HPLC-ESI-MS/MS Profiling of Phenolic Acids, Flavonoids And Sesquiterpene Lactones from Xanthium spinosum

Bathurst burr (Xanthium spinosum L.) is used worldwide in traditional medicine to treat a diverse range of health problems including urinary problems associated with various prostate diseases. The aim of this study was to complete the identification and structural characterization of the chemical constituents from the aerial part of X. spinosum by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. From the unequivocally detected and characterized compounds of aqueous-methanol extract, protocatechuic acid, 4-O-caffeoylquinic acid and 3,5-di-O-caffeoylquinic acid were found for the first time in X. spinosum. Besides these compounds, 25 phenolics (including 7 hydroxybenzoic derivatives, 13 hydroxycinnamic derivatives, one benzyl alcohol-hexose-pentose, and 4 flavonoids), 6 sesquiterpenes and 3 diterpenes were tentatively identified.


1.Introduction
Bathurst burr (Xanthium spinosum L.), from the family Asteraceae is an annual herb originated from South America. The herb is used traditionally in Romania for urinary problems and various prostate diseases [1]. The pharmacological profile of X. spinosum includes the anti-inflammatory activity attributed to its flavonoids. The sesquiterpene lactone xanthatin from the leaves has antibacterial and antifungal properties [2,3]. Xanthatin extracted from the aerial parts of the plant shows inhibitory activity against a wide variety of viruses. Also, it was revealed that xanthatin has a strong anti-angiogenesis capacity in vitro [4]. The infusion and tincture obtained from X. spinosum aerial parts are efficient in the treatment of rats with induced benign prostate hypertrophia [5,6].

Plant material
The flowering aerial parts of Xanthium spinosum L. were collected at the end of October 2014 from the Botanical Garden belonging to the University of Medicine, Pharmacy, Sciences and Technology "George Emil Palade" from Târgu Mureș, Romania (46°33'16.91"N, 24°35'0.51"E). The plant was identified and the voucher specimen (GB/H/0162/2014) is deposited at the Faculty of Pharmacy, Department of Pharmaceutical Botany.

Chemicals and solvents
The chemical reference substances: 4-caffeoylquinic acid, 1,3-dicaffeoylquinic acid (cynarin), 3,5dicaffeoylquinic acid, caffeic acid, and protocatechuic acid were purchased from Sigma-Aldrich (St. Louis, USA), while xanthatin was bought from ChemFaces (Wuhan, China). These standards were chosen, after the phytochemical exploring of the Xanthium genus. Acetonitrile and methanol were of HPLC super gradient grade (Sigma-Aldrich). Water was purified with Millipore (Billerica, MA, USA) Milli-Q equipment. All other chemicals were of analytical reagent grade.

Extraction
The powdered herb of X. spinosum (2 g) was extracted twice with 20 mL of 8:2 (v/v) methanol:water by sonication (Braun Labsonic U, Melsungen, Germany) for 2 × 5 min. The extracts were separated from the plant powder by centrifugation at 6000 rpm (2500 g) for 10 min. The two methanolic fractions were combined and evaporated to dryness under reduced pressure (Rotavapor, R-200, Büchi, Flawil, Switzerland) below 60°C before purification by solid-phase extraction (SPE).
SPE was performed on Supelclean LC-18 micro-columns (500 mg, 3 mL), supplied by Supelco (Bellefonte, PA, USA) using a 12-port vacuum manifold processor (LiChrolut extraction unit; Merck). Samples were dissolved in 80% (v/v) aqueous methanol and were loaded on to the SPE micro columns previously activated by 5 mL methanol, 5 mL water and 2.5 mL methanol. The collected eluate was completed by a further portion of the extract obtained by loading 2.5 mL of 80% methanol on to the SPE column. Finally, the samples obtained were evaporated to dryness and were dissolved in 2 mL of 80% methanol.
Electrospray conditions were as follows: drying gas (N2) temperature, 350 °C; flow rate, 9 L/min; nebulizer pressure, 45 psi (N2); fragmentor voltage, 100V; capillary voltage, 3500V. High purity nitrogen was used as collision gas, and collision energy was changed between 5 and 60 eV according to differences in molecule structures. Full scan mass spectra were recorded in positive and negative ion mode over an m/z range of 50-1000.

Results and discussions
The HPLC-DAD-ESI-MS/MS analysis of the aqueous-methanolic extract of the aerial part of Xanthium spinosum revealed the presence of the following unequivocally detected constituents: protocatechuic acid (3), 4-O-caffeoylquinic acid (13), caffeic acid (15), 3,5-di-O-caffeoylquinic acid (22), and xanthatin (35) ( Table 1). Three of the five unequivocally detected compounds are mentioned for the first time in X. spinosum: protocatechuic acid, 4-O-caffeoylquinic acid, and 3,5-di-Ocaffeoylquinic acid. Protocatechuic acid and 3,5-di-O-caffeoylquinic acid were also detected in the aerial part of X. strumarium, with a yield of 25.3 mg and 5.8 mg, respectively [8]. According to their study, 3,5-di-O-caffeoylquinic acid has an inhibitory effect on α-glucosidase (antihyperglycemic activity), while protocatechuic acid presents inhibitory effect on ABTS + radical scavenging activity. Caffeoylquinic acids derivatives are the major active principles in the fruits of X. strumarium, and are known for their antioxidant, antibacterial and anti-inflammatory effects [26]. Therefore, further analyses are necessary to determine the content of the detected constituents, and to test their biological effects.  Besides these compounds, 25 phenolics (including 7 hydroxybenzoic derivatives, 13 hydroxycinnamic derivatives, one benzyl alcohol-hexose pentose, and 4 flavonoids), 6 sesquiterpenes and 3 diterpenes were tentatively identified. The detected compounds with the main chromatographic and mass spectrometric data are listed in Table 1