Ceratonia siliqua leaves exert a strong ROS-scavenging effect in human neutrophils, inhibit myeloperoxydase in vitro and protect against intestinal fluid and electrolytes secretion in rats

Abstract

Chronic inflammation and oxidative stress are induced by biological, chemical and physical factors which are, in turn, associated with an increased risk of several human diseases. Plants are a large source of new bioactive molecules with therapeutic potential. In this respect, the present study was designed to investigate the effects of a Ceratonia siliqua L. leaf aqueous extract (CSLAE) on human neutrophil reactive oxygen species (ROS) production, in vitro myeloperoxidase (MPO) activity and expression as well as the small intestinal fluid and electrolyte secretion. Neutrophils were isolated from whole human blood of healthy volunteers using the ficoll–dextran method and ROS generation and H₂O₂ were measured by luminol amplified chemiluminescence. Superoxide anion generation was also detected by chemiluminescence using the lucigenin method. MPO activity and quantity were measured by the tetramethylbenzidine oxidation method and Western blotting analysis. Concerning the in vivo part, fasted male rats received by gavage either the vehicle (NaCl, 0.9%), the extract at various doses (50, 100 and 200 mg kg⁻¹) or clonidine (1 mg kg⁻¹). An activated charcoal suspension was administered by oral gavage. Thirty minutes after receiving the charcoal meal, rats were euthanized and the small intestine was removed. The length of the small intestine and the distance traveled by the charcoal were recorded. Castor oil-induced hypersecretion in Wistar rats was treated with administration of CSLAE (50, 100 and 200 mg kg⁻¹) and antidiarrheal drug, Atropine (0.1 mg kg⁻¹ i.p.). CSLAE inhibited luminol-amplified chemiluminescence of (PMA)-stimulated neutrophils in a concentration-dependent manner and is able to scavenge superoxide anions and hydrogen peroxide. The CSLAE also reduces significantly and dose-dependently MPO activity and expression. On the other hand, in vivo studies showed that the CSLAE decreased notably and dose-dependently the GIT activity, intestinal fluid and electrolyte concentration. The chemical analysis using a HPLC technique showed that the CSLAE is rich in phenolic compounds, especially kaempferol, tannic acid and catechin hydrate. The intended neutrophil inhibition is introduced as a part of a new strategy for pharmacological modulation of chronic inflammatory and oxidative stress processes. On the basis of these findings, it can be also assumed that CSLAE could be a potential source for novel discovery for antidiarrhoeal drug development.

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1. Introduction

Inflammation and oxidative stress are intimately involved in the development of many chronic diseases including cancer,¹ and cardiovascular² and neurodegenerative diseases³ as well as diabetes and their complications.⁴ Oxidative stress is a biochemical dysregulation of the redox status caused by an imbalance of reactive oxygen or nitrogen species and antioxidative stress defense systems in cells, which triggers activation of numerous signaling pathways resulting in inflammation. The nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (or NOX) are a unique family of enzymes dedicated to the production of reactive oxygen species (ROS) such as the superoxide anion (O₂˙⁻) and hydrogen peroxide (H₂O₂), through single-electron reduction of molecular oxygen with NADPH as the electron donor.⁵ During this process, the cytosolic phox proteins (phox: phagocyte oxidase) p47^phox, p67^phox, p40^phox and Rac2 translocate to the plasma membrane or to membranes of specific granules, where they associate with the membrane-bound components (p22^phox, gp91^phox) to assemble the catalytically active oxidase.⁶ Superoxide anion is the precursor of other ROS and is immediately transformed into hydrogen peroxide (H₂O₂), spontaneously or through enzymatic dismutation by superoxide dismutase. Interaction between H₂O₂ and superoxide anion can give rise to the hydroxyl radical, one of the most powerful oxidants. Moreover, hydrogen peroxide is a substrate of myeloperoxidase (MPO), which catalyses its transformation into highly toxic molecules such as hypochlorous acid, chloramines and tyrosyl radicals.⁷ Recently, MPO has been found to be implicated in a multitude of diseases, including atherosclerosis,⁸ myocardial infarction,⁹ atrial fibrillation,¹⁰ multiple sclerosis,¹¹ lung cancer,¹² transplant rejection¹³ and rheumatoid arthritis.¹⁴

Hypersecretion is a prevalent symptom and sign of patients with inflammatory bowel diseases (IBDs) and affects most patients.¹⁵ The effects of castor oil are mediated by ricinoleic acid, a hydroxylated fatty acid released from castor oil by intestinal lipases. The triglyceride present in castor oil is hydrolyzed in the small bowel by the action of lipases into glycerol and the active agent, ricinoleic acid.¹⁶ Ricinoleic acid induces diarrhoea by hypersecretory response.¹⁷ Due to the polar nature of ricinoleic acid, it is poorly absorbed and its presence in the small intestine results a modification in permeability of the intestinal mucosa to electrolytes and stimulates peristaltic activity in the intestine which in turn produces hypersecretion, and fluid accumulation occur.¹⁸ The ricinoleic acid produces many irritating and inflammatory actions on the intestinal mucosa leading to the release of prostaglandins¹⁹ by colonic cells²⁰ or stimulates adenyl cyclase in the intestinal epithelial cells. Prostaglandins, thus released, promote vasodilatation, smooth muscle contraction, and mucus secretion in the small intestines and thereby produces diarrhoea.²¹ Ceratonia siliqua L. (Fabacae) has been widely cultivated in Mediterranean countries for years. This tree was distributed by Arabs in the Mediterranean area.²² Leaves are 3–7 cm long, alternate, pinnate, with or without a terminal leaflet. Carob does not shed its leaves in the autumn but only in July every second year, and it only partially renews leaves in spring.²³ The fruits are more important in food industry and are a source of many products such as gum, sugar, and alcohol.²⁴ Thanks to its richness in the tannin compounds, the carob tree is used in traditional medicine as an antidiarrheal and diuretic.²⁵ Leaves and pods of carob exerted diverse physiological functions such as its antioxidant activity.^26–33 Indeed, the extracts of the carob tree showed significant radical scavenging activity³⁴ and a remarkable ability to inhibit tumor cell proliferation.²⁶ Carob tree extracts contain anti-proliferative agents that could be of practical importance in the development of functional foods and/or chemopreventive drugs.³³ In addition, leaves and pods of carob are rich in polyphenols and flavonoids.³⁵

The aim of the present study was to examine the effect of carob tree leaves aqueous extract (CSLAE) on ROS production and MPO activity and expression in vitro as well as its protective effect on castor oil-induced small intestinal hypersecretion in rat.

2. Materials and methods

2.1. Chemicals and reagents

Phorbolmyristate acetate (PMA), luminol, lucigenin, and Horseradish peroxidase (HRPO) were from Sigma-Aldrich (Saint-Quentin Fallavier, France). HBSS and HEPES were from Gibco. Ficoll and dextran T₅₀₀ were from GE Healthcare. Atropine and methanol were purchased from Sigma-Aldrich Co. (Germany). All other chemicals and reagents used were of analytical grade.

2.2. Ethics statement

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

Animals were used in accordance with the local ethics committee of Tunis University for the use and care of animals in accordance with the NIH recommendations.³⁶

The necessary permits for the field studies and collection of Ceratonia siliqua leaves 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 Beja-Tunisia. The voucher specimens have been deposited with the herbarium of the Higher Institute of Biotechnology of Beja.

2.3. Ceratonia siliqua leaves aqueous extract preparation

The Ceratonia siliqua leaves were collected from the region of Tabarka (North-West of Tunisia) during June 2015. Briefly, the plant material was later dried in an incubator at 37 °C during 72 hours and powdered in an electric blender (MoulinexOvatio 2, FR). Powder of leaves was dissolved in bi-distilled water (1/10; w/v) and incubated at room temperature for 24 h in a shaking bath. The sample was filtered through a colander (0.5 mm mesh size). Finally, the lyophilized CSLAE (extraction yield = 10%) was immediately used for in vitro and in vivo experiments.

2.4. Phytochemical investigation

A total polyphenol compound in extract was determined by the Folin–Ciocalteu assay according to the method described by Singleton.³⁷ The absorbance was read at 725 nm UV-Vis spectrophotometer and all the experiments were performed in triplicate. The content of totalpolyphenol is reported as gallic acid equivalents (EqGA) by reference to standard curve. The total flavonoid content was determined by aluminum chloride colorimetric method.³⁸

In brief, 0.5 ml of extract was mixed with 1.5 ml of methanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water. After incubation at room temperature for 30 min, the absorbance of the reaction mixture was measured at 415 nm with a UV spectrophotometer. The amount of 10% aluminum chloride was substituted by the same amount of distilled water in the blank. Total flavonoid contents were calculated as catechin equivalent. Contents of total tannins were determined spectrophotometrically using the method of Folin–Ciocalteu reaction utilizing tannic acid as standard.³⁹ We take use of dinitrosaIicyIic acid (DNS) reagent for the determination of total and reducing sugars.⁴⁰ Finally, the fiber content was determined by AOAC method 991.42.⁴¹

2.5. Characterization of phenolic compounds by HPLC technique

Dried samples from leaves were hydrolyzed according to the slightly modified method of Proestos et al.⁴² 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. Twenty milliliters of methanol was 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 to 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 kept 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.

2.6. Isolation of human neutrophils and measurement of ROS production by chemiluminescence

Venous blood was collected from healthy adult volunteers and neutrophils were isolated by dextran sedimentation and density gradient centrifugation. Isolated human neutrophils was counted and incubated with extract at concentrations of 0, 25, 50, 100, 200 and 250 μg ml⁻¹ during 30 min and their viability was determined with the trypan blue exclusion method.⁴³ Isolated cells were resuspended in HBSS at a concentration of 1 million per ml. Cells suspensions (5 × 10⁵) in 0.5 ml of HBSS containing 10 mM luminol in the presence or absence of CSLAE were added after dilution using a micropipette, preheated at 37 °C in the thermostated chamber of a lumino-meter (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.⁴⁴

2.7. Measurement of superoxide and H₂O₂ inhibition using chemiluminescence assay

The generation of superoxide anion is measured by the chemiluminescence of lucigenin-method.⁴⁵ 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).⁴⁶

2.8. Measurement of MPO activity and quantity after azurophilic granules preparation

Neutrophils (5 × 10⁵) from healthy donors were pretreated or not with CSLAE for 30 min, stimulated with 100 ng ml⁻¹ of phorbol 12-myristate 13-acetate at 37 °C during 3 min. Cells were lysed by nitrogen cavitation and the granule fraction was purified by Percol gradient centrifugation. The granules were sonicated in 0.2 CTAB. The supernatant of samples was assayed for MPO activity by measuring the hydrogen peroxide (H₂O₂)-dependent oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB). In its oxidized form, TMB has a blue color, which was measured spectrophotometrically at 650 nm. The reaction mixture for analysis consisted of a 25 μl tissue sample, 25 μl of TMB (final concentration 0.16 mM) dissolved in dimethylsulfoxide, and 200 μl of H₂O₂ (final concentration 0.30 mM) dissolved in phosphate buffer (0.08 M, pH 5.4). The reaction mixture was incubated for 3 minutes at 37 °C and the reaction was stopped by adding 1 ml of sodium acetate (0.2 M, pH 3.0), after that, absorbance at 650 nm was measured.⁴⁷ MPO expression was monitored by immunoblotting using monoclonal antibodies.

2.9. Gastrointestinal motility and hypersecretion tests

Healthy adult male Wistar rats (weighing 220–250 g; housed five per cage) were purchased from Society of Pharmaceutical Industries of Tunisia (SIPHAT, Ben-Arours, TN). They were provided with standard food (standard pellet diet-BadrUtique-TN) and water ad libitum and maintained in animal house at controlled temperature (22 ± 2 °C) with a 12 h light–dark cycle.

The rats were divided into half a dozen groups.

Gastrointestinal transit (motility) was investigated in rats according to the method of Aye-Than et al.,⁴⁸ as described by Odetola and Akojenu.⁴⁹ Adult male rats were divided into five groups of ten animals each to determine the effect of CSLAE on normal intestinal transit of a marker meal (10% charcoal suspension in 5% gum acacia). Group I received 10 ml kg⁻¹ of distilled water, while animals in groups II–IV were treated orally with the graded doses of the aqueous extract (50, 100 and 200 mg kg⁻¹ respectively) and those in group V received the clonidine (1 mg kg⁻¹, b.w., i.p.). Two hours after the administration of distilled water, extract or reference molecule, each animal was given the standard charcoal meal (10% activated charcoal suspension in 5% gum acacia). The rats were sacrificed 30 min after the administration of the charcoal meal, the abdomen were opened and the small intestine was immediately isolated. The length of the intestine from pylorus to the caecum (LSI) and the distance traveled by the charcoal (LM) were measured. The peristaltic index (PI) for each animal was calculated, expressed as percentage of the distance traveled by the charcoal meal relative to the total length of the small intestine. The percentage inhibition relative to the control was also assayed as:

PI = LM/LSI × 100%

where PI = peristaltic index, LM = length of charcoal meal; LSI = length of small intestine

% inhibition = (control − test)/control × 100

To evaluate the effect of extract on hypersecretion, male Wistar rats fasted for 16 h were randomly allocated to five groups of ten animals each. Group I received 10 ml kg⁻¹ of distilled water, group II, III and IV received 50, 100 and 200 mg kg⁻¹ of body weight of the aqueous Ceratonia siliqua leaf extract orally while, group V was given atropine (0.1 mg kg⁻¹ i.p.). After 1 h of treatment with extract, distilled water or standard drug, diarrhoea was induced by administration of castor-oil (5 ml kg⁻¹, b.w., p.o.) to each rat and observed for 4 h. The characteristic diarrhoeal droppings were noted in the absorbent paper placed beneath the individual rat perforated cages.⁵⁰ Concerning the intraluminal fluid, the small intestine was removed, tied with thread at the pyloric end and the ileo-caecal junction, and weighed. The intestinal content was milked into a graduated tube and their volume measured. The intestine was reweighed and the difference between full and empty intestines was calculated. The Na⁺ and K⁺ concentrations in the supernatant after centrifuging the intraluminal fluid were measured by flame photometry.⁵¹

2.10. Statistical analysis

The data were analyzed by one-way analysis of variance (ANOVA) and were expressed as means ± standard error of the mean (S.E.M.). The data are representative of 3–10 independent experiments. All statistical tests were two-tailed, and a p value of 0.05 or less was considered significant.

3. Results

3.1. RP-HPLC analysis and phytochemical investigation

The use of HPLC technique (Fig. 1) revealed the identification of various phenolic compounds in CSLAE, as shown in Table 1. The principal compounds are: kaempferol (77 ± 2.43%), tannic acid (13 ± 0.45%), catechin hydrate (4.30 ± 0.34%) and polydatin (0.85 ± 0.22%).

Fig. 1 Chromatographic profile of aqueous extract of Ceratonia siliqua leaves (CSLAE).

Table 1 Identification and quantification of phenolic compounds in Ceratonia siliqua L. leaves by HPLC analysis^a

	Phenolic compounds	Retention time (min)	Relative abundance (%)
a Values are expressed as mean ± SEM (n = 3).
1	Kaempferol	3.63	77 ± 2.43
2	Tannic acid	4.60	13 ± 0.45
3	Catechin hydrate	4.71	4.30 ± 0.34
4	Gallic acid	5.20	0.43 ± 0.02
5	Polydatin	5.40	0.85 ± 0.22
6	Isorhamnetin	5.67	0.64 ± 0.05
7	Chlorogenic acid	6.35	0.32 ± 0.03
8	A-2, hydroxyphenyl acetic	6.70	0.08 ± 0.01
9	Fraxidin	6.86	0.20 ± 0.04
10	Resorcinol	7.26	0.20 ± 0.03
11	Daidzein	8.90	0.08 ± 0.07
12	Morin	18.30	0.03 ± 0.01
13	Flavonol	20.50	0.084 ± 0.01

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The results of the quantitative evaluation of polyphenol compounds, sugars and fibers are presented in Table 2. We firstly showed that CSLAE is rich in these compounds. In fact, total polyphenol content was 62.5 ± 6.52 mg GAE per g DM, a higher rate of dietary fiber (16.2 ± 1.12% of DM), whereas total sugar level was 80.00 ± 3.24 g l⁻¹.

Table 2 Phytochemical compounds in Ceratonia siliqua leaves^a

Composition	Contents
a The data are expressed as means ± standard error of the mean (SEM) (n = 5).
Polyphenols (mg per g DM)	62.5 ± 6.52
Flavonoïdes (mg per g DM)	21.32 ± 2.22
Tannins (mg per g DM)	38.50 ± 4.33
Dietary fibers (% of DM)	16.2 ± 1.12
Totals sugars (g l^−1)	80.00 ± 3.24
Reduced sugars (g l^−1)	45.00 ± 4.4

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3.2. Effect of CSLAE on ROS production and neutrophil viability

Neutrophils (5 × 10⁵) were incubated with different CSLAE concentrations (0–40 μg ml⁻¹) for 30 min, treated in the presence or absence of PMA and the production of ROS were measured by luminol-amplified chemiluminescence. Our results show that the CSLAE significantly and dose-dependently inhibited luminol-amplified chemiluminescence in neutrophils stimulated with PMA.

To assess specifically the effect of the extract on superoxide anion production, we studied its effect using a lucigenin-dependent chemiluminescence assay. We showed that the CSLAE significantly reduces PMA-induced superoxide anion production at low concentrations in a dose dependent manner suggesting that the extract was more effective in scavenging of superoxide anions. Further, the effect of the CSLAE on H₂O₂ production was tested in a cell free system using horseradish peroxidase (HRPO). Results show also that CSLAE significantly and dose dependently decreased the luminol-amplified chemiluminescence (Fig. 2).

Fig. 2 Effect of CSLAE on luminol-amplified chemiluminescence of human neutrophils. Human neutrophils (5 × 10⁵) were incubated in the presence or absence CSLAE, stimulated with PMA (100 ng ml⁻¹) and luminol-amplified chemiluminescence was measured during 30 min (mean ± S.E.M. of five experiments,*: p < 0.05 compared to control group and ^#: p < 0.05 compared to PMA group).

To verify that the inhibitory effect of the CSLAE was not due to its toxic activity on neutrophils, we examined its effect on cell viability using trypan blue exclusion assay. The result shows that after 30 min of neutrophils incubation with increasing concentrations of extract, cell viability was greater than 96% (Fig. 3). Thus cell viability was not affected by Ceratonia siliqua leaves extract.

Fig. 3 Effect of the CSLAE on neutrophils viability. Isolated human neutrophils were exposed to extract at concentrations. Viability was evaluated with adding Trypan Blue, and blue cells were counted. % of viable cells was expressed compared to control conditions (without CSLAE). Results are expressed as mean ± SEM, n = 5, *p < 0.05.

3.3. Effect of CSLAE on MPO activity and expression from azurophilic granules

Myeloperoxidase (MPO) released from azurophilic granules catalyses the transformation of H₂O₂ in the presence of a halogen (Cl⁻, Br⁻, I⁻) into highly toxic molecules such as hypochloric acid (HOCl). Other reactions between OCl⁻ and H₂O₂ can lead to the formation of singlet oxygen (¹O₂). Most of the hypochloric acid thus generated is converted into toxic chloramines. Thus NADPH oxidase activation and MPO degranulation act in synergy during the inflammatory process. To test whether CSLAE could also affect neutrophil degranulation, human neutrophils were treated or not with extract and MPO release was assessed by measuring its activity and expression. As shown in Fig. 4, CSLAE 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 CSLAE co-treatment significantly abolished PMA-induced MPO expression in a dose dependent manner (Fig. 5).

Fig. 4 Human neutrophils (5 × 10⁵) from healthy donors were pretreated or not with CSLAE for 30 min, 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. (Mean ± SEM of five experiments, *: p < 0.05 compared to control group and ^#: p < 0.05 compared to PMA group).

Fig. 5 Human neutrophils (5 × 10⁵) from healthy donors were pretreated or not with CSLAE for 30 min, stimulated with PMA (100 ng ml⁻¹) for 3 min. Cells were centrifuged and MPO quantity was determined with western blotting analysis. 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 compared to control group and ^#: p < 0.05 compared to PMA group).

3.4. Effect of CSLAE on small intestinal transit, enteropooling and electrolytes secretion

The aqueous extract of carob tree leaves and standard drug treatments significantly slowed down the propulsion of charcoal meal through the gastrointestinal tract. Indeed, the extract treatment at various doses (50, 100 and 200 mg kg⁻¹) significantly (p < 0.05) slowed down the propulsion of charcoal meal through the gastrointestinal tract when compared to the control (Table 3). The CSLAE was found to be effective in a dose dependent manner against castor oil induced diarrhoea on experimental rats at all tested doses. At various doses, the extract produced a significant decrease in the severity of diarrhoea in terms of reduction in the rate of defecation and consistency of faeces in rats. At the dose of 200 mg kg⁻¹ body weight, the extract showed significant antidiarrhoeal activity showing 82.14 ± 2.04% reduction in hypersecretion comparable to that of the standard drug atropine that showed 84.44 ± 3.34% reduction in diarrhoea (Table 3). In this respect, CSLAE was also found to possess anti-enteropooling activity. Indeed, the oral administration of castor oil produced a significant increase in the intestinal fluid (3.8 ± 0.33 ml) as compared to normal rats (0.12 ± 0.03 ml). CSLAE, when given orally 1 h before castor oil, significantly inhibited the enteropooling (1.6 ± 0.621 ml) and the volume of intestinal fluid was comparable to that obtained with the standard drug (1.4 ± 0.12 ml) (Table 4). On another hand, treatment of rats with castor oil significantly increased the Na⁺ concentration to 12.21 ± 1.23 meq. l⁻¹ as compared to the control group (8.32 ± 0.41 meq. l⁻¹). The reference molecule pretreatment did not alter the Na⁺ concentration in intestinal fluid as compared to the castor oil treated group. However, the CSLAE induced a decrease of Na⁺ concentration in a dose dependent manner. None of the treatments produced a significant change in the K⁺ concentration although it was low in atropine pretreated rats (Table 4).

Table 3 Effect of Ceratonia siliqua leaves aqueous extract (CSLAE) on GIT and hypersecretion in rat^a

		Peristaltic index (%)	% of inhibition	Mean defecation (4 h)	% of decrease
a Animals were pre-treated with various doses of CSLAE (50, 100 and 200 mg kg^−1 b.w., p.o.), reference molecule (clonidine, 1 mg kg^−1), two hours after, rats received the standard charcoal meal (10% charcoal in 5% gum arabic) or vehicle (NaCl, 0.9%). Concerning the hypersecretion, one hour after, animals received castor oil (5 ml kg^−1, b.w., p.o.), reference molecule atropine (0.1 mg kg^−1 i.p.) or vehicle (NaCl, 0.9%) by gavage and observed for defecation up to 4 h. Results are expressed as mean ± SEM; n = 10 in each group. Data was analyzed by Statview ANOVA. Values with different superscripts differ significantly from each other (P < 0.05).
Control (H2O, 10 ml kg^−1)		78.62 ± 2.13^a	—	—	—
Clonidine (2 mg kg^−1)		34.42 ± 3.21^b	56.22	—	—
Atropine (0.1 mg kg^−1 i.p.)		—	—	2.10 ± 0.34^a	84.44 ± 3.34
Castor-oil (5 ml kg^−1)		—	—	13.5 ± 1.65^b	—
CSLAE	(50 mg kg^−1)	69.42 ± 2.22^c	12.00	6.00 ± 1.21^c	55.56 ± 2.44
	(100 mg kg^−1)	56.00 ± 1.50^d	29.00	4.34 ± 0.45^c	69.50 ± 2.23
	(200 mg kg^−1)	42.11 ± 1.32^e	46.44	2.42 ± 0.62^a	82.14 ± 2.04

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Table 4 Effect of Ceratonia siliqua leaves aqueous extract (CSLAE) on intestinal fluid accumulation and electrolyte concentration in rat^a

		Intestinal fluid (ml)	% of reduction	Na^+ (meq. l^−1)	K^+ (meq. l^−1)
a Animals were pre-treated with various doses of CSLAE (50, 100 and 200 mg kg^−1 b.w., p.o.), reference molecule atropine (0.1 mg kg^−1 i.p.) or vehicle (NaCl, 0.9%). One hour after, animals received castor oil (5 ml kg^−1, b.w., p.o.) and observed enteropooling up to 4 h. Results are expressed as mean ± SEM; n = 10 in each group. Data was analyzed by Statview ANOVA. Values with different superscripts differ significantly from each other (P < 0.05).
Control (H2O, 10 ml kg^−1)		0.12 ± 0.03^a	96.84	8.32 ± 0.41^a	37.34 ± 3.45^a
Atropine (0.1 mg kg^−1 i.p.)		1.4 ± 0.12^b	63.15	12.21 ± 1.23^b	26.26 ± 3.72^b
Castor-oil (5 ml kg^−1)		3.8 ± 0.33^c	—	12.02 ± 0.72^b	36.63 ± 2.22^a
CSLAE	(50 mg kg^−1)	2.9 ± 0.21^d	23.7	11.45 ± 0.34^b	35.82 ± 2.66^a
	(100 mg kg^−1)	2.2 ± 0.34^d	42.10	10.6 ± 0.44^b	34.52 ± 2.91^a
	(200 mg kg^−1)	1.6 ± 0.62^e	58.1	8.54 ± 0.55^a	32.33 ± 1.11^a

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4. Discussion

A novel strategy of anti-inflammatory and antioxidant therapy is based upon pharmacological natural agents capable to enhance the treatment of inflammation and oxidative stress as well as their complications. In this respect, we investigated the effect of carob tree leaves aqueous extract on human neutrophils myeloperoxidase, in vitro ROS-scavenging activity as well as the small intestinal fluid and electrolytes secretion.

Firstly, our phytochemical study revealed that CSLAE is rich in total polyphenols, tannins and favonoids. In addition, we found that carob tree leaves are richer in fiber than the carob pods.⁵² However, the carob extract is more richer in total and reduced sugars compared to CSLAE.⁵² The use of the HPLC technique revealed the identification of many phenolic compounds with kaempferol, tannic acid, catechin hydrate and polydatin in the carob tree leaves. Though, the pyrogallol, catechin, tannic acid, gallic acid and ferulic acid as the main compounds in the carob.³⁴

The variability in chemical composition can be attributed to the climatic conditions and to different parts of the same plant species as well as the solvent and the mode of extraction.⁵³

Using the luminol-amplified chemiluminescence, a technique which detects the total ROS production. Results show that, in PMA stimulated neutrophils, the CSLAE notably inhibits total ROS production in a dose-dependent manner. Secondly, using the lucigenin-dependent chemiluminescence assay, a specific method to measure superoxide anions (O₂˙⁻) production, we found that the CSLAE at low concentrations highly inhibited neutrophils O₂˙⁻ production. In addition, we showed also that the extract significantly inhibited the intracellular production of H₂O₂, in a dose-dependent manner. In contrast, these effects were not due to a toxic effect of the extract since cell viability was not affected. Superoxide anion plays an important role in the formation of other ROS such as hydrogen peroxide, hydroxyl radical, and singlet oxygen, which induce oxidative damage in lipids, proteins and DNA.⁵⁴ It has been found that polyphenol compounds are highly absorbed by human cells⁵⁵ and stored in vivo in some tissues.^56–58 In addition, most interest has been devoted to the antioxidant activity of flavonoids, which is due to their ability to reduce free radical formation and to scavenge free radicals. The capacity of flavonoids to act as antioxidants in vitro has been the subject of several studies in the past years, and important structure–activity relationships of the antioxidant activity have been established. Most ingested flavonoids are extensively degraded to various phenolic acids, some of which still possess a radical-scavenging ability. Both the absorbed flavonoids and their metabolites may display an in vivo antioxidant activity.⁵⁴ For example, the kaempferol can be metabolized in the small intestine (to glucuronides and sulfoconjugates) by intestinal conjugation enzymes.⁵⁹ After absorption, kaempferol is extensively metabolized in the liver to form glucurono- and sulfo-conjugated forms.^59,60 These conjugated forms of kaempferol can reach systemic circulation and tissues.⁶¹ Numerous in vitro and some animal studies support a role of kaempferol in the prevention and/or treatment of various diseases, such as inflammation. In addition, others studies have shown that the presence of a double bond at C₂–C₃ in conjugation with an oxo group at C₄, and the presence of hydroxyl groups at C₃, C₅ and C_4′, are important structural features involved in the antioxidant activity of kaempferol.⁶² Similar to many polyphenols, tannic acid has been shown to possess antioxidant.^63,64 The antioxidant mechanism of tannic acid is still far from being fully understood; therefore, it requires further investigation. For example, in the presence of copper ions, tannic acid acts either as a prooxidant, promoting DNA damage,^65,66 or as an antioxidant, suppressing hydroxyl radical formation.⁶⁴ On the other hand, Kondo et al.,⁶⁷ show that the (−)-epigallocatechin gallate was found to be the most effective scavenger among tea catechins for the superoxide anion, hydroxyl radical and 1,1-diphenyl-3-picrylhydrazyl radical.

The CSLAE is a rich source of antioxidants and could be beneficial in many diseases due to its inhibitory action on neutrophil ROS production and also by scavenging superoxide anion and H₂O₂ which is the most diffusible ROS. Thus the CSLAE can protects tissues of different organs from H₂O₂, limiting its bystander toxic effects. These results suggest that the CSLAE is able also to affect NADPH oxidase activation, probably by interfering with the neutrophil signaling pathways involved in NADPH oxidase activation. In this context, we examined the effect of carob pods aqueous extract and its ability to inhibit the phosphorylation of p47^phox Ser-328 which induces the inhibition of NADPH oxidase activity.³⁴

Add to that, our present results showed that the incubation of neutrophils with CSLAE caused a strong reduction of MPO activity and expression in stimulated human cells induced by PMA stimulation in a dose dependent manner. MPO generates several reactive species, including hypochlorous acid (HOCl). It functions as the major enzymatic catalysts for initiation of LDL oxidation.⁶⁸ Immunohistochemically studies have demonstrated the presence of MPO and MPO-derived HOCl in human atherosclerotic lesions. Like other flavonoids, kaempferol has anti-inflammatory properties. Indeed, numerous reports have shown that kaempferol, kaempferol glycosides and/or kaempferol-containing plants have anti-inflammatory activity not only in vitro but also in vivo.⁶⁹ However, several studies have been shown recently the inhibition of ROS production and MPO activity by many plants extracts and molecules as punicic acid;⁷⁰ aqueous pomegranate peels extract;⁷¹ Myrtle berry seed aqueous extract.⁷² In part, a recent study shows that a higher consumption of dietary fiber is associated with lower hs-CRP levels in persons with type 1 diabetes. High dietary fiber intake (>30 g per day) may play a role in reducing inflammation in this population.⁷³

The aqueous extract of carob tree leaves inhibited gastrointestinal propulsion, fluid accumulation and electrolyte concentration in small intestine. This makes it beneficial as a preventive agent. This is indicative of the ability of the plant to alter normal peristaltic movement and, hence, decrease the movement of materials in the intestinal tract allowing greater time for absorption.⁷⁴ Flavonoids has been ascribed with ability to inhibit intestinal motility and hydro-electrolytic secretion; flavonoids have been ascribed the ability to inhibit contractions induced by spasmogenics.⁷⁵ It is possible that the flavonoids present in the aqueous extract may be responsible for these effects. In addition, tannic acid present in many of the plant extracts are shown to form a complex with the luminal proteins which then precipitate and form a coat over the intestinal line and reduce secretion in a model of charcoal induced hyper peristalsis.⁷⁶

5. Conclusion

Our results show that Ceratonia siliqua leaves exert a strong antioxidant effect via scavenging of reactive oxygen species and they could have an anti-inflammatory effect by inhibiting neutrophil MPO activity and expression, thus limiting their toxic effects. The results indicate also that CSLAE possesses significant anti-diarrhoeal activity due to its inhibitory effect on gastrointestinal propulsion, fluid and electrolytes secretion. Carob tree leaves could be used to extract for medicinal applications.

Conflict of interest

Only the authors are responsible for the content of this paper.

Abbreviations

CSLAE	Ceratonia siliqua leaves aqueous extract
HPLC	High performance liquid chromatography
HRP	Horseradish peroxidase
H2O2	Hydrogen peroxide
MPO	Myeloperoxidase
PMA	Phorbolmyristate acetate
ROS	Reactive oxygen species

Acknowledgements

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

References

1. S. Reuter, S. C. Gupta, M. M. Chaturvedi and B. B. Aggarwal, Oxidative stress, inflammation, and cancer: How are they linked?, Free Radical Biol. Med., 2010, 49, 1603–1616.
2. E. J. Reverri, B. M. Morrissey, C. E. Cross and F. M. Steinberg, Inflammation, oxidative stress, and cardiovascular disease risk factors in adults with cystic fibrosis, Free Radical Biol. Med., 2014, 76, 261–277.
3. B. Uttara, A. V. Singh, P. Zamboni and R. T Mahajan, Oxidative Stress and Neurodegenerative Diseases: A Review of Upstream and Downstream Antioxidant Therapeutic Options, Curr. Neuropharmacol., 2009, 7, 65–74.
4. F. Giacco and M. Brownlee, Oxidative stress and diabetic complications, Circ. Res., 2010, 107, 1058–1070.
5. J. D. Lambeth, NOX enzymes and the biology of reactive oxygen, Nat. Rev. Immunol., 2004, 4, 181–189.
6. J. El-Benna, P. M. C. Dang and A. Périanin, Peptide-based inhibitors of the phagocyte NADPH oxidase, Biochem. Pharmacol., 2010, 80, 778–785.
7. J. M. Robinson, Phagocytic leukocytes and reactive oxygen species, Histochem. Cell Biol., 2009, 131, 465–469.
8. R. Zhang, M. L. Brennan, X. Fu, R. J. Aviles, G. L. Pearce, M. S. Penn, E. J. Topol, D. L. Sprecher and S. L. Hazen, Association between myeloperoxidase levels and risk of coronary artery disease, JAMA, J. Am. Med. Assoc., 2001, 286, 2136–2142.
9. M. Nahrendorf, D. Sosnovik, J. W. Chen, P. Panizzi, J. L. Figueiredo, E. Aikawa, P. Libby, F. K. Swirski and R. Weissleder, Activatable magnetic resonance imaging agent reports myeloperoxidase activity in healing infarcts and noninvasively detects the anti-inflammatory effects of atorvastatin on ischemia-reperfusion injury, Circulation, 2008, 117, 1153–1160.
10. V. Rudolph, R. P. Andrie, T. K. Rudolph, K. Friedrichs, A. Klinke, K. Sydow, M. Didié, A. Seniuk, E. C. von Leitner, K. Szoecs, J. W. Schrickel, H. Treede, U. Wenzel, T. Lewalter, G. Nickenig, W. H. Zimmermann, T. Meinertz, R. H. Böger, H. Reichenspurner, B. A. Freeman, T. Eschenhagen, H. Ehmke, S. L. Hazen, S. Willems and S. Baldus, Myeloperoxidase acts as a profibrotic mediator of atrial fibrillation, Nat. Med., 2010, 16, 470–474.
11. E. Gray, T. L. Thomas, S. Betmouni, N. Scolding and S. Love, Elevated myeloperoxidase activity in white matter in multiple sclerosis, Neurosci. Lett., 2008, 444, 195–198.
12. M. B. Schabath, M. R. Spitz, W. K. Hong, G. L. Delclos, W. F. Reynolds, G. B. Gunn, L. W. Whitehead and X. Wu, A myeloperoxidase polymorphism associated with reduced risk of lung cancer, Lung Cancer, 2002, 37, 35–40.
13. F. K. Swirski, M. Wildgruber, T. Ueno, J. L. Figueiredo, P. Panizzi, Y. Iwamoto, E. Zhang, J. R. Stone, E. Rodriguez, W. J. Chen, J. M. Pittet, R. Weissleder and M. Nahrendorf, Myeloperoxidase-rich Ly-6C⁺ myeloid cells infiltrate allografts and contribute to an imaging signature of organ rejection in mice, J. Clin. Invest., 2010, 120, 2627–2634.
14. L. K. Stamp, I. Khalilova, J. M. Tarr, R. Senthilmohan, R. Turner, R. C. Haigh, P. G. Winyard and A. J. Kettle, Myeloperoxidase and oxidative stress in rheumatoid arthritis, Rheumatology, 2012, 51, 1796–1803.
15. H. Heimo and M. D. Wenzl, Diarrhea in Chronic Inflammatory Bowel Diseases, Clin. Gastroenterol., 2012, 41, 651–675.
16. B. C. Semwal, A. Goyal and V. Varshney, Antidiarrheal activity of Rhododendron arborium leaves in Wistar rats, Int. J. Pharm. Res. Bio-Sci., 2014, 3, 591–600.
17. P. Sharma, G. Vidyasagar, S. Singh, S. Ghule and B. Kumar, Antidiarrhoeal activity of leaf extract of celosia argentea in experimentally induced diarrhoea in rats, J. Adv. Pharm. Technol. Res., 2010, 1, 41–48.
18. K. M. Prasanth, V. Suba, B. Ramireddy and B. P. Srinivas, Evaluation of antidiarrhoeal activity of ethanolic extract of celtis timorensis leaves in experimental rats, Asian J. Pharm. Clin. Res., 2014, 7, 185–188.
19. M. K. Rahman, M. A. U. Chowdhury, M. T. Islam, M. A. Chowdhury, M. E. Uddin and C. D. Sumi, Evaluation of Antidiarrheal Activity of Methanolic Extract of Maranta arundinacea L. Leaves, Adv. Pharmacol. Sci., 2015 DOI:10.1155/2015/257057.
20. F. Awouters, C. J. E. Niemegeers, F. M. Lenaerts and P. A. J. Janseen, Delay of castor oil in rats; a new way to evaluate inhibitors of prostaglandin biosynthesis, J. Pharm. Pharmacol., 1978, 30, 4–45.
21. M. Asadujjaman, A. U. Mishuk, M. A. Hossain and U. K. Karmakar, Medicinal potential of Passiflora foetida L. plant extracts: biological and pharmacological activities, J. Integr. Med., 2014, 12, 121–126.
22. S. Kumazawa, M. Taniguchi, Y. Suzuki, M. Kwon and T. Nakayama, Antioxidant activity of polyphenols in carob pods, J. Agric. Food Chem., 2002, 50, 373–377.
23. R. Fletcher, Carob agroforestry in Portugal and Spain. The Australian New Crops Newsletter., 1997, 7,http//:www.newcrops.uq.edu.au/newslett/ncnl17-13.htm.
24. H. El Hajaji, N. Lachkar, K. Alaoui, Y. Cherrah, A. Farah, A. Ennabili, B. El Bali and M. Lachkar, Antioxidant activity, phytochemical screening, and total phenolic content of extracts from three genders of carob tree barks growing in Morocco, Arabian J. Chem., 2011, 4, 321–324.
25. H. Loeb, Y. Vandenplas, P. Wursch and P. Guesry, Tannin-rich carob pod for the treatment of acute-onset diarrhea, J. Pediatr. Gastroenterol. Nutr., 1989, 8, 480–485.
26. L. Corsi, R. Avallone, F. Cosenza, F. Farina, C. Baraldi and M. Baraldi, Anti-proliférative effects of Ceratonia siliqua L. on mouse hepatocellular carcinoma cell line, Fitoterapia, 2002, 73, 674–684.
27. L. Custódio, E. Fernandes, A. L. Escapa, R. Aligué, F. Alberício and A. Romano, Antioxidant activity and in vitro inhibition of tumor cell growth by leaf extracts from the carob tree (Ceratonia siliqua L.), Pharm. Biol., 2009, 47, 721–728.
28. L. Custódio, E. Fernandes, A. L. Escapa, R. Aligué, F. Alberício and A. Romano, In vitro cytotoxic effects and apoptosis induction by a methanol leaf extract of carob tree (Ceratonia siliqua), J. Med. Plants Res., 2011a, 5, 1987–1996.
29. L. Custódio, E. Fernandes, A. L. Escapa, R. Aligué, F. Alberício, N. Neng, J. M. F. Nogueira and A. Romano, Phytochemical profile, antioxidant and cytotoxic activities of carob tree (Ceratonia siliqua L.) germ flour extracts, Plant Foods Hum. Nutr., 2011b, 66, 78–84.
30. L. Custódio, E. Fernandes, A. L. Escapa, R. Aligué, F. Alberício, N. Neng, J. M. F. Nogueira and A. Romano, Antioxidant and cytotoxic activities of carob tree fruit pulps are strongly influenced by gender and cultivar, J. Agric. Food Chem., 2011c, 59, 7005–7012.
31. L. Custódio, A. L. Escapa, J. Patarra, R. Aligué, F. Alberício, N. R. Neng, J. M. F. Nogueira and A. Romano, Sapwood of carob tree (Ceratonia siliqua L.) as a potential source of bioactive compounds, Rec. Nat. Prod., 2013, 7, 225–229.
32. L. Custódio, J. Patarra, F. Alberício, N. R. Neng, J. M. F. Nogueira and A. Romano, In vitro antioxidant and inhibitory activity of water decoctions of carob tree (Ceratonia siliqua L.) on cholinesterases, α-amylase and α-glucosidase, Nat. Prod. Res., 2015, 29, 2155–2159.
33. S. Klenow, F. Jahns, Z. B. L. Pool and M. Glei, Does an extract of carob (Ceratonia siliqua L.) have chemopreventive potential related to oxidative stress and drug metabolism in human colon cells?, J. Agric. Food Chem., 2009, 57, 2999–3004.
34. K. Rtibi, M. A. Jabri, S. Selmi, A. Souli, H. Sebai, J. El-Benna, M. Amr and L. Marzouki, Carob pods (Ceratonia siliqua L.) inhibit human neutrophils myeloperoxidase and in vitro ROS-scavenging activity, RSC Adv., 2015, 5, 84207–84215.
35. M. G. A. Rasheed, phytochemical study of certain Egyptian plants with antioxidant activity, M. Sc. thesis, Faculty of Pharmacy, Cairo University, 2006, pp. 97–105.
36. National Research Council, Guide for the care and the use of laboratory animals, National Institute of Health, Bethesda, Md., USA, 1985, vol. 20, p. 85.
37. V. L. Singleton and J. A. Rossi, Colorimetry of total phenolics with phosphomolybdic-phosphotungstic reagents, Am. J. Enol. Vitic., 1965, 16, 144–158.
38. C. Chang, M. Yang, H. Wen and J. Chern, Estimation of total flavonoid content in propolis by two complementary colorimetric methods, J. Food Drug Anal., 2002, 10, 178–182.
39. F. Pizzaro, M. Olivares, E. Hertrampf and T. Walter, Factors which modify the nutritional status of iron: tannic content of herbal teas, Arch. Latinoam. Nutr., 1994, 44, 277–280.
40. L. M. Gail, Use of DinitrosaIicyIic Acid Reagent for Determination of Reducing Sugar, Anal. Chem., 1959, 31, 3.
41. AOA, Method 991.42. Official Methods of Analysis of AOAC International, The Association, Gaithersburg, MD, 16th edn, 1995b.
42. C. Proestos, I. S. Boziaris, G. J. E. Nycha and M. Komaitis, Analysis of flavonoids and phenolic acids in Greek aromatic plants: Investigation of their antioxidant capacity and antimicrobial activity, Food Chem., 2006, 95, 664–671.
43. J. El-Benna and P. M. Dang, Analysis of protein phosphorylation in human neutrophils, Methods Mol. Biol., 2007, 412, 85–96.
44. P. M. Dang, A. Stensballe, T. Boussetta, H. Raad, C. Dewas, Y. Kroviarski, G. Hayem, O. N. Jensen, M. A. Gougerot-Pocidalo and J. El-Benna, A specific p47^phox–serine phosphorylated by convergent MAPKs mediates neutrophil NADPH oxidase priming at inflammatory sites, J. Clin. Invest., 2006, 116, 2033–2043.
45. A. K. Papadakis and K. A. Roubelakis-Angelakis, The generation of active oxygen species differs in Nicotiana and Vitis plant protoplasts, Plant Physiol., 1999, 121, 197–205.
46. A. Navas-Diaz, F. Garcia-Sanchez and J. A. Gonziilez-Garcia, Hydrogen peroxide assay by using enhanced chemiluminescence of the luminol H₂O₂–horseradish peroxidase system: Comparative studies, Anal. Chim. Acta, 1996, 327, 161–165.
47. L. F. Li, C. T. Yang, C. C. Huang and Y. Y. Liu, Low molecular-weight heparin reduces hyperoxia augmented ventilator-induced lung injury via serine/threonine kinase protein kinase B, Respir. Res., 2011, 12, 90.
48. H. J. Aye-Than, W. Kukarni and S. J. Tha, Antidiarrhoeal Efficacy of Some Burmese Indigenous Drugs Formations in Experimental Diarrhoeal Test Models, J. Crude Drug Res., 1989, 27, 195–200.
49. A. A. Odetola and S. M. Akojenu, Antidiarrhoeal and gastrointestinal potentials of the aqueous extract of Phylluanthus amarus (Euphorbiaceae), Afr. J. Med. Sci., 2000, 29, 119–122.
50. P. K. Mukherjee, J. Das, R. Balasubramanian, K. Saha, M. Pal and B. P. Saha, Antidiarrhoeal evaluation of Nelumbo nucifera rhizome extract, Indian J. Pharmacol., 1995, 27, 262–264.
51. R. Boominathan, B. P. Devi, S. Dewanjee and S. C. Manda, Studies on antidiarrhoeal activity of Ionodium suffruticosam ging. (Violaceae) extract in rats, Recent Prog. Med. Plants, 2005, 10, 375–380.
52. K. Rtibi, S. Selmi, M. A. Jabri, G. Mamadou, N. Limas-Nzouzi, H. Sebai, J. El-Benna, L. Marzouki, B. Eto and M. Amri, Effects of aqueous extracts from Ceratonia siliqua L. pods on small intestinal motility in rats and jejunal permeability in mice, RSC Adv., 2016, 6, 44345–44353.
53. H. Sebai, A. Souli, L. Chehimi, K. Rtibi, M. Amri, J. El-Benna and M. Sakly, In vitro and in vivo antioxidant properties of Tunisian carob (Ceratonia siliqua L.), J. Med. Food, 2013, 7, 85–90.
54. P. G. Pietta, Flavonoids as antioxidants, J. Nat. Prod., 2000, 63, 1035.
55. F. Visioli, A. Poli and C. Gall, Antioxidant and other biological activities of phenols from olives and olive oil, Med. Res. Rev., 2002, 22, 65–75.
56. A. Parzonko and M. Naruszewicz, Silymarin inhibits endothelial progenitor cells' senescence and protects against the antiproliferative activity of rapamycin: preliminary study, J. Cardiovasc. Pharmacol., 2010, 56, 610–618.
57. K. S. Shivashankara and S. N. Acharya, Uptake of Polyphenols in Tissues. Bioavailability of Dietary Polyphenols and the Cardiovascular Diseases, Open Nutraceuticals J., 2010, 3, 227–241.
58. A. Scalbert and G. Williamson, Dietary intake and bioavailability of polyphenols, J. Nutr., 2000, 130, 2073S–2085S.
59. V. Crespy, C. Morand, C. Besson, N. Cotelle, H. Vezin, C. Demigne and C. Remesy, The splanchnic metabolism of flavonoids highly differed according to the nature of the compound, Am. J. Physiol.: Gastrointest. Liver Physiol., 2003, 284, G980–G988.
60. S. Yodogawa, T. Arakawa, N. Sugihara and K. Furuno, Glucuronoandsulfo-conjugation of kaempferol in rat liver subcellular preparations and cultured hepatocytes, Biol. Pharm. Bull., 2003, 26, 1120–1124.
61. A. Barve, C. Chen, V. Hebbar, J. Desiderio, C. L. Saw and A. N. Kong, Metabolism, oral bioavailability and pharmacokinetics of chemopreventive kaempferol in rats, Biopharm. Drug Dispos., 2009, 30, 356–365.
62. C. Rice-Evans, Flavonoid antioxidants, Curr. Med. Chem., 2001, 8, 797–807.
63. L. T. Wu, C. C. Chu, J. G. Chung, C. H. Chen, L. S. Hsu, J. K. Liu and S. C. Chen, Effects of tannic acid and its related compounds on food mutagens or hydrogen peroxide-induced DNA strands breaks in human lymphocytes, Mutat. Res., 2004, 556, 75–82.
64. R. G. Andrade, L. T. Dalvi, J. M. C. Silva, G. K. B. Lopes, A. Alonso and M. Hermes-Lima, The antioxidant effect of tannic acid on the in vitro copper-mediated formation of free radicals, Arch. Biochem. Biophys., 2005, 437, 1–9.
65. L. R. Ferguson, Role of plant polyphenols in genomic stability, Mutat. Res., 2001, 75, 89–111.
66. N. S. Khan, A. Ahmad and S. M. Hadi, Anti-oxidant, pro-oxidant properties of tannic acid and its binding to DNA, Chem.-Biol. Interact., 2000, 125, 177–189.
67. K. Kondo, M. Kurihara, N. Miyata, T. Suzuki and M. Toyoda, Scavenging mechanisms of (-)-epigallocatechin gallate and (-)-epicatechingallate on peroxyl radicals and formation of superoxide during the inhibitory action, Free Radical Biol. Med., 1999, 27, 855–863.
68. Z. Huang, M. J. Fasco and L. S. Kaminsky, Inhibition of estrone sulfatase in human liver microsomes by quercetin and other flavonoids, J. Steroid Biochem. Mol. Biol., 1997, 63, 9–15.
69. H. P. Kim, K. H. Son, H. W. Chang and S. S. Kang, Anti-inflammatory plant flavonoids and cellular action mechanisms, J. Pharmacol. Sci., 2004, 96, 229–245.
70. T. Boussetta, H. Raad, P. Lettéron, M.-A. Gougerot-Pocidalo, J.-C. Marie, F. Driss and J. El-Benna, Punicic Acid a Conjugated Linolenic Acid Inhibits TNFα-Induced Neutrophil Hyperactivation and Protects from Experimental Colon Inflammation in Rats, PLoS One, 2009, 4, e6458.
71. R. Bachoual, W. Talmoudi, T. Boussetta, F. Braut and J. El-Benna, An aqueous pomegranate peel extract inhibits neutrophil myeloperoxidase in vitro and attenuates lung inflammation in mice, Food Chem. Toxicol., 2011, 49, 1224–1228.
72. M.-A. Jabri, K. Rtibi, H. Tounsi, K. Hosni, A. Souli, J. El-Benna, L. Marzouki, M. Sakly and H. Sebai, Myrtle berry seed aqueous extract inhibits human neutrophil myeloperoxidase in vitro and attenuates acetic acid-induced ulcerative colitis in rats, RSC Adv., 2015, 5, 64865–64877.
73. F. S. R. Bernaud, M. V. Beretta, C. do Nascimento, F. Escobar, J. L. Gross, M. J. Azevedo and T. C. Rodrigues, Fiber intake and inflammation in type 1 diabetes, Diabetol. Metab. Syndr., 2014, 6, 66.
74. A. Karim, H. Makhfi, A. Ziyyat, A. Legssyer and M. Bnouham, Antidiahoeal activity of crude aqueous extract of Rubia tinctorum. Roots in rodent, J. Smooth Muscle Res., 2010, 46, 119–123.
75. G. C. Akuodor, I. Muazzam, M. Usman-Idris, U. A. Megwas, J. L. Akpan, K. C. Chilaka, D. O. Okoroafor and U. A. Osunkwo, Evaluation of the antidiarrheal activity of methanol leaf extract of Bombax buonopozense in rats, Ibnosina J. Med. Biomed. Sci., 2011, 3, 15–20.
76. P. K. Mukherjee, K. Saha and T. Murugesam, Screening of Anti Diarrhoeal Profile of Some Plant Extract of A Specific Region of West Bengal, India, J. Ethnopharmacol., 1998, 60, 85–89.

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