Polyphenol content and bioactivity of Achillea moschata from the Italian and Swiss Alps

2.1 Plant material

The two analyzed samples were collected, at the flowering stage, in Val Mustair (Switzerland, 2300 m a.s.l.) (VMS) and in Valchiavenna (Italy, 2200 m a.s.l.) (VCI), air dried exsiccated and kept in paper bags until analyses. Voucher specimens were deposited at the Department of Agricultural and Environmental Sciences of the Milan State University (Milan, Italy) after the identification according to Flora d’Italia [2].

2.2 Extraction

Powdered aerial parts of A. moschata VMS and VCI (26.5 g each) were defatted with petroleum ether (PET) and successively extracted with dichloromethane (DCM) and methanol (MeOH) in a Soxhlet apparatus. The extracts were evaporated at 30°C (rotary evaporator) to dryness and then stored at 4°C until analyses.

2.3 High-performance liquid chromatography (HPLC)

The HPLC analysis was performed on a Waters HPLC 600 liquid chromatograph (Milan, Italy) system equipped with a photo diode array detector, DAD 2828 Waters. Data were processed with the Empower™ 2 Waters Software. The analyses were running on a Gemini C18 (Phenomenex) column (250×4.6 mm internal diameter; 5 μm particle size). The mobile phase was water with 0.1% (v/v) formic acid (A) and acetonitrile with 0.1% (v/v) of formic acid (B). The linear gradient started from 10% B and reached to 60% B in 60 min. The flow rate was 1 mL/min. UV spectra of the samples were conventionally recorded at 210, 270, 310 and 350 nm. All analyses were run in triplicate. An aliquot of 20 μL of each polar extract was injected for each run. For quantitative analysis, a five-level calibration curve was obtained by injection of 10–250 μg/mL concentrations of different standards such as luteolin-6-C-glucoside (Extrasynthese, 99% purity; y=44,625x+119,464; R²=0.9992) and 5-O-caffeoylquinic acid (y=32,345x–58,693; R²=0.9999).

2.4 Electrospray ionization-tandem mass spectrometry (ESI-MS/MS)

Flow injection MS analyses were performed on a 1100 Series Agilent LC/MSD Trap System VL. An Agilent Chemstation (LC/MSD TrapSoftware 4.1, Agilent Technologies, Santa Clara, CA, USA, 2002) was used for the data acquisition and processing. All analyses were carried out using an ESI ion source in the negative mode with the following settings: capillary voltage, 4000 V; nebulizer gas (N₂), 15 psi; drying gas (N₂), 350″C, 5 L/min. Full-scan spectra were acquired over the range of 100–2200 m/z with a scan time of 13,000 m/z/s. Automated ESI-MS/MS was performed by isolating the base peaks (molecular ions) using an isolation width of 4.0 m/z, threshold set at 100 and ion charge control on, with a max acquire time set at 300 ms. Different collision energies were conventionally used (1.0, 10 and 30 V) for MS/MS fragmentation. Polar samples were dissolved in MeOH at the concentration of 20–30 ppm and injected at a flow rate of 10 μL/min (KD Scientific Syringe Pump).

2.5 Antiradical activity

2.6 Antibacterial activity

The disk diffusion agar test was performed to assess antibacterial properties of the A. moschata extracts (VMS and VCI) according to Vitalini et al. [12]. Sterile paper disks (6 mm in diameter) impregnated with 10 μL of each plant extract (MeOH, DCM and PET), extraction solvents as negative controls or ceftazidime (30 μg) and erythromycin (5 μg) as references for Gram-negative and Gram-positive bacteria, respectively, were placed onto the surface of the media inoculated with Bacillus cereus ATCC 999091, Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 25923, as well as Escherichia coli ATCC 35218, Proteus mirabilis ATCC 12453, Pseudomonas aeruginosa ATCC 27853 strains. After18 h of incubation at 37°C, the plates were read and the bacterial growth inhibition zones were measured according to the following scale: no activity (−); <10 mm, low activity (+); 10–20 mm, moderate activity (++); >20 mm, high activity (+++) [14]. Each extract or solvent or antibiotic/microorganism combination was tested in triplicate.

3.1 Qualitative and quantitative analysis of the MeOH extracts

The phytochemical composition in the phenolic compounds of A. moschata VMS and VCI methanolic extracts was achieved by a combination of analytical data from HPLC-DAD (diode-array detection), ultraviolet (UV) and ESI-MS/MS and spiking with authentic available compounds. Both samples were characterized by the presence of a prevalent compound (peaks 9 and 4, respectively) (Figure 1A and B) with a UV spectrum typical of the isorhamnetin and apigenin derivatives. The HPLC profile of A. moschata VCI showed another remarkable phenolic compound, apigenin as a free aglycone (peak 8, Figure 1B).

Figure 1:

HPLC chromatograms (310 nm) of the methanolic extracts of (A) A. moschata VCI and (B) VMS: (1) 5-O-caffeoylquinic acid, (2) di-succinilcaffeoylquinic acid, (3) luteolin 7-O-glucoside, (4) apigenin 7-O-glucoside, (5) 4,5-O-di-caffeoylquinic acid, (6) kaempferol-3-O-glucoside, (7) luteolin, (8) apigenin, (9) isorhamnetin-3-O-glucoside and (10) isorhamnetin-3-O-rutinoside.

The A. moschata VMS and VCI extracts showed different fragmentation patterns, suggesting the presence of flavonoids and phenolic acids (Figure 2A and B). This information was supported by the spectroscopic data (Figure 3A and B). The UV spectra of the HPLC eluted components show two major absorption bands: band I absorption in the 330–350 nm range due to the B-ring cinnamoyl system; band II in the range 240–280 due to the A-ring benzoyl system. A closer inspection of the spectroscopic data suggested that they were derivatives of four different aglycones (luteolin, apigenin, kaempferol and isorhamnetin). Compounds with absorption ranges at 324.7–329.3 and 246.1–250.0 nm plus a diagnostic sharp shoulder at 290–300 nm were unequivocally identified as chlorogenic acids.

Figure 2:

Typical fragmentation patterns of flavonoids (A) and phenolic acids (B).

Figure 3:

UV spectra showing typical behavior of flavonoids (A) and chlorogenic acids (B).

Quantitative analyses (Tables 1 and 2) showed that the extracts of A. moschata VCI and VMS were characterized by flavonoids as major compounds (85.20 and 80.27 μg/mg DW, respectively) followed by phenolic acids (33.03 and 40.69 μg/mg DW, respectively). Among the flavonoid derivatives, apigenin was the predominant aglycone, free and glycosylated, followed by isorhamnetin, kaempferol and luteolin. The A. moschata VCI extract was characterized by apigenin as the main compound (60.36 μg/mg DW) followed by di-succinilcaffeoylquinic acid (25.23 μg/mg DW) and isorhamnetin-3-O-glucoside (12.29 μg/mg DW). Apigenin 7-O-glucoside was the principal metabolite of A. moschata VMS (44.66 μg/mg DW) followed by 4.5-O-di-caffeoylquinic acid (26.42 μg/mg DW), apigenin (19.22 μg/mg DW) and 5-O-caffeoylquinic acid (14.27 μg/mg DW).

In agreement with previous phytochemical studies, our data confirmed that in the genus Achillea, flavonoids mainly occur as flavones and flavonols and their derivatives. They are usually mono- and diglycosides of apigenin, luteolin and quercetin. Like A. moschata, many Achillea species also accumulate free flavonoid aglycons [15], [16], [17]. In the same way, in addition to flavonoids, phenolic acids including caffeoylquinic acids were also quantified in other Achillea species [18], [19], [20], [21].

Achillea species including A. moschata are rich in polyphenols, which are partially responsible of their biological activities [16].

3.2 Antiradical activity

The scavenging ability of the different extracts against the DPPH˙ stable radical, expressed as IC₅₀, is reported in Table 3. In general, as expected, MeOH extracts from both samples – VCI and VMS – showed a higher activity in comparison with the DCM (~10-fold lower) and PET (40- to 50-fold lower) residues. Their removal action was similar to that of quercetin and four- to fivefold stronger than that of Trolox^®, used as reference compounds (Table 3). Equally, the ABTS˙+assay demonstrated that the antiradical activity was noted only in the polar extracts (MeOH) (Table 3). Low values were obtained for the DCM and PET extracts (Table 3). No significant difference was reported in the two A. moschata samples. The obtained results are in agreement with those previously reported for A. moschata [12].

According to the literature, phenolic content and antioxidant activity of the Achillea species are particularly investigated in relation to the traditional use of these plants in order to identify the active compounds responsible for the wide range of their pharmacological activities [17].

Although there may be significant inter- and intra-specific differences in the polyphenol content of the Achillea species due to factors such as their origin, the plant growth stage, the analyzed plant part, the extraction solvent and the extraction method, previous data suggested that phenolic acids and flavonoids may be the major contributors for the radical scavenging activity of the Achillea extracts [17], [18], [19], [22], [23], [24].

3.3 Antibacterial activity

The antibacterial results displayed as inhibition diameters are summarized in Table 4. MeOH extracts were inactive against almost all tested microorganisms. Only the Italian A. moschata VCI extract was weakly effective on B. cereus (inhibition diameter, 11 mm), and A. moschata VMS on B. cereus and S. aureus (8 mm, respectively). Otherwise, both DCM extracts showed different moderate activity against all three Gram-positive bacteria (B. cereus, E. faecalis and S. aureus) and P. mirabilis (Gram-negative), with inhibition diameters ranging from 10 to 16 mm. In particular, DCM extracts inhibited the growth of B. cereus similarly to the reference antibiotic erythromycin (16-mm diameter). Moreover, the DCM extract from A. moschata VMS was slightly active against P. aeruginosa (8 mm).

PET extracts were found to be active against all tested bacteria (inhibition diameters ranging from 8 to 15 mm), with the exception of P. aeruginosa, which was not inhibited by the extract of the VCI sample. On the other hand, it is a type of bacterium – Gram-negative – against which, in general, A. moschata was less effective (Table 4), which is in agreement with previous results from other Achillea species [12], [20]. Disks only impregnated with extraction solvents used as negative controls showed no bacterial growth inhibition. The ceftazidime reference disks showed inhibition diameters ranging from 17 to 23 mm against E. coli, P. mirabilis and P. aeruginosa, while erythromycin disks inhibited B. cereus and S. aureus growth with diameters ranging from 16 to 20 mm. Erythromycin was not able to be effective toward E. faecalis.

A. moschata, as other Achillea species (e.g. A. millefolium L.), is traditionally used against bacterial infections [7], [12], [25]. Some in vitro studies demonstrated sufficient evidence for their ethnopharmacological use. The extracts from aerial parts of different Achillea species were found to possess a broad spectrum of antimicrobial activity against several pathogenic organisms, including specific strains both of Gram-positive and Gram-negative bacteria [25], [26]. In some cases, the reported data confirmed our results showing the greater efficacy of the less polar extracts than the methanolic extracts [12], [26].