Effects of an Aqueous Extract from Leaves of Ligustrum vulgare on Mediators of Inflammation in a Human Neutrophils Model

• Introduction
• Materials and Methods
• Chemicals
• Plant material
• Extract preparation and characterization by the HPLC-DAD- MSn method
• Isolation of human neutrophils
• Evaluation of ROS production by human neutrophils
• Myeloperoxidase release by human neutrophils
• Elastase release by human neutrophils
• MMP-9 and IL-8 production by human neutrophils
• Expression of adhesion molecules CD62L and CD11b/CD18 in human neutrophils
• Cytotoxicity assays
• Statistical analysis
• Results
• Discussion
• References

Abstract

Leaves of Ligustrum vulgare (common privet) have been used for treatment of oropharyngeal inflammations or as antirheumatic, diuretic, and hypotensive agents in folk medicine in southern Europe. Taking into account that neutrophils are involved in the inflammation, the aim of the study was to determine the effect of an aqueous extract prepared from leaves of Ligustrum vulgare on neutrophil functions. The extract was characterized by the HPLC-DAD-MSⁿ method. The inhibition of reactive oxygen species production by formyl-met-leu-phenylalanine- or phorbol 12-myristate 13-acetate-stimulated neutrophils was determined using luminol- or lucigenin-dependent chemiluminescence. The effect on myeloperoxidase, metalloproteinase 9, and interleukin 8 production by neutrophils was measured by an enzyme-linked immunosorbent assay. Neutrophil elastase release was established spectrophotometrically. The expression of adhesion molecules on neutrophils was analyzed with flow cytometry. The main compounds detected were flavonoids, phenylpropanoids, hydroxycinnamates, and secoiridoids. The inhibition of oxidative burst by the extract was comparable in both stimuli models (formyl-met-leu-phenylalanine: IC₅₀ = 18.2 ± 4.0 µg/mL; phorbol 12-myristate 13-acetate: IC₅₀ = 19.8 ± 3.0 µg/mL). The extract in the concentration range of 5–50 µg/mL inhibited neutrophil elastase release by 23.9–34.1 % and myeloperoxidase release by 24.2–37.4 %. The inhibitory effect on metalloproteinase 9 and interleukin 8 production was around 20 %. The extract in the highest concentration modulated the expression of L-selectin and β ₂ integrin. Our results partly support the traditional use of common privet leaves as an anti-inflammatory agent.

Introduction

The genus Ligustrum (Oleaceae) encompasses species which are distributed in Europe and Asia. Most plants of this genus are used by traditional physicians for their tonic, immunomodulatory, anti-inflammatory, and antiaging effects [1].

Among the species of the genus Ligustrum, common privet (Ligustrum vulgare L.) is one of the most popular decorative shrubs in Europe. The fresh leaves were once chewed against smooth oropharyngeal inflammations or aphthae, and the decoctions of leaves were considered to be active against burns and headaches [2]. Nowadays in some regions, they are still used for their anti-inflammatory, antirheumatic, diuretic, and hypotensive activities [3], [4], [5]. Results of recent in vitro studies confirmed significant anti-lipoxygenase activity and a complement-inhibiting effect of water infusion, methanolic and ethyl acetate leaves extracts, and isolated compounds, such as flavonoids and iridoids, from privet leaves [2], [6]. The ethanolic extract showed weak antibacterial activity against gram-positive bacteria [7] and cytotoxic activity against HeLa cells [8]. Different, aqueous and organic, extracts from leaves and fruits of Ligustrum vulgare had the capacity to scavenge DDPH radicals. Among the extracts screened, water infusions had the strongest activity. The authors recognized flavonoids as the main active principles [9]. Among them, apigenin, luteolin, apigenin-7-O-glucoside, apigenin-7-O-rutinoside, ligustroflavone (apigenin-7-O-dirhamnopyranosyl-glucopyranoside), luteolin-7-O-glucoside, kaempferol, and quercetin glycosides (3-O-rhamnosides, 3-O-glucosides, 3-O-glucosides 7-O-rhamnosides, 3,7-dirhamnosides) were identified in the Ligustrum vulgare leaves [2], [10], [11]. However, secoiridoids, such as oleuropein, ligstroside and oleacein (dialdehydic form of oleuropein aglycon), were also identified in this plant material [2], [10], [12]. The genus Ligustrum was also characterized by the presence of phenylpropanoids, echinacoside and verbascoside, as well as other hydroxycinnamates represented by p-coumaric acid [6], [13].

Neutrophils, also known as PMN, constitute the first line of defence of the innate immune system. In a situation of inflammation, the production of neutrophils increases and once activated contributes to the generation of ROS and to the release of proteases, chemokines, and cytokines [14]. Neutrophils trap infectious agents and kill and eliminate them into the phagocytic vacuole by intense ROS production and release of MPO and proteases from neutrophil granules [14], [15]. Upon resolution of inflammation, PMNs die by apoptosis and are phagocyted by macrophages. However, during chronic inflammatory processes, neutrophils are activated by a great variety of stimuli, and an increase in the neutrophilsʼ attributes is observed. The activation and recruitment of dysregulated neutrophils result in severe damage of normal tissues [14], [16]. Therefore, neutrophil models are useful for searching plant extracts and natural compounds of therapeutic interest in the regulation of chronic and acute inflammatory diseases [17], [18], [19], [20], [21], [22], [23], [24].

In order to confirm the traditional use of privet leaves as an anti-inflammatory agent, we investigated the inhibitory activity of the phytochemically characterized (HPLC-DAD-MSⁿ) aqueous extract from privet leaves not only against oxidative burst and myeloperoxidase release in human neutrophils but also on other mediators of inflammation such as elastase, MMP-9, IL-8 production, and expression of adhesion molecules in neutrophils.

Materials and Methods

Chemicals

Luminol (lot No. 2083094, 98.0 % HPLC purity), PMA (lot No. 049K1246; 99 % TLC purity), f-MLP (lot No. 129K2056; 97 % HPLC purity), LPS (from Escherichia coli 0111:B4), cytochalasin B (lot No. 012M4012 V, 98 % HPLC purity), MTT (lot No. 23396AJ; 98 % purity), HEPES, L-glutamine and N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (lot No. SLBB1034V; 98.0 % HPLC purity), (S)-(+)-camptothecin (lot No. SLBB9623V; 95.0 % HPLC purity), ascorbic acid (reagent grade), chlorogenic acid (95 % titration purity), quercetin (95 % HPLC purity), and pentagalloyl glucose (96 % HPLC purity) were purchased from Sigma-Aldrich Chemie GmbH. Gallic acid (SH purity) was obtained from ChromaDex and lucigenin (Lot No. 219–023–4, 97 % purity) from Carl Roth. FBS was purchased from Gibco. Penicillin-streptomycin was obtained from PAA and propidium iodide from BD Biosciences.

PBS was purchased from Biomed. Hanksʼ balanced salt solution and RPMI 1640 medium was purchased from Sigma-Aldrich Chemie GmbH.

Plant material

Ligustrum vulgare leaves were collected in June 2012 in Legionowo near Warsaw. The plant was identified by Dr. W. Szypuła, Department of Botany and Pharmaceutical Biology, Warsaw Medical University. A voucher specimen (No LV062012) of the leaves is available from the plant collection in the Department of Pharmacognosy and Molecular Basis of Phytotherapy, Medical University of Warsaw.

Extract preparation and characterization by the HPLC-DAD- MSⁿ method

Extract preparation: 400 g of powdered plant material were macerated in water (1 : 10) for 2 h and then extracted four times with water (1 : 10) in an ultrasonic bath (30 °C) for 30 min. The obtained aqueous extract was concentrated under reduced pressure at 40 °C and lyophilized. The Folin–Ciocalteu method was used to determine the total phenol content which was expressed as gallic acid equivalents.

HPLC-DAD- MSⁿ analysis was performed on a UHPLC-3000 RS system (Dionex) with DAD detection and an AmaZon SL ion trap mass spectrometer with an ESI interface (Bruker Daltonik GmbH). Separation was performed on a Zorbax SB C18 column (150 × 2.1 mm, 1.9 µm) (Agilent); the column temperature was 25 °C. The mobile phase A was water/acetonitrile/formic acid (95 : 5 : 0.1, v/v/v), and the mobile phase B methanol. A linear gradient system was used: 0–60 min, 1–60 % B. The flow rate was 0.2 mL/min. The column was equilibrated for 10 min between injections. UV spectra were recorded over a range of 200–450 nm; chromatograms were acquired at 240 nm, 280 nm, 325 nm, and 350 nm. The LC eluate was introduced directly into the ESI interface without splitting. The nebulizer pressure was 40 psi; dry gas flow, 9 L/min; dry temperature, 300 °C; and capillary voltage, 4.5 kV. Analysis was carried out using a scan from m/z 200 to 2200. Compounds were analyzed in the negative ion mode. The MS² fragmentation was obtained for the most abundant ion at the time.

Isolation of human neutrophils

Peripheral venous blood was obtained from healthy adult volunteers (< 35 years old) from the Warsaw Blood Donation Centre. Donors declared that they were nonsmokers and were not taking any medication. They were clinically confirmed to be healthy, and routine laboratory tests showed values within a normal range. Neutrophils were isolated by dextran sedimentation and centrifugation in a Ficoll Hypaque gradient. Erythrocytes were removed by hypotonic lysis. The purity of the neutrophil preparation was > 97 % [25]. Following isolation, the cells were resuspended in an appropriate medium, such as (Ca²⁺)-free Hanksʼ balanced salt solution, (Ca²⁺)-free PBS buffers at pH 7.4, or RPMI 1640 medium and were maintained at 4 °C before use.

Evaluation of ROS production by human neutrophils

The evaluation of the ROS production by f-MLP- or by PMA- stimulated neutrophils was performed with slight modifications as described by OʼDowd et al. and Van den Worm et al. [20], [21]; it was determined using luminol- or lucigenin-dependent chemiluminescence, respectively. Following isolation, cells were resuspended in Hanksʼ balanced salt solution. Cell suspension (3.5 × 10⁵) was incubated with 50 µL of tested extract at a final concentration range of 5–50 µg/mL and luminol (20 mM) or lucigenin (0.4 mM) in a 96-well plate. The ROS production was initiated by the addition of f-MLP (0.1 µg/mL) or PMA (1 µg/mL) to obtain a total volume of 200 µL/well. Changes in chemiluminescence were measured over a 40-min period at intervals of 2 min in a microplate reader. Background chemiluminescence produced by non-stimulated cells was also determined.

The percentage of inhibition was calculated in comparison to the control without the tested extract replaced by Hanksʼ balanced salt solution buffer in the maximum luminescence (the height of the peak).

Myeloperoxidase release by human neutrophils

The cell suspension (2 × 10⁶) in PBS buffer with Ca²⁺ and Mg²⁺ was preincubated with 50 µL of the tested extract at a final concentration range of 5–50 µg/mL for 15 min at 37 °C and then stimulated with cytochalasin B (5 µg/mL) and f-MLP (0.1 µg/mL) for 15 min as described by Wanikiat et al. [22]. After centrifugation (1000 × g; 10 min; 4 °C), the released myeloperoxidase into cell supernatants was measured by ELISA following the indications of the manufacturer (R&D Systems). The effect on myeloperoxidase release was calculated as the percentage of released enzyme in comparison with the stimulated control without the tested extract replaced by PBS buffer with Ca²⁺ and Mg²⁺.

Elastase release by human neutrophils

Neutrophil elastase release was determined with slight modifications as described by Wanikiat et al. [22] using N-succinyl-alanine-alanine-valine p-nitroanilide (SAAVNA) as a substrate, and p-nitrophenol was measured spectrophotometrically. The cell suspension (5 × 10⁵) was preincubated with 50 µL of the tested extract at a final concentration range of 5–50 µg/mL for 15 min at 37 °C and then stimulated with cytochalasin B (5 µg/mL) and f-MLP (0.1 µg/mL) for 15 min. After the addition of SAAVNA (100 µM), the absorbance was measured spectrophotometrically for 1 h at intervals of 20 min at 412 nm using a microplate reader (BioTek). The effect on elastase release was calculated as the percentage of released enzyme in comparison with the stimulated control without the tested extract replaced by HBSS buffer.

MMP-9 and IL-8 production by human neutrophils

Neutrophils (2 × 10⁶/mL) were cultured in a 24-well plate in RPMI 1640 medium with 10 % FBS, 10 mM HEPES, and 2 mM L-glutamine for 24 h at 37 °C with 5 % CO₂ in the absence or presence of the extract at a final concentration range of 5–50 µg/mL added 1 h before the stimulation with LPS (100 ng/mL). The amount of released MMP-9 and IL-8 into cell supernatants was measured by ELISA following the indications of the manufacturer (R&D Systems). The effect on MMP-9 and IL-8 production was calculated as the percentage of released agent in comparison with the stimulated control without the tested extract replaced by PBS buffer.

Expression of adhesion molecules CD62L and CD11b/CD18 in human neutrophils

The 400 µL of cell suspension (1 × 10⁶) in PBS buffer was preincubated with 100 µL of extract at final concentrations of 10 and 50 µg/mL or 100 µL of PBS buffer for 30 min at 37 °C, and then cells were stimulated by LPS (1 µg/mL) for additional 30 min [23]. Neutrophils were marked with monoclonal antibody against CD62L-(FITC)-conjugate (eBioscience,) or CD11b-(PE)-conjugate (eBioscience) and incubated for 30 min at 4 °C in the dark. The cells were analyzed by flow cytometry FACSCalibur (Becton Dickinson), and data from 5000 events were recorded.

Cytotoxicity assays

Cytotoxicity of the extract for neutrophils was tested by the MTT colorimetric assay. The suspension of neutrophils (5 × 10⁵ in RPMI 1640 medium) with 50 µL of the tested extract at a final concentration range of 5–50 µg/mL or 50 µL of PBS buffer was incubated in a 96-well plate for 1 h and 24 h at 37 °C, and then MTT (1 mg/mL) was added and incubated at 37 °C for an additional 1 h. The insoluble formazan product was dissolved in 200 µL 0.04 M HCl in isopropanol and measured spectrophotometrically at 570 nm using a microplate reader.

Neutrophils cytotoxicity was also assessed by flow cytometry with the propidium iodide assay [26]. The suspension of neutrophils (2 × 10⁵/mL in PBS or RPMI 1640 medium) with 50 µL of the tested extract at final concentrations of 10 and 50 µg/mL, or 50 µL of PBS buffer or RPMI 1640 medium was incubated for 1 h and 24 h at 37 °C, respectively, and then centrifuged at 1000 × g for 10 min at 4 °C. PMNs were resuspended in 400 µL of PBS containing 5 µL of propidium iodide (50 µg/mL) and left for 15 min in the dark. The cells were analyzed by flow cytometry FACSCalibur (Becton Dickinson), and data from 10 000 events were recorded. Cells that displayed high permeability to propidium iodide were expressed as a percentage of IP (+) cells.

Statistical analysis

The results are expressed as the mean ± SEM of the indicated number of experiments. The IC₅₀ values of the tested extract were obtained by interpolation from linear regression. All analyses were performed using Statistica 8 software. Statistical significance of differences between means was established by ANOVA with Dunnettʼs post hoc test. P values below 0.05 were considered statistically significant.

Results

The total phenolic content in the extract was 170.0 ± 14.8 mg/g. The HPLC-DAD-MSⁿ analysis allowed to characterize about 40 compounds belonging to the group of flavonoids, phenylpropanoids, hydroxycinnamates, and secoiridoids. A part of these compounds were previously identified in Ligustrum vulgare ([Table 1]). Based on UV-Vis spectra and MSⁿ, we were able to identify the presence of rare glucarates of p-coumaric and caffeic acid, as well as more common depsides of quinic acid and hydroxycinnamates. The peaks of dominating p-coumaroyl glucarates had [M – H]⁻ 355. The MS² fragmentation showed a cleavage of p-coumaroyl [M – H – 146]⁻ resulting in m/z 209, indicating the presence of glucaric acid ([Table 1]). The dominating compounds in the extract were as follows: oleoside, p-coumaroyl glucarate isomer (Rt = 11.3), secoiridoids derivative (Rt = 22.6), p-coumaroyl quinic acid isomer (Rt = 27.1), echinacoside, secoiridoids derivative (Rt = 37.8), ligustaloside A, verbascoside, luteolin glucoside, quercetin rutinoside, oleacein, ligustroflavone, and oleuropein ([Fig. 1]).

The extract inhibited ROS generation in human neutrophils in a dose-dependent manner. The inhibitory effect was comparable in both stimuli models ([Fig. 2 A] and [B]). The values of IC₅₀ were 19.8 ± 3.0 µg/mL for PMA stimulation and 18.2 ± 4.0 µg/mL for f-MLP stimulation. At the same time, the IC₅₀ values for positive control vitamin C were 4.9 ± 2.0 µg/mL (PMA) and 1.0 ± 0.4 µg/mL (f-MLP), respectively. The aqueous extract inhibited MPO release in a concentration-dependent manner ([Fig. 3]). It inhibited MPO release from 24.2 ± 4.9 % for the concentration of 10 µg/mL to 37.4 ± 3.1 % for the concentration of 50 µg/mL. The positive control gallic acid [24] at the concentration of 10 µg/mL showed a reduction of MPO release by 80.6 ± 4.4 %. It also inhibited elastase release from 23.9 ± 12.4 % for the lowest concentration of 5 µg/mL to 34.1 ± 8.6 % for the concentration of 50 µg/mL ([Fig. 4]). The positive control quercetin [27] at a concentration of 10 µg/mL showed a reduction of elastase release by 54.4 ± 4.8 %. Activation of neutrophils by LPS resulted in the release of MMP-9 to the level of 694 ± 25 ng/2 × 10⁶ cells in comparison to the level of 270 ± 44 ng/2 × 10⁶ cells in the untreated control. Incubation for 24 h of LPS-stimulated neutrophils with the aqueous extract from Ligustrum vulgare leaves (5–50 µg/mL) resulted in a weak reduction in MMP-9 release by the cell ([Fig. 5]). The extract at the highest concentration reduced MMP-9 release from the cell culture by 19.4 ± 4.1 % in a statistically significant manner. The positive control chlorogenic acid [28] at a concentration of 10 µg/mL showed the reduction of MMP-9 release by 22.3 ± 6.0 %. Activation of the neutrophils by LPS resulted in release of IL-8 to the level of 1486 ± 43 pg/2 × 10⁶ cells in comparison to the level of 13 ± 6 pg/2 × 10⁶ cells in the untreated control. Incubation for 24 h of LPS-stimulated neutrophils with the aqueous extract (5–50 µg/mL) resulted in a statistically significant reduction of IL-8 release by the cell culture ([Fig. 6]). The extract inhibited IL-8 release from 20.3 ± 3.5 % for the concentration of 10 µg/mL to 27.1 ± 0.3 % for the concentration of 50 µg/mL. The positive control pentagalloyl glucose [29], [30] at the concentration of 10 µg/mL showed a reduction of IL-8 production by 68.5 ± 4.4 %. The treatment of neutrophils with LPS (1 µg/mL) alone decreased the expression of CD62L (left shift of histogram) and increased the expression of CD11b (right shift of histogram). The pretreatment of neutrophils with aqueous extract in the concentration of 50 µg/mL slightly increased CD62L expression by shifting the histogram on the right, whereas the expression of CD11b decreased and histogram was shifted on the left in the direction of non-stimulated controls ([Fig. 7]). However, the increase of CD62L expression was less spectacular than the decrease of CD11b/CD18 expression in comparison with cells treated with LPS. At the same time, the extract in the concentration range used in the experiments had no adverse effect on the neutrophils viability after 1 h incubation and after 24 h incubation (not shown). The cytotoxic effect after 1 h and 24 h incubation was also performed using the propidium iodide assay. At the concentration of 50 µg/mL after 1 h of incubation, the extract has shown statistically significant diminished membrane integrity in comparison with the control (11.4 ± 1.4 % vs. 17.7 ± 4.3 % PI (+) cells). After 24 h of incubation, no cytotoxic effect compared to the control cells was observed ([Fig. 8 A]). The positive control camptothecin [31] at the concentration of 10 µg/mL showed increased cytotoxicity to the level of 25.3 ± 6.2 % and 29.0 ± 1.2 % after 1 h and 24 h incubation, respectively ([Fig. 8 B]).

Discussion

The biological effects of the aerial parts of species from the genus Ligustrum are suggested to be determined by the presence of glycosides and aglycones of flavonoids, phenylpropanoids, and secoiridoids [1]. Our phytochemical analysis using HPLC-DAD-MSⁿ confirmed the presence of these phenolic compounds in the aqueous extract from privet leaves. The occurrence of rare glucarates of p-coumaric and caffeic acid, as well as more common depsides of quinic acid and hydroxycinnamates was detected. The presence of hydroxycinnamates glucarates has been previously reported in Eupatorium perfoliatum L. [32] and Mercurialis perennis L. [33].

In the present study, we investigated the influence of phytochemically characterized aqueous extract from Ligustrum vulgare leaves on the functions of human neutrophils. Our results show for the first time its effect on oxidative burst, myeloperoxidase, elastase, IL-8, MMP-9 production, and adhesion molecules (L-selectin, β ₂ integrin) expression in neutrophils. We focused our attention on this plant material in reference to the usage of water infusions and decoctions from privet leaves in the folk medicine.

During inflammation, activated PMNs generate high amounts of ROS. In our experiment, we stimulated PMNs with bacterial derived peptide f-MLP, which acts by a specific receptor, or with PMA, which is a direct protein kinase C activator [34]. We used the oxidation of luminol or lucigenin to detect PMNs burst. Depending on the stimulator, cells produce ·O₂ ⁻, H₂O₂, and HOCl that oxidize luminol or only ·O₂ ⁻, H₂O₂ that oxidize lucigenin. The f-MLP-mediated stimulation in contrary to PMA-mediated stimulation led to the release of active myeloperoxidase which is responsible for the HOCl production from H₂O₂ [15], [35]. For this reason, luminol, which has more specificity to enhance HOCl and also ·O₂ ⁻, H₂O_2-mediated luminescence, was used together with f-MLP, while lucigenin with PMA, was used for detection of extracellular release of ·O₂ ⁻ [15]. In both stimuli models, the inhibitory effect was comparable (PMA: IC₅₀ = 19.8 ± 3.0 µg/mL; f-MLP: 18.2 ± 4.0 µg/mL). Our results suggest an important scavenging activity of the extract from privet leaves against intracellular and extracellular ROS produced by neutrophils independently of the stimulating model. Among the antimicrobial systems present in the phagosomes, the MPO-H₂O₂-Cl⁻ system plays a crucial role. The initial product of MPO-H₂O₂-Cl⁻ is HOCl, a potent antimicrobial agent [36]. However under pathological conditions, the excessive activation of the MPO-H₂O₂-Cl⁻ system is responsible for adverse effects on tissues and oxidation of arachidonic acid, which initiates the inflammatory cascades [36], [37]. For this reason, in order to further elucidate the antioxidant and anti-inflammatory profile of the aqueous extract from privet leaves, we determined its direct effect on MPO release. Our results show that the privet leaves extract by inhibiting MPO production (37.4 % for the concentration of 50 µg/mL) may reduce the amount of MPO products and in this way prevent the inflammatory injury of tissue.

By releasing some chemotactic factors, neutrophils potentiate their recruitment and that of other leukocytes. Moreover, several cytokines and proteases released by neutrophils promote a systemic inflammatory response, modulate endothelial permeability and affect endothelial and smooth muscle cell response [38]. Neutrophils adherence to the vascular endothelium is the first step and an essential event of neutrophils accumulation. Adhesion molecules, such as L-selectin and β ₂ integrin (CD11b/CD18), are responsible for the rolling of neutrophils along the endothelium and their firm adhesion and migration to vascular endothelial cells and other inflamed sites. LPS-stimulated neutrophils demonstrate downregulation of CD62L and upregulation of CD11b [23].

The effect of privet extract on elastase (reduction by 34.1 ± 8.6 % for the concentration of 50 µg/mL) and IL-8 release (reduction by 27.1 ± 0.3 % for the concentration of 50 µg/mL) was stronger than the effect on MMP-9 release (reduction by 19.4 ± 4.1 % for the concentration of 50 µg/mL). Taking into account that neutrophil elastase directly activates metalloproteinases, such as MMP-9 and the inflammatory cytokines, such as IL-8 [39], the whole modulating effect of privet leavesʼ extract on extracellular matrix degradation and chemotaxis during acute inflammation might be more spectacular. Additionally, strong antioxidant activity of the aqueous extract from privet leaves may also reduce levels of oxidant-activated MMPs [40]. Coincubation of stimulated neutrophils with aqueous extract from privet leaves upregulates CD62L expression and downregulates CD11b/CD18 expression. In this manner, by inhibition of extravascular migration of neutrophils, the local inflammation might be alleviated.

In conclusion, our results offer a pharmacological explanation of the activity of aqueous extract from Ligustrum vulgare. We present further evidence that aqueous extract may play an important role against ROS-induced injury of human biomolecules and tissue dysfunctions and in this manner diminish the risk of chronic inflammatory diseases. The modulating effect of the extract on proteases and cytokines release may contribute to the tissue repair and to healing improvement. However, it is not possible to predict the in vivo anti-inflammatory effect after oral administration of Ligustrum extracts. Nevertheless, our results partly support traditional use, especially external, of privet extract against wounds and smooth inflammations and indicate this species as a potential source of a natural anti-inflammatory agent.

Conflict of Interest

The authors have declared that there is no conflict of interest.

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• 19 Ciz M, Denev P, Kratchanova M, Vasicek O, Ambrozova G, Lojek A. Flavonoids inhibit the respiratory burst of neutrophils in mammals. Oxid Med Cell Longev 2012; 2012: 181295
  PubMedSearch in Google Scholar
• 20 OʼDowd Y, Driss F, Dang PMC, Elbim C, Gougerot-Pocidalo M-A, Pasquier C, El-Benna J. Antioxidant effect of hydroxytyrosol, a polyphenol from olive oil: scavenging of hydrogen peroxide but not superoxide anion produced by human neutrophils. Biochem Pharmacol 2004; 68: 2003-2008
  CrossrefPubMedSearch in Google Scholar
• 21 Van den Worm E, Beukelman CJ, Van den Berg AJJ, Kroes BH, Labadie RP, Van Dijk H. Effects of methoxylation of apocynin and analogs on the inhibition of reactive oxygen species production by stimulated human neutrophils. Eur J Pharmacol 2001; 433: 225-230
  CrossrefPubMedSearch in Google Scholar
• 22 Wanikiat P, Panthong A, Sujayanon P, Yoosook C, Rossi AG, Reutrakul V. The anti-inflammatory effects and the inhibition of neutrophil responsiveness by Barleria lupulina and Clinacanthus nutans extracts. J Ethnopharmacol 2008; 116: 234-244
  CrossrefPubMedSearch in Google Scholar
• 23 Liu JJ, Song CW, Yue Y, Duan CG, Yang J, He T, He YZ. Quercetin inhibits LPS-induced delay in spontaneous apoptosis and activation of neutrophils. Inflamm Res 2005; 54: 500-507
  CrossrefPubMedSearch in Google Scholar
• 24 Kroes BH, Van den Berg AJJ, Quarles van Ufford HC, Van Dijk H, Labadie RP. Anti-inflammatory activity of gallic acid. Planta Med 1992; 58: 499-504
  Thieme ConnectPubMedSearch in Google Scholar
• 25 Böyum A. A one-stage procedure for isolation of granulocytes and lymphocytes from human blood. General sedimentation properties of white blood cells in a 1 g gravity field. Scand J Clin Lab Invest 1968; 97: 51-76
  PubMedSearch in Google Scholar
• 26 Schinella G, Aquila S, Dade M, Giner R, Recio Mdel C, Spegazzini E, de Buschiazzo P, Tournier H, Ríos JL. Anti-inflammatory and apoptotic activities of pomolic acid isolated from Cecropia pachystachya . Planta Med 2008; 74: 215-220
  Thieme ConnectPubMedSearch in Google Scholar
• 27 Kanashiro A, Souza JG, Kabeya LM, Azzolini AE, Lucisano-Valim YM. Elastase release by stimulated neutrophils inhibited by flavonoids: importance of the catechol group. Z Naturforsch C 2007; 62: 357-361
  PubMedSearch in Google Scholar
• 28 Jin UH, Lee JY, Kang SK, Kim JK, Park WH, Kim JG, Moon SK, Kim CH. A phenolic compound, 5-caffeoylquinic acid (chlorogenic acid), is a new type and strong matrix metalloproteinase-9 inhibitor: isolation and identification from methanol extract of Euonymus alatus . Life Sci 2005; 77: 2760-2769
  CrossrefPubMedSearch in Google Scholar
• 29 Kiss AK, Filipek A, Czerwińska M, Naruszewicz M. Oenothera paradoxa defatted seeds extract and its bioactive component penta-O-galloyl-β-D-glucose decreased production of reactive oxygen species and inhibited release of leukotriene B₄, interleukin-8, elastase and myeloperoxidase in human neutrophils. J Agric Food Chem 2010; 58: 9960-9966
  CrossrefPubMedSearch in Google Scholar
• 30 Oh GS, Pae HO, Choi BM, Lee HS, Kim IK, Yun YG, Kim JD, Chung HT. Penta-O-galloyl-β-D-glucose inhibits phorbol myristate acetate-induced interleukin-8 gene expression in human monocytic U937 cells through its activation of nuclear factor-κB. Int Immunopharmacol 2004; 4: 377-386
  CrossrefPubMedSearch in Google Scholar
• 31 Shellhaas JL, Zuckerman SH. In vitro detection of apoptotic stimuli by use of the HL-60 myeloid leukemic cell line. Clin Diagn Lab Immunol 1995; 2: 598-603
  PubMedSearch in Google Scholar
• 32 Maas M, Petereit F, Hensel A. Caffeic acid derivatives from Eupatorium perfoliatum L. Molecules 2009; 14: 36-45
  PubMedSearch in Google Scholar
• 33 Lorenz P, Conrad J, Bertrams J, Berger M, Duckstein S, Meyer U, Stintzing FC. Investigations into the phenolic constituents of Dogʼs mercury (Mercurialis perennis L.) by LC-MS/MS and GC-MS analyses. Phytochem Anal 2012; 23: 60-71
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• 34 Sheppard FR, Kelher MR, Moore EE, McLaughlin NJ, Banerjee A, Silliman CC. Structural organization of the neutrophil NADPH oxidase: phosphorylation and translocation during priming and activation. J Leukoc Biol 2005; 78: 1025-1042
  CrossrefPubMedSearch in Google Scholar
• 35 Franck T, Kohnen S, de la Rebière G, Deby-Dupont G, Deby C, Niesten A, Serteyn D. Activation of equine neutrophils by phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine induces a different response in reactive oxygen species production and release of active myeloperoxidase. Vet Immunol Immunopathol 2009; 130: 243-250
  CrossrefPubMedSearch in Google Scholar
• 36 Malle E, Furtmüller PG, Sattler W, Obinger C. Myeloperoxidase: a target for new drug development?. Br J Pharmacol 2007; 152: 838-854
  CrossrefPubMedSearch in Google Scholar
• 37 Zhang R, Brennan ML, Shen Z, MacPherson JC, Schmitt D, Molenda CE, Hazen SL. Myeloperoxidase functions as a major enzymatic catalyst for initiation of lipid peroxidation at sites of inflammation. J Biol Chem 2002; 277: 46116-46122
  CrossrefPubMedSearch in Google Scholar
• 38 Baetta R, Corsini A. Role of polymorphonuclear neutrophils in atherosclerosis: current state and future perspectives. Atherosclerosis 2010; 210: 1-13
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• 39 Alam SR, Newby DE, Henriksen PA. Role of the endogenous elastase inhibitor, elafin, in cardiovascular injury: from epithelium to endothelium. Biochem Pharmacol 2012; 83: 695-704
  CrossrefPubMedSearch in Google Scholar
• 40 Yager DR, Nwomeh BC. The proteolytic environment of chronic wounds. Wound Repair Regen 1999; 7: 433-441
  CrossrefPubMedSearch in Google Scholar

Correspondence

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  CrossrefPubMedSearch in Google Scholar
• 21 Van den Worm E, Beukelman CJ, Van den Berg AJJ, Kroes BH, Labadie RP, Van Dijk H. Effects of methoxylation of apocynin and analogs on the inhibition of reactive oxygen species production by stimulated human neutrophils. Eur J Pharmacol 2001; 433: 225-230
  CrossrefPubMedSearch in Google Scholar
• 22 Wanikiat P, Panthong A, Sujayanon P, Yoosook C, Rossi AG, Reutrakul V. The anti-inflammatory effects and the inhibition of neutrophil responsiveness by Barleria lupulina and Clinacanthus nutans extracts. J Ethnopharmacol 2008; 116: 234-244
  CrossrefPubMedSearch in Google Scholar
• 23 Liu JJ, Song CW, Yue Y, Duan CG, Yang J, He T, He YZ. Quercetin inhibits LPS-induced delay in spontaneous apoptosis and activation of neutrophils. Inflamm Res 2005; 54: 500-507
  CrossrefPubMedSearch in Google Scholar
• 24 Kroes BH, Van den Berg AJJ, Quarles van Ufford HC, Van Dijk H, Labadie RP. Anti-inflammatory activity of gallic acid. Planta Med 1992; 58: 499-504
  Thieme ConnectPubMedSearch in Google Scholar
• 25 Böyum A. A one-stage procedure for isolation of granulocytes and lymphocytes from human blood. General sedimentation properties of white blood cells in a 1 g gravity field. Scand J Clin Lab Invest 1968; 97: 51-76
  PubMedSearch in Google Scholar
• 26 Schinella G, Aquila S, Dade M, Giner R, Recio Mdel C, Spegazzini E, de Buschiazzo P, Tournier H, Ríos JL. Anti-inflammatory and apoptotic activities of pomolic acid isolated from Cecropia pachystachya . Planta Med 2008; 74: 215-220
  Thieme ConnectPubMedSearch in Google Scholar
• 27 Kanashiro A, Souza JG, Kabeya LM, Azzolini AE, Lucisano-Valim YM. Elastase release by stimulated neutrophils inhibited by flavonoids: importance of the catechol group. Z Naturforsch C 2007; 62: 357-361
  PubMedSearch in Google Scholar
• 28 Jin UH, Lee JY, Kang SK, Kim JK, Park WH, Kim JG, Moon SK, Kim CH. A phenolic compound, 5-caffeoylquinic acid (chlorogenic acid), is a new type and strong matrix metalloproteinase-9 inhibitor: isolation and identification from methanol extract of Euonymus alatus . Life Sci 2005; 77: 2760-2769
  CrossrefPubMedSearch in Google Scholar
• 29 Kiss AK, Filipek A, Czerwińska M, Naruszewicz M. Oenothera paradoxa defatted seeds extract and its bioactive component penta-O-galloyl-β-D-glucose decreased production of reactive oxygen species and inhibited release of leukotriene B₄, interleukin-8, elastase and myeloperoxidase in human neutrophils. J Agric Food Chem 2010; 58: 9960-9966
  CrossrefPubMedSearch in Google Scholar
• 30 Oh GS, Pae HO, Choi BM, Lee HS, Kim IK, Yun YG, Kim JD, Chung HT. Penta-O-galloyl-β-D-glucose inhibits phorbol myristate acetate-induced interleukin-8 gene expression in human monocytic U937 cells through its activation of nuclear factor-κB. Int Immunopharmacol 2004; 4: 377-386
  CrossrefPubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
• 33 Lorenz P, Conrad J, Bertrams J, Berger M, Duckstein S, Meyer U, Stintzing FC. Investigations into the phenolic constituents of Dogʼs mercury (Mercurialis perennis L.) by LC-MS/MS and GC-MS analyses. Phytochem Anal 2012; 23: 60-71
  CrossrefPubMedSearch in Google Scholar
• 34 Sheppard FR, Kelher MR, Moore EE, McLaughlin NJ, Banerjee A, Silliman CC. Structural organization of the neutrophil NADPH oxidase: phosphorylation and translocation during priming and activation. J Leukoc Biol 2005; 78: 1025-1042
  CrossrefPubMedSearch in Google Scholar
• 35 Franck T, Kohnen S, de la Rebière G, Deby-Dupont G, Deby C, Niesten A, Serteyn D. Activation of equine neutrophils by phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine induces a different response in reactive oxygen species production and release of active myeloperoxidase. Vet Immunol Immunopathol 2009; 130: 243-250
  CrossrefPubMedSearch in Google Scholar
• 36 Malle E, Furtmüller PG, Sattler W, Obinger C. Myeloperoxidase: a target for new drug development?. Br J Pharmacol 2007; 152: 838-854
  CrossrefPubMedSearch in Google Scholar
• 37 Zhang R, Brennan ML, Shen Z, MacPherson JC, Schmitt D, Molenda CE, Hazen SL. Myeloperoxidase functions as a major enzymatic catalyst for initiation of lipid peroxidation at sites of inflammation. J Biol Chem 2002; 277: 46116-46122
  CrossrefPubMedSearch in Google Scholar
• 38 Baetta R, Corsini A. Role of polymorphonuclear neutrophils in atherosclerosis: current state and future perspectives. Atherosclerosis 2010; 210: 1-13
  CrossrefPubMedSearch in Google Scholar
• 39 Alam SR, Newby DE, Henriksen PA. Role of the endogenous elastase inhibitor, elafin, in cardiovascular injury: from epithelium to endothelium. Biochem Pharmacol 2012; 83: 695-704
  CrossrefPubMedSearch in Google Scholar
• 40 Yager DR, Nwomeh BC. The proteolytic environment of chronic wounds. Wound Repair Regen 1999; 7: 433-441
  CrossrefPubMedSearch in Google Scholar