Inhibition of Neutrophil Pholasin Chemiluminescenceby Cleome arabica Leaf Extract

Abstract

The antioxidative activity of Cleome arabica L. leaf extract was studied toward superoxide anion radical generating systems. The extract at 10 µg/ml has no scavenger effect on superoxide anion radicals generated by the xanthine-xanthine oxidase system, as determined by either the reduction of cytochrome c or pholasin luminescence. Also, this extract, up to 50 µg/ml, did not affect the uric acid production by xanthine oxidase. However, the extract showed a great inhibition activity, in a dose-dependent manner, of neutrophil pholasin luminescence stimulated by formyl-methionyl-leucyl-phenylalanine (fMLP). Indeed, the inhibition of pholasin luminescence was 80.22±2.9%, 90.22 ± 4.0%, and 101.0±1.0% in presence of the extract at concentrations of 1, 2, and 10 µg/ml, respectively. Thereby, it is likely that Cleome arabica leaf extract showed an inhibitory effect on enzyme(s) involved in signaling pathways of fMLP-stimulated neutrophils. Effectively, the extract exerted a significant inhibitory effect (p < 0.05), in a dose-dependent manner, on elastase release by fMLP/cytochalasin B–stimulated neutrophils.

Keywords:

• Chemiluminescence
• flavonoids
• Cleome arabica
• degranulation
• neutrophils
• Pholasin
• superoxide anion radicals

Introduction

The genus Cleome (Capparidaceae) is abundantly distributed in the north of Africa. Most species of Cleome are glandular annual herbs (Wollenweber & Dorr, 1992). The plant leaves of some Cleome species are well reported in folk medicine to have an almost immediate effect on relieving abdominal and rheumatic pains (Sharaf et al., 1992). Many components of plant origin are investigated as possible antioxidant or anti-inflammatory agents (Packer et al., 1999; Morebise et al., 2002; Braca et al., 2003; Selloum et al., 2003; Peng et al., 2003). Among plant compounds, a growing number of reports deal with the antioxidative, anti-inflammatory, and antitumoral activities of flavonoids (Inoue & Jackson, 1999; Kobuchi et al., 1999; Middleton et al., 2000). Our previous results showed that Cleome arabica L. leaf extract has a large content of flavonoids (Bouriche et al., 2003). It is likely that this extract may possess potent antioxidative activity. In fact, many flavonoids have been reported to be potent inhibitors of some enzymes such as phospholipase A₂ (Chang et al., 1994), xanthine oxidase (Cos et al., 1998), protein kinase C (PKC), and phosphatidylinositol 3-kinase (PI 3-kinase) (Gamet-Payrastre et al., 1999; Selloum et al., 2001). The antioxidative activity of flavonoids has been reported in many reports either in cellular (Limasset et al., 1993; Pietta, 2000) or in noncellular systems (Hu et al., 1995; Robak & Marcinkiewicz, 1995).

Polymorphonuclear neutrophils (PMNs) play an important role in both acute and chronic inflammatory processes. The antimicrobial function of PMNs is based on their phagocytic capacity and ability to release proteolytic enzymes and reactive oxygen species (ROS) into phagosome (Hampton et al., 1998). Stimulated neutrophils generate superoxide anion radicals (O₂^√−), a reaction catalyzed by the enzymatic system NADPH-O₂ oxidoreductase (NADPH oxidase). Subsequently, O₂^√− is converted to other reactive compounds such as hydrogen peroxide (H₂O₂), hydroxyl radical (^√OH), hypochlorous acid (HOCl), and N-chloramins (R-NHCl) (Weiss, 1989). The rate of production of ROS may exceed the capacity of cell antioxidant defences, thereby resulting in substantial tissue damage (Weiss, 1989; Kasai, 1997; Wahn & Hammerschmidt, 2001).

In the current work, we investigated the antioxidative activity of Cleome arabicaL. leaf extract toward superoxide anion radicals generated either by the xanthine-xanthine oxidase system or by chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated human neutrophils, using pholasin chemiluminescence probe. Furthermore, we studied the effect of this extract on the degranulation of fMLP/cytochalasin B–stimulated neutrophils by determination of the rate of released elastase.

Materials and methods

Chemicals

Bovine erythrocytes superoxide dismutase (SOD), allopurinol, horse heart cytochrome c, fMLP, cytochalasin B, phenylmethylsulfonyl fluoride (PMSF), Histopaque-1077, bovine xanthine oxidase, and N-succinyl-Ala-Ala-Ala-p-nitroanilide were obtained from Sigma (Germany). Pholasin was a product from Knight Scientific (Plymouth, UK). Solvents were from Prolabo (France). All other reagents were from Sigma.

Preparation of plant extract

Cleome arabica was collected from Boussaada, Algeria, in March 2002. A voucher specimen is deposited in the laboratory of Botany, Department of Natural Sciences, University of Setif (Algeria).

The air-dried, powdered leaves (100 g) were extracted using methanol/water (7/3, v/v) and ethyl acetate, successively. The quantitative estimation of total flavonoid contents present in the extract was determined using AlCl₃ reagent (Bouriche et al., 2003).

Effect of the extract on the activity of xanthine oxidase

The enzyme activity was determined by measuring the rate of oxidation of 100 µM xanthine to uric acid spectrophotometrically at 295 nm, using ε₂₉₅ = 9600 M⁻¹cm⁻¹(Robak & Gryglewski, 1988). Assays were performed at room temperature in air-saturated 50 mM phosphate buffer, pH 7.4, containing 0.1 mM EDTA. The reaction was started by the addition of 0.04 U/ml xanthine oxidase. One unit of xanthine oxidase activity was defined as the amount yielding 1 µM of uric acid per min. The inhibitory effect of the extract was studied by adding 50 µg/ml of the extract dissolved in 1% ethanol (v/v), in both reaction and reference mixtures. All concentrations are final ones. The percentage of inhibition was calculated by comparing the reaction rate with the control solution in the absence of the extract.

Effect of the extract on the generation of superoxide anion radicals by the xanthine-xanthine oxidase system

Cytochrome c test

The reaction mixture contained 100 µM xanthine, 25 µM horse heart cytochrome c, and 10 µg/ml of the extract dissolved in ethanol 1% (v/v), in air-saturated sodium phosphate buffer (50 mM, pH 7.4) supplemented with 0.1 mM EDTA (Robak & Gryglewski, 1988). The reaction was started by adding 0.04 U/ml xanthine oxidase. All concentrations indicated are final ones. After 1 min, reduced cytochrome c was determined at 550 nm against enzyme-free mixture. The amount of generated superoxide was calculated using ε₅₅₀ = 21,100 M⁻¹ cm⁻¹. The sensitivity of the reaction was determined by using bovine erythrocyte superoxide dismutase (330 U/ml, final concentration).

Pholasin-enhanced luminescence

Xanthine (10⁻⁷M) and the extract dissolved in DMSO 0.5% (v/v) were incubated with 7 × 10⁻⁸ M pholasin for 2 min. The generation of superoxide anion radicals was induced by the addition of 50 µl xanthine oxidase (0.01U/ml) to 200 µl of sample solution (Reichl et al., 2000). The resulting chemiluminescence was followed at 37°C for 5 min. All luminescence measurements were carried out using a microtiter plate luminometer (MicroLumat LB 96 P, EG&G Berthold, Wildbad, Germany).

Isolation of neutrophils

Human polymorphonuclear leukocytes (PMNs) were isolated from freshly heparinized (10 U/ml) blood from healthy donors as previously described (Selloum et al., 2001). Briefly, PMNs were prepared using standard dextran sedimentation and gradient separation on Histopaque. This procedure yields a PMN population that is 95–98% viable, as determined by trypan blue exclusion test.

Effect of the extract on the respiratory burst of fMLP-stimulated neutrophils

PMNs (10⁵ cells/ml) were incubated with the extract and dissolved in DMSO 0.5% (v/v) for 2 min at 37°C in the presence of 10⁻⁵M KCN and 7 × 10⁻⁸M pholasin (Reichl et al., 2000). PMNs were stimulated with 50 µl of 10⁻⁶ M fMLP using an injection device. All concentrations are final ones. The total measuring volume was 250µl. The resulting chemiluminescence was followed for 15 min. In the control mixture, the extract was replaced by Hank's balanced salt solution (HBSS). The sensitivity of the reaction was determined by using SOD (100 U/ml, final concentration).

Effect of the extract on neutrophil degranulation

The degranulation of fMLP/cytochalasin B–stimulated neutrophils was determined by measuring the elastase release in presence or absence of Cleome arabica leaf extract. PMNs (5 × 10⁶ cells/ml) were equilibrated in HBSS for 10 min at 37°C. Various concentrations of the extract (1, 10, and 25 µg/ml) dissolved in DMSO 0.5% (v/v) were added for an additional 10 min. fMLP (10⁻⁶ M) and cytochalasin B (10⁻⁵ M) were then added, and the incubation was continued for 30 min. Supernatants were separated by centrifugation at 400 × g for 10 min at 4°C. Elastase exocytosis was measured using the synthetic substrate N-succinyl-Ala-Ala-Ala-p-nitroanilide (Bieth et al., 1974). Seventy-five microliters of each supernatant was mixed, in a microtiter plate well, with 4.5 µg of substrate dissolved in 50 µl of 0.1 M HEPES, 0.5 M NaCl, pH 7.4. The plate was incubated at 37°C for 2 h. All concentrations indicated are final ones. The absorbance of released p-nitroanilide was measured at 405 nm with microplate reader (Bio-Tek Instruments) against the appropriate enzyme-free reference. Results were expressed as the percentage of released elastase compared with control without extract set at 100%.

The effect of the extract on elastase activity was determined in a cell-free system. Elastase was produced by incubating PMNs (5 × 10⁶ cells/ml) for 30 min at 37°C in presence of 10⁻⁶ M fMLP and 10⁻⁵ M cytochalasin B. Then, the activity of elastase was determined in the cell-free supernatant with or without extract. The release of p-nitroanilide was measured as mentioned above.

Data analysis

Statistical analysis of the dose effect of Cleome arabica leaf extract on neutrophil degranulation was performed by analysis of variance.

Results

Effect of the extract on the activity of xanthine oxidase

The oxidation of xanthine to uric acid by xanthine oxidase was measured in the absence or presence of Cleome arabica leaf extract. The specific inhibitor of xanthine oxidase allopurinol (50µM, final concentration) was used as control (Fig. 1). Under our experimental conditions, the amount of uric acid produced was 41.12±5.4 µM. Results obtained showed that the extract up to 50 µg/ml did not present any inhibitory effect on the uric acid production, whereas the activity of the enzyme was reduced by 94.4±0.5% in the presence of allopurinol.

Figure 1 Effect of the extract (50 µg/ml) and allopurinol (50 µM) on the activity of xanthine oxidase as measured by the production of uric acid spectrophotometrically at 295 nm. Xanthine oxidase (0.04 U/ml) was added to xanthine (100 µM) preincubated with the extract for 2 min. All concentrations are final ones. Enzyme activity of the control sample without extract was set to 100%. Values represent the mean±SD, in triplicate.

Effect of the extract on the generation of superoxide anion radicals using the xanthine-xanthine oxidase system

The effect of the extract, at 10 µg/ml final concentration, was studied for its ability to scavenge superoxide anion radicals (O₂^√−) generated by the xanthine-xanthine oxidase system. The amount of generated O₂^√−was determined by measuring the reduction of horse heart cytochrome c. In these assay conditions, the amount of O₂^√−generated in the absence of the extract was 16.1±1.3 µM. The reduction of cytochrome c was almost totally inhibited by SOD at 330 U/ml (final concentration).

Under our experimental conditions, we observed no inhibitory effect of the extract on the reduction of cytochrome c. In fact, the inhibition of the generation of superoxide anion radicals by the extract at 10 µg/ml was only 5.58±0.6% (Fig. 2). In a control experiment, we observed that reduced cytochrome c was not reoxidized by the extract added at 10 µg/ml when cytochrome c reduction in the control sample reached the half of its maximum.

Figure 2 Inhibition of the generation of superoxide anion radicals from xanthine-xanthine oxidase by the extract as measured by the cytochrome c test. Xanthine oxidase (0.04 U/ml) was added to xanthine (100 µM) preincubated with extract (10 µg/ml) and cytochrome c (25 µM) for 2 min. All concentrations are final ones. Amount of superoxide anion radicals in the control sample without extract was set to 100%. Values represent the mean±SD of three independent experiments.

To confirm the results of the cytochrome c test, we used another method to detect O₂^•−. Pholasin, the photoprotein of the common piddock Pholas dactylus L., emits an intense luminescence in the presence of superoxide anion radicals (Reichl et al., 2000). The addition of xanthine oxidase to a solution of pholasin and xanthine induced a light emission that is totally abolished by SOD (100 U/ml), indicating the dependence of the signal on superoxide anion radicals. Figure 3, illustrating the kinetic of pholasin luminescence, showed that the luminescence intensity in presence of the extract at 10 µg/ml was quite similar to the control. Results of the pholasin assay were similar to those obtained with cytochrome c test (Fig. 4).

Figure 3 Kinetics of pholasin luminescence induced by superoxide anion radicals in the absence (▴) or presence (•) of the extract (10 µg/ml). Xanthine (10⁻⁷ M) was incubated with pholasin (7 × 10⁻⁸ M). Generation of superoxide anion radicals was induced by addition of xanthine oxidase (0.01 U/ml). Experiments were also performed in the presence of 100 U/ml of superoxide dismutase (o). All concentrations are final ones. Symbols were used to mark kinetic traces; they do not correspond to measuring points.

Figure 4 Effect of the extract (10 µg/ml) on pholasin luminescence induced by superoxide anion radicals. Total luminescence yield was determined as the integral for 5 min after the addition of xanthine oxidase. The luminescence of the control sample without extract was set to 100%. All other experimental conditions are the same as in Figure 3. Data represent the mean ± SD of three experiments.

Effect of Cleome arabica extract on the respiratory burst of fMLP-stimulated human neutrophils

The inhibitory effect of Cleome arabica extract on the respiratory burst of human neutrophils stimulated with fMLP was investigated using the pholasin-enhanced chemiluminescence. Under these assay conditions, the extract, up to 100 µg/ml final concentration, did not induce cell toxicity as assessed by lactate dehydrogenase (LDH) release. Furthermore, in a control experiment, the extract did not affect the activity of LDH in a cell-free system. PMNs (10⁵cells/ml) were preincubated at 37°C with the extract during 2 min, in the presence of pholasin. Neutrophils were then stimulated with 10⁻⁶M fMLP, and the chemiluminescence was monitored for 15 min. Potassium cyanide (10⁻⁵ M) was previously added to cells in order to prevent a light emission caused by released myeloperoxidase. Because SOD (100 U/ml) inhibited most of the pholasin luminescence (Fig. 5), superoxide anion radicals generated by fMLP-stimulated neutrophils can be very sensitively detected by pholasin.

Figure 5 Kinetics of pholasin luminescence from neutrophils stimulated with fMLP (10⁻⁶ M,) in the presence of 2 µg/ml of the extract (▴), or 100 U/ml of SOD (o). Neutrophils (10⁵ cells/ml) were incubated with the extract in the presence of KCN (10⁻⁵ M) and pholasin (7 × 10⁻⁸ M) for 2 min at 37°C before stimulation. All concentrations are final ones. Symbols were used to mark kinetic traces; they do not correspond to measuring points.

Figure 5 shows that stimulation of neutrophils by fMLP induced a strong light emission lasting about 3 min. The extract at 2 µg/ml has a strong inhibitory effect on this luminescence. Indeed, the inhibition of pholasin chemiluminescece was 80.32±2.9%, 90.22±4.0%, and 101.0±1.0% in presence of the extract at 1, 2, and 10 µg/ml, respectively (Fig. 6).

Figure 6 Effect of the extract at different concentrations (1, 2, and 10 µg/ml) on pholasin luminescence of fMLP-stimulated neutrophils. Total luminescence yield was determined as the integral for 15 min after the addition of fMLP. The luminescence of the control sample containing SOD (100 U/ml) was set to 100%. All other experimental conditions are the same as in Figure 5. Data represent the mean ± SD of three experiments.

Finally, the effect of Cleome arabica leaf extract on elastase release by fMLP-stimulated neutrophils, in the presence of cytochalasin B, was investigated. The inhibition of elastase release was 50.93±5.8% in the presence of the extract at 10 µg/ml. The extract exerted a significant inhibitory effect (p < 0.05), in dose-dependent manner, on elastase exocytosis (Fig. 7).

Figure 7 Effect of the extract at different concentrations and PMSF (10 mM) on elastase release by fMLP/cytochalasin B–stimulated neutrophils. PMNs (5 × 10⁶ cells/ml) were incubated with the extract in the presence of fMLP (10⁻⁶ M) and cytochalasin B (10⁻⁵ M). The activity of elastase released in the supernatant was measured at 405 nm. The residual activity in the control sample without extract was set to 100%. All concentrations are final ones. Values represent the mean ± SD of three experiments. A significant dose response is seen (p < 0.05).

In our experimental conditions, we observed that the elastase release is about 14 times greater in cells stimulated with fMLP/cytochalasin B than with fMLP alone. In a control experiment, the extract up to 25 µg/ml did not inhibit the activity of elastase in a cell-free system.

Discussion

There is a growing interest in the antioxidative activity of plant compounds, especially flavonoids. The quantitative estimation of flavonoids present in the crude extract of Cleome arabica showed that this extract contains 19.33% of dry matter, using the natural flavonol rutin as standard. In this study, the antioxidative activity of Cleome arabica leaf extract was investigated toward superoxide anion radicals either generated by xanthine-xanthine oxidase system or by fMLP-stimulated neutrophils and was assessed by pholasin chemiluminescence. It has been reported that pholasin is a very sensitive detector of superoxide anion radicals, and the luminescence of pholasin can be abolished by superoxide dismutase (Reichl et al., 2000). The extract exerted different effects on superoxide anion radicals generated by both systems.

The xanthine-xanthine oxidase is a suitable system in vitro to produce superoxide anion radicals (Robak & Gryglewski, 1988; Cos et al., 1998). The extract showed no scavenging activity on superoxide anion radicals generated by this system as assessed by the reduction of cytochrome c (Fig. 2). These results were confirmed by the more sensitive pholasin luminescence method (Fig. 4). Furthermore, the extract up to 50 µg/ml did not influence uric acid production by xanthine oxidase (Fig. 1). It seems that, in spite of the high flavonoid content, the extract has no scavenger effect on superoxide anion radicals.

In contrast to the above results, the extract showed a great inhibitory effect on pholasin luminescence produced by fMLP-stimulated neutrophils (Fig. 6). Under these assay conditions, Cleome arabica leaf extract did not induce cell toxicity as assessed by LDH release. Therefore, the activity of the extract could not be due to a cytotoxic effect. KCN, a specific inhibitor of myeloperoxidase (MPO), was used to prevent reaction of pholasin with MPO. It has been reported that MPO/H₂O₂/Cl⁻ reacts with pholasin to produce oxypholasin (Reichl et al., 2000). The inhibition of fMLP-stimulated neutrophil chemiluminescence by superoxide dismutase indicated that superoxide anion radicals contributed to this light emission.

These results showed a possible inhibition activity of the extract toward enzyme(s) involved in signaling pathways. Our conclusion is based on (a) the extract did not show any scavenger effect on superoxide anion radicals, otherwise (b) it has a great inhibitory effect on neutrophil respiratory burst. Indeed, many studies reported the inhibitory effect of flavonoids, especially flavonols, on PI 3-kinases and PKC (Vlahos et al., 1994; Gamet-Payrastre et al., 1999). There are several routes to activate the NADPH-oxidase in fMLP-stimulated neutrophils that give rise to the generation of ROS (Roos, 1991). PI 3-kinase activity is directly stimulated by the G-protein–linked fMLP receptor, and this activity appears to be upstream of the protein kinases that phosphorylate p47^phox (Vlahos et al., 1994). These authors reported that a synthetic chromone (LY294002), using quercetin as a model, completely and specifically abolished PI 3-kinase activity with a very selective structure-activity relationship. In previous work (Selloum et al., 2001), we have found that the flavonol rutin, which has no scavenger effect on superoxide anion radicals, inhibited pholasin luminescence of fMLP-stimulated neutrophils. Furthermore, this flavonol exhibited in vitro an inhibitory effect on PI 3-kinase γ activity. It is well-known that PI 3-kinases play a key role in neutrophil degranulation (Payrastre et al., 2001). Effectively, we have observed that the extract exerted a remarkable inhibitory effect on neutrophil degranulation stimulated by fMLP/cytochalasin B. Thus, the inhibitory effect of the extract on neutrophil respiratory burst may be due to a possible presence of rutin among its flavonoid contents, as we reported previously for the natural flavonol rutin (Selloum et al., 2001, 2003).

Our results showed that the extract, which has no scavenger effect on superoxide anion radicals, exerted a great inhibition activity on neutrophil respiratory burst. Indeed, we previously reported that this extract has anti-inflammatory activity in vivo as well as in vitro. These results may contribute to elucidate the pharmacological properties of Cleome arabica leaf extract. Molecular components of the crude extract have not been identified as yet. Their isolation and characterization is now under investigation in our laboratory.

Acknowledgments

The authors are most grateful for the encouragement and facilities provided by PD Dr. J. Arnhold in whose laboratory at the University of Leipzig (Germany) some of these studies were carried out. This study was supported by grants from OPCW, The Netherlands (grant no. L/ICA/ICB/ 68016/03), from NATO, Brussels (Collaborative Linkage grant no. 980199) and from the Ministry of High Education and Scientific Research, Algeria (F 1901/01/2001).