Effects of Phytochemically Characterized Extracts From Syringa vulgaris and Isolated Secoiridoids on Mediators of Inflammation in a Human Neutrophil Model

Aim of the study: The aim of the present study was to investigate the effects of phytochemically characterized extracts connected with the traditional use (infusions and ethanolic extracts) of different parts of Syringa vulgaris (common lilac) on the pro-inflammatory functions of neutrophils. Active compounds were isolated from the most promising extract(s) using bioassay-guided fractionation, and their activity and molecular mechanisms of action were determined. Methods: The extracts were characterized using a HPLC-DAD- MSn method. The effects on ROS, MMP-9, TNF-α, IL-8, and MCP-1 production by neutrophils were measured using luminol-dependent chemiluminescence and enzyme-linked immunosorbent assay (ELISA) methods. The effects on p38MAPK, ERK1/2, JNK phosphorylation, and NF-kB p65 translocation were determined using western blots. Results: The major compounds detected in the extracts and infusions belong to structural groups, including caffeic acid derivatives, flavonoids, and iridoids. All extracts and infusions were able to significantly reduce ROS and IL-8 production. Bioassay-guided fractionation led to the isolation of the following secoiridoids: 2″-epiframeroside, oleonuezhenide, oleuropein, ligstroside, neooleuropein, hydroxyframoside, and framoside. Neooleuropein appeared to be the most active compound in the inhibition of cytokine production by attenuating the MAP kinase pathways. Conclusion: The present study demonstrated that common lilac, which is a traditionally used medicinal plant in Europe, is a valuable source of active compounds, especially neooleuropein.

Scientific data supporting the traditional use of S. vulgaris are connected with its antioxidant, anti-inflammatory, antinociceptive and antipyretic properties (Bálinet, 1971;Tóth et al., 2015Tóth et al., , 2016Dudek et al., 2017). Moreover, information about the single compounds responsible for each of these activities is scarce.
The aim of the present study was to investigate the effects of phytochemically characterized (HPLC-DAD-MS n ) extracts connected with the traditional use (infusions and ethanolic extracts) of different parts of the common lilac (bark, fruits, leaves and flowers) on the pro-inflammatory functions of neutrophils, such as reactive oxygen species (ROS) production, metalloproteinase-9 (MMP-9), interleukin 8 (IL-8), and tumor necrosis factor (TNF-α) release. We isolated active compounds from the most promising extracts using bioassay-guided fractionation. Finally, we focused on the activity and molecular mechanisms of action of the isolated compounds that were able to regulate neutrophil inflammation. The biological activities of single compounds were compared with oleuropein and ligstroside, characteristic compounds from the Oleacea family (Jensen et al., 2002), and with positive control clarithromycin, a macrolide antibiotic, which has been shown to inhibit the pro-inflammatory function of neutrophils (Simpson et al., 2008;Cervin et al., 2009).
NMR spectra were recorded on a Bruker Advance III 600 MHz spectrometer (Bruker Biospin, Germany) in methanold 4 . Preparative HPLC was performed with a Shimadzu LC-20AP instrument (Japan) using a Zorbax SB-C18 column (150 × 21.2 mm, 5 µm) (Agilent, USA) at a flow rate of 20.0 mL/min. TLC was performed on Merck silica gel 60 F 254 (0.25 mm) plates with a dichloromethane/methanol/formic acid/water (80:25:1.5:4; v/v/v/v) solvent system. Chromatograms were visualized by spraying with sulfuric acid (5% in methanol) followed by heating at 105 • C for 10 min. All solvents used for chromatography were of gradient grade. HPLC-DAD-MS n analysis was performed on a UHPLC-3000 RS system (Dionex, Germany) with DAD detection and an AmaZon SL ion trap mass spectrometer with an ESI interface (Bruker Daltonik GmbH, Germany). Separation was performed on a Zorbax SB-C18 column (150 × 2.1 mm, 1.9 µm) (Agilent, USA). The mobile phase consisted of 0.1% HCOOH in water (A) and 0.1% HCOOH in MeCN (B) using the following gradients: 0-60 min, 5-40% B. The LC eluate was introduced into the ESI interface without splitting, and compounds were analyzed in negative ion mode with the following settings: nebulizer pressure of 40 psi; drying gas flow rate of 9 L/min; nitrogen gas temperature of 300 • C; and a capillary voltage of 4.5 kV. The mass scan ranged from 100 to 2,200 m/z. UV spectra were recorded in the range of 200-400 nm.
The absorbance and luminescence were measured using a BioTek microplate reader (Highland Park, USA). Flow cytometry was performed using a BD FACSCalibur apparatus (BD Biosciences, USA).

Plant Material
Bark from the branches, leaves, flowers, and fruits of S. vulgaris L. were collected in May and August (fruits) 2016, from a native plant growing in Legionowo, Mazovian district, Poland (52 • 23 ′ 17.647 ′′ N; 20 • 55 ′ 53.171 ′′ E). The plant material was authenticated according to Flora Europaea (Tutin et al., 1972) by Anna K. Kiss. A voucher specimen (no. SV052015) has been deposited in the Plant Collection, Department of Pharmacognosy and Molecular Basis of Phytotherapy, Medical University of Warsaw, Poland ( Figure 1S).

Infusions (Aqueous Extracts)
Air-dried plant materials (5 g) were poured with boiling water (250 mL), covered, and allowed to stand for 15 min. Extracts were then filtered and lyophilized, resulting in the following aqueous extract masses: bark-0.52 g, fruit-0.48 g, flower-1.69 g, and leaf-1.04 g.

Ethanolic Extracts
The air-dried plant materials (2 g) were extracted with 60% ethanol (v/v, 1:20) in a water bath (70 • C) for 1 h. Then, extracts were filtered, the ethanol was evaporated, and the water residues were lyophilized, resulting in the following ethanolic extract masses: bark-0.25 g, fruit-0.21 g, flower-0.58 g and leaf-0.51 g.
The extracts were characterized using an HPLC-DAD-MS/MS method. The presence of substances in extracts was confirmed by comparing the retention times and spectra (UV, MS, MS/MS) with standards and/or published data.

Preparation of Solutions of Extracts, Fractions, and Compounds for Bioassay
Tested extracts were dissolved in PBS or HBSS (1 mg/mL). Fractions and compounds were dissolved in DMSO (10 mg/mL or 10 mM stock solutions) and then diluted with RPMI 1640 medium. The extracts were tested in a concentration range of 25-100 µg/mL. Compounds were tested at concentrations of 10-50 µM. The concentration of DMSO used (<0.1%) did not influence the assays.

Isolation of Human Neutrophils
Peripheral venous blood was taken from healthy human donors (18-35 years old) in the Warsaw Blood Donation Centre. Donors did not smoke or take any medications. They were clinically recognized to be healthy and a routine laboratory tests showed all values to be within the normal ranges. The study conformed to the principles of the Declaration of Helsinki.
Neutrophils were isolated by dextran sedimentation and centrifugation in a Ficoll Hypaque gradient (Böyum, 1968) and then re-suspended in HBSS buffer or RPMI 1640 medium.

Cytotoxicity
Cytotoxicity was determined by a standard flow cytometric probe using propidium iodide (PI) staining. After 24 h of incubation with extracts/compounds, the neutrophils were harvested and centrifuged (1500 RPM; 10 min; 4 • C), washed once with cold PBS and re-suspended in 400 µL of PBS. Five microliters of PI (50 µg/mL) solution was added to the cell suspensions. After 15 min of incubation at room temperature, cells were analyzed by flow cytometry, and 10,000 events were recorded per sample. Cells that displayed high permeability to PI were expressed as a percentage of PI(+) cells.

ROS Production
Neutrophils (3.5 × 10 5 ) were incubated in HBSS buffer with 50 µL of the tested extracts and 50 µL of luminol (20 mM) in a 96-well plate. ROS production was initiated by the addition of f-MLP (0.1 µg/mL) to obtain a total volume of 200 µL/well. Chemiluminescence changes were measured over 40 min at intervals of 2 min in a microplate reader. The background chemiluminescence produced by non-stimulated cells was also determined. The percentage of inhibition was calculated by comparison to the control without the tested extracts at the maximum luminescence.
IL-8, MMP-9, MCP-1, and TNF-α Production Neutrophils (2 × 10 6 ) were cultured in 24-well plates in RPMI 1640 medium with 10% FBS, 10 mM HEPES, and 2 mM Lglutamine in the presence or absence of LPS (100 ng/mL) for 24 h at 37 • C with 5% CO 2 in the presence or absence of test extracts/fractions/compounds. After 24 h, the neutrophils were harvested and centrifuged (2000 RPM; 10 min; 4 • C). The amount of released cytokines was measured by enzyme-linked immunosorbent assay (ELISA) following the manufacturer's instructions (BD Biosciences, USA). The effects on IL-8, MMP-9, MCP-1, and TNF-α production were calculated by comparing the percentages of the released agents to the stimulated control, which lacked the test extracts/fractions/compounds.

Western Blotting
Neutrophils (4 × 10 6 ) were suspended in RPMI 1640 medium and incubated for 40 min at 37 • C in the presence or absence of LPS (100 ng/mL) and in the presence or absence of the test compounds. They were then centrifuged at 1500 RPM for 10 min at 4 • C. Cells were lysed in ice-cold buffer containing PBS, 5 mM EDTA, 1% Triton X-100, phosphatase and protease inhibitors, and the resulting lysates were centrifuged at 8,000 RPM for 15 min at 4 • C. The proteins were separated by 12% SDS-PAGE. Proteins were transferred to nitrocellulose filters and immunoblotted with rabbit anti-p38MAPK, anti-ERK1/2, and anti-JNK at 1:1000 dilutions and a rabbit anti-actin polyclonal antibody at a 1:2000 dilution. Peroxidase-conjugated affinipure goat anti-rabbit antibody was used as a secondary antibody at a dilution of 1:10000. Finally, the blots were incubated with chemiluminescent substrate for the detection of HRP (Thermo-Scientific, USA) for 10-15 min. Western blots were quantified using the ImageJ 1.38 software after densitometric scanning of the bands.

Statistical Analysis
The results were expressed as the mean ± SEM of three independent experiments performed at least in duplicate. All analyses were performed using Statistica 9 software. The statistical significance of the differences between means was established by ANOVA with Dunnett's or Tukey's post hoc test. P-values below 0.05 were considered statistically significant.

Effect of Bark, Fruit, Flower and Leaf Infusions and Ethanolic Extracts of S. vulgaris on the Pro-inflammatory Function of Stimulated Neutrophils
In all concentrations of the tested extracts, no significant reduction in membrane integrity was observed in comparison to the control using a propidium iodide assay (Figures 2A,B). Activation of neutrophils by f-MLP results in degranulation and a significant release of ROS compared to the untreated control (Figures 2C,D). Additionally, stimulation with LPS resulted in a significant release of proteinase MMP-9, chemokine IL-8, and cytokine TNF-α (Figure 3). Incubation of stimulated neutrophils with extracts at concentration ranges of 25-100 µg/mL resulted in a statistically significant and dose dependent reduction of ROS production by all extracts over all tested concentrations. There was no statistically significant difference between infusions and ethanolic extracts (Figures 2C,D).
All infusions and extracts were able to inhibit the release of IL-8 in a dose-dependent manner (Figures 3A,B). The effect Frontiers in Pharmacology | www.frontiersin.org   Data were expressed as the mean ± SEM; at least three independent experiments were conducted, and they were assayed in duplicate. Experiments were performed using cells from different donors. Statistical significance #P < 0.01 compared to the non-stimulated control; *P < 0.05, **P < 0.01, ***P < 0.001 decrease compared to the stimulated control.
was significant (p < 0.001) for all concentrations tested of the flower infusion and flower and leaf extracts. The effect on TNFα production/release was less pronounced and was statically significant only at the highest concentration of 100 µg/mL for bark and leaf ethanolic extracts and the flower infusion (Figures 3C,D). MMP-9 release was inhibited the most by bark and fruit ethanolic extracts (Figures 3E,F). In general, ethanolic extracts were slightly more active than infusions, and flower and leaf preparations were more active than bark and fruit preparations. In a previous study, we isolated 29 compounds that were hydroxycinnamoyl and secoiridoid derivatives (Dudek et al., 2017) from the flower extract with moderate antiinflammatory activity. In this study, we performed a bioassayguided isolation of active compounds from the leaf extract.

Effect on Cytokine/Chemokine Production, MAP Kinases and NF-kB Activation
Lipopolysaccharide stimulation of human neutrophils resulted in the induction of the production/release of chemokines IL-8 (neutrophil chemotactic factor) and MCP-1 (monocyte chemoattractant protein 1) along with cytokine TNF-α in comparison to cells that were not stimulated. Isolated compounds I, II, V, VI, and VII were tested at concentrations of 10, 25, and 50 µM, and their activity was compared with oleuropein and ligstroside, the characteristic compounds of the Oleacea family that were also isolated from active fraction F6, and with the positive control clarithromycin, a macrolide antibiotic which has been shown to inhibit the pro-inflammatory function of neutrophils. At all concentrations of the tested compounds, no significant reduction in membrane integrity ; Data were expressed as the mean ± SEM; at least three independent experiments were conducted, and they were assayed in duplicate. Experiments were performed using cells from different donors. Statistical significance #P < 0.01 compared to the non-stimulated control; *P < 0.05, **P < 0.01, ***P < 0.001 decrease compared to the stimulated control.
was observed in comparison to the control using a propidium iodide assay ( Figure 6A). Neooleuropein was the most active compound toward inhibiting IL-8 secretion ( Figure 6B); at concentrations of 25 and 50 µM, the release was reduced to 50.2% (p < 0.001) and 31.4% (p < 0.001) in comparison with LPS-stimulated cells (100% of release), respectively. The effect of neooleuropein at 50 µM was more significant than the effects of 2 ′′ -epiframeroside (p < 0.001), oleonuezhenide (p < 0.001), ligstroside (p < 0.001), and oleuropein (p < 0.001) at the same concentration. Framoside and neooleuropein were the most active in reduction of TNF-α release at all concentrations tested ( Figure 6C). The effect of framoside at 50 µM was more significant than the effect of 2 ′′ -epiframeroside (p < 0.001), oleonuezhenide (p < 0.001), and ligstroside (p < 0.001) at the same concentration. Additionally, neooleuropein was statistically more active than the positive control clarithromycin (p < 0.05). All isolated compounds except oleonuezhenide significantly inhibited the release of MCP-1 from stimulated neutrophils ( Figure 6D). However, only neooleuropein was active at all concentrations tested ( Figure 6D). The effect of neooleuropein at 10 µM was more significant than the effects of framoside (p < 0.05) and hydroxyframoside (p < 0.05); at 25 µM, the effect was more significant than the effects of framoside (p < 0.01), hydroxyframoside (p < 0.01) and oleonuezhenide (p < 0.01), while at 50 µM, the effect was more significant than the effect of oleonuezhenide (p < 0.001) and the positive control clarithromycin (p < 0.01) at the same concentration. In general, neooleuropein was as effective or more effective in comparison with oleuropein and clarithromycin in reducing the release of pro-inflammatory agents. The molecular mechanism of the observed effect was related to MAP kinases and NF-kB activation. Lipopolysaccharide stimulation of human neutrophils resulted in the rapid phosphorylation of proteins, including p38 MAPK, p42/44 extracellular signal-regulated kinase (ERK), and c-Jun NH 2terminal kinase (JNK), as well as the translocation of NF-kB-p65 from the cytoplasm to the nucleus. Our data show that the LPS-induced phosphorylation of p38 MAPK and ERK1/2 FIGURE 4 | Effect of S. vulgaris leaf extract fractions on IL-8 production by stimulated neutrophils [%] (A) on TNF-α production by stimulated neutrophils [%] (B); scheme of fractionation and compounds isolation from leaf extract (C). Data were expressed as the mean ± SEM; at least three independent experiments were conducted, and they were assayed in duplicate. Experiments were performed using cells from different donors. Statistical significance #P < 0.01 compared to the non-stimulated control; *P < 0.05, **P < 0.01, ***P < 0.001 decrease compared to the stimulated control.
was decreased by neooleuropein and oleuropein, while JNK phosphorylation was decreased by all tested compounds at 50 µM ( Figure 7A). The inhibition of the translocation of NF-kB-p65 from the cytoplasm to the nucleus was observed most significantly for oleuropein; however, the activity of neooleuropein was evident but more modest (Figure 7B).

DISCUSSION
S. vulgaris preparations have been traditionally used in Europe to treat several ailments connected with inflammation and, together with Asian Syringa sp., appear to be an interesting source of diverse bioactive structures (Su et al., 2015). The aim of the present study was to investigate the effects of phytochemically characterized (HPLC-DAD-MS n ) extracts that have been used in traditional medicine (infusions and ethanolic extracts) from different parts of the common lilac (bark, fruits, leaves, and flowers) on the pro-inflammatory functions of neutrophils and to isolate the most active compounds using bioassay-guided fractionation. Finally, we focused on the activity and molecular mechanisms of action of the isolated compounds able to regulate neutrophil inflammation.
In the neutrophil model of the pro-inflammatory state, all extracts and infusions were able to significantly reduce ROS production (Figures 2C,D). This is probably related to the presence of compounds that contain caffeic acid, pcoumaric acid, hydroxytyrosol or tyrosol phenolic moieties, which display antioxidant properties (Bi et al., 2011;Dudek et al., 2017). All extracts and infusions were especially active in decreasing IL-8 production (Figures 3A,B) and, to a lesser extent, MMP-9 and TNFα release (Figures 3C-F). However, the leaf extract fractionation revealed that fraction F2 increased TNFα production, while fractions F5 and F6 significantly decreased the production of this cytokine (Figure 4). A similar phenomenon was observed for Echinacea purpurea extracts (Todd et al., 2015). Further bioassay-guided fractionation of fraction F6 led to the isolation of the following secoiridoids: 2 ′′ -epiframeroside, oleonuezhenide, oleuropein, ligstroside, neooleuropein, hydroxyframoside, and framoside ( Figure 5). We compared the anti-inflammatory activity of 2 ′′ -epiframeroside, oleonuezhenide, neooleuropein, hydroxyframoside, and framoside with oleuropein and ligstroside, two well-known compounds that are widespread in the Oleaceae family (Jensen et al., 2002). Apart from oleonuezhenide, a dimeric secoiridoid, all compounds exhibited significant inhibition of pro-inflammatory cytokine and chemokine release/production. 2 ′′ -epiframeroside primarily inhibited MCP-1 release, while neooleuropein appeared as the most active compound (Figure 6). Neooleuropein inhibited the secretion of IL-8 more significantly than oleuropein ( Figure 6B) and secretion of TNF-α and MCP-1 more significantly than the positive control clarithromycin (Figures 6C,D).
The mechanism of LPS stimulation of human neutrophils is connected with a functional response through the activation of mitogen-activated protein kinases (MAPKs): p38 kinase, p42/44 extracellular signal-regulated kinase (ERK) and c-Jun NH 2 -terminal kinases (JNKs) (Nick et al., 1999;Arndt et al., 2004;Simard et al., 2015). Neooleuropein and oleuropein significantly inhibited the phosphorylation of the MAP kinases ERK1/2, p38 and JNK after LPS stimulation (Figure 7A), as well as the translocation of NF-kB p65 to the nucleus ( Figure 7B). Neooleuropein was more active toward the inhibition of p38 phosphorylation, while oleuropein more significantly inhibited NF-kB p65 translocation, and all tested compounds inhibited JNK phosphorylation (Figures 7A,B). The activation of p38 MAPK in neutrophils is connected with the synthesis of TNFα and IL-8 (Nick et al., 1999). ERK activation also led to the elevated expression of pro-inflammatory cytokines in human neutrophils (Simard et al., 2015). In our study, neooleuropein was the most active compound in inhibiting p38 phosphorylation, which correlated with a significant inhibition of IL-8 production. However, oleuropein was more active in NF-kB inhibition, which has been shown to affect cytokine gene expression in human neutrophils, although it has a less marked effect on IL-8 gene expression (Cloutier et al., 2007). The inhibition of MCP-1 production by all tested compounds was correlated with the inhibition of JNK phosphorylation, which has been strictly correlated with MCP-1 expression but not IL-8 or TNFα expression (Arndt et al., 2004). Interestingly, framoside was able to significantly decrease the TNF-α and IL-8 secretion (Figures 6B,C) without affecting MAPKs nor NF-kB activation (Figure 7). It appears that framoside, the most lipophilic compounds, displays a different mode of action. As in human neutrophils other pathways such as PI3K are also involved in IL-8 production and acts downstream of p38 MAPK (Fortin et al., 2011).
The observed significant inhibition of TNF-α and chemokine (IL-8 and MCP-1) production is of special interest for treating inflammatory diseases such as rheumatic arthritis, respiratory diseases, and arteriosclerosis. The pro-inflammatory effect of TNF-α mainly results from its capacity to stimulate the expression of adhesive molecules in endothelial cells and promote neutrophil attachment to the vascular endothelium in addition to their degranulation and pro-oxidative activity (Witko-Sarsat et al., 2000). MCP-1 (CCL2) is able to stimulate chemotaxis of monocytes and cellular events associated with chemotaxis and integrin expression. At high concentrations, MCP-1 elicits a respiratory burst leading to the generation of ROS (Palomino and Marti, 2015). IL-8 (CXCL8), one of the most important cytokines produced by neutrophils, mediates chemotaxis, releases granule enzymes, and promotes integrin expression and adhesion to endothelial cells (Gabrilovich, 2013). Although the anti-inflammatory and antioxidant activity of oleuropein and ligstroside was intensively studied, this is the first report concerning the biological activity of 2 ′′ -epiframeroside, neooleuropein, hydroxyframoside, and framoside. In particular, neooleuropein appears to an interesting compound for further in vitro and in vivo study.

CONCLUSIONS
The present study demonstrated that the common lilac, which has been traditionally used in Europe as a medicinal plant, is a valuable source of active compounds, especially neooleuropein, for further research regarding their use in treating inflammatory diseases that result from the excessive activation of neutrophils. The observed decreases in the production of cytokines, such as TNF-α, IL-8, MCP-1, depend on the inhibition of the phosphorylation of MAP kinases.