Polyphenolic composition, antioxidant and antibacterial activities for two Romanian subspecies of Achillea distans Waldst. et Kit. ex Willd.

The aim of this work was to study the chemical composition, antioxidant and antibacterial properties of Achillea distans Waldst. et Kit. subsp. distans and Achillea distans Waldst. et Kit. subsp. alpina Rochel, from the Rodna Mountains (Romania). The identification and quantification of major phenolic compounds was performed by a HPLC-MS method. The total polyphenolic and flavonoid content was determined spectrophotometrically. The antioxidant activity was evaluated using the DPPH bleaching method, trolox equivalent antioxidant capacity assay (TEAC), hemoglobin ascorbate peroxidase activity inhibition (HAPX) assay, and an Electron Paramagnetic Resonance (EPR) spectroscopy method. A data indicated that A. distans subsp. alpina extract has more antioxidant activity than A. distans subsp. distans extract. Luteolin, apigenin, quercetin, caffeic and chlorogenic acids were present in the two extracts of A. distans, but in different amounts. Three flavonoids were detected only in A. distans subsp. alpina. The polyphenol-richer A. distans subsp. alpina extract showed a higher antioxidant activity than A. distans subsp. distans extract. A. distans subsp. distans extract showed inhibitory activity for Gram-positive bacteria, as evaluated with four species. The quantitative and qualitative differences between the two subspecies of Achillea distans could be used as a potential taxonomic marker in order to distinguish the species.

1. Introduction

The genus Achillea (Asteraceae) is represented by about 85 species throughout the World, and 23 species and 10 varieties or subspecies can be found in the Romanian flora. Some Achillea species have ethnopharmacologic importance and are known to be used in folk remedies for various purposes [1,2]. Achillea distans Waldst. et Kit. ex Willd. (Alps yarrow) is an Alpino-Carpatho-Balkan species that vegetates on the upper limit of mountain forests and in subalpine shrubs. According to the length of ligulate florets and to their color, two subspecies are recognized: Achillea distans Waldst. et Kit. subsp. Distans, with white flowers and about 2 mm length of ligulae and Achillea distans Waldst. et Kit. subsp. alpina (Rochel) Soó, with pink flowers and about 3 mm length of ligulae [1,2,3]. Due to the differences in the chemical composition of the essential oils of A. distans subsp. distans and A. distans subsp. alpine, these were considered as infraspecific chemical taxa or chemovarieties of Achillea distans [3]. Achillea distans Waldst. et Kit. ex Willd., found in the Rodna Mountains (a subdivision of the Eastern Carpathians in Northern Romania), was confirmed as a native species of the Romanian flora [1]. The flower heads of A. distans species contain essential oils, polyphenolic compounds, terpenoids; the essential oil and the tincture showed antimicrobial and anti-inflammatory activities [4,5,6,7]. The roots of A. distans subsp. distans also contain essential oil [8].

Phenolic compounds are a major group of compounds acting as primary antioxidants or free radical scavengers; flavonoids are a ubiquitous group of the polyphenolic substances which are present in all higher plants [9]. The total phenolic compounds and flavonoid content are also related to the antioxidant activity. Over the past decade, major advances have been made to investigate the antioxidant and antimicrobial properties of different Achillea species: A. alexandri-regis Bornm. & Rudsky [10], A. biebersteinii Afan. [11], A. moschata Wulf., A. distans Waldst. & Kit. ex Willed. [12], A. micrantha Willd., A. filipendula Lam., A. tenuifolia Lam., A. vermicularis Trin., and A. wilhelmsii C. Koch. [13], A. santolina [14], A. ptarmica L., A. nobilis L. [15], A. millefolium L. [13,16,17,18,19], A. ligusta All. [20], A. collina Becker, A. pannonica Scheele [21,22], A. teretifolia Waldst. and Kitt, A. schischkinii Sosn. [23]. Only one of the studies evaluated the antioxidant activity of Achillea distans from northern Italy, showing that methanolic extracts from the flowering parts possess antioxidant activity, by employing DPPH and LDL oxidation assays [12]. Scientific data on the two subspecies (alpina and distans) of A. distans are very limited.

The aim of the present paper was to characterize the polyphenolic composition of hydroalcoholic extracts from the flowers of two subspecies of Achillea distans Waldst. et Kit. ex Willd.: Achillea distans Waldst. et Kit. subsp. distans and Achillea distans Waldst. et Kit. subsp. alpina (Rochel) Soó, and to evaluate their in vitro antioxidant and antibacterial activities.

2. Results and Discussion

2.1. HPLC Analysis of Polyphenols

HPLC coupled with MS is a very powerful analytical technique, due to its high sensitivity and the structural information that can be obtained about the analytes. A high-performance liquid chromatographic (HPLC) method has been developed for the determination of 19 phenolic compounds (eight phenolic acids, four quercetin glycosides, and seven flavonol and flavone aglycones) from plant material. The applicability of the proposed analytical method and the qualitative and quantitative determination of the standard phenolic compounds have already been verified [4,24,25,26]. The method allows a simultaneous analysis of different classes of polyphenols by a single pass column (the separation of all examined compounds was carried out in 35 min). The concentrations of identified polyphenolic compounds in both analyzed samples are presented in Table 1. They were shown in the order of their retention time. The HPLC chromatogram of A. distans subsp. distans sample is presented in Figure 1 and the HPLC chromatogram of A. distans subsp. alpina sample is presented in Figure 2. The quantitative determination was performed using the external standard method (Table 2).

Table 1

Polyphenolic compounds content in Achillea distans subspecies (mg/100 g plant material).

Polyphenolic compounds	m/zvalue	tR ± SD(min)	Achillea distans subsp. distans	Achillea distans subsp. alpina
Caffeic acid	179	5.60 ± 0.04	<0.2	<0.2
Chlorogenic acid	353	5.62 ± 0.05	<0.2	<0.2
Hyperoside	463	18.60 ± 0.12	NF	2.05 ± 0.10
Isoquercitrin	463	19.60 ± 0.10	NF	<0.2
Rutin	609	20.20 ± 0.15	NF	<0.2
Quercetin	301	26.80 ± 0.15	1.44 ± 0.06	1.11 ± 0.09
Luteolin	285	29.10 ± 0.19	763.12 ± 1.88	52.65 ± 0.85
Apigenin	279	33.10 ± 0.15	264.84 ± 1.16	13.47 ± 0.53

Note: NF - not found, below limit of detection. Values are the mean ± SD (n = 3).

Figure 1

HPLC chromatogram of A. distans subsp. distans.

Figure 2

HPLC chromatogram of A. distans subsp. alpina.

Table 2

Retention times (RT) of polyphenolic compounds (min).

Peak no.	Phenolic compounds	m/z	tR ± SD (min)
1.	Caftaric acid	311	3.54 ± 0.05
2.	Gentisic acid	179	6.52 ± 0.04
3.	Caffeic acid	179	5.60 ± 0.04
4.	Chlorogenic acid	353	5.62 ± 0.05
5.	p-Coumaric acid	163	9.48 ± 0.08
6.	Ferulic acid	193	12.8 ± 0.10
7.	Sinapic acid	223	15.00 ± 0.10
8.	Cichoric acid	473	15.96 ± 0.13
9.	Hyperoside	463	18.60 ± 0.12
10.	Isoquercitrin	463	19.60 ± 0.10
11.	Rutin	609	20.20 ± 0.15
12.	Myricetin	317	21.13 ± 0.12
13.	Fisetin	285	22.91 ± 0.15
14.	Quercitrin	447	23.64 ± 0.13
15.	Quercetin	301	26.80 ± 0.15
16.	Patuletin	331	29.41 ± 0.12
17.	Luteolin	285	29.10 ± 0.19
18.	Kaempferol	285	32.48 ± 0.17
19.	Apigenin	279	33.10 ± 0.15

Note: SD, standard deviation.

In the ethanolic extract of A. distans subsp. distans flowers, luteolin was the compound found in the largest amount (763.12 ± 1.88 mg/100 g) followed by apigenin (264.84 ± 1.16 mg/100 g). We detected quercetin at lower levels than major flavonoides (1.44 ± 0.06 mg/100 g). Caffeic acid and chlorogenic acid were also identified in this extract, but they were in too low concentration to be quantified (Table 1).

Notes: Chromatographic conditions were as given in the Experimental section. The identified compounds: 1, Quercetin;2, Luteolin 3, Apigenin.

Notes: Chromatographic conditions were as given in the Experimental section. The identified compounds: 1, Hyperoside; 2, Quercetin; 3, Luteolin; 4, Apigenin.

In the extract of A. distans subsp. alpina flowers, the two free aglycones, i.e., apigenin and luteolin, were found in much smaller amounts than in “distans” subspecies (13.47 ± 0.27, and 52.65 ± 0.85 mg/100 g, respectively). Quercetin was detected in small amounts (1.11 ± 0.09 mg/100 g). In this extract, three flavonoid glycosides were identified. Hyperoside (quercetin 3-O-galactoside) was determined in quantities of 2.05 ± 0.10 mg/100 g plant material. Rutin (quercetin 3-O-rutinoside) and isoquercitrin (quercetin 3-O-glucoside) were identified in the ethanolic extract, but they were in too low concentration to be quantified. The three glycosides of quercetin detected only in A. distans subsp. alpina, could be used as a potential taxonomic marker in order to distinguish the subspecies. Caffeic acid and chlorogenic acid were also identified in the extract of A. distans subsp. alpina (Table 1). Considering the 19 standard compounds used in this study (Table 2), some other peaks were not identified.

Thus, the comparative study showed large differences, especially quantitative, between the two taxa of Achillea distans. The richest subspecies in aglycones of flavonoids (luteolin, apigenin, quercetin) was A. distans subsp. distans, but the flavonoid glycosides were not found.

2.2. Polyphenolic and Flavonoid Contents of the Extracts

Flavonoids and phenolic acids make up one of the most pervasive groups of plant phenolics. The plants and herbs consumed by humans may contain various amounts of different phenolic acid and flavonoid components. The effect of dietary phenolics is currently of great interest due to their antioxidant and possible anticarcinogenic activities. Phenolic acids and flavonoids also function as reducing agents, free radical scavengers, and quenchers of singlet oxygen. In addition, flavonoids and phenolic acids components play important roles in the control of cancer and other human diseases. Due to their importance in plants and human health, it would be useful to know the concentration of the polyphenolic compounds and biological activities that could indicate their potentials as therapeutic agents, and also for predicting and controlling the quality of medicinal herbs [27].

The total polyphenolic content (TPC) values summarized for Achillea extracts in Table 3 were quantified based on the linear equation obtained from gallic acid standard calibration curve. Thus, TPC values were expressed as gallic acid equivalent (mg GAE/g sample). The calculation of total flavonoid content of plant extracts was carried out using the standard curve of rutin and presented as rutin equivalents (mg RE/g sample). The highest amount of the total polyphenols was determined in the extract of A. distans subsp. alpina flowers (174.75 ± 1.47 mg·g−1) followed by A. distans subsp. distans extract (101.61 ± 1.24 mg·g−1). Concerning the content of flavonoids, contrary, the extract of A. distans subsp. distans (37.26 ± 0.71 mg·g−1) was richer in flavonoids, than the extract of A. distans subsp. alpina (33.18 ± 0.60 mg·g−1).

Table 3

The content of total polyphenols and flavonoids in A. distans extracts.

Samples	TPC (mg GAE/g plant material)	Flavonoids (mg RE/g plant material)
A. distans subsp. distans	101.61 ± 1.24	37.26 ± 0.71
A. distans subsp. alpina	174.75 ± 1.47	33.18 ± 0.60

Each value is the mean ± SD of three independent measurements. GAE: Gallic acid equivalents; RE: rutin equivalents.

2.3. Antioxidant Activity

The antioxidant capacity of the ethanolic extracts of flowers heads of A. distans subsp. distans and A. distans subsp. alpina was determined by several methods: DPPH bleaching assay, the Trolox equivalent antioxidant capacity (TEAC), Electron Paramagnetic Resonance (EPR) method, and the hemoglobin ascorbate peroxidase activity inhibition (HAPX) assay (Table 4).

Table 4

Antioxidant capacity parameters obtained using several methods for studied Achillea samples.

Samples	DPPH (% decolorization)	IC50 (µg·mL−1)	TEAC (µmol Trolox/mg plant material)	HAPX (%)
A. distans subsp. distans	52.73 ± 0.77	204.85 ± 2.93	42.14 ± 0.48	4.91 ± 1.24
A. distans subsp. alpina	93 ± 1.47	83.80 ± 1.20	46.72 ± 0.35	10.27 ± 1.81
Quercetin	-	5.60 ± 0.35	-	-
BHT	-	16 ± 0.54	-	-

Each value is the mean ± SD of three independent measurements.

The antioxidant activity of the ethanol extracts was further assessed by the DPPH radical bleaching method. The DPPH scavenging ability of the subspecies alpina was 1.76 times larger than that of the subspecies distans at the same concentration (218.75 µg plant product/mL extract) (Table 4). The highest radical scavenging activity was showed by the extract of A. distans subsp. alpina with IC50 = 83.80 ± 1.20 µg·mL−1, followed by the extract of A. distans subsp. distans (IC50 = 204.85 ± 2.93). This is in good agreement with the TPC values listed in Table 3. Compared to the reference compounds, quercetin (IC50 = 5.60 ± 0.35 µg·mL−1) and BHT (IC50 = 16 ± 0.54 µg·mL−1), the ethanol extracts of A. distans showed lower antioxidant capacity. According to this method, A. distans subsp. alpina extract exhibited a moderate antioxidant capacity (50 µg/mL < IC50 ≤ 100 µg·mL−1), and the ethanol extract of A. distans subsp. distans has no relevant antioxidant activity (IC50 > 200 µg·mL−1). In the TEAC assay, the stable radical is dissolved in an aqueous solution, thus expecting a different mechanism of interaction between antioxidants molecule and the radical since TEAC assesses the more hydrophilic components while DPPH describes all components. This can be observed by the final TEAC values (Table 4), even though the antioxidant power is significantly higher in A. distans subsp. alpina extract, the difference in absolute value is not so great as in the case of DPPH method. Thus, it can be speculated that the A. distans subsp. distans extract contain antioxidant compounds which better act in aqueous solution than in organic solvent (ethanol). In the HAPX assay one measures, in a physiological relevant manner, the capability of the extract components to quench the HbFeIV resulted by hydrogen peroxide-induced damage upon HbFeIII. The results further indicate that by this assay too, A. distans subsp. alpina extract is double more antioxidant than A. distans subsp. distans extract. Figure 3 illustrates the results of an antioxidant assay using electron paramagnetic spectroscopy (EPR). This assay also indicate that A. distans subsp. alpina extract has a comparable activity to that of gallic acid and A. distans subsp. distans extract has three times less activity than gallic acid. A. distans subsp. alpina extract is much more antioxidant than A. distans subsp. distans extract.

Figure 3

TEMPO consumption (1.4 mM) by A. distans subsp. distans (ADD) and A. distans subsp. alpina (ADA). The experimental data were fitted with exponential decay function according to the equation I(t) = I0 + I1e−kt, and the kinetic constants are kADA = 0.126; kADD = 0.04. Gallic acid was used as control, kgallic acid = 0.131, at a concentration of 6 mg/mL.

2.4. Antibacterial Activity

The antibacterial activity of the ethanol extracts of A. distans is shown in the Table 4. The antibacterial activity is ranked from no activity (-: inhibition diameter < 10 mm), low (+: inhibition diameter between 10 and 15 mm), moderate (++: inhibition diameter between 15 and 20 mm) and high activity (+++: diameter inhibition ≥ 20 mm). The extracts were investigated for their in vitro antimicrobial properties using a disk-diffusion method against S. aureus, L. monocytogenes, E. coli, S. typhymurium [28,29] (Table 5).

Table 5

Antibacterial activity (inhibition zone expressed in mm) a of two investigated extracts of A. distans.

Samples	Inhibition zone in diameter (mm)
	Staphylococcus aureus	Listeria monocytogenes	Escherichia coli	Salmonella typhymurium
A. distans subsp. distans	13.0 ± 0.05	17.0 ± 0.1	10.0 ± 0.05	7.0 ± 0.00
A. distans subsp. alpina	12.0 ± 0.07	12.0 ± 0.00	8.0 ± 0.05	6.0 ± 0.02
Gentamicin	19 ± 0.05	18 ± 0.1	22 ± 0.00	18 ± 0.05

Notes: a The values represent the average of three determinations ± standard deviations. Gentamicin (10 µg/disk) was used as a positive control.

The extract of A. distans subsp. distans showed a moderate activity towards L. monocytogenes (inhibition diameter between 15 and 20 mm), a low antibacterial effect towards S. aureus and E. coli(inhibition diameter between 10 and 15 mm), and inactive against S. typhymurium (inhibition diameter < 10 mm). The extract of A. distans subsp. alpina showed a low antibacterial activity towards S. aureus, L. monocytogenes, and it did not show antibacterial activity against the tested gram-negative bacterial strains.

3. Experimental

3.1. Plant Material and Extraction Procedure

The flowers of Achillea distans Waldst. et Kit. ex Willd. subsp. distans (voucher No. 953) and the flowers of Achillea distans Waldst. et Kit. subsp. alpina (Rochel) Soo (voucher No.954) were collected in August 2011 (Rodna Mountains, in the north of the Eastern Carpathians, near Iezer Lake, at 1700–1750 m altitude, Romania) in the blossom period. Voucher specimens were deposited in the Herbarium of the Department of Pharmaceutical Botany of the Faculty of Pharmacy, Cluj-Napoca, Romania. The plant material (flowers) was reduced to a proper degree of fineness. 2.0 g of the material was extracted with 20 mL of 70% ethanol (Merck, Darmstadt, Germany), for 30 min on a water bath, at 60 °C. The samples were then cooled down and centrifuged at 4,500 rpm for 15 min, and the supernatant was recovered [24,26].

3.2. Chemicals

Chlorogenic acid, p-coumaric acid, caffeic acid, rutin, apigenin, quercetin, isoquercitrin, quercitrin, hyperoside, kaempferol, myricetol, fisetin from Sigma (St. Louis, MO, USA), ferulic acid, sinapic acid, gentisic acid, gallic acid, patuletin, luteolin from Roth (Karlsruhe, Germany), cichoric acid, caftaric acid from Dalton (Toronto, ON, Canada). HPLC grade methanol, analytical grade orthophosphoric acid, hydrochloric acid and Folin-Ciocalteu reagent were purchased from Merck, hydrogen peroxide, ABTS (2,2′-azinobis-3-ethylbenzotiazoline-6-sulphonic acid) were from Sigma (St. Louis, MO, USA); aluminum chloride, sodium acetate, sodium carbonate, ethanol (Merck), DPPH (2,2-diphenyl-1-picrylhydrazyl) and BHT (butylated hydroxytoluene) were obtained from Alfa-Aesar (Karlruhe, Germany). Bovine hemoglobin was purified following the general protocol of Antonini and Brunori [30]. The met forms of hemoglobin were prepared by ferricyanide treatment as previously described [31]. All microorganism products were distributed by MicroBioLogics®: Staphylococcus aureus ATCC 49444 (Gram+), Listeria monocytogenes ATCC 13076 (Gram+), Escherichia coli ATCC 25922 (Gram-), and Salmonella typhymurium ATCC 14028 (Gram-).

3.3. HPLC/MS Analysis

3.3.1. Apparatus and Chromatographic Conditions

The experiment was carried out using an Agilent Technologies 1100 HPLC Series system (Agilent, Santa Clara, CA, USA) equipped with G1322A degasser, G13311A binary gradient pump, column thermostat, G1313A autosampler and G1316A UV detector. The HPLC system was coupled with an Agilent 1100 mass spectrometer (LC/MSD Ion Trap VL). For the separation, a reverse-phase analytical column was employed (Zorbax SB-C18 100 × 3.0 mm i.d., 3.5 μm particle); the work temperature was 48 °C. The detection of the compounds was performed on both UV and MS mode. The UV detector was set at 330 nm until 17.5 min, then at 370 nm. The MS system operated using an electrospray ion source in negative mode. The chromatographic data were processed using ChemStation and DataAnalysis software from Agilent. The mobile phase was a binary gradient: methanol and acetic acid 0.1% (v/v). The elution started with a linear gradient, beginning with 5% methanol and ending at 42% methanol, for 35 min; then 42% methanol for the next 3 minutes [24,25,26]. The flow rate was 1 mL·min−1 and the injection volume was 5 µL.

The MS signal was used only for qualitative analysis based on specific mass spectra of each polyphenol. The MS spectra obtained from a standard solution of polyphenols were integrated in a mass spectra library. Later, the MS traces/spectra of the analysed samples were compared to spectra from library, which allows positive identification of compounds, based on spectral mach. The UV trace was used for quantification of identified compounds from MS detection. Using the chromatographic conditions described above, the polyphenols eluted in less than 40 min (Table 2). Four polyphenols cannot be quantified in current chromatographic conditions due overlapping (caftaric acid with gentisic acid and caffeic acid with chlorogenic acid). However, all four compounds can be selectively identified in MS detection (qualitative analysis) based on differences between their molecular mass and MS spectra. For all compounds, the limit of quantification was 0.5 μg/mL, and the limit of detection was 0.1 μg·mL−1. The detection limits were calculated as minimal concentration producing a reproductive peak with a signal-to-noise ratio greater than three. Quantitative determinations were performed using an external standard method. Calibration curves in the 0.5–50 μg·mL−1 range with good linearity (R2 > 0.999) for a five point plot were used to determine the concentration of polyphenols in plant samples [24,25,26].

3.3.2. Identification and Quantification of Polyphenols

The detection and quantification of polyphenols was performed in UV assisted by mass spectrometry detection. Due to peak overlapping, four polyphenol-carboxylic acids (caftaric, gentisic, caffeic, chlorogenic) were determined only based on MS spectra, whereas for the rest of the compounds the linearity of the calibration curves was very good (R2 > 0.998), with detection limits in the range of 18 to 92 ng·mL−1. The detection limits were calculated as the minimal concentration yielding a reproducible peak with a signal-to-noise ratio greater than three. Quantitative determinations were performed using an external standard method; retention times were determined with a standard deviation ranging from 0.04 to 0.19 min (Table 2). For all compounds, the accuracy was between 94.13% and 105.3%. Accuracy was checked by spiking samples with a solution containing each polyphenol in a 10 μg·mL−1 concentration. In all analyzed samples the compounds were identified by comparison of their retention times and recorded electrospray mass spectra with those of standards in the same chromatographic conditions.

3.4. Determination of Total Polyphenols and Flavonoids Content

The total phenolic content (TPC) of the extracts was determined by the Folin-Ciocalteau method with some modifications [32,33,34,35]. Each ethanolic extract (2 mL) diluted 25 times was mixed with Folin-Ciocalteu reagent (1.0 mL) and distilled water (10.0 mL) and diluted to 25.0 mL with a 290 g/L solution of sodium carbonate. The samples were incubated in the dark for 30 min. The absorbance was measured at 760 nm. Gallic acid was used as standard for the calibration curve and was plotted at 0.02, 0.04, 0.06, 0.08, and 0.10 mg·mL−1, prepared in methanol-water (50:50, v/v). TPC values were determined using an equation obtained from the calibration curve of gallic acid graph (R2 = 0.9990).

The spectrophotometric aluminum chloride method was used for flavonoids determination. Each extract (5 mL) was mixed with sodium acetate (5.0 mL, 100 g·L−1), aluminum chloride (3.0 mL, 25 g·L−1, and filled up to 25 mL by methanol in a calibrated flask. The absorbance was measured at 430 nm [36]. Total flavonoids content values was determined using an equation obtained from calibration curve of the rutin graph (R2 = 0.9996).

3.5. Antioxidant Activity Test

3.5.1. DPPH• Radical Scavenging Assay

The free radical scavenging activity of the ethanolic extracts of the two subspecies of A. distans was measured in terms of hydrogen donating or radical scavenging ability using the stable DPPH radical method. A DPPH solution (0.1 g·L−1) in methanol was prepared and this solution (4.0 mL) was added to of extract solution (or standard) in methanol at different concentrations ((4.0 mL, 10–50 μg·mL−1). After 30 min of incubation at 40 °C in a thermostatic bath, the decrease in the absorbance (n = 3) was measured at 517 nm. The percent DPPH scavenging ability was calculated as: DPPH scavenging ability = (Acontrol − A sample/Acontrol) × 100, where Abscontrol is the absorbance of DPPH radical + methanol (containing all reagents except the sample) and Abssample is the absorbance of DPPH radical + sample extract. Afterwards, a curve of % DPPH scavenging capacity versus concentration was plotted and IC50 values were calculated. IC50 denotes the concentration of sample required to scavenge 50% of DPPH free radicals [16,21,34,37,38,39,40]. The positive controls were those using the standard solution quercetin and butylated hydroxytoluene (BHT). IC50 value is related with the antioxidant capacity. So, IC50 ≤ 50 µg/mL value means a high antioxidant capacity; 50 µg/mL < IC50 ≤ 100 µg·mL−1 value means a moderate antioxidant capacity and IC50 > 200 µg·mL−1 value means no relevant antioxidant capacity [39].

3.5.2. Trolox Equivalent Antioxidant Capacity (TEAC) Assay

In a quartz cuvette, to phosphate buffer saline (PBS, 955 μL) the following were added: Achillea extracts (20 μL, diluted 100 times), and ABTS+● (25 μL, from 74 mM stock solution). The experiments were done in duplicates, with a relative standard deviation of less than 6%. The Achillea extract in the assay mixture was 8.24 mg·L−1. The content of the generated ABTS●+ radical was measured at 734 nm after 600 s reaction time and was converted in Trolox equivalents by the use of a calibration curve (R2 = 0.9987) constructed with 0, 2, 4, 6, 8, 10 mg·L−1 Trolox standards [41,42].

3.5.3. Hemoglobin/Ascorbate Peroxidase Activity Inhibition (HAPX) Assay

In a quartz cuvette sodium acetate buffer (956 μL, 50 mM, pH 5.5), was mixed with ascorbic acid (7 μL, 50 mM), hydrogen peroxide (20 μL, 50 mM) and 10-times diluted extracts of A. distans ssp. distans and A. distans ssp. alpina (10 μL, final concentration of 82.4 mg L−1). After 12–15 s, met-hemoglobin (met-Hb) from a 1.4 mM stock solution (7 μL) was added to the reaction mixture and the 290 nm absorbance was further monitored. A measurable significant inhibition of the ascorbic acid consumption was observed compared to the reference (run in four different experiments) in which the extract was replaced by an equal amount of extraction solvent [31,43]. The slope of each sample was calculated at the tested concentration and also without the tried sample (blank). The inhibition of the ascorbic acid consumption was determined as follows: HAPX = 100 − [(slope of the sample/slope of the blank) × 100]. All the spectroscopic measurements were performed using a Jasco V-530 UV-Vis spectrophotometer (Jasco International Co., Ltd., Tokyo, Japan).

3.5.4. EPR (Electron Paramagnetic Resonance) Spectroscopy Method

The EPR spectra were measured using an EMX Micro spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). EPR instrument conditions were as follows: microwave frequency 9.43 GHz, microwave power 15.89 mW, modulation frequency 100 kHz, modulation amplitude 3 G, sweep rate 10 G/s; time constant 10.24 ms, average of three sweeps for each spectrum, room temperature. For the 2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) radical scavenging by the extract monitored by EPR, 3.43 mM TEMPO (20 µL) was quickly mixed with extract (30 µL) and transferred with a syringe to an an EPR micro tube. The EPR signal is registered at defined time interval and the double integrals are calculated. The kinetic profile obtained is fitted with a first order exponential decay function and the kinetic constant is considered an antioxidant parameter [44]. Gallic acid was used as control.

3.6. Antibacterial Activity Test

The ethanolic extracts of A. distans were tested for antimicrobial activity against two Gram-positive bacterial strains: Staphylococcus aureus (ATCC 49444), Listeria monocytogenes (ATCC 13076), and against two Gram-negative bacterial strains: Escherichia coli (ATCC 25922), Salmonella typhymurium (ATCC 14028) by a previously described disc diffusion method, in Petri dishes [28,29]. Each microorganism was suspended in Mueller Hinton (MH) broth and diluted approximately to 10E6 colony forming unit (cfu)/mL. They were “flood-inoculated” onto the surface of MH agar and MH Dextroxe Agar (MDA) and then dried. Six-millimeter diameter wells were cut from the agar using a sterile cork-borer, and 60 μL of each extract were delivered into the wells. The plates were incubated at 37 °C and the diameters of the growth inhibition zones were measured after 24 h. Gentamicin (10 μg/well) was used as positive control. The controls were performed with only sterile broth and with only overnight culture and 10 μL of 70% ethanol [28,29]. All tests were performed in triplicate, and clear halos greater than 10 mm were considered as positive results.

3.7. Statistical Analysis

All the samples were analyzed in triplicate, except those for EPR method wich were analyzed in duplicate; the average and the relative SD were calculated using the Excel software package.

4. Conclusions

We have determined the phenolic profile, the antioxidant and antibacterial activities for two indigenous subspecies of Achillea distans and we have completed the literature data with new information concerning the polyphenolic compounds and their bioactivity. The simultaneous determination of a wide range of polyphenolic compounds was performed using a rapid, highly accurate and sensitive HPLC method assisted by mass spectrometry detection. The antioxidant activity evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) bleaching method, Trolox equivalent antioxidant capacity assay (TEAC), hemoglobin ascorbate peroxidase activity inhibition (HAPX) assay, and an Electron Paramagnetic Resonance (EPR) spectroscopy method, indicate that A. distans subsp. alpina extract is more antioxidant than A. distans subsp. distans extract, related with the polyphenolic total content.

The comparative study showed significant differences, both qualitative and especially quantitative, between the two taxa of Achillea distans. This study suggests that the flowers of A. distans subsp. alpina and A. distans subsp. distans from Romania may be considered a source of important polyphenols with bioactive properties, a source that could be pharmaceutically exploited.