Inhibition of Neutrophil Functions and Antibacterial Effects of Tarragon (Artemisia dracunculus L.) Infusion—Phytochemical Characterization

The aim of the study was to characterize phytochemicals in an infusion of the aerial parts of tarragon (Artemisia dracunculus L.) using ultra-high-performance liquid chromatography diode array detector electrospray ionisation tandem mass spectrometry UHPLC‐DAD‐ESI‐MS/MS method, as well as an evaluation of its effects on mediators of the inflammation in an in vitro model of human neutrophils, and antimicrobial activity on selected pathogens. Flavonoids and caffeoylquinic acids were the main phenolic components of the extract of tarragon’s aerial parts. The infusion was able to inhibit reactive oxygen species (ROS), interleukin 8 (IL-8), and tumour necrosis factor α (TNF-α) production. The antimicrobial assay was performed with the use of nine strains of bacteria, both Gram-negative and Gram-positive. Three human pathogens, Staphylococcus aureus ATCC6538, Staphylococcus epidermidis ATCC14990, and Staphylococcus aureus MRSA (methicyllin-resistant Staphylococcus aureus) ATCC43300, proved to be the most sensitive to tarragon infusion. Our study demonstrated the antiinflammatory and antimicrobial properties of tarragon (Artemisia dracunculus L.), meaning the common spice may be a prospective source of health-promoting constituents.


INTRODUCTION
The popularity of spices as ingredients in food for the prevention and treatment of disease has increased in recent years. Tarragon (Artemisia dracunculus L.), also known as estragon, dragon wormwood, false tarragon, or dragon's wort, is a species of a perennial herb of the Asteraceae family. The species name dracunculus is associated with the shape of the leaves, which is reminiscent of a dragon's tongue (Aglarova et al., 2008). Informal names for distinguishing the main variety include French tarragon and Russian tarragon. While French tarragon is well-described in the recognized Western scientific literature, considerable information on Russian tarragon is covered in only a few publications (Govorko et al., 2007;Eisenman et al., 2011;Obolskiy et al., 2011). French tarragon is often used as a culinary herb, whereas Russian tarragon is bitter and more often used for medicinal preparations.
Tarragon comes from eastern and central Europe, southern Russia, and western Asia (Raghavan, 2006;Obolskiy et al., 2011). In the wild, tarragon grows on alkaline soils, near birch groves, near rivers and old fallow land, in steppe areas, and in the area of hills and in mountains. Tarragon grows well in any soil and under any temperature and lighting and tolerates spring and autumn frosts. Tarragon is most often propagated by seeds, but also by division of plants, as well as cuttings and root slips (Aglarova et al., 2008). A. dracunculus is cultivated for use of the leaves as an aromatic culinary herb and for medicinal purposes. The herb has a long history of use as a carminative, for stimulating the appetite, and detoxification of the liver (Obolskiy et al., 2011). The plant helps to alleviate the pain associated with dental diseases and also acts as an antiinflammatory agent, and may also be useful in the treatment of microbial infections (Raghavan, 2006;Obolskiy et al., 2011;Eidi et al., 2016). Tarragon has also been used in the management of dysregulalated glucose metabolism, including hyperglycaemia, diabetes, and related metabolic syndromes (Obolskiy et al., 2011). A. dracunculus herb preparation has been applied widely for the treatment of skin wounds, irritations, allergic rashes, and dermatitis. A preliminary phytochemical study of A. dracunculus revealed the presence of essential oils (methyl eugenol, estragol, elemicin, terpinolene, and others), flavonoids (quercetin, luteolin, patuletin, kaemferol, isorhamnetin, naringenin, pinocembrin, and estragonoside and their glycosides), phenolic acids (cichoric acid, hydroxybenzoic acid, chlorogenic acid, caffeic acid, 5-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, and others), coumarins (herniarin, coumarin, esculetin, esculin, capillarin, 8-hydroxycapillarin, artemidin, 8-hydroxyartemidin, artemidinol, and others) and alkamides (pellitorin, neopellitorin A, and neopellitorin B) (Sayyah et al., 2004;Logendra et al., 2006;Govorko et al., 2007;Aglarova et al., 2008;Eisenman et al., 2011;Miron et al., 2011;Obolskiy et al., 2011;Lin and Harnly, 2012). The chemical composition of A. dracunculus was investigated using specific reactions, UV/Vis spectroscopy, nuclear magnetic resonance spectroscopy NMR, chromatographic techniques (gas chromatography GC, high performance liquid chromatography HPLC), and mass spectrometry MS.
The aim of this work was to evaluate an infusion of Russian tarragon as a potential food product for the prevention and treatment of inflammation and bacterial infections. We investigated the effects of infusion on proinflammatory functions of human neutrophils, such as ROS production and IL-8 and TNF-a release. The anti-microbial activity was also checked using several pathogens. The comprehensive analysis of infusion A. dracunculus' aerial parts (ADI) in our studies has never been performed using the UHPLC-DAD-ESI-MS/ MS method.

Plant Material
The aerial parts of Russian tarragon were collected in August 2014 from the experimental field of the Department of Vegetables and Medicinal Plants in Wilanoẃ, Warsaw, Mazovian district, Poland (21°0099109 E 52°162209 N). The plant material was authenticated by Prof. Ewa Osinśka (Warsaw University of Life Sciences, Poland) according to a guidebook (Rutkowski, 2006). Voucher specimens no. 121 were deposited at the herbarium of the Department of Vegetable and Medicinal Plants, Warsaw University of Life Sciences.

Preparation of ADI and Its Phytochemical Characterization by UHPLC-DAD-MS/MS Method
A 3 g portion of air-dried plant material was poured into boiling water (250 mL), covered, and allowed to stand for 15 min (3×) in a tea infuser (Ambition, Warsaw, Poland). Extracts were then filtered and lyophilized (lyophilizer Telstar Cryodos 50, Telstar International, S.L., Terrassa, Spain), resulting in the following yields: sample 1 -1.32 g, sample 2 -0.98 g, sample 3 -1.24 g. UHPLC-DAD-MS analysis was conducted using a Dionex Ultimate 3000RS system coupled with an Amazon SL ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany). The ion trap AmazonSL mass spectrometer was equipped with an ESI interface. The eluate was introduced into the ESI interface of the mass spectrometer without splitting. The parameters for the ESI source were as follows: nebulizer pressure 40 psi; dry gas flow 9 L/ min; dry temperature 300°C; and capillary voltage 4.5 kV. Analysis was carried out using scanning from m/z 70 to 2200. Mass spectra were recorded in positive-and negative-ion modes. A sample of the crude plant extract dissolved in methanol (20 mg/mL) was filtered through a 0.45 mm syringe filter and subjected to UHPLC-DAD-MS analysis. The separation was carried out on a Kinetex XB-C 18 (150 mm × 3.0 mm × 2.6 mm, Phenomenex, Torrance, CA, USA) column maintained at 25°C. The mobile phases were 0.1% HCOOH in water (A) and 0.1% HCOOH in acetonitrile (B), and elution was conducted with the following gradient: 0 min -0% B; 60 min -26% B; and 80 min, 90% B. The flow rate was 0.4 mL/min, and the injection volume was 3 mL of the prepared extract. The UV−Vis spectra of the detected compounds were recorded over the 190 -450 nm range. The chromatogram was recorded at 280 nm and 350 nm. Compounds were characterized based on the maxima observed in their UV−Vis spectra and on their MS spectra.

Isolation of Human Neutrophils
The buffy coats were prepared from peripheral venous blood collected from healthy human donors (< 35 years old) at the Warsaw Blood Donation Centre. Donors were confirmed to be healthy and all tests carried out showed values within a normal range. Donors did not smoke or take any medications. The study conformed to the principles of the Declaration of Helsinki. Neutrophils were isolated using a standard method by dextran sedimentation and centrifugation in a Pancoll gradient (Böyum, 1968). After isolation, cells were suspended in (Ca 2+ )-free HBSS or RPMI 1640 culture medium.

Evaluation of ADI Cytotoxicity
Cytotoxicity was determined by flow cytometry using propidium iodide (PI) staining. After 24 h of incubation in the standard conditions (37°C, 5% CO 2 ) with extracts or standards used as positive controls in tests, the neutrophils were harvested and centrifuged (1500 RPM; 10 min; 4°C), washed once with cold PBS, and re-suspended in 500 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 10000 events were recorded per sample. Cells that displayed high permeability to PI were expressed as a percentage of PI (+) cells. Triton X was used as positive control.

Measurement of ROS Production
The ROS production by f-MLP-stimulated neutrophils was determined using luminol-dependent chemiluminescence. ADI were tested at concentrations of 12.5, 25, 50, and 100 mg/mL. Following isolation, cells were suspended in 70 mL (Ca 2+ )-free HBSS. Cell suspension (3.0×10 5 /mL) was incubated with 50 mL of the samples with tested concentrations of extract and 50 mL of luminol (100 mM). ROS production was initiated by the addition of 30 mL of f-MLP (0.1 mg/mL). Changes in the chemiluminescence were measured over a 40 min period at intervals of 2 min in a microplate reader (BioTek, Synergy 4) at 37°C. Background chemiluminescence produced by nonstimulated cells was also checked. The tested extract did not interfere with the chemiluminescence signal. As a positive control, quercetin was used at a concentration of 20 mM. The percentage of ROS production was calculated in comparison to the control without investigated tarragon water extract.

IL-8 and TNF-a Release
Neutrophils (2 × 10 6 cells/mL) were cultured in RPMI 1640 medium with 10% FBS, 10 mM HEPES, and 2 mM L-glutamine for 24 h at 37°C with 5% CO 2 in the absence or presence of extract at final concentrations of 12.5, 25, 50, and 100 mg/mL (96-well plates, 1 mL per well) 1 h before stimulation LPS (100 ng/mL). After 24 h, plates were centrifuged (2000 RPM; 10 min; 4°C) and supernatants were collected. The release of cytokines by stimulated neutrophils was evaluated by enzyme-linked immunosorbent (ELISA) tests following the manufacturer's instructions (BD Biosciences, San Jose, CA, USA or R&D Systems, Minneapolis, MN, USA). Dexamethasone at concentrations of 12.5, 25, and 50 mM and quercetin at concentration of 50 mM were used as a positive control for the release of IL-8 and TNF-a.

Statistical Analysis
The results were expressed as the mean ± SEM of three independent experiments performed in triplicate. The statistical significance of differences between means and control was determined by ANOVA with Tukey's post hoc test. P values below 0.05 were considered statistically significant with Statistica 10 software (Statsoft, Poland).

Antimicrobial Assay
The antibacterial activity was assessed against Gram-positive (Staphylococcus aureus ATCC6538, Staphylococcus aureus MRSA ATCC43300, Staphylococcus epidermidis ATCC14990, Enterococcus hirae ATCC10541, and Corynebacterium diphtheriae) and Gram-negative (Escherichia coli ATCC8739, Klebsiella pneumoniae ATCC13883, Proteus vulgaris NCTC4635, and Helicobacter pylori ATCC43504) reference strains. All bacteria were obtained from the Department of Pharmaceutical Microbiology, Medical University of Gdanśk's collection. Brainheart infusion broth (BHI, Becton Dickinson) was used for breeding of E. hirae and supplemented with 10% bovine serum for C. diphtheriae (grown in aerobic conditions at 37°C for 48 h) (Lekogo et al., 2010). Mueller-Hinton broth (MH cation-adjusted, Becton Dickinson) was used for: S. aureus, E. coli, K. pneumoniae, and P. vulgaris (grown in aerobic conditions at 37°C for 48 h). BHI (Becton Dickinson) supplemented with 5% horse serum was used for H. pylori (grown in microaerophilic conditions, GENbag microaer, BioMerieux at 37°C for 72 to 96 h). The antibacterial assay was performed according to a previously established method (Kula et al., 2013). Dry extract (1 g) was dissolved in the sterile distilled water (2 mL). The final concentrations of the extracts used for the antimicrobial activity ranged from 0.004 to 94.000 mg/mL. The lowest concentration at which no visible growth was taken as the MIC (minimal inhibitory concentration).

Functions of Stimulated Neutrophils and Cytotoxicity
Neutrophils, also known as polymorphonuclear cells (PMNs), after infiltration to the inflammation site, generate ROS. Stimulation by f-MLP (bacterial derived factor) results in degranulation and the significant release of ROS compared to the non-stimulated control ( Figure 2). Incubation of stimulated neutrophils with ADI in the concentration range of 12.5 -100 mg/mL turned out to inhibit the production of ROS (Figure 2). Quercetin at a concentration of 20 µM was used as a positive control in performed experiments. The most significant activity was noticed for ADI at a concentration of 100 µg/mL with the decrease of ROS production down to 33.6 ± 1.4% compared with the stimulated control for f-MLP (p < 0.001). Furthermore, LPS-stimulated neutrophils were used for the assessment of IL-8 and TNF-a release inhibition ( Figures 3A, B). Tarragon (Artemisia dracunculus L.) infusion at concentrations of 50 and 100 µg/mL was able to inhibit the secretion of IL-8 by human neutrophils (45.4 ± 4.0% and 37.3 ± 4.8% compared to LPS stimulated control 99.7 ± 2.6%, p<0.001). At concentrations of 50 and 100 mg/mL, a significant decrease of the production of TNF-a from stimulated PMNs was also observed (52.5 ± 7.8% and 29.7 ± 5.2% compared to LPS stimulated control 99.9 ± 1.8%, p<0.001). However, the observed effects for the TNF-a release were less relevant than for the positive control (dexamethasone at concentrations 12.5, 25, 50 µM, Figure 3B). ADI at lower concentrations of 12.5 and 25 mg/mL did not affect the release of IL-8 and TNF-a from LPS-stimulated neutrophils (no statistically significant differences were observed). The effect is probably connected with the presence of polyphenolic compounds such as caffeoylquinic acids and flavonoids in ADI. Tarragon infusion at concentrations of 12.5, 25, 50, and 100 mg/mL have shown no statistically significant reduction in cells' membrane integrity in comparison to the control cells in a propidium iodide assay ( Figure  4). At higher concentrations no differences between samples treated with ADI and LPS-stimulated cells were observed. A common antiinflammatory drug used in this studydexamethasone -at all tested concentrations did not influence the viability of cells compared to LPS control.

Antibacterial Activity
The antimicrobial activity of ADI was evaluated against nine strains of human pathogenic bacteria compared to the ampicillin (

CONCLUSION
In the present study, the chemical composition of Artemisia dracunculus aerial part infusion was elucidated. This common spice is a source of compounds from the groups of phenolic acid derivatives and flavonoids. The potential anti-inflammatory activity of tarragon was evaluated in the human neutrophils model for the first time. The water extract obtained from Artemisia dracunculus herb was able to inhibit ROS, IL-8, and TNF-a production by PMNs in the imitated inflammation caused by neutrophil-stimulating bacterial factors such as f-MLP or LPS. Our results provide the background for the hypothesis that ADI, which is non-toxic for PMNs, exerts two direct activities needed in the case of bacterial infections. The first one is the resolution of bacteria-derived inflammation through the inhibition of neutrophils' functions, which are the first line of immune defense. The second one is the direct activity against pathogens. ADI was characterized by moderate antibacterial activity against selected pathogenic Gramnegative and Gram-positive bacteria, including several Staphylococcus aureus and Staphylococcus epidermidis strains, which are the most common skin pathogens. Staphylococcus and their resistance to antibacterial drugs are one of the most important problems in hospital infections. The obtained results indicate the inhibitory effect of water A. dracunculus extract on Corynebacterium diphtheria and Helicobacter pylori, bacteria causing diphtheria and stomach ulcers, respectively. The investigated infusion from tarragon herb demonstrated a therapeutic potential to some extent, showing both the inhibition of cytokines' secretion and antibacterial properties. Obtained results partially support the traditional usage of Artemisia dracunculus herb in the treatment of symptoms of bacterial infections-related inflammatory disorders. We believe that these properties may be used in the treatment of respiratory tract and skin infections. However, as flavonoids and phenolic acids usually undergo metabolic changes when administered orally, further research is needed and in vivo experiments are required to verify the extent of their systemic and topical activity.

DATA AVAILABILITY STATEMENT
The data are available on request to the corresponding author.

FUNDING
This project was carried out with the use of CePT infrastructure financed by the European Union's European Regional Development Fund within the Operational Program "Innovative economy" for 2007-2013. The access publication fees were financially supported by Medical University of Warsaw.