Investigation of optimal extraction, antioxidant, and antimicrobial activities of Achillea biebersteinii and A. wilhelmsii

Abstract

Objective: Achillea species are endowed with multiple biological activities including antioxidant properties. However, no study has yet investigated the impact of extraction method and pH on the biological activities of these plants. The present study aimed to investigate the antioxidant and antimicrobial effects of methanol extracts from the aerial parts of the species Achillea biebersteinii Afan and Achillea wilhelmsii C. Koch (Asteraceae). In addition, the impact of extraction method and pH on these biological activities was evaluated.

Materials and methods: Methanol extracts of A. biebersteinii and A. wilhelmsii were prepared using classical maceration and high-intensity ultrasound methods. Ultrasound-assisted extraction was performed at three different pH values: 5.7, 6.3 and 6.9.

Results: Total phenolic compounds (range: 20.16–108.54 vs. 17.18–59.61 mg gallic acid equivalent/g sample in A. biebersteinii and A. wilhelmsii, respectively), total flavonoids (range: 8.33–12.97 vs. 7.79–9.41 mg catechin equivalent/g sample), 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (IC₅₀: 40.63–346.34 vs. 84.02–462.41) and reducing power (IC₅₀: 504.44–4406.67 vs. 1710.00–5501.67) were significantly higher in A. biebersteinii vs. A. wilhelmsii and ultrasound-assisted vs. classical maceration extracts of both species. The aforementioned items were higher at pH = 6.3, followed by pHs of 6.9 and 5.7, respectively. Overall, A. biebersteinii extracts were more active against all of the tested microorganisms than A. wilhelmsii. Sensitivities of Gram-positive bacteria were higher for both Achillea extracts than the Gram-negative bacteria. No observable inhibitory activity was found from different extracts against Aspergillus niger.

Conclusion: The findings of the present study suggest that methanol extracts of A. biebersteinii and A. wilhelmsii possess antioxidant and antimicrobial activity, being higher in the former. Ultrasound-assisted extraction and pH of 6.3 have significant augmenting impact on the total phenolic and flavonoid content as well as antioxidant activities of both species.

Keywords::

• Ultrasound
• extract
• oxidative stress
• phenolics
• flavonoid
• antifungal

Introduction

Plant-derived drugs constitute a considerable fraction of world’s total medicines. To date, numerous herbs have been reported for their medicinal properties (Tajkarimi et al., 2010; Martinia et al., 2009). Moreover, global awareness about harmful side effects of chemical drugs has promoted investigation on the therapeutic effects of herbal products. Besides their various medicinal features, the antimicrobial and antioxidant effects of plants are well investigated (Martinia et al., 2009; De et al., 2009; Entezari et al., 2009; Tajkarimi et al., 2010).

Microorganisms, including bacteria and fungi, are the main causes of various human infections (Matasyoh et al., 2009) and are often responsible for the loss of food safety and quality. These microorganisms are increasingly the focus of investigations due to the outbreaks of food-borne diseases. Importance of herbal antimicrobial agents can be more appreciated, because about 30% of people in industrialized countries suffer from a food-borne disease each year (Nedorostova et al., 2009). Furthermore, it has been estimated that 70% of bacterial infections in US hospitals involves strains that are resistant to at least one drug (Cushnie et al., 2005). Natural antimicrobials, especially those obtained from herbal materials, have received great attention in recent decades because they are able to control microbial growth, reduce the need for antibiotics, delay microbial spoilage and decrease development of antibiotic resistance by pathogenic microorganisms (Tajkarimi et al., 2010).

In addition, it has been indicated that free radicals generated in human body can cause oxidative damage, resulting in critical diseases such as atherosclerosis, coronary heart disease, aging and cancer (Bajpai et al., 2009). Natural antioxidants obtained mainly from herbal materials are effective agents in the prevention of these adverse effects of free radicals.

The genus Achillea is one of the most famous genera of the Asteraceae family. Species included in this genus are widespread around the world and many of them display medicinal properties. There are different species belonging to Achillea in different parts of Iran. Achillea species have been used in folk medicine for a long time (Bozin et al., 2008; Tajik et al., 2008). Some applications of Achillea in folk medicine include treatment of pain, inflammation, headache and spasmodic diseases (Tajik et al., 2008). The therapeutic importance of this genus is so high that it has been included in national pharmacopoeias of some European countries (Tajik et al., 2008). In recent years, several studies have been reported dealing with antioxidant and antimicrobial properties of Achillea (Konyalioglu and Karamenderes, 2005; Bozin et al., 2008).

Once the medicinal, including antimicrobial and antioxidant, effects of a given plant extract are confirmed, it is reasonable to seek the optimum and efficient extraction method. All extraction methods have been set up to facilitate release of intra-cellular compounds into extraction medium. The more the violation and cell wall is broken, the higher efficiency of extraction method will be achieved. Many extraction methods have been used for the extraction of plant metabolites. Classical maceration (Entezari et al., 2009; Stanisavljevi et al., 2009), supercritical fluid extraction (Benelli et al., 2010), ultrasound-assisted extraction (Hromadkova et al., 2008; Stanisavljevi et al., 2009) and hydrodistillation (Bajpai et al., 2009) can be mentioned.

Ultrasound-assisted extraction is a new method of high efficacy in extracting plant bioactives. This technique enhances mass transfer rates by cavitation forces that generate high pressure and consequently cause plant tissues to rupture. This procedure improves the release of intracellular components into extraction solvent which promotes extraction efficiency (Corrales et al., 2008).

Different extraction methods have different efficacy in extracting bioactive materials of plant cells and tissues. Depending on this, the antioxidant and antimicrobial effects of the extracts obtained by these different methods would vary. This is because these effects are proportional to amounts of intra-cellular compounds released in the extraction procedure.

The present study aimed to investigate antioxidant and antimicrobial effects of extracts obtained from two Achillea species namely A. biebersteinii Afan and A. wilhelmsii C. Koch. In addition, the impact of extraction method and pH on these biological activities was evaluated.

Materials and methods

Plant materials

Aerial parts of A. biebersteinii were collected from farms of Astan Qods e Razavi in Toroq, Mashhad in Northeast of Iran and the aerial parts of A. wilhelmsii were collected from Gonabad in West of Iran in May 2010. Specimens were identified by M. R. Joharchi, Ferdowsi University of Mashhad Herbarium Iran, using Flora Iranica (Rechinger, 1963) and voucher specimens were deposited at the Herbarium of the Department of Botany in the cited institute [FUMH–Voucher no. ED 22004 (A. biebersteinii) and ED 42232 (A. wilhelmsii)]. The plants were dried at room temperature in a shaded place under air flow and powdered using an electrical mill. The powder was classified by particle size distribution with sieve (no:.100) (Lachman et al., 1986). The particle size was less than 149 μ. The powder was stored in a dark, dry and cool place.

Chemicals

Folin-Ciocalteu reagent, butylated hydroxyanisole (BHA, high-performance liquid chromatography (HPLC) grade), sodium carbonate and methanol (HPLC grade) were purchased from Merck, Germany, and 2,2-diphenyl-1-picrylhydrazil (DPPH) from Sigma-Aldrich, USA.

Extraction of plant materials

Maceration and ultrasound-assisted extraction methods were used to obtain leaf and flower extracts. For both methods, 80% methanol was used as solvent.

Maceration extraction

One hundred mL of 80% methanol as extraction solvent was added to 5 g of powdered sample (in a 1:20 w/v ratio) through two 24 h phases using a shaker set at room temperature. After the first phase, the mixture was filtered by Whatman filter paper No.1 and stored in a refrigerator. The part of the mixture remaining on the paper was dried, scratched, weighed and again mixed with methanol (1:20 w/v) and placed on the shaker for 24 h to get thoroughly mixed. After the second 24 h phase, filtration was again performed. The two extracts from the first and second phases were blended. Methanol removal and extract concentration were then performed at 35°C using rotary condenser. Final removal of the solvent was accomplished in vacuum oven under temperature of 35°C and partial vacuum of 60 cm Hg until achieving fixed weight. The dried sample was scraped and stored in dessicator until the time of experiments.

Ultrasound-assisted extraction

Ultrasound-assisted extraction was applied by means of a high intensity ultrasound probe system of 200 W and 24 kHz (model UP 200H, Dr.Hielscher GmbH, Germany) with a horn fitted of microtip: 2 mm (S2) which was immersed in a water bath in which a precipitate glass with the sample (1 g) was placed (internal dimensions: 280:195:135 mm). Acoustic power and intensity of ultrasonic vibrations were 0.171402 W and 21.8346 W/cm², respectively. Ultrasonic intensity was determined calorimetrically by measuring the time–temperature increase of the solvent under adiabatic conditions (Margulis et al., 2003). The extractions were performed at 35°C (ensured by a temperature controller coupled to the ultrasonic bath) and three different pHs: 5.7, 6.3 and 6.9. After extraction the insoluble part was removed by refrigerated centrifuge (model 2-16 KC, Sigma, Laborzentrigugen GmbH, Germany). Rotary evaporator (Heidolph Laborota 4001 efficient, Germany) was used to remove the solvent after extraction. Soluble part was dried by means of a vacuum oven (Labtech, Korea). A digital scale (HR-200, A&D, Germany) with 0.1 mg accuracy was used for measurement.

Total phenolic determination

Total amount of phenolics in both classic and ultrasound methods was determined according to the Folin-Ciocalteau method (Hayouni et al., 2007). To 100 µL of extract having concentration of 2000 ppm in a test tube, 500 µL of diluted solution of Folin-Ciocalteau reagent (1:10 ratio) was added. One min later, 1.5 mL of 20% sodium carbonate was added and stirred. The mixture contained in the test tube was kept for 2 h in darkness and room temperature. Afterwards, absorption of the solution was determined at 760 nm using a Shimadzu UV-Vis spectrophotometer. Distilled water was used as control. Total amount of phenolics was expressed as gallic acid equivalent defined as mg gallic acid per g dried extract. Analyses were performed in triplicate.

Total flavonoid determination

Total flavonoid content was measured using an aluminium chloride (AlCl₃)-based colorimetric assay (Zhishen et al., 1999). Briefly, plant extracts and catechin (used as the standard compound), were transferred into 10 mL volumetric flasks and the volume was set to 10 mL by distilled water. At zero time, 0.3 mL of sodium nitrite (5% w/v) was added to the flask. After 5 min, 0.6 mL of AlCl₃ (10% w/v) was added. Afterwards, at the 6^th min, 2 mL of NaOH (1 m) was also added to the mixture followed by 2.1 mL of distilled water. Finally, absorbance was read at 510 nm. Total flavonoid content was expressed as mg catechin equivalents per g dried extract. Analyses were performed in triplicate.

Antioxidant activity (DPPH radical scavenging activity)

Antioxidant activity was determined using 2,2-diphenyl-1-picrylhydrazyl (DPHH) free radical scavenging assay (Siger et al., 2008). To volumes of methanol extract between 100–1000 µL with the concentration of 200 ppm (0.020 g/100 mL) in the test tube, 80% methanol was added to get total volume of 4 mL. Then, 1000 µL of DPPH with concentration of 120 ppm was added and stirred for 30 s. The resultant solution was kept in darkness for 115 min and then absorption was determined at 517 nm by a Shimadzu spectrophotometer. Absorption rate of control sample containing 4 mL methanol and 1 mL DPPH (120 ppm) was also determined after 115 min. Antioxidant activity was calculated according the following formula:

Results were reported as IC₅₀ values (concentration of sample required to scavenge 50% of free radicals). BHA was used as control.

Reducing power assay

Evaluation of the reducing power was carried out based on the method of Yildirim et al. (2001). Briefly, different concentrations (50–200 μg/mL) of extracts and BHA (used as the standard compound) were prepared in 1 mL of methanol and then mixed with 2.5 mL of phosphate buffer (0.2 M, pH = 6.6) and 2.5 mL of potassium ferricyanide (10 g/L). Following incubation (50°C; 30 min), 2.5 mL of 100 g/L trichloroacetic acid solution was added to the mixture and then centrifuged at 3000 rpm for 10 min. Afterwards, 2.5 mL of the upper layer was mixed with distilled water (2.5 mL) and 1 g/L FeCl₃ (0.5 mL) and the absorbance was read at 700 nm. Analyses were performed in triplicate.

Minimum inhibitory concentration testing

Eight microorganisms including Escherichia coli (PTCC 1399; Persian Type Culture Collection), Escherichia coli 0157 (PTCC 5011), Listeria monocytogenes (PTCC 1165), Salmonella typhi (PTCC 1609) and Pseudomonas aeruginosa (PTCC 1707), as Gram-negative bacteria, Bacillus cereus (PTCC 1247) and Staphylococcus aureus (PTCC 1431), as Gram-positive bacteria and Aspergilus niger (PTCC 5011) and Candida albicans (PTCC 5027) as fungal strains were used. Microorganisms were purchased from PTCC, Iranian Research Organization for Science and Technology, Iran.

The antimicrobial screening tests were performed using 24 h growth culture of bacteria at 37°C and 48 h at 25°C for C. albicans on tryptone soy agar (Himedia, India) which was adjusted to approximately 10⁶ CFU/mL with tryptone soy broth (Himedia, India). For each desired concentration to be tested, a separate solution was prepared by dissolving the dried extract in a few milliliters of methanol and then increasing the volume by Muller Hinton broth until achieving each desired concentration. Each well of 96-well plates were filled with 200 µL of each extract and 20 µL of 10⁶ cell suspension. Culture media alone (to show the sterility of media), gentamicin (5 µg/mL) (as positive control for bacteria) and ketoconazole (250 µg/mL) (as positive control for fungi) were also used. The plates were incubated for 24 h at 37°C for bacteria and 48 h at 25°C for C. albicans.

The growth or no growth of the microorganisms was assessed by methylthiazol tetrazolium (MTT) assay. The amount of 100 µL MTT (5 mg/mL, Sigma) was added to each well and plates were incubated again for about 1 h at 37°C. Minimum inhibitory concentration (MIC) was defined as the lowest concentration of the extract which was able to inhibit the red dye formation.

To determine the MIC of the extracts against A. niger, the poisoned food technique was used with some modifications (Patra et al., 2002). The required quantity of extract was added to sabouraud dextrose agar medium (SDA, Himedia, India). Mycelia discs of 5 mm diameter of A. niger was cut out from the periphery of seven day old culture and aseptically inoculated upside down on the agar surface of the culture medium. Plates were incubated at 25°C and the observations were recorded on the seventh day. Percentage of mycelia growth inhibition (MGI) was calculated using the following formula:

where dc = fungal colony diameter in control (medium without extract), dt = fungal colony diameter in plates containing different concentrations of extract.

The MICs were ascertained by the method of Garber and Huston (1959). This was done by reinoculation of the inhibited fungal discs at MICs on SDA medium. Observations were recorded after 7 days of inoculation. Fungal growth on the seventh day indicated a fungistatic nature, while the absence of fungal growth denoted fungicidal action of the extract.

Statistical analysis

All experiments were run in triplicate. Analyses were performed using statistical software package SPSS (version 11.5). Group comparisons were made using one-way analysis of variance or independent samples t-test. A two-sided p-value of <0.05 was considered statistically significant.

Results

Effect of extraction method on total phenolic content

Overall, the extraction of phenolic compounds was significantly higher in the ultrasound compared to the classical maceration method and this was true at all three assessed pHs for both plants (p < 0.001). With respect to different pHs in the ultrasound method, total phenolic content was significantly higher at pH = 6.3 compared to both pHs of 5.7 and 6.9 in both species (p < 0.001). Total phenolics were also higher at pH = 6.9 compared to pH = 5.7 in both tested species (p < 0.001) (Figure 1).

Figure 1.  Effect of extraction method, plant species and pH on total phenolic content. GAE: gallic acid equivalent; UAE: ultrasound-assisted extraction.

Effect of plant species on total phenolic content

Total phenolics were found to be higher in the extracts obtained from A. biebersteinii compared to A. wilhelmsii and this was true for both maceration (p = 0.003) and ultrasound (at all assessed pHs) methods (p < 0.001) (Figure 1).

Effect of extraction method on total flavonoid content

Overall, the extraction of flavonoid compounds was significantly higher in the ultrasound compared to the classical maceration method and this was true at all three assessed pHs for both plants (p < 0.001). With respect to different pHs in the ultrasound method, total flavonoid content was significantly higher at pH = 6.3 compared to both pHs of 5.7 and 6.9 in both A. biebersteinii (p < 0.001 and p = 0.009 for pH = 5.7 and pH = 6.9, respectively) and A. wilhelmsii (p < 0.001). Total flavonoids were higher at pH = 6.9 compared to pH = 5.7 in both tested species (p < 0.001) (Figure 2).

Figure 2.  Effect of extraction method, plant species and pH on total flavonoid content. CE: catechin equivalent; UAE: ultrasound-assisted extraction.

Effect of plant species on total flavonoid content

Total flavonoids were found to be higher in A. biebersteinii compared to A. wilhelmsii and this was true for both maceration (p = 0.01) and ultrasound-assisted extracts at pH = 5.7 (p < 0.001), pH = 6.3 (p = 0.02) and pH = 6.9 (p < 0.001) (Figure 2).

Effect of extraction method on radical scavenging activity

In line with the results of phenolic and flavonoid assays, ultrasound-assisted extraction was associated with higher radical scavenging activity at all three assessed pHs, compared to the classical maceration extraction for both evaluated species (p < 0.001). With respect to the effect of pH, the highest radical scavenging activity was observed at pH = 6.3 which was significantly higher than activities at pHs of 5.7 and 6.9 for both species (p < 0.001). The activity of extract at pH = 5.7 was found to be lower than that at pH = 6.9 in both A. biebersteinii (p < 0.001) and A. wilhelmsii (p = 0.035) (Figure 3).

Figure 3.  Effect of extraction method, plant species and pH on DPPH radical scavenging activity. BHA was used as standard compound (IC₅₀ = 178.21 ± 2.89). UAE: ultrasound-assisted extraction.

Effect of plant species on radical scavenging activity

Significantly higher radical scavenging activity in DPPH test was observed from methanol extract of A. biebersteinii compared to A. wilhelmsii, irrespective of applied extraction method and pH (p < 0.001). In comparison with BHA as the positive control, ultrasound extracts of A. biebersteinii and A. wilhelmsii were found to be significantly more potent at all assessed pHs (p < 0.001). In contrast, BHA had significantly higher activity compared to the maceration extracts of both species (p < 0.001) (Figure 3).

Effect of extraction method on reducing power

Overall, ultrasound-assisted extraction was associated with a significantly higher reducing capacity at all assessed pHs and in both tested plant species (p < 0.001). As for the effect of pH, the reducing power was found to be significantly lower at pH = 5.7 compared to pHs of 6.3 and 6.9 in both A. biebersteinii (p < 0.001 and p = 0.003 for comparison with pH = 6.3 and pH = 6.9, respectively) and A. wilhelmsii (p = 0.006 and p = 0.021). The IC₅₀ values at pH = 6.3 were lower than those at pH = 6.9 in both species. However, unlike A. wilhelmsii (p < 0.001), this difference did not reach statistical significance in A. biebersteinii (p > 0.05) (Figure 4).

Figure 4.  Effect of extraction method, plant species and pH on reducing power. Ascorbic acid was used as standard compound (IC₅₀ = 47.58 ± 2.18). UAE: ultrasound-assisted extraction.

Effect of plant species on reducing power

In consistence with previous findings, ferric reducing capacity was found to be significantly higher in classical maceration (p = 0.001) as well as ultrasound-assisted (p = 0.014, 0.001 and p < 0.001 at pH = 5.7. 6.3 and 6.9, respectively) extracts of A. biebersteinii compared to those of A. wilhelmsii. The reducing activity of all tested extracts from both species was less than ascorbic acid which was used as the standard compound (Figure 4).

Antimicrobial activity

MICs of extracts of A. biebersteinii and A. wilhelmsii on all tested bacteria and yeast are reported in Table 1. The MIC values against the bacteria were from 0.6 mg/mL to > 40 mg/mL.

Table 1.  Minimum inhibitory concentration (mg/mL) of A. biebersteinii and A. wilhelmsii extracts against different bacterial and fungal strains.

Plant extract	pH	Extraction technique	PTCC number Bacteria and yeast^a
			E.c. 1399	E.c. 0157	L.m.	P.a.	S.a.	S.t.	B.c.	C.a.
A. biebersteinii	6.3	Ultrasound	20	10	5	10	1.2	5	1.2	2.5
A. biebersteinii	5.7	Ultrasound	20	10	5	5	0.6	2.5	0.6	0.6
A. biebersteinii	6.9	Ultrasound	5	5	20	5	5	10	2.5	5
A. biebersteinii	6.3	Maceration	40	40	5	10	1.2	10	1.2	5
A. wilhelmsii	6.3	Ultrasound	40	40	20	> 40	5	20	2.5	2.5
A. wilhelmsii	6.9	Ultrasound	40	40	10	5	5	40	5	5
A. wilhelmsii	5.7	Ultrasound	40	40	10	5	5	40	2.5	1.2
A. wilhelmsii	6.3	Maceration	40	> 40	20	> 40	5	40	5	2.5

E.c.: Escherichia coli, L.m.: Listeria monocytogenes, P.a.: Pseudomonas aeruginosa, S.a.: Staphylococcus aureus, S.t.: Salmonella typhi, B.c.: Bacillus cereus, C.a.: Candida albicans.

PTCC, persian type culture collection.

As shown in Table 1, different extracts of A. biebersteinii were more active against all of the tested microorganisms than A. wilhelmsii. Sensitivities of Gram-positive bacteria were higher for both Achillea extracts than the Gram-negative bacteria. There was not any observable MGI for different extracts against A. niger, as even at highest concentration of the extract, dc was equal to dt.

Discussion

The main issues that were investigated by this research were antioxidant and antimicrobial properties of two endemic Achillea species namely A. biebersteinii and A. wilhelmsii, and the impact of extraction method (classical maceration vs. ultrasound-assisted) and pH on these biological effects. The results of DPPH radical scavenging activity and ferric reducing power tests consistently indicated that ultrasound extracts were of higher activity compared to classical maceration extracts in both tested plant species. As for the activity at different pHs, radical scavenging activity decreased in both species in the following order: 6.3 > 6.9 > 5.7. Since pH = 6.3 is near to the natural pH of the plant, higher activity at this pH could be attributed to the preservation of structure and activity of plant’s active phytochemicals as confirmed by the results of total phenolic and flavonoid assays. The same reason could be stated for the higher activity at pH = 6.9 vs. pH = 5.7 as destruction of phytochemicals is lower in the near-neutral pH compared to acidic pH. Interestingly, the present findings from DPPH radical scavenging and reducing power assays were all in line with the findings of phenolic and flavonoid assays and further confirm the significant impact of phenolic and flavonoid compounds in the total antioxidant activity of a given plant. These phytochemicals have been repeatedly reported for their antioxidant properties and are present in most plant species including those of genus Achillea (Stojanovic et al., 2005; Trifunovic et al., 2006; Tuberoso et al., 2009). Scavenging of free radicals such as reactive oxygen species by these phytochemicals could lead to the prevention of LDL-oxidation, DNA and membrane damage, and other sequeale of oxidative stress such as aging, mutagenesis and carcinogenesis (Rice-Evans et al., 1997; Heim et al., 2002).

Ultrasound-assisted extraction has attracted considerable attention due to its positive impacts on extraction efficiency, extraction time, solvent consumption and particle size (Mason et al., 1996; Salisová et al., 1997; Vinatoru et al., 1997; Valachovic et al., 2001). As mentioned before, these positive improvements by ultrasound waves are mainly attributed to the acoustic cavitation phenomonen. To date, this method has been successfully employed for the extraction of a variety of herbal products and phytochemicals that are of industrial as well as medicinal importance such as pectin, xylans, soybean protein, phenolics, flavonoids, antioxidants, polysaccharides, hydrocarbons, fatty acid esters, etc. (Panchev et al., 1988; Hromadkova et al., 1999).

There have been a number of previous reports on the antioxidant activity of Achillea species (Trouillas et al., 2003; Candan et al., 2003; Ozgen et al., 2004; Sökmen et al., 2004; Kundakovic et al., 2005a,b; Conforti et al., 2005; Baris et al., 2006, 2011; Vitalini et al., 2006; Wojdylo et al., 2007; Ardestani et al., 2007; Kizil et al., 2010) (Table 2). In two investigations conducted by Kundaković et al. (2005a) ethyl acetate and buthanolic extracts of A. alexandri-regis were found to possess hydroxyl radical scavenging activity (Kundakovic et al., 2005a), and methanol extract was reported to be active in DPPH radical scavenging test (Kundakovic et al., 2005b). In another study, infusions of 15 Achillea species (including A. biebersteinii) from Turkey were evaluated for their antioxidant protection against H₂O₂-induced oxidative damage. The results indicated that all of the tested species were effective and could positively modulate the activity of antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase), increase glutathione and decrease lipid peroxide levels (Konyalioglu and Karamenderes 2005). Methanol extracts from the aerial parts of A. distans and A. moschata were also reported to possess antioxidant capacity in DPPH as well LDL-oxidation assays (Sökmen et al., 2004). With respect to A. ligustica, the methanol extract and phenolic fraction from the flowered parts of A. ligustica were reported to exert radical scavenging activity on DPPH (Conforti et al., 2005). In another study, ethanol extracts from the flowered tops of A. ligustica were found effective antioxidants against cellular oxidative stress as well as in DPPH and linoleic acid autoxidation assays (Tuberoso et al., 2009).

Table 2.  Studies on the antioxidant effects of Achillea species.

Study	Species	Type of extract	Antioxidant activities
Baris et al. (2011)	A. aleppica D.C. subsp. aleppica A. aleppica D.C. subsp. zederbaueri (Hayek) Hub.-Mor A.biebersteinii	Ethanol	Total antioxidant activity DPPH radical scavenging Reducing power Deoxyribose assay Metal chelating activity
Baris et al. (2006)	A.biebersteinii	Methanol	DPPH radical scavenging Linoleic acid oxidation
Ozgen et al. (2004)	A. biebersteinii A. Wilhelmsii	Water Methanol	Total antioxidant activity, Reducing power DPPH radical scavenging activity
Kizil et al. (2010)	A. aleppica D.C. subsp. aleppica A. aleppica D.C. subsp. zederbaueri (Hayek) Hub.-Mor A.biebersteinii	Ethanol	Lipid peroxidation Protein oxidation DNA damage
Tuberoso et al. (2009)	A. ligustica	Ethanol	DPPH radical scavenging activity Lipid peroxidation Protection against cellular oxidative stress
Kundaković et al. (2005a)	A. alexandri-regis	Ethyl acetate Butanol	Inhibition of hydroxyl and superoxide radicals
Kundaković et al. (2005b)	A. alexandri-regis	Methanol	DPPH radical scavenging activity
Vitalini et al. (2006)	A. distans A. moschata	Methanol	DPPH radical scavenging activity LDL-oxidation
Conforti et al. (2005)	A. ligustica	Methanol	DPPH radical scavenging activity
Sökmen et al. (2004)	A. biebersteinii	Methanol	DPPH radical scavenging activity Inhibition of hydroxyl and superoxide radicals Lipid peroxidation
Konyalioglu and Karamenderes (2005)	15 Achillea species	Infusion	Protection against H2O2-induced oxidative damage modulation of antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase) Effect on glutathione and lipid peroxide levels
Wojdylo et al. (2007)	A. millefolium	Methanol	DPPH radical scavenging activity 2,2′azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging activity Reducing power
Candan et al. (2003)	A. millefolium subsp. Millefolium	Water-soluble fraction of the methanol extract	DPPH radical scavenging activity Inhibition of hydroxyl and superoxide radicals Lipid peroxidation Total antioxidant activity
Ardestani and Yazdanparast (2007)	A. santolina	Hydroalcoholic	DPPH, superoxide and hydroxyl radical scavenging activity Lipid peroxidation Protein oxidation Reducing power
Trouillas et al. (2003)	A. millefolium	Water-soluble fraction of the hydroalcoholic extract	DPPH sradical scavenging activity Inhibition of hydroxyl and superoxide radicals

As for the A. biebersteinii, apart from the study of Konyalioglu and Karamenderes, (2005), there has been some other reports showing the antioxidant activity of: a) polar subfraction of the methanol extract in the DPPH, inhibition of superoxide and hydroxyl radicals, and inhibition of the lipid peroxidation assays (Vitalini et al., 2006); b) ethanolic extract in lipid peroxidation, protein oxidation, DNA damage, DPPH, metal chelating, reducing power and deoxyribose assays (Kizil et al., 2010; Baris et al., 2011); c) methanol extract in DPPH and linoleic acid oxidation assays (Baris et al., 2006); and d) water extract in lipid peroxidation, DPPH free radical scavenging and reducing power assays (Ozgen et al. 2004). Nevertheless, studies on the antioxidant activity of A. wilhelmsii have been much fewer with only one report by Ozgen et al., (2004). In addition, no previous investigation has been performed on the biological activities of the ultrasound-assisted extracts from Achillea spp. and their comparison with simple maceration extracts.

In the present study, antibacterial and antifungal activities of Achillea extracts were also investigated. Gram-positive bacterial strains were found to be more susceptible to Achillea extracts. In addition, similar to the findings of antioxidant activity, A. biebersteinii was found to possess stronger antibacterial activity on tested strains. With respect to the antimicrobial properties of phenolics and flavonoids (Cushnie et al., 2005), this observation could be partly attributed to the higher phenol and flavonoid content of this species. Data regarding antibacterial and/or antifungal activities of Achillea species are scant with only one previous report by Baris et al. (2006) on A. biebersteinii. In this latter study, weak antibacterial and strong antifungal activity was reported form A. biebersteinii essential oil while the extract did not exert any antimicrobial activity.

Conclusion

Findings of the present study suggest that methanol extracts of A. biebersteinii and A. wilhelmsii possess antioxidant activity, being higher in the former species. Furthermore, current results further confirm the application of high-intensity ultrasound to increase the extraction of phenolics and flavonoids and hence antioxidant effects. Aside from the extraction method, observed antioxidant effects were dependent on the pH of extract and the highest activity was observed at pH = 6.3. These findings might serve as a base for the design of future studies on the optimization of extraction from Achillea species. Finally, further studies are warranted to confirm these results using additional antioxidant activity assays and also to elucidate the effect of extraction method, pH and species on other biological activities of these plants.

Declaration of interest

The authors declare no conflicts of interest.