THE INVESTIGATION OF BIOACTIVE SECONDARY METABOLITES OF THE METHANOL EXTRACT OF ERYNGIUM AMETHYSTINUM

Eryngium amethystinum L. belonging to the Apiaceae family, is a perennial plant distributed in Southeast Europe. Ev en though this plant is used in traditional medicine, its phytochemical characteriz ation is still incomplete. In this study composition of bioactive constituents of the m thanol extract are reported for the first time. By means of the UPLC-LTQ-Orbitr ap-MS method, altogether sixty-three constituents were characterized: eight hydroxybenzoic acid derivatives (7-13, 32), fifteen cinnamic acid derivatives (14, 17-19, 214-26, 28, 30, 39-42 and 44), four flavonoid aglycones (45, 51, 52, 54), twenty-four flavonoid derivatives (23, 27, 29, 31, 33-38, 43, 46-50, 53, 5-59, 61 and 62), three coumarin derivatives (15, 16 and 22) and nine other compounds (1-6, 20, 60 and 63).


INTRODUCTION
The genus Eryngium L. is distributed all around the world, and with more than 200 species represents the taxonomically most complex genus of the family Apiaceae (WORZ, 2004;CALVINO et al., 2007).
Eryngium amethystinum L., commonly known as amethyst sea holly, is distributed in western and central Serbia on calcareous and arid soil, up to 1600 m above the sea level. In folk medicine of Southeast Europe E. amethystinym is used for its diuretic and laxative properties, in treatment of edemas, urinary ailments, and acidosis. It is also useful as an aid to digestion (FLAMINI et al., 2008).
Linear ion trap quadrupole-Orbitrap-mass spectrometry (LTQ-Orbitrap-MS) delivers single-stage mass analysis providing molecular mass information, two-stage mass analysis (MS 2 ) and multi-stage mass analysis (MS n ) delivering structural information. Exact mass measurements and elemental composition assignment are essential for the characterization of small molecules. Accurate mass measurement of the product ions formed in MS n experiments facilitates the elucidation of the structures of unknown compounds (TCHOUMTCHOUA et al., 2013).
The aim of the present study was to characterize the secondary bioactive metabolites in the methanol extract of E. amethystinum by using UPLC-HRMS n . The chemical structures of the identified compounds and their glycoside derivatives are important to reveal information on possible bioactive effect of examined plant.
In this paper we presented for the first time chemical profiling and levels of antioxidant activitiy of the methanol extracts of wild growing E. amethystinum.

Chemicals and reagents
LC-MS grade formic acid and acetonitrile were purchased from Thermo Scientific Pierce (Thermo Fisher Scientific, Pierce Biotechnology, Rockford, IL, USA). Water was treated in a Milli-Q water purification system (TGI Pure Water Systems, Brea, CA, USA).

Plant material and sample preparation
Eryngium amethystinum aerial parts were collected (Jun 2014) in the region of Raška (south Serbia, altitude 350 m, 43º30'53''N, 20º37'07''E) and identified at the Department of Biology and Ecology, Faculty of Science in Kragujevac, University of Kragujevac (Serbia). Voucher specimen (17088, BEOU) was deposited at the Department of Botany, Faculty of Biology, University of Belgrade, Serbia.
The collected plant material was air-dried in darkness at ambient temperature. A portion of the sample (20 g) was ground to a thin powder, and extracted three times (at room temperature, 24h) with HPLC grade methanol (200 mL). Methanol extracts were combined and evaporated under reduced pressure by means of rotary evaporator at 40 ºC. A portion of crude extract (50 mg) was dissolved in methanol, filtered through a membrane filter and used directly for LC-MS analysis.

UPLC-LTQ-Orbitrap-MS for metabolomic analysis
Chromatographic separations were performed using an ultrahigh-performance liquid chromatography (UPLC) system consisting of a quaternary Accela 600 pump and Accela Autosampler (ThermoFisher Scientific, Bremen, Germany). Analytical column used for separations was a Syncronis C18 column (100 x 2.1 mm, 1.7 µm particle size, ThermoFisher Scientific). The mobile phase consisted of (A) ultrapure water with 1% formic acid and (B) acetonitrile (MS grade). The injection volume for the sample was 10 µL, elution gradient programme was 5-95 % B for 20 min, with the flow rate of 0.3 mL/min.
The UPLC system was coupled to a linear-trap quadrupole (LTQ) orbitrap hybrid mass spectrometer equipped with a heated-electrospray ionisation probe (HESI-II, ThermoFisher Scientific, Bremen, Germany). The mass spectra were obtained in negative and positive ion modes. Operation parameters were as follows: source voltage 4.5 kV (4.2 kV in positive mode), capillary voltage -10 V (42 V in positive mode), tube lens voltage -35 V (110 V in positive mode), capillary temperature 300 ºC, sheath and auxiliary gas flow (N2) 32 and 8 (arbitrary units), respectively. The MS spectra were acquired by full range acquisition covering 100-1000 m/z, and for the fragmentation study, a data dependent scan was performed by deploying collision-induced dissociation (CID): the normalized collision energy of the CID cell was set to 35 eV.
Compounds were identified according to the corresponding spectral characteristics: mass spectra, accurate mass, characteristic fragmentation and characteristic retention time. ThermoFisher Scientific Xcalibur software (Version 2.1) was used for instrument control, data acquisition and data analysis.

UPLC-LTQ-Orbitrap-MS n identification of secondary metabolites of E. amethystinum
In the present work, a total of sixty-three compounds were tentatively identified in the sample, mainly phenolic compounds such as: simple phenols, flavonoids, coumarins and other compounds. UPLC-LTQ-Orbitrap MS n chromatograms were given in Figure 1 and Figure 2. The tentatively identified metabolites are summarized in Table 1

Benzoic acid and hydroxycinnamic derivatives
As can be seen from Table 1, twenty-three compounds were identified. Among them, eight hydroxybenzoic acid and fifteen cinnamic acid derivatives were found.

Flavonoid aglycones and their derivatives
Among twenty-eight compounds, four aglycones and twenty-four flavonoid derivatives were identified in methanol extract of E. amethystinum.

CONCLUSIONS
In this study we performed for the first time UPLC-LTQ-Orbitrap-MS n non-targeted metabolic analysis of methanol extract of E. amethystinum. The approach used allowed identification of sixty-three secondary metabolites, most of them belonging to the phenolics class (simple phenolics, flavonoids and coumarins). These data suggest that this medicinal plant might be a valuable source of bioactive secondary metabolites from classes of phenolic acids, flavonoids and coumarins with beneficial proprieties, and a promising source of health products for functional food or nutraceutical industries.