Profiling of Polyphenol Composition and Antiradical Capacity of Erica cinerea

The aim of the current study was to determine the profile and content of polyphenols present in Erica cinerea, an important plant species from Northern Portuguese flora and often reported as having anti-inflammatory, antioxidant, and anti-radical activity. The analysis of polyphenols was performed by HPLC-DAD/UV-Vis, and the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+) method was used to evaluate its radical scavenging activity. HPLC analysis showed that both plants presented a great diversity of compounds, with 33% flavones, 28% flavanols, and 26% hydroxycinnamic acids. The antiradical activity was dose-dependent, and the IC50 values were 0.251 mg mL−1. Based on our study, E. cinerea presented interesting bioactive compounds and it can be used to extract and purify bioactive polyphenols to be used in pharmaceutical or agro-food industries.


Introduction
Erica cinerea L. (Ericaceae) is largely present in the mountains of west and middle Europe, and is traditionally used in folk medicine to treat inflammatory diseases [1,2]. Several studies [3][4][5] have shown that Ericaceae contain a large number of bioactive compounds, including polyphenols. Additionally, recent studies with another medicinal Ericaceae species plant-Erica australis [6,7]-have shown that this plant is a rich source of polyphenols often associated with the prevention of degenerative and inflammatory diseases, as well as associated to antiradical processes [6,7]. In this context, we present this study in which we characterize the polyphenol composition of E. cinerea from Portuguese endemic flora by high performance liquid chromatography system coupled with a diode array type of UV/Vis detector (HPLC-DAD/UV-Vis) and determine its antiradical scavenging activity. The information will help to clarify if this plant species can be used to extract bioactive polyphenols.

Plant Material
One kilogram (fresh weight) of Erica cinerea (leaves and flowers) was collected in April 2015 in the natural open fields in Northern Portugal, Vila Real Region (400 m altitude) near the Natural Park of Alvão (N 41 • 17 35.538 , W 7 • 44 29.6268 ). The samples were properly and botanically identified. After harvested and when in laboratory, the samples were dried in a freeze-drier system (Ultra Dry Systems TM, Warminster, PA, USA) and milled in a commercial blender and stored in dark flasks at 4 • C until extraction. The fresh and dry weights were recorded, and dry matter was determined. Three replications were taken.

Polyphenol Composition by HPLC-DAD/UV-Vis
The quantification of polyphenols present in E. cinerea extracts was performed using a HPLC-DAD-UV/Vis [8] system equipped with a C18 column (250 × 46 mm, 5 µm) (ACE ® HPLC columns, Advanced Chromatography Technologies, Ltd., Aberdeen, Scotland), an eluent composed by 1% of trifluoroacetic acid (TFA) in water (solvent A) and 1% TFA in acetonitrile (solvent B). The elution was performed with a flow rate of 1 mL min −1 , a gradient starting with 100% water, and an injection volume of 10 µL. The chromatograms were recorded at 254, 280, 320, 370, and 520 nm. The polyphenols were identified and quantified using peak retention time, UV spectra, and UV max absorbance bands and trough comparison with external commercial standards (Extrasynthese, CEDEX, France, and Sigma-Aldrich, Tauferkichen, Germany), as well as by comparing with published literature [9,10]. The external standards were freshly prepared in 70% methanol (70 methanol:30 water) at a concentration of 1.0 mg mL −1 and running in HPLC-DAD-UV-Vis before the samples. The results were expressed as µg g −1 dry weight (dw). All solvents were HPLC-grade solvents.

ABTS (2,2 -Azinobis-(3-Ethylbenzothiazoline-6-Sulfonic Acid)) Radical Scavenging Activity
The ABTS radical scavenging activity was evaluated using the (ABTS•+) radical-scavenging activity colorimetric method [11] conducted in a 96-well microplate. Ten different methanolic concentrations (ranging from 0.195 to 10.0 mg mL −1 ) of E. cinerea extracts were prepared and used in the antiradical assay. A radical ABTS solution was freshly prepared by mixing 7 mM of ABTS at pH 7.4 (5 mM NaH 2 PO 4 , 5 mM Na 2 HPO 4 , and 154 mM NaCl) with 2.5 mM potassium persulfate, followed by storage in the dark at room temperature for 16 h. The mixture was then diluted with ethanol to give an absorbance of 0.70 ± 0.02 units at 734 nm, using a multiscan microplate reader (Multiskan™ FC Microplate Photometer, Waltham, MA, USA). After that, to each microplate well 15 µL of extract was added followed by addition of 285 µL fresh ABTS solution. The microplates were then incubated at room temperature in the dark for 10 min. The absorbance values were then measured in a multiscan microplate reader at 734 nm. Simultaneously, a curve with commercial standard ascorbic acid was used and served as positive control of antiradical activity. The results were expressed as percentage (%) of ABTS radical scavenging activity, using the following formula: ABTS radical scavenging (%) = [(absorbance solvent − absorbance sample)/absorbance solvent) × 100]. The concentration of antioxidants which scavenge the free radical ABTS•+ about 50% (IC 50 ) for plant extract and ascorbic acid was also determined. In order to classify the antiradical activity, we selected the following scale: IC 50 < 0.1 mg mL −1 -strong/high antiradical activity; 0.1 mg mL −1 < IC 50 < 1 mg mL −1 -moderate; and >1 mg mL −1 -weak antiradical activity.

Statistical Analysis
All determinations were carried out in triplicate, and the results were expressed as mean values ± standard deviation (SD). The Software SPSS v.17 (SPSS-IBM, Orchard Road-Armonk, New York, NY, USA) was used to carry out these analysis.

Results
The analysis by HPLC-DAD/UV-Vis of E. cinerea methanolic extracts revealed the presence of different classes of polyphenols (Table 1). The quantity of each polyphenol identified in each plant extract is presented in Table 1. E. cinerea exhibited an average level of 1646 µg g −1 dw, in which the highest values were found for chlorogenic acid + neochlorogenic acid (22%), luteolin-3-O-rutinoside (11%), luteolin-3-O-galactoside (10%), apigenin-7-O-rutinoside (9%), and quercetin-3-O-glactoside (6%). These polyphenols represented more than 50% of the total polyphenols identified.  1 Values expressed as mean ± standard deviation (SD) of three replicates. Figure 1 shows the variation of antiradical activity by ABTS method, including the respective IC 50 values (samples with lower IC 50 values correspond to those with stronger antiradical activity), and it was possible to observe that the extracts of E. cinerea presented lower antiradical activity when compared to ascorbic acid. However, when compared to other medicinal and aromatic plants [12][13][14], and with the scale adopted, we considered the antiradical activity moderate.

Discussion
Ericaceae plants have been presented as having health protective effects due to their richness in polyphenols-particularly phenolic acids and flavonoids, highly associated with high antioxidant capacity, antiradical activity, and anti-inflammatory effect [6,7]. Among these compounds, polyphenols like chlorogenic acid, caffeic acid, kaempferol, myricetin, quercetin, and luteolin glycosides [15][16][17][18][19] have been reported as having important health potential, since they present high capacity to scavenge different oxidizing molecules (such as superoxide anion, hydroxyl radical, or peroxy radicals, and as quenchers of singlet oxygen) or to increase powerful intracellular antioxidant enzymes and act as pro-active cell protectors [15][16][17][18][19]. Thus, a high content of such polyphenols should mean high bioactivities. Therefore, based on our results, since E. cinerea contains high levels of such important bioactive polyphenols, we may state that E. cinerea shows high antioxidant potential, which may partially explain the ethnobotanical use of this plant as a treatment for inflammation-related conditions.

Conclusions
The richness of Erica cinerea in polyphenols like hydroxycinnamic acids, flavanols, flavonols, and flavones justify their use in traditional and folk medicine, and they can be used to extract and purify important bioactive compounds for the nutraceutical and food supplement market. More studies measuring seasonal or geographical variations in the polyphenols profile and content of E. cinerea and exploring if this plant can be a good medical crop plant should be done in the future.

Discussion
Ericaceae plants have been presented as having health protective effects due to their richness in polyphenols-particularly phenolic acids and flavonoids, highly associated with high antioxidant capacity, antiradical activity, and anti-inflammatory effect [6,7]. Among these compounds, polyphenols like chlorogenic acid, caffeic acid, kaempferol, myricetin, quercetin, and luteolin glycosides [15][16][17][18][19] have been reported as having important health potential, since they present high capacity to scavenge different oxidizing molecules (such as superoxide anion, hydroxyl radical, or peroxy radicals, and as quenchers of singlet oxygen) or to increase powerful intracellular antioxidant enzymes and act as pro-active cell protectors [15][16][17][18][19]. Thus, a high content of such polyphenols should mean high bioactivities. Therefore, based on our results, since E. cinerea contains high levels of such important bioactive polyphenols, we may state that E. cinerea shows high antioxidant potential, which may partially explain the ethnobotanical use of this plant as a treatment for inflammation-related conditions.

Conclusions
The richness of Erica cinerea in polyphenols like hydroxycinnamic acids, flavanols, flavonols, and flavones justify their use in traditional and folk medicine, and they can be used to extract and purify important bioactive compounds for the nutraceutical and food supplement market. More studies measuring seasonal or geographical variations in the polyphenols profile and content of E. cinerea and exploring if this plant can be a good medical crop plant should be done in the future.
Author Contributions: Alfredo Aires conceived the work, designed the experiments and organized the written manuscript; Alfredo Aires and Rosa Carvalho performed the extraction, HPLC analysis and antioxidant activity bioassays; Alfredo Aires analyzed the data, wrote and revised the paper.

Conflicts of Interest:
The authors declare no conflict of interest.