Biological Activities and Biochemical Composition of Endemic Achillea fraasii

In this study, we investigated the antimicrobial, antioxidant, and antibiofilm activities and the biochemical composition of Achillea fraasii. The antimicrobial activity of A. fraasii ethanol extract (AFEt) was tested against 48 strains, and this is the first study testing the antimicrobial activity of this plant to this extent. The antioxidant activity was determined using the DPPH assay, and the antibiofilm activity of A. fraasii aqueous extract (AFAq) against five strains was assessed. The chemical composition of the plant extract was determined using GC-MS with artemisia ketone (19.41%) as the main component. The findings indicated that AFEt displayed antimicrobial activity against 38 strains, with a particular efficacy observed against various Staphylococcus aureus strains, such as S. aureus ATCC 25923, clinically isolated, multidrug resistant (MDR), and methicillin-resistant (MRSA) strains. In addition, the highest activity was observed against Enterococcus faecium. Moreover, the extract demonstrated activity against Candida strains. The plant extract also showed relatively good antioxidant activity compared to ascorbic acid, with an EC50 value of 55.52 µg/mL. However, AFAq acted as a biofilm activator against Escherichia coli ATCC 25922, increasing the biofilm formation by 2.63-fold. In conclusion, our study demonstrates the potential of A. fraasii as a source of antimicrobial and antioxidant agents.


Introduction
The rise of multidrug resistance (MDR) is a major global health concern and has led to significant challenges in the treatment of infectious diseases [1]. MDR often occurs due to the overuse and misuse of antibiotics, which can give rise to the selection and spread of resistant strains. The World Health Organization (WHO) predicts that resistance to antibiotics will pose a severe risk to public health within the twenty-first century [2].
One of the mechanisms that can cause antibiotic resistance is the formation of biofilms. Biofilm is a matrix in which bacteria are embedded and is extremely resistant to external stresses such as UV light, chemical biocides, host immunological responses, and antibiotics [3,4]. Biofilm development is the primary virulence factor of many bacteria that cause chronic infectious diseases [5]. The current antimicrobial treatment methods and materials often fail to remove the biofilm structure at the site of infection [6].

Antibacterial and Antifungal Activity Test
Using Andrews' disk diffusion method, the antibacterial and antifungal effects of the AFEt were assessed [16]. A total of 50, 100, and 200 µL (3.77 mg, 7.55 mg, and 15.10 mg, respectively) of AFEt were loaded onto Oxoid Antimicrobial Susceptibility Test Disks with a 6 mm radius. To evaporate the ethyl alcohol in the extract, which could alter the test results, the disks were left to dry overnight at 30 • C under sterile conditions. After drying, standardized microorganisms were inoculated onto Petri dishes containing Mueller Hinton Agar (BD Difco, East Rutherford, NJ, USA). The extract-loaded disks were placed on the culture media, and the Petri dishes were incubated according to the time and temperature combinations reported in Section 2.2 on the inoculum preparation. At the end of the incubation period, the diameters of the inhibition zones formed around the disks were measured in mm and recorded. In this study, sterile blank disks and ethyl-alcohol-loaded disks were used as negative controls. As positive controls, we used Gentamicin (Gen) for Gram-negative bacterial strains, Ampicillin (Amp) for Gram-positive bacterial strains, and Tobramycin (Tob) for fungal strains in order to compare the results obtained.

Antioxidant Activity Test
The DPPH method is based on the assessment of the DPPH radical scavenging properties of antioxidant compounds found in plant extracts. To prepare the DPPH solution, 0.0039 g of 2,2-diphenyl-1-picrylhydrazyl (DPPH) was added to 50 mL of ethanol, and it was kept in the dark until further use [19]. A 96-well plate containing DPPH solution and different concentrations of AFEt ranging between 1.075 and 200 µg/mL was then incubated at room temperature for 30 min in the dark. A plate reader (Biotek Microplate Spectrophotometer, Winooski, VT, USA) was used to measure the absorbances of the wells at 515 nm after the incubation time. In this study, ascorbic acid was used as the positive control.
The EC 50 value was calculated according to the following equation: Hill coe f f icient 2.6. Antibiofilm Activity Test The method previously proposed by Karaca et al. [20] was used with modifications in the antibiofilm activity test. This method has two stages, namely, the determination of the conditions for biofilm formation and determination of the antibiofilm activity of AFAq.
After the incubation period, 200 µL of crystal violet was transferred into each well and incubated for 15 min before being rinsed with distilled water (dH 2 O). The crystal violet was drained after the incubation period, and all the wells were then cleaned with dH 2 O once again. Finally, 200 µL of ethyl alcohol: acetone (30:70% (v/v)) solution was pipetted into all the wells, which were then incubated for 15 min. At the end of the incubation period, the ethyl alcohol: acetone solution was collected from each well and transferred to the wells of clean microplates. The absorbance of each well was determined at 550 nm. As a result, the optimal parameters for biofilm formation were obtained as 48 h of incubation time and a 1.5% glucose concentration for all the strains used in the test.
Determination of antibiofilm activity: A concentration range of AFAq was tested for its antibiofilm potential against the five bacterial strains used in the first step. The same steps carried out in the first part of the test were followed, but the bacteria were only incubated for 48 h at 37 • C in TSB containing 1.5% glucose. The antibiofilm potentials of different concentrations of AFAq were determined at 550 nm.

Gas Chromatography-Mass Spectroscopy Method (GC-MS)
A gas chromatography-mass spectrometry device is used to separate the compounds present in a solution, and mass spectroscopy is used to structurally define the compounds [21]. In this study, the Agilent 8890 GC-MS instrument was used. The injector temperature was 350 • C, and He gas was used as the carrier (1 mL/min). The injector mode is 10:1 split, and the injector volume is 1 microliter. The furnace temperature was increased from 40 • C to 150 • C at an increment of 4 degrees per minute, from 150 • C to 180 • C at 3 degrees per minute, from 180 • C to 230 • C at 2 degrees per minute, and from 230 • C to 280 • C at 1 degree per minute. The ions formed as a result of electron ionization by the GC-MS technique were separated according to their mass/charge ratios and recorded with the detector. The compounds were determined by cross-matching the data of the compounds in the most recent Nist and Wiley data libraries.

Statistics
All disk diffusion tests for antimicrobial and antifungal activities were performed in triplicate. The Pearson correlation coefficient was calculated to represent any correlation between the amount and biological activity of the extract, and the ANOVA test was used to determine whether the differences between the replicates were statistically significant or not. For the statistical analysis, R Studio 3.3.2 was used.
AFEt demonstrated antimicrobial activity against 38 out of the 48 strains (Table 1). Among them, three showed high susceptibility (≥15 mm), twenty showed moderate susceptibility (10-14 mm), and fifteen showed low susceptibility (7-9 mm) [2]. Notably, all the strains of S. aureus were susceptible to AFEt, and the most susceptible Grampositive bacterial strain was FI2, with a 28 mm inhibition zone, being higher than those for Gentamicin and Tobramycin. The most susceptible Gram-negative bacterial strain was MDR11, with a 13 mm inhibition zone, with AFEt exhibiting a higher activity than all the antibiotics. All the fungal strains were susceptible to AFEt, with CI12 and CI13 exhibiting more effective results than the positive controls.

Antioxidant Activity of AFEt
The results for the antioxidant activity of AFEt and ascorbic acid are provided in Table 2. According to the results, AFEt showed lower antioxidant activity than the positive control, ascorbic acid. The EC 50 value of AFEt, determined to be 55.52 µg/mL, represents the concentration at which it scavenges 50% of the DPPH radicals. The statistical analysis of the DPPH test results for the AFEt extract indicates no significant difference between the test replicates (p = 0.9925). In addition, the correlation test suggests a strong negative correlation between the increase in the extract concentration and DPPH absorbance (Pearson correlation coefficient = −0.9319768). This strongly negative correlation suggests that as the extract concentration increases, DPPH radical scavenging activity also increases, leading to a decrease in DPPH absorbance. Consequently, this finding highlights the dose-dependent antioxidant activity of the AFEt extract.

Antibiofilm Activity of AFAq
The effects of AFAq on biofilm formation among the tested microorganisms were investigated in the study, and the fold increases in biofilm formation were calculated (Table 3). According to the fold increases in biofilm, it can be observed that the highest fold increase was observed in E. coli ATCC 25922, being 2.63.

Analysis of the Biochemical Composition of AFEt
The biochemical composition of AFEt and its percentages are given in Table 4, according to the data obtained from the GC-MS analysis.

Analysis of the Biochemical Composition of AFEt
The biochemical composition of AFEt and its percentages are given in Table 4, according to the data obtained from the GC-MS analysis. According to the fold increases in biofilm, it can be observed that the highest fold increase was observed in E. coli ATCC 25922, being 2.63.

Analysis of the Biochemical Composition of AFEt
The biochemical composition of AFEt and its percentages are given in Table 4, according to the data obtained from the GC-MS analysis. According to the fold increases in biofilm, it can be observed that the highest fold increase was observed in E. coli ATCC 25922, being 2.63.

Analysis of the Biochemical Composition of AFEt
The biochemical composition of AFEt and its percentages are given in Table 4, according to the data obtained from the GC-MS analysis. According to the fold increases in biofilm, it can be observed that the highest fold increase was observed in E. coli ATCC 25922, being 2.63.

Analysis of the Biochemical Composition of AFEt
The biochemical composition of AFEt and its percentages are given in Table 4, according to the data obtained from the GC-MS analysis. According to the fold increases in biofilm, it can be observed that the highest fold increase was observed in E. coli ATCC 25922, being 2.63.

Analysis of the Biochemical Composition of AFEt
The biochemical composition of AFEt and its percentages are given in Table 4, according to the data obtained from the GC-MS analysis. According to the fold increases in biofilm, it can be observed that the highest fold increase was observed in E. coli ATCC 25922, being 2.63.

Analysis of the Biochemical Composition of AFEt
The biochemical composition of AFEt and its percentages are given in Table 4, according to the data obtained from the GC-MS analysis. According to the fold increases in biofilm, it can be observed that the highest fold increase was observed in E. coli ATCC 25922, being 2.63.

Analysis of the Biochemical Composition of AFEt
The biochemical composition of AFEt and its percentages are given in Table 4, according to the data obtained from the GC-MS analysis. According to the fold increases in biofilm, it can be observed that the highest fold increase was observed in E. coli ATCC 25922, being 2.63.

Analysis of the Biochemical Composition of AFEt
The biochemical composition of AFEt and its percentages are given in Table 4, according to the data obtained from the GC-MS analysis.  According to Table 4, artemisia ketone (19.41%), aromadendrene (5.11%), and (R)-lavandulyl (R)-2-methylbutanoate (4.86%) are major compounds in the biochemical composition of AFEt.

Discussion
AFEt was tested against 48 strains to determine its antibacterial and antifungal activities and showed activity against 38 strains. It is possible that the antimicrobial activity of AFEt is produced by some major compounds found in the extract, such as artemisia ketone, aromadendrene, copaene, ledol, neophytadiene, pentadecane, and heneicosane, which were revealed in the results of the GC-MS analysis and whose antibacterial and antifungal activities were determined in previous studies.

Discussion
AFEt was tested against 48 strains to determine its antibacterial and antifungal activities and showed activity against 38 strains. It is possible that the antimicrobial activity of AFEt is produced by some major compounds found in the extract, such as artemisia ketone, aromadendrene, copaene, ledol, neophytadiene, pentadecane, and heneicosane, which were revealed in the results of the GC-MS analysis and whose antibacterial and antifungal activities were determined in previous studies.

Discussion
AFEt was tested against 48 strains to determine its antibacterial and antifungal activities and showed activity against 38 strains. It is possible that the antimicrobial activity of AFEt is produced by some major compounds found in the extract, such as artemisia ketone, aromadendrene, copaene, ledol, neophytadiene, pentadecane, and heneicosane, which were revealed in the results of the GC-MS analysis and whose antibacterial and antifungal activities were determined in previous studies.

Discussion
AFEt was tested against 48 strains to determine its antibacterial and antifungal activities and showed activity against 38 strains. It is possible that the antimicrobial activity of AFEt is produced by some major compounds found in the extract, such as artemisia ketone, aromadendrene, copaene, ledol, neophytadiene, pentadecane, and heneicosane, which were revealed in the results of the GC-MS analysis and whose antibacterial and antifungal activities were determined in previous studies.

Discussion
AFEt was tested against 48 strains to determine its antibacterial and antifungal activities and showed activity against 38 strains. It is possible that the antimicrobial activity of AFEt is produced by some major compounds found in the extract, such as artemisia ketone, aromadendrene, copaene, ledol, neophytadiene, pentadecane, and heneicosane, which were revealed in the results of the GC-MS analysis and whose antibacterial and antifungal activities were determined in previous studies.
The antibacterial activity results of the standard, clinically isolated, multidrug-resistant (MDR), and methicillin-resistant (MRSA) S. aureus strains used in the

Discussion
AFEt was tested against 48 strains to determine its antibacterial and antifungal activities and showed activity against 38 strains. It is possible that the antimicrobial activity of AFEt is produced by some major compounds found in the extract, such as artemisia ketone, aromadendrene, copaene, ledol, neophytadiene, pentadecane, and heneicosane, which were revealed in the results of the GC-MS analysis and whose antibacterial and antifungal activities were determined in previous studies.

. Discussion
AFEt was tested against 48 strains to determine its antibacterial and antifungal activities and showed activity against 38 strains. It is possible that the antimicrobial activity of AFEt is produced by some major compounds found in the extract, such as artemisia ketone, aromadendrene, copaene, ledol, neophytadiene, pentadecane, and heneicosane, which were revealed in the results of the GC-MS analysis and whose antibacterial and antifungal activities were determined in previous studies.
The antibacterial activity results of the standard, clinically isolated, multidrug-resistant (MDR), and methicillin-resistant (MRSA) S. aureus strains used in the study were remarkable. S. aureus is a bacterium that causes various organ and tissue abscesses; skin, urinary tract, lung, and central nervous system infections; endocarditis; and osteomyelitis diseases. Therefore, antimicrobial activity against S. aureus is essential, since this bacterium increases the number of hospital deaths and is a severe public health threat. Compounds with antibacterial activity, as determined by the results of GC-MS analysis, support the antibacterial effect of A. fraasii [32,33].
The results of this study showed that AFEt exhibited antifungal activity against the three Candida strains tested. The antifungal activity of AFEt may be due to the presence of artemisia ketone, which has previously been shown to be an antifungal agent [28]. Fungal infections caused by Candida strains continue to be a major health problem, resulting in high mortality rates [34]. Mortality rates remain high even when antifungal drugs are used against Candida infections, which results in expensive medical costs for patients and governments. The antifungal activity exhibited by A. fraasii is important in today's world, in which the need for antifungal agents is increasing [35].
Magiatis et al. [17] previously tested the essential oils of A. fraasii against C. albicans ATCC 10231, C. glabrata ATCC 28838, C. tropicalis ATCC 13801, E. coli ATCC 25922, Enterobacter cloacae ATCC 13047, K. pneumoniae ATCC 13883, P. aeruginosa ATCC 227853, S. aureus ATCC 25923, and S. epidermidis ATCC 12228 by minimum inhibitory concentration (MIC) tests. As a result, they observed that the essential oils of A. fraasii presented antibacterial activities against all the bacteria tested, with MIC values ranging between 3.21 and 6.87 mg/mL. When these results are compared to our results, it can be observed that only the results regarding S. epidermidis are different in terms of the common bacteria used in these two studies. On the other hand, AFEt exhibited antifungal activity against C. albicans, C. glabrata, and C. tropicalis, whereas the essential oils of A. fraasii did not show any antifungal activities against these yeast strains. The possible reasons for these differences may be based on both the strains and extracts used in these two studies.
In addition, there are several studies on the antibacterial activities of ethanol extracts from various Achillea species. For instance, Baris et al. [36] investigated the antibacterial activities of three Achillea species, namely, Achillea aleppica subsp. aleppica (AA), Achillea aleppica subsp. zederbaueri (AZ), and Achillea biebersteinii (AB), against C. albicans, E. aerogenes, E. cloacae ATCC 23355, E. coli ATCC 25922, Klebsiella oxytoca, K. pneumoniae ATCC 13883, P. aeruginosa ATCC 27853, S. typhimurium ATCC 14028, S. aureus, S. epidermidis ATCC 12228, and Streptococcus pyogenes. The results obtained in these two studies were similar, excepting those for E. aerogenes, K. pneumoniae, and S. epidermidis. The differences between the results may be attributed to the different strains and plants used in the two studies.
In comparison to the Achillea aleppica subsp. aleppica investigated by Çolak et al. [37], our study focused on a different species within the same genus. Çolak et al. observed a weaker antimicrobial effect against E. faecium relative to our findings, while reporting stronger effects against S. aureus strains. The observed discrepancies in antimicrobial activities might be attributed to both the differences in plant species and the choice of solvents employed.
E. faecium (FI) demonstrated a significant antimicrobial effect in our study, with zone diameters of 20 mm for 50 µL, 26 mm for 100 µL, and 28 mm for 200 µL. This finding is particularly important considering the role of E. faecium in healthcare-associated infections (HAIs). According to the World Health Organization (WHO), HAIs affect 3.5% to 12% of hospitalized patients in high-income countries and result in excess deaths [38]. E. faecium is a major cause of infection in healthcare settings, and vancomycin-resistant E. faecium (VREfm) poses a serious threat to immunocompromised patients [39]. The high prevalence of vancomycin resistance in E. faecium clinical isolates in countries like Australia highlights the need for alternative treatment options. Our findings of strong antimicrobial activity against E. faecium (FI) may contribute to the development of new therapeutic options for combating HAIs caused by this pathogen.
In general, it is expected that an increase in extract concentration would lead to larger zones of inhibition, indicating a stronger antimicrobial activity. However, in our study, some exceptions were observed for certain strains, including E. coli ATCC 25922, S. boydii (CI), C. glabrata (CI), E. coli (MDR), K. pneumoniae (MDR), and P. rustigianii (MDR), where smaller or no zones of inhibition were seen at higher levels of added extract. This phenomenon may be attributed to the presence of multiple compounds within the extract, which can exhibit different mechanisms of action and may interact either synergistically or antagonistically. It is hypothesized that the decrease in antimicrobial activity despite the increase in extract concentration may be due to the occurrence of an antagonistic effect, where one or more compounds within the extract suppress the antimicrobial activity of other components. Further research into the individual compounds and their interactions is needed to confirm this hypothesis and elucidate the mechanisms underlying these observations.
Living organisms are constantly exposed to reactive oxygen species generated as a result of respiratory, metabolic, or disease stress [40]. It is important to eliminate oxidation caused by reactive oxygen species, which cause many diseases, and to neutralize free radicals [41]. As a result of the antioxidant activity test, AFEt showed relatively strong antioxidant activity compared to ascorbic acid, which was used as a positive control, with the EC 50 at 55.52 µg/mL. It is believed that this effect may be due to the copaene and neophytadiene compounds in the chemical content of the plant, the effects of which have been demonstrated previously [25,32]. Baris et al. [39] also tested the DPPH scavenging potentials of AA, AZ, and AB and identified EC 50 values of 33, 33, and 32 µg/mL, respectively. The variations observed in the results are due to differences between the plant samples used in the two studies.
As a result of the data obtained from the antibiofilm test, it was determined that the plant extract did not exhibit antibiofilm activity against any of the microorganisms used in the assay. Contrary to expectations, AFAq was found to act as an activator of biofilm formation in E. coli ATCC 25922, increasing the biofilm formation by 2.63-fold. Samoilova et al. [42] previously proved that water extracts of Zea mays, Betula pendula, Tilia cordata, Vaccinium vitis-idaea, and Arctostaphylos uva-ursi and low concentrations of Camellia sinensis stimulate biofilm formation in E. coli, with an increase of up to 3-fold. They also proposed that the biofilm-triggering activity of these extracts may be due to their pro-oxidant properties. Nevertheless, further research is still needed in order to understand the biofilm activator activity of the extract in the E. coli ATCC 25922 strain.
As a result of GC-MS analysis, twenty-two different compounds were identified in AFEt. The biological activities of eight of these compounds had been demonstrated in previous studies. It was seen that the compounds with known biological activities support the antibacterial, antifungal, and antioxidant activities of AFEt obtained as a result of this study. Magiatis et al. [17] also determined the major constituents of the essential oils of A. fraasii by GC-MS, but the compounds determined in this study were completely different from the compounds determined in our study. The observed differences can primarily be attributed to the different extracts used in these two studies.
To investigate the biological activities of the primary compounds in AFEt and whether AFEt exhibits a more potent antibacterial effect than the sum of its individual components, we recommended analyzing the synergistic effects of the main compounds. This will help to elucidate the potential synergy between different compounds in AFEt and provide insight into their combined mechanism of action.

Conclusions
As a result of this study, it was observed that AFEt showed antibacterial, antifungal, and antioxidant activities, whereas AFaq exhibited biofilm-triggering activity. Biofilms can have both beneficial and detrimental effects depending on their location and composition. In some cases, biofilms can be useful for industrial and medical purposes, such as wastewater treatment, bioremediation, and medical implants. For example, biofilms can help to degrade organic pollutants in wastewater and can provide a stable surface for the growth of beneficial bacteria on medical implants. However, in other cases, biofilms can be harmful, as in the formation of dental plaque, chronic infections, and biofouling in water systems.
Therefore, purification and further studies are required to determine whether the antibacterial, antifungal, and antioxidant activities are due to a single known biochemical compound, such as artemisia ketone, aromadendrene, or copaene, or to a synergistic effect.