Echinacea purpurea (L.) Moench: Chemical Characterization and Bioactivity of Its Extracts and Fractions

Echinacea purpurea (L.) Moench is widely known for its medicinal properties, being one of the most used medicinal plants for its immunostimulant properties. Nevertheless, there is still scarce information on its cytotoxic activity. Thus, this study aims at evaluating the cytotoxicity and antimicrobial activity of several aqueous and organic extracts of the aerial parts of this plant and chemically characterizing the obtained extracts. The analysis was performed by HPLC–DAD–ESI/MS. Fifteen compounds were identified; of these, seven were phenolic acids and eight were flavonoids. Non-polar compounds were evaluated by GC/MS, with a total of sixty-four compounds identified, and the most abundant groups were the sterols, fatty acids and long-chain hydrocarbons. The highest antimicrobial activity was exhibited by the dichloromethane, ethyl acetate, and acetone extracts. Dichloromethane and n-hexane extracts showed the highest cytotoxic activity. Therefore, they were fractionated, and the obtained fractions were also assessed for their cytotoxicity. Notwithstanding, the cytotoxicity of the extracts was superior to that of the obtained fractions, evidencing a possible synergistic effect of different compounds in the whole extracts.


Introduction
Taking advantage of the traditional ethnomedicinal application of a wide diversity of plants, they are now being used as a powerful tool for disease prevention [1].Nowadays, approximately 30% of the pharmaceutical market and 11% of essential drugs (considered drugs intended for primary care) are plant-based [2].Echinacea purpurea (L.) Moench is a perennial plant native to eastern North America that belongs to the Asteraceae family [3].This plant species is considered a safe herbal medicine, thus, usually being used in a self-medication manner, mainly through aqueous or ethanolic extracts of the dried aerial parts or roots [4,5].E. purpurea is considered one of the most known and used medicinal plants against a variety of treatments, such as snake bites and wound infections, and also for its anti-inflammatory, antioxidant and antitumor properties [6][7][8][9].This plant can be used as an infusion or tincture, and it is available on the market in standardized preparation solutions (fluid forms) or in the form of capsules (containing dried E. purpurea) [10,11].According to previous studies, the most common phytochemicals found in E. purpurea are alkamides, polysaccharides, lipoproteins, betaine, sesquiterpenes, polyacetylene, saponins and phenolic compounds (echinacoside and other caffeic acid derivatives, and chicoric acid) [6,11,12].These classes of bioactive compounds have been described as being responsible for the mentioned biological properties.The multiple activities of this plant species indicate that several compounds may contribute to its medicinal benefits, which are also dependent on the plant part used, since the roots have been described as having more alkamides while the leaves are rich sources of flavonoids [13].Nevertheless, so far, most studies on the bioactive properties of E. purpurea aerial parts have mainly focused on the evaluation of their immunostimulant capacity [14][15][16][17][18]. Thus, the present work aims at assessing the phenolic compounds profile of a commercial sample of E. purpurea aerial parts, and also at evaluating the antimicrobial and antiproliferative activity of different aqueous and organic extracts.Moreover, the most active extracts in terms of cytotoxic effects were fractionated by gradient elution through column chromatography on silica gel and the resulting fractions were assessed for their antiproliferative potential.

HPLC-DAD-ESI/MS Analysis of Phenolic Compounds
Table 1 presents the data (retention time, λ max , pseudomolecular ions, main fragment ions in MS 2 , tentative identification and quantification) obtained from the HPLC-DAD-ESI/MS analysis of the EtOAc, acetone, MeOH, infusion and decoction extracts of E. purpurea.An exemplificative chromatogram of the phenolic profile recorded at 280 and 370 nm of the methanolic extract is shown in Figure 1 (the extract that presented most of the phenolic compounds tentatively identified).The n-hexane and DCM extracts were not considered for this analysis, since in non-polar or lower polarity extracts, it is not expected to detect phenolic compounds in significant amounts.Fifteen phenolic compounds were tentatively identified, namely seven phenolic acids (mainly caffeic, chicoric, p-coumaric, ferulic, and caftaric acid derivatives, corresponding to peaks 1, 2, 3, 4, 5, 8 and 14) and eight flavonoids (mainly quercetin, kaempferol, and diosmetin glycosylated derivatives, corresponding to peaks 6, 7, 9, 10, 11, 12, 13 and 15).Peaks 2, 3 and 7 were identified as 5-O-caffeoylquinic acid, caffeic acid and quercetin-3-O-rutinoside, respectively, according to their retention time, mass and UV-Vis characteristics in comparison with commercial standards.Other authors also found similar compounds in different species [19].All the remaining compounds were tentatively identified according to their mass and UV-Vis characteristics in comparison with information found in the literature.Peaks 1 ([M-H] − at m/z 311) and 4 and 5 (both presenting [M-H] − at m/z 473) were tentatively identified as caftaric acid and cis and trans chicoric acid, respectively, according to their pseudomolecular ion, MS 2 fragmentation and UV-Vis spectra.trans-Chicoric acid was the main compound found in the methanolic extract; however, in the infusion and the decoction, the cis form was found in higher amounts.Peaks 8 ([M-H] − at m/z 487) and 14 ([M-H] − at m/z 501) were tentatively identified as feruloylcaffeoyltartaric acid and p-coumaroylsinapoyltartaric acid, respectively, following the identification described by Ma et al. [20].These type of compounds have been previously reported in E. purpurea water:methanol:formic acid (80:20:0.1,v/v/v) extracts [21].
Regarding the flavonoids identified in E. purpurea samples, kaempferol derivatives were the main compounds found in these samples.Peaks 9, 10, 11, and 13 ([M-H] − at m/z 607) presented a unique MS 2 fragment at m/z 285, corresponding to kaempferol aglycone, which was confirmed by its characteristic UV-Vis spectrum.Peaks 9, 10 and 11 showed a pseudomolecular ion [M-H] − at m/z 593, all being tentatively identified as kaempferol-O-deoxyhexosyl-hexoside, while compound 10 was identified as kaempferol-3-O-rutinoside, showing a similar retention time to the commercial standard.Peak 13 presented a pseudomolecular ion [M-H] − at m/z 607 and was tentatively identified as kaempferol-O-deoxyhexosyl-glucuronide.Regarding the quercetin derivatives, peak 6 presented the same chromatographic characteristics as peak 7, except for its retention time, and was tentatively identified as quercetin-O-deoxyhexosylhexoside.Peak 12 ([M-H] -at m/z 549) revealed MS 2 fragments at m/z 505 (44 u), 463 (42 u), and 301 (162 u, quercetin aglycone), and was tentatively identified as quercetin-O-malonylhexoside.Finally, peak 15 ([M-H] -at m/z 577) showed an MS 2 fragment at m/z 299 (diosmetin moiety).Given its higher value of retention time, the −146 u should correspond to the loss of a p-coumaroyl moiety and the −132 u to a pentosyl moiety, being tentatively identified as diosmetin-O-p-coumaroyl-pentoside.
Comparing the profile obtained for the different extracts, the methanolic extract was the one that presented the highest number of phenolic compounds identified, being also the extract richest in phenolic acids (53 ± 1 mg per g of extract, mainly due to trans-chicoric acid) and flavonoids (8.6 ± 0.2 mg per g of extract, mainly due to quercetin-3-O-rutinoside). The infusion and decoction showed a similar qualitative profile, with the only difference being the presence of 5-O-caffeoylquinic acid in the former, despite being in low amounts.Only two compounds were identified in the acetone extract, with the particularity of this extract being the only one exhibiting the presence of diosmetin-O-p-coumaroyl-pentoside.The EtOAc extract was found to have only minute amounts of one compound (caffeic acid), possibly due to the low polarity of the extracting solvent.According to Brown et al. [22], methanolic extracts of the E. purpurea are rich in chicoric acid and caftaric acid.Regarding the quercetin derivatives, peak 6 presented the same chromatographic characteristics as peak 7, except for its retention time, and was tentatively identified as quercetin-O-deoxyhexosyl-hexoside.Peak 12 ([M-H] − at m/z 549) revealed MS 2 fragments at m/z 505 (44 u), 463 (42 u), and 301 (162 u, quercetin aglycone), and was tentatively identified as quercetin-O-malonylhexoside.Finally, peak 15 ([M-H] − at m/z 577) showed an MS 2 fragment at m/z 299 (diosmetin moiety).Given its higher value of retention time, the −146 u should correspond to the loss of a p-coumaroyl moiety and the −132 u to a pentosyl moiety, being tentatively identified as diosmetin-O-p-coumaroyl-pentoside.
Comparing the profile obtained for the different extracts, the methanolic extract was the one that presented the highest number of phenolic compounds identified, being also the extract richest in phenolic acids (53 ± 1 mg per g of extract, mainly due to trans-chicoric acid) and flavonoids (8.6 ± 0.2 mg per g of extract, mainly due to quercetin-3-O-rutinoside). The infusion and decoction showed a similar qualitative profile, with the only difference being the presence of 5-O-caffeoylquinic acid in the former, despite being in low amounts.Only two compounds were identified in the acetone extract, with the particularity of this extract being the only one exhibiting the presence of diosmetin-O-p-coumaroyl-pentoside.The EtOAc extract was found to have only minute amounts of one compound (caffeic acid), possibly due to the low polarity of the extracting solvent.According to Brown et al. [22], methanolic extracts of the E. purpurea are rich in chicoric acid and caftaric acid.

GC/MS Analysis of Non-Polar Extracts
Table 2 presents the data obtained from the GC/MS analysis of the n-hexane and DCM extracts, which enabled the identification of 61-63% of the compounds, corresponding to a total of 64 compounds.Among those, 35 compounds were found to be present in only one of the extracts, 17 being identified in the n-hexane extract and 18 in the DCM extract.In general, the extracts revealed the presence of various bioactive compounds.Regarding the terpenes group (mono, di and sesquiterpenes), they were present in the highest amounts in the n-hexane extract, with oxygen-containing terpenes being found only in this extract.Phytol was present in both of the n-hexane and DCM extracts, but in different quantities (1.1% and 0.55%, respectively).The most abundant compounds identified in the n-hexane extract were fatty acids (25.8% of the extract) including hexadecanoic acid (palmitic acid, C16:0), 9,12-octadecadienoic acid (linoleic acid, C18:3), octadecanoic acid (stearic acid, C18:0), followed by long-chain hydrocarbons (14.6%) and sterols (13.9%).The obtained results are in agreement with those that reported the prevalence of unsaturated fatty acids, mainly linoleic acid, in the fatty-oil of E. purpurea fruits obtained by extraction with n-hexane [23].Regarding the DCM extract, the most abundant class of identified compounds were long-chain hydrocarbons (27.1%) and fatty acids (27.8%).a LRI, linear retention index determined on a SH-RXi-5ms fused silica column relative to a series of n-alkanes (C8-C40).* identified as TMS derivative.b relative % is given as mean ± SD, n = 3.In each row, p values < 0.05 means significant differences.

Antimicrobial Activity
The results concerning the ability of all seven extracts of E. purpurea to inhibit the growth or kill the assayed microorganisms are presented in Table 3.All tested extracts did not evidence a microbicide activity at the tested concentrations.Nevertheless, several were able to inhibit the growth of bacteria, with MICs varying between 2.5 to 20 mg/mL.The exception was the infusion extract, which did not present any activity, even at the highest tested concentration (20 mg/mL).Additionally, the decoction and the methanolic extract also evidenced low activity, with the former being able to inhibit only the growth of K. pneumoniae and the last only S. aureus (both susceptible and resistant strains).In general, the DCM, EtOAc, and acetone extracts were the ones evidencing the best antimicrobial activity, since they were capable of inhibiting the growth of all microorganisms.DCM extract showed particularly good results against Gram-positive bacteria, with Enterococcus faecalis and Listeria monocytogenes being the most susceptible ones, with a MIC value of 2.5 mg/mL.Concerning S. aureus, the strain MSSA was mostly inhibited by the acetone extract, with a MIC value of 2.5 mg/mL, while the strain MRSA was most sensitive to the DCM, EtOAc, and acetone extracts (all presenting a similar MIC value of 5 mg/mL).According to Hudson et al. [10], preparations of E. purpurea have relatively little effect on the growth of MRSA or MSSA, which was not demonstrated in the present work, since all organic extracts were able to inhibit the growth of both S. aureus strains, with the best results being obtained for the acetone extract.The n-hexane extract also evidenced the capacity of inhibiting some Gram-positive bacteria, although requiring higher MICs in general.Among the Gram-negative bacteria, the most susceptible were Morganella morganii and Escherichia coli, namely due to the DCM and EtOAc extracts, both with a MIC value of 5 mg/mL.Sharma, et al. [24] evaluated the antimicrobial activity of E. purpurea commercial extracts (corresponding to a mixture of roots and aerial parts extracted with 40% of EtOH) and concluded that several bacteria, including E. coli, K. pneumoniae, P. aeruginosa and E. faecalis, were relatively insensitive to the extracts, as only a minute reduction in colony forming units was obtained.In the present work, these four bacteria showed variable MIC values according to the assayed extract, with all of them having lower sensitivity (in general, MIC > 20 mg/mL) for the aqueous and methanolic extracts, while presenting better results for the less polar extracts.Regarding antifungal activity, C. albicans was most susceptible to the DCM and acetone extracts, showing a MIC of 5 mg/mL.This yeast also showed some susceptibility against the n-hexane and EtOAc extracts, with MIC values of 10 mg/mL.According to Barnes et al. [7], n-hexane extracts of E. purpurea roots show high activity against several yeast strains, including Saccharomyces cerevisiae and Candida albicans, which is in good agreement with the results obtained in the present work.

Cytotoxic Properties of Extracts and Fractions
The results obtained regarding the cytotoxic activity of the seven extracts of E. purpurea are shown in Table 4. Almost all extracts showed activity against the four human tumor cell lines, with DCM being the extract that showed the best results [GI 50 = 48 ± 4 µg/mL (NCI H460), GI 50 = 36.7 ± 0.6 µg/mL (HepG2), GI 50 = 51 ± 4 µg/mL (HeLa) and GI 50 = 21 ± 2 µg/mL (MCF-7)].Nevertheless, all the organic extracts, with the exception of MeOH, displayed also toxicity against the non-tumor hepatic cell line (PLP2), although presenting higher GI 50 values compared to those of the tumor cell lines (Table 4).Despite the MeOH extract being only effective against the HeLa (GI 50 = 111 ± 9) µg/mL and MCF-7 tumor cell lines (GI 50 = 76 ± 5 µg/mL), as mentioned, it presented the advantage of not showing toxicity against the PLP2 primary cell line at the maximum concentration assayed (>400 µg/mL).The infusion was able to inhibit the growth of the MCF-7 cell line and both aqueous extracts inhibit the growth of HeLa cells, however, requiring a higher GI 50 .In addition, none presented cytotoxicity against non-tumoral PLP2 cells.This result is worth noting since the aerial parts E. purpurea are frequently consumed in the form of infusion when used in traditional medicine.
The obtained results correlate well with the ability to inhibit the growth of cancer cell lines, reported by Aarland et al. [6], who investigated the cytotoxic capacity of the hydroalcoholic extract prepared with the aerial parts of E. purpurea against MCF-7, HeLa and HCT-15 cells and showed a higher toxicity for the HeLa cell line.Tsai et al. [25] tested a hydroethanolic extract prepared with the flower of E. purpurea, and compared it with the commercial standard compound of chicoric acid.The results showed significant inhibition of proliferation, in a dose-and time-dependent manner, in human colon cancer cells Caco-2 and HCT-116.Chicoric acid was found to decrease the telomerase activity in HCT-116 cells, inducing apoptosis in colon cancer cells.In the present work, despite chicoric acid being also the main compound in the aqueous extracts, these extracts were the ones showing the lower cytotoxic activity.However, better results were obtained for the MeOH compared to both aqueous extracts, which can be related to its significantly higher concentration in trans-chicoric acid.Previously, Chicca, et al. [26] reported that one constituent of Echinacea pallida roots, namely the acetylenic compound pentadeca-(8Z,13Z)-dien-11-yn-2-one, revealed a concentration-dependent cytotoxicity on several human cancer cell lines, including leukemia (Jurkat and HL-60), breast carcinoma (MCF-7), and melanoma (MeWo) cells.Nevertheless, in the present work, this compound was not identified in the extracts from the aerial parts of E. purpurea.According to the obtained results (Table 4), in the present work, DCM and n-hexane extracts were the ones that showed the most promising cytotoxic activity.Therefore, these two E. purpurea extracts were selected for fractionation, and the obtained fractions were further investigated for their cytotoxicity.A total of fourteen fractions from the n-hexane extract (FH 1-14) and fifteen fractions from the DCM (FD 1-15) were obtained, with the results of their cytotoxicity being shown in Table 4. Almost all fractions from the n-hexane extract showed activity against the four human tumor cell lines, nevertheless showing a marked reduction in activity when compared to the whole extract, as the GI 50 values were much higher.The same was observed for the DCM fractions, with the reduction in activity being more evident in this case since, besides higher GI 50 values, five fractions did not present any cytotoxic activity at all.In both cases, the obtained results suggest a high synergic effect among the compounds present in the whole extract.Therefore, considering that all DCM and n-hexane fractions exhibited a lower cytotoxic activity compared to the whole extract, they were not further analyzed for its chemical composition.In what concerns the two whole extracts, DCM and n-hexane, by comparing their chemical composition obtained by GC-MS analysis (Table 2), it can be observed that the composition regarding the major group of compounds is somehow similar, comprising fatty acids, sterols, and long-chain hydrocarbons, thus, not justifying the differences observed in the cytotoxic assays.However, it should be noticed that, for both extracts, several compounds were not possible to be identified by the used technique (corresponding to 37.2% and 38.9% for DCM and n-hexane extracts, respectively).Most possibly, those non-identified compounds can be related with both the cytotoxic effects exhibited against the tumor and hepatic cell lines as well as the synergic effects suggested by the results obtained with the fractions.
Todd et al. [16] evaluated the effect of a 75% ethanolic extract of E. purpurea roots, and thereof, fractions on the suppression of cytokines and observed that both the fractions that contained alkylamides as well as those without detectable alkylamides displayed similar suppressive effects, allowing the authors to conclude that the ethanolic E. purpurea extract probably contains multiple constituents that differentially regulate cytokine production by macrophages.

Plant Material
Echinacea purpurea (L.) Moench (aerial parts) (150 g) were acquired from Cantinho das Aromáticas, Vila Nova de Gaia, Portugal, in September 2017.According to the supplier, E. purpurea was from a biological production, harvested when flowering and dried at 40-45 • C for three days in a dryer with controlled ventilation.The dried sample was ground to a fine powder, mixed to obtain a homogeneous sample and stored at room temperature protected from light and humidity.

Preparation of Extracts
The extracts were prepared from the powdered plant using different solvents with increasing polarities, as described by Graça et al. [27].The extracts were separated into organic (n-hexane, dichloromethane-DCM, ethyl acetate-EtOAc, acetone, and methanol-MeOH) and aqueous (decoction and infusion) extracts.

Organic Extracts
For the preparation of the mentioned organic extracts, 7 g of dried plant material were submitted to a sequential extraction process based on increasing the solvent's polarity.The plant was extracted twice with each organic solvent (500 mL) for 48 h, under vigorous stirring (150 rpm), at room temperature.The solutions were filtered under reduced pressure through a sintered Buchner glass funnel, the combined organic extracts were evaporated to dryness under reduced pressure at 40 • C (Büchi R-20, Flawil, Switzerland), and the obtained residue was further extracted with another solvent following the same procedure.

Aqueous Extracts
Two aqueous extracts were prepared: decoction and infusion.For the former, the dried sample (1 g) was added to 100 mL of distilled water and boiled for 5 min; the infusion extract was obtained by adding the dried sample (1 g) in 100 mL of boiling water and left to stand for 5 min at room temperature.The mixtures were filtered under reduced pressure through a sintered glass Buchner funnel, and further frozen and lyophilized.

Fractionation of the Extracts
Fractionation was carried out in the extracts that showed promising results in the bioactivity assays, namely DCM and n-hexane extracts.The procedure was conducted based on the methodology described by Graça et al. [28], with some modifications.

n-Hexane Extracts
For the n-hexane extract, a fractionation procedure similar as described for the DCM extract was used.The gradient elution using the same solvents/mixture of solvents allowed collecting of a total of eight hundred and thirty-six samples (~23 mL each), which were collected and grouped in fourteen fractions (FH 1-14), according to the similarity of their TLC profiles [silica gel, CH 2 Cl 2 /MeOH-(99:1), stained with 50% H 2 SO 4 in MeOH, heating].The solvent of these final fractions was removed under reduced pressure until complete dryness.

Analysis of Phenolic Compounds by HPLC-DAD-ESI/MS
The phenolic compounds present in E. purpurea extracts were analyzed as described in Bessada et al. [29], with minor modifications.The EtOAc and acetone extracts were dissolved in MeOH, while the methanol extract was dissolved in MeOH/H 2 O (1:4, v/v), and the infusion and the decoction extracts were dissolved in distilled H 2 O, at a final concentration of 5 mg/mL, and filtered through a 0.22 µm disposable LC filter disk.All the extracts were analyzed using a Dionex Ultimate 3000 UPLC (Thermo Scientific, San Jose, CA, USA) system equipped with a diode array detector coupled to an electrospray ionization mass detector (LC-DAD-ESI/MS n ), a quaternary pump, an autosampler (kept at 5 • C), a degasser and an automated thermostatized column compartment.Chromatographic separation was achieved with a Waters Spherisorb S3 ODS-2 C18 (3 µm, 4.6 × 150 mm, Waters, Milford, MA, USA), column thermostatized at 35 • C. The solvents used were: (A) 0.1% formic acid in water, (B) acetonitrile.The elution gradient established was the following: 15% B (5 min), 15% B to 20% B (5 min), 20-25% B (10 min), 25-35% B (10 min), 35-50% B (10 min), and re-equilibration of the column, using a flow rate of 0.5 mL/min.Double online detection was carried out in the DAD (using 280, 330 and 370 nm as preferred wavelengths) and in a mass spectrometer (MS) connected to a HPLC system via the DAD cell outlet.MS detection was performed in the negative mode, using a Linear Ion Trap LTQ XL mass spectrometer (Thermo Finnigan, San Jose, CA, USA) equipped with an ESI source.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, and 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 (Thermo Finnigan, 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, 7-level calibration curves for each available phenolic standard was constructed based on the UV signal of caffeic acid (y = 388345x + 406369, R 2 = 0.999, LOD = 0.78 µg/mL and LOQ = 1.97 µg/mL), ferulic acid (y = 633126x − 185462, R 2 = 0.999, LOD = 1.85 µg/mL and LOQ = 5.61 µg/mL), p-coumaric acid (y = 301950x + 6966.7,R 2 = 0.9999, LOD = 1.10 µg/mL and LOQ = 3.32 µg/mL), and quercetin-3-O-rutinoside (y = 13343x + 76751, R 2 = 0.999, LOD = 0.21 µg/mL and LOQ = 0.71 µg/mL).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 (Figure S1, Supplementary Material).The results were expressed as mg per g of extract.

Analysis of Non-Polar Compounds by GC-MS
E. purpurea n-hexane and DCM extracts were chemically characterized by GC-MS after sample derivatization.A portion of the N-hexane and DCM extracts (50 mg) was dissolved in 600 µL of bis-(trimethylsilyl) trifluoroacetamide (BSTFA; PanReac AppliChem, Germany) and the mixture was heated at 70 • C for 1 h.The derivatized sample was analyzed by GC-MS using a GC-2010 Plus (Shimadzu, USA) gas chromatography system equipped with a AOC-20iPlus (Shimadzu) automatic injector, a SH-RXi-5ms column (30 m × 0.25 mm × 0.25 µm; Shimadzu, USA) and a mass spectrometry detector, operating under the conditions previously described by Spréa et al. [30], with minor modifications, namely the oven program, was as follows: initial oven temperature of 45 • C increasing at 3 • C/min to 175 • C, then at 15 • C/min to 300 • C, and finally, was held isothermal for 15 min.The compounds were identified based on the comparison of the obtained spectra with those from the NIST17 mass spectral library, and confirmed by determining the linear retention index (LRI) based on the retention times of an N-alkanes mixture (C8-C40, Supelco).Whenever possible, comparisons were also performed with commercial standards and with published data.Compounds were quantified as relative percentage using relative peak area values obtained directly from the total ion current (TIC) values.

Antimicrobial Activity
The organic and aqueous extracts were evaluated for their antimicrobial potential based on the methodology presented by Alves, et al. [31], with some modifications.The used microorganisms were clinical isolates from patients hospitalized in various departments of the Centro Hospitalar de Trás-os-Montes e Alto Douro (Vila Real and Bragança) and comprised five Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Morganella morganii, Proteus mirabilis and Pseudomonas aeruginosa), three Gram-positive bacteria (Enterococcus faecalis, Listeria monocytogenes, Methicillin-resistant Staphylococcus aureus (MRSA), and Methicillin-Susceptible Staphylococcus aureus (MSSA)) and a yeast (Candida albicans).All the microorganisms were incubated at 37 • C in an appropriate fresh medium for 24 h before analysis to maintain the exponential growth phase.The minimum inhibitory concentration (MIC), corresponding to the lowest concentration of the E. purpurea extracts able to inhibit microbial growth, was determined against each microorganism using a colorimetric assay.The extracts were dissolved in 5% (v/v) dimethyl sulfoxide (DMSO)/Mueller-Hinton Broth (MHB) or Tryptic Soy Broth (TSB) to give a final concentration of 100 mg/mL for the stock solution.Then, the samples were serially diluted, obtaining a concentration range from 20 to 0.15 mg/mL.For the determination of the minimal bactericide concentration (MBC) or minimal fungicidal concentration (MFC) for the yeast, 10 µL of each well that showed no change in color was plated on blood agar (7% sheep blood) solid medium and incubated at 37 • C for 24 h.The lowest concentration that yielded no growth was set as the MBC or MFC.A negative control was prepared with 5% (v/v) dimethyl sulfoxide (DMSO)/Mueller-Hinton Broth (MHB) or Tryptic Soy Broth (TSB).One growth control was prepared with MHB and each inoculum.For the Gram-negative bacteria, antibiotics, such as ampicillin and Imipenem, were used as positive controls, while for the Gram-positive bacteria, ampicillin and vancomycin were selected.

Cytotoxic Activity
The evaluation of cytotoxicity was in human tumor cell lines, namely: NCI-H460 (lung cancer); MCF-7 (breast adenocarcinoma); HepG2 (hepatocellular carcinoma); HeLa (cervical carcinoma) was determined according to the procedure used in Barros et al. [32].A phase-contrast microscope was used to monitor the growth of cell cultures, which were subcultured and plated in 96-well plates (density of 5.0 × 10 4 cells/mL) using Dulbecco's modified Eagle's medium (DMEM) supplemented with FBS (10%), 1% penicillin/streptomycin.The cell growth inhibition was measured using the sulforhodamine B (SRB) assay, where the quantity of pigmented cells is directly proportional to the total protein content and, therefore, to the number of bounded cells.The samples were dissolved in water or DMSO (1%) depending on the extract, at 8 mg/mL and then, submitted to further dilutions (400-3.125µg/mL).The results were expressed as GI 50 values (sample concentration that inhibited 50% of the net cell growth, in µg/mL).Ellipticine was used as the positive control.

Hepatotoxicity
For the hepatotoxicity assay, a cell culture was prepared from a freshly harvested porcine liver (obtained from a local slaughterhouse) and designated as PLP2 [33].Before reaching confluence, cells were subcultured and plated in 96-well plates at a density of 1.0 × 10 4 cells/well and cultivated in commercial DMEM medium supplemented with 10% FBS, 1% penicillin/streptomycin.The samples were dissolved in water or DMSO (1%) depending on the extract, at 8 mg/mL, and then, submitted to further dilutions (400-3.125µg/mL).Ellipticine was used as a positive control and results were expressed in GI 50 values corresponding to the sample concentration achieving 50% of growth inhibition in liver primary culture PLP2.

Statistical Analysis
The described experiments were performed in triplicate and the results were expressed as the mean ± standard deviation (SD).The differences between the different extracts were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's honest significant difference post hoc test with α = 0.05, coupled with Welch's statistic.A Student's t-test was used to determine the significant difference among two different samples, with α = 0.05.Statistical analysis was carried out using the SPSS v. 23.0 program (SPSS v. 23.0;IBM Corp., Armonk, NY, USA).

Conclusions
The work reported herein highlights the difference of the biological activity in the extracts of E. purpurea prepared with solvents of different polarity as well as their chemical characterization.The highest antimicrobial activity was observed for the DCM, EtOAc, and acetone extracts, while the highest cytotoxicity was evidenced by the DCM and n-hexane extracts.Despite exhibiting lower activity, it is worth noting that the infusion, which is frequently used by consumers, was able to inhibit the growth of MCF-7 and HeLa cell lines.In general, the cytotoxicity of the DCM and n-hexane extracts was superior compared to the corresponding fractions, which points to a possible synergistic effect of the mixture of compounds present in the initial extracts.

Figure 1 .
Figure 1.Phenolic profile of the MeOH extract of E. purpurea recorded at 280 nm (A) and 370 nm (B).

Figure 1 .
Figure 1.Phenolic profile of the MeOH extract of E. purpurea recorded at 280 nm (A) and 370 nm (B).

Table 1 .
Retention time (Rt), wavelengths of maximum absorption in the visible region (λ max ), mass spectral data, tentative identification and quantification (mg/g extract) of the phenolic compounds present in five different extracts of E. purpurea.

Table 2 .
Chemical composition of n-hexane and DCM extracts of E. purpurea obtained by GC-MS analysis.

Table 3 .
Antimicrobial activity of all seven E. purpurea extracts against clinical isolates of Gram-negative and Gram-positive bacteria and one yeast strain.
MIC and MBC are expressed in mg/mL.nt-not tested.The highest tested concentration was 20 mg/mL.

Table 4 .
Cytotoxicity of organic and aqueous extracts and obtained fractions (n-hexane and DCM, µg/mL) of E. purpurea against human tumor cell lines and non-tumor cell line (PLP2).