule.²⁰ The use of the HPLC technique revealed the identification of phenolic compounds in carob pods with pyrogallol and catechin as main compounds in the pulp and tannic acid in the seeds. However, previous studies have reported that these molecules are well known for their antioxidant properties.^34,35 The variability in chemical composition as well as in phenolic compounds content can be attributed to the climatic conditions as well as the solvent and the mode of extraction.²⁰ Importantly, we showed that the aqueous carob pods extract (pulp and seeds) inhibits ROS, superoxide anion and H₂O₂ production in a concentration-dependent manner.

We also examined the effect of CPAE and its ability to inhibit the phosphorylation of p47phox–Ser-328. We found that p47phox phosphorylation on p47phox–Ser-328 phosphorylation was modulated by CPAE, which induces the inhibition of NADPH oxidase activity. In addition, we showed that CPAE inhibits the MPO activity in a concentration-dependent manner. MPO inhibition by carob extracts has therefore the ability to reduce the production of hypochlorous acid (HOCl) from H₂O₂ and could attenuate the inflammatory reactions.

More importantly, our present results showed that incubation of neutrophils with CPAE caused a significant inhibition of lactoferrin release from stimulated human cells induced by PMA stimulation in a dose dependent manner. Lactoferrin (LF), an iron-binding protein found in specific granules of neutrophils, enhances OH˙ generation by human neutrophils, by a xanthine oxidase system that generates oxygen radicals.³⁶ In addition, to enhance OH˙ production, previous reports have suggested that LF may play a role in neutrophils bactericidal activity. Moreover, Wang-Iverson et al.³⁷ have shown a defect in bactericidal activity at high bacteria-to-cell ratios with neutrophils depleted of specific granules and LF by pretreatment with PMA.

These results also corroborate those of Corsi et al.,³⁸ who identified mainly gallic acid, (−)-epigallocatechin-3-gallate (EGCG) and (−)-epicatechin-3-gallate (ECG) in an infusion of carob pods with boiling water. Other investigations demonstrated that carob pods contain a large spectrum of hydrolysable tannins such as gallotannins and ellagitannins, which explain the high antioxidant capacity of this fruit.^39–41 However, Temiz et al.⁴² recently concluded that carob extract showed a hepatoprotective effect and antioxidant capacity in rats with ethanol toxicity, probably acting by promoting the antioxidative defense systems.

Several studies provide an overview of the currently available NADPH oxidase inhibitors derived from natural extracts such as polyphenols. Indeed, resveratrol is a naturally occurring polyphenol, which has vasoprotective effects in diabetic animal models and inhibits high glucose (HG)-induced oxidative stress in endothelial cells. It has been reported that HG induces endothelial cells apoptosis through a NF-ΚB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol.^43,44

A previous study using Ginkgo biloba extract was tested for its effect on ROS release during human neutrophils stimulation by a soluble agonist.⁴⁵ However, this extract slowed down the oxygen consumption (respiratory burst) of the stimulated cells by its inhibitory action on NADPH oxidase. Moreover, Ginkgo biloba also had a higher free radical scavenging activity.⁴⁶ Another study used tea polyphenols to demonstrate a beneficial effect against the development of tumor promotion and progression through both ROS scavenging and NADPH oxidase.⁴⁷ On the other hand, Pastene et al.⁴⁸ studied the effects of a standardized extract of apple peel (60% of total polyphenols, 58% of flavonoids) with regard to the intra- and extra-cellular production of ROS in human neutrophils stimulated by Helicobacter pylori, PMA, or fMLP. Indeed, apple-peel extract inhibited the respiratory burst of neutrophils induced by all three activators in a concentration-dependent manner. A better understanding of phenomena involved in the regulation of NADPH oxidase could help to develop novel therapeutic agents for inflammatory diseases, involving abnormal superoxide production by neutrophils. However, a therapeutic goal could lead to lowering the oxidant activity of stimulated neutrophils and the MPO activity. Therefore, the novel strategy of anti-inflammatory and antioxidant therapy is based upon pharmacological agents capable of enhancing the resolution of inflammation and oxidative stress.^49–51

5. Conclusion

In conclusion, our data clearly demonstrate that the aqueous extracts of carob pods (pulp and seeds) inhibit human neutrophils myeloperoxidase and in vitro ROS-scavenging activity owing to, in part, their antioxidant properties.

Declaration of interest

The authors alone are responsible for the content of this paper.

Acknowledgements

The authors would like to thank all the members of U1149, Center for Research on Inflammation, Paris, France, for assistance and helpful discussion. Financial support of the INSERM, the Tunisian Ministry of Higher Education and the Scientific Research are gratefully acknowledged. Financial disclosures: none declared.

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