Phenolic profile and antioxidant activity of Coleostephus myconis ( L . ) Rchb . f . : an underexploited and highly disseminated species

Coleostephus myconis (L.) Rchb.f. (Asteraceae) is a species with ruderal growth and persistence in abandoned soils, being characterized for its plentiful yellow flowering between March and August. Despite its botanical relevance, C. myconis had never been studied neither for its antioxidant activity, nor individual phenolic compounds. Herein, the antioxidant activity of different botanical parts: stems and leaves (green parts), floral buds, flowers in anthesis and senescent flowers, was studied in selected extracts (ethanol, ethanol:water 1:1 and water) through different chemical and biochemical assays. In addition, the phenolic profiles of the hydroethanolic extracts of each botanical part were also characterized by liquid chromatography with dioade array detection and electrospray ionization tandem mass spectrometry (LC-DAD-ESI/MS). The antioxidant activity was significantly modulated by the extract type, with the hydroethanolic extracts showing the highest antioxidant activity, especially those obtained from the senescent flowers and floral buds. The phenolic profiles were the same for all flowering stages (with quantitative differences), but that characterized in the green parts was quite different. Floral buds gave the highest contents in phenolic compounds, mainly due to the contribution of 3,5-O-dicaffeoylquinic acid and myricetin-O-methyl-hexoside. Overall, C. myconis showed an interesting potential to be included in different industrial applications.


Introduction
Some researchers suggest that two-thirds of the world's plant species have medicinal value. Actually, medicinal plants used in folk medicine are being increasingly studied and used in pharmaceutical and nutraceutical fields. Furthermore, the so called phytomedicines are playing a progressively higher role in human health care system. In fact, in a society increasingly concerned with health and nutrition, these products are emerging as a strong alternative to the synthetic ones (Phillipson, 2007, Krishnaiah et al., 2011. The Asteraceae family has a worldwide distribution (with special relevance in the Mediterranean, Eastern Europe and Asia Minor), being acknowledged about 25 000 species integrated in approximately 1000 genera. In addition to the anti-inflammatory, analgesic and antipyretic potential of some of these species, their high antioxidant power, as proven in research works with extracts (of roots, stems, bark, leaves, flowers, fruits and seeds) should be highlighted (Bessada et al., 2015;Cabral et al., 2013;Krishnaiah et al., 2011). In Portugal, there are nearly 314 Asteraceae species, having a large representation in the Portuguese flora. Coleostephus myconis (L.) Rchb.f. belongs to Asteraceae family and is characterized as being a species with ruderal growth and persistence in abandoned soils. C. myconis is available throughout all the territory (mainly in the north) and has a seasonal growth, with plentiful yellow flowering between March and August.
Some of the biological properties of plant-derived products are related to their antioxidant activity. Oxidative stress, which results from a lack of balance between reactive species (and their metabolites) and antioxidant defense, plays a pivotal role in the development of human diseases and skin aging (López-Alarcón and Denicola, 2013).
The antioxidant activity of plants is often related to its individual phenolic compounds.
These compounds occur frequently conjugated with glycosides, being usually located in the cell vacuolar structures. It is generally accepted that solvent extraction is the most commonly used procedure to extract and liberate phenolic compounds (Proestos et al., 2008). However, the effectiveness of the solid-liquid extraction is significantly influenced by the type of solvent, mainly due to the varying polarity or solvents' proportions, being also swayed by the chemical composition and physical characteristics of the samples (Radojkovi et al., 2012). Accordingly, the process of extraction should be standardized for each material vis-a-vis with the solvent, which is the underlying reason for the different solvents tested in this work.
As far as we know, C. myconis species was not previoulsy studied for its antioxidant activity and individual phenolic compounds, since no related references could be found in literature. Besides the innovative character, studying C. myconis is also relevant for its high dissemination in the Portuguese territory (mainly in the northern region).
The phenolic compound standards were from Extrasynthese (Genay, France). All other chemicals and solvents were of analytical grade and purchased from common sources.
Water was treated in a Milli-Q water purification system (TGI Pure Water Systems, Greenville, SC, USA).

Samples
C. myconis plants were collected in the northwest of Portugal (Riba de Mouro, Minho) in June (2014) and, after taxonomical identification, were further divided in i) green parts (stems and leaves); ii) floral buds; iii) flowers in anthesis (fully open and functional flowers); iv) senescent flowers. The vegetal material was then frozen, lyophilized (48 h, -78 ºC, 0.015 mbar) (Telstar Cryodos-80, Terrassa, Barcelona), reduced to powder, mixed to obtain homogenized samples and stored in plastic tubes at room temperature for subsequent use.

Preparation of extracts
For the extracts preparation, a fine dried powder (20 mesh; ~0.5 g) of each sample was stirred (150 rpm) with 50 mL of one of three different solvents: ethanol, water or ethanol:water (1:1 v/v), at 25 °C for 1 h. The residues obtained in each case were then extracted with additional 50 mL portions of each solvent under the same conditions.
The combined extracts were filtered through Whatman No. 4 paper, evaporated at 35 °C under reduced pressure (rotary evaporator Büchi R-210, Flawil, Switzerland), redissolved in the specific solvent at 10 mg/mL (stock solution), and stored (4 °C) for further evaluation of the antioxidant activity. From the 10 mg/mL solution, several sequential dilutions were made (0.08-5.00 mg/mL).

DPPH radical-scavenging activity
This methodology was performed using an ELX800 microplate reader (Bio-Tek Instruments, Inc.) (Barreira et al., 2013). The reaction mixture in each one of the 96wells consisted of one of the different concentrations of the extracts (30 µL) and methanolic solution (270 µL) containing DPPH radicals (6 × 10 -5 mol/L). The mixture was left to stand for 30 min in the dark. The reduction of the DPPH radical was determined by measuring the absorption at 515 nm.
The radical scavenging activity (RSA) was calculated as a percentage of DPPH discoloration using the equation: % RSA = [(A DPPH -A S )/A DPPH ] × 100, where A S is the absorbance of the solution when the sample extract has been added at a particular level, and A DPPH is the absorbance of the DPPH solution. The concentration providing 50% of radicals scavenging activity (EC 50 ) was calculated from the graph of RSA percentage against extract concentration.

Reducing power
This methodology was performed using the microplate reader described above. The different concentrations of the extracts (0.5 mL) were mixed with sodium phosphate buffer (200 mmol/L, pH 6.6, 0.5 mL) and potassium ferricyanide (1% w/v, 0.5 mL).
The mixture was incubated at 50 ºC for 20 min, and trichloroacetic acid (10% w/v, 0.5 mL) was added. The mixture (0.8 mL) was poured in the 48-wells, as also deionized water (0.8 mL) and ferric chloride (0.1% w/v, 0.16 mL), and the absorbance was measured at 690 nm. The concentration providing 0.5 of absorbance (EC 50 ) was calculated from the graph of absorbance at 690 nm against extract concentration (Barreira et al., 2013).
A solution of β-carotene was prepared by dissolving β-carotene (2 mg) in chloroform (10 mL). Two milliliters of this solution were pipetted into a round-bottom flask. The chloroform was removed at 40 ºC under vacuum and linoleic acid (40 mg), Tween 80 emulsifier (400 mg), and distilled water (100 mL) were added to the flask with vigorous shaking. Aliquots (4.8 mL) of this emulsion were transferred into test tubes containing extracts with different concentrations (0.2 mL). As soon as the emulsion was added to each tube, the zero time absorbance was measured at 470 nm (AnalytikJena 200 spectrophotometer, Jena, Germany). The tubes were shaken and incubated at 50 ºC (2 h) in a water bath and the absorbance was measured again. β-Carotene bleaching inhibition was calculated using the following equation: (β-carotene content after 2h of assay/initial β-carotene content) × 100. The concentration providing 50% antioxidant activity (EC 50 ) was calculated by interpolation from the graph of β-carotene bleaching inhibition percentage against extract concentration (Barreira et al., 2013).

Inhibition of lipid peroxidation using thiobarbituric acid reactive substances (TBARS)
Brain porcine tissue was homogenized in Tris-HCl buffer (20 mM, pH 7.4) 1:2 (w/v), and further centrifuged at 3000g for 10 min. An aliquot (0.1 mL) of the supernatant was incubated with the extracts at different concentrations (0.2 mL) in the presence of FeSO 4 (10 mM; 0.1 mL) and ascorbic acid (0.1 mM; 0.1 mL) at 37 °C for 1 h. The reaction was stopped by the addition of trichloroacetic acid (28%, w/v, 0.5 mL), followed by thiobarbituric acid (TBA, 2%, w/v, 0.38 mL), and the mixture was then heated at 80 °C for 20 min. After centrifugation at 3000g for 10 min to remove the precipitated protein, the color intensity of the malondialdehyde (MDA-TBA) complex in the supernatant was measured by its absorbance at 532 nm.
The inhibition ratio (%) was calculated using the following formula: Inhibition ratio (%) = [(A -B)/A] × 100%, where A and B were the absorbance of the control and the sample solution, respectively. The concentration providing 50% antioxidant activity (EC 50 ) was calculated by interpolation from the graph of TBARS formation inhibition percentage against sample concentration (Barreira et al., 2013). MS detection was performed in negative mode, using a Linear Ion Trap LTQ XL mass spectrometer (ThermoFinnigan, San Jose, CA, USA) equipped with an ESI source.

Characterization of phenolic compounds by LC-DAD-ESI/MS
Nitrogen served as the sheath gas (50 psi); the system was operated with a spray voltage of 5 kV, a source temperature of 325 °C, a capillary voltage of -20 V. The tube lens offset was kept at a voltage of -66 V. The full scan covered the mass range from m/z 100 to 1500. The collision energy used was 35 (arbitrary units). Data acquisition was carried out with Xcalibur® data system (ThermoFinnigan, San Jose, CA, USA).
The phenolic compounds were identified by comparing their retention times, UV-vis and mass spectra with those obtained from standard compounds, when available.
Otherwise, compounds were tentatively identified comparing the obtained information with available data reported in the literature. For quantitative analysis, a calibration curve for each available phenolic standard was constructed based on the UV signal. For the identified phenolic compounds for which a commercial standard was not available, the quantification was performed through the calibration curve of the most similar available standard. The results were expressed as µg/g of extract.

Statistical analysis
For each botanical part, three independent experiments were performed, and each of them was analyzed in triplicate. The results were expressed as mean values±standard deviation (SD). The statistical differences represented by letters were obtained through one-way analysis of variance (ANOVA) followed by Tukey's honestly significant difference post hoc test (homoscedastic distributions) or Tamhane's T2 test

Antioxidant activity of the C. myconis extracts
The solvent type had some influence on the antioxidant potential, as it was exemplified by the higher activity measured in the hydroethanolic extracts, when compared to the remaining assayed extraction solvents ( Table 1) Since there are no similar reports on C. myconis antioxidant activity, the determined values cannot be directly compared to those obtained in parallel laboratorial conditions.

Characterization of phenolic compounds in the C. myconis hydroethanolic extracts
The characterization of the phenolic compounds, as performed by LC-DAD/ESI-MS n analysis, was conducted only in the hydroethanolic extracts, owing their best results in the antioxidant activity assays. Data of the retention time, λ max , pseudomolecular ion, main fragment ions in MS 2 and tentative identification of phenolic acid and flavonoid derivatives are presented in Table 2. As examples, the HPLC phenolic profiles, recorded at 280 nm, of the senescent flowers (A) and green parts (B) are presented in  Clifford et al. (2003Clifford et al. ( , 2005. Peak 3 was identified as 5-Ocaffeoylquinic acid by comparison with the commercial standard. Peak 1, with the same fragments as peak 3, presented a deprotonated quinic acid (m/z 191), as the base peak, and a caffeate (m/z 179) with a relative percentage higher than 50% (comparing to the base peak) and was identified as 3-O-caffeoylquinic acid, considering that these are common features for 3-acyl chlorogenic acids (Clifford et al., 2003(Clifford et al., , 2005. The predominant phenolic compound (Table 3) in all C. myconis parts (stems and leaves: 62 µg/g extract; floral bud: 271 µg/g extract; flower in anthesis: 102 µg/g extract; senescent flower: 190 µg/g extract) was detected as peak 15, which corresponds to 3,5-O-dicaffeoylquinic acid, according to its pseudomolecular ion [M-H]at m/z 515, fragmentation pattern, and relative abundances of the fragment ions as described by Clifford et al. (2003;2005). Considering the same criteria, peak 16 was identified as 4,5-O-dicaffeoylquinic acid. A fifth phenolic acid was detected as peak 2, identified from its retention time and spectral characteristics by comparison with a commercial standard. These same criteria allowed identifying peaks 6, 10 and 12 as luteolin-6-C- In general, phenolic acids, especially caffeoylquinic acid derivatives, represent the majority of phenolic compounds in C. myconis samples. The floral bud showed significantly higher amounts of all phenolic compounds, except protocatechuic acid (higher in stems and leaves). On the other hand, the green parts showed the lowest contents in phenolic acids (99 µg/g extract) and flavonoids (18.4 µg/g extract).
The antioxidant activity is frequently modulated by the phenolic profile of a determined matrix (Cheung et al., 2003;Li et al., 2014). The present results are generally in agreement with this principle, since the floral buds and the senescent flowers, which showed the highest contents in phenolic acids and flavonoids contents, exhibited the most powerful antioxidant activity. On the other hand, the extracts from stems and leaves presented the weakest antioxidant activity (DPPH scavenging activity: EC 50 = 0.51-3.9 mg/mL, reducing power: EC 50 = 0.30-0.78 mg/mL, β-carotene bleaching inhibition: EC 50 = 1.4-1.5 mg/mL; and TBARS inhibition: EC 50 = 0.09-0.20 mg/mL), which is also in agreement with their lower amounts of phenolic compounds.

Conclusion
The antioxidant activity exhibited by each of the extracts obtained from different botanical parts of C. myconis showed to be modulated by the extraction solvent, with the best results being obtained for the hydroethanolic mixture. Furthermore, stems and leaves (green parts) showed the lowest values of this bioactivity indicator, whilst the floral buds and the senescent flowers presented the highest antioxidant activity. The phenolic profiles of the hydroethanolic extracts revealed high predominance of phenolic acids (mainly 3,5-O-dicaffeoylquinic acid). The same compounds were detected throughout the flowering stages (despite the significant quantitative differences), but the profile of the green parts was quite different (7 of the detected compounds were only detected in the stems and leaves).