Acta agriculturae Slovenica, 117/2, 1–10, Ljubljana 2021 doi:10.14720/aas.2021.117.2.2072 Original research article / izvirni znanstveni članek Essential oil content, chamazulene content and antioxidative properties of Achillea millefolium agg. extracts from Slovenia Boris TURK 1, 2 , Dea BARIČEVIČ 1 , Franc BATIČ 1 Received February 02, 2021; accepted April 29, 2021. Delo je prispelo 2. februarja 2021, sprejeto 29. aprila 2021. Essential oil content, chamazulene content and antioxidative properties of Achillea millefolium agg. extracts from Slovenia Abstract: The study aimed to clarify some biochemi- cal properties, important for the phytopharmaceutical use of yarrow from the A. millefolium agg.. The study comprised 41 populations from Slovenia. The most abundant taxa were in- cluded: Achillea millefolium L., A. roseoalba Ehrend., A. collina (Wirtg.) Becker ex Rchb., A. distans Waldst. & Kit. ex Willd., A. pannonica Scheele, A. pratensis Saukel & R.Länger and A. nobilis L. Assessment of essential oil content with the steam dis- tillation method showed no significant difference between taxa. Essential oil content was the lowest in A. collina (6.50 ml kg -1 of dry matter), followed by A. pannonica (7.75 ml kg -1 ), A. distans (8.50 ml kg -1 ), A. nobilis (9.40 ml kg -1 ), A. pratensis (9.65 ml kg - 1 ), A. nobilis × A. millefolium (12.25  ml  kg -1 ), A. roseoalba (12.75 ml kg -1 ) and A. millefolium (13.50 ml kg -1 ). The content of azulenes was determined by photometrical measurement of chamazulene in essential oil extracts. Chamazulene was only present in the diploid taxon and one tetraploid taxon, i.e., A. roseoalba (0.16 % of dry plant mass) and A. collina (0.05 %). The differences in antioxidative capacity of extracts from differ - ent taxa were not statistically significant, so we can assume that specific antioxidative capacity is not bound to a specific taxon or ploidy level. Key words: Achillea; yarrow; chamazulene; essential oils; antioxidants 1 University of Ljubljana, Biotechnical Faculty, Agronomy Department, Ljubljana, Slovenia 2 Corresponding author, e-mail: boris.turk@bf.uni-lj.si Vsebnost eteričnih olj in hamazulena ter antioksidativne la- stnosti izvlečkov taksonov Achillea millefolium agg. v Slove- niji Izvleček: Raziskava je skušala razjasniti nekatere bioke- mijske lastnosti, pomembne za uporabo različnih vrst rmana (Achillea millefolium agg.). V raziskavo je bilo vključenih 41 populacij rmana iz Slovenije. Zajete so bile najpogostejše vrste: Achillea millefolium L., A. roseoalba Ehrend., A. collina (Wirtg.) Becker ex Rchb., A. distans Waldst. & Kit. ex Willd., A. pan- nonica Scheele, A. pratensis Saukel & R.Länger in A. nobilis L. Vsebnost eteričnih olj, določena z metodo parne destilacije, ni pokazala statistično značilnih razlik med taksoni. Vsebnost ete- ričnih olj je bila najmanjša pri A. collina (6,50 ml kg -1 suhe sno- vi), sledijo A. pannonica (7,75 ml kg -1 ), A. distans (8,50 ml kg -1 ), A. nobilis (9,40 ml kg -1 ), A. pratensis (9,65 ml kg -1 ), A. nobilis × A. millefolium (12,25 ml kg -1 ), A. roseoalba (12,75 ml kg -1 ) in A. millefolium (13,50 ml kg -1 ). Vsebnost azulenov je bila določena s fotometričnimi meritvami hamazulena v izvlečku eteričnih olj. Hamazulen je bil prisoten le pri diploidni vrsti in eni tetraploidni vrsti, to sta A. roseoalba (0,16 % suhe snovi) in A. collina (0,05 %). Razlike v antioksidativni kapaciteti izvleč- kov različnih taksonov niso bile statistično različne, zato lahko sklepamo, da antioksidativne lastnosti niso vezane na določen takson ali ploidnostno stopnjo. Ključne besede: Achillea; rman; hamazulen; eterična olja; antioksidanti Acta agriculturae Slovenica, 117/2 – 2021 2 Boris TURK et al. 1 INTRODUCTION The genus Achillea (yarrow) belongs to the family Asteraceae and subfamily Anthemideae, and currently includes around 130 species (Guo et al., 2004; Ehrendor- fer & Guo, 2020). Its center of diversity is southeastern Europe (Ehrendorfer & Guo, 2006), although its rep- resentatives are spread all over Eurasia and the North American continent. Some species, for example Achil- lea millefolium, were spread throughout the northern hemisphere by humans. The genus shows great ecologi- cal plasticity, with different species inhabiting dry desert areas, subalpine mountainous regions and anthropogeni- cally modified ruderal habitats. Different species of yarrow, used in folk medicine and phytopharmaceutical products, originate from natu- ral populations, collected in natural habitats, or from cul- tivation (Vitkova et a., 2005; Edreva et al., 2017; Edreva et al., 2019). They are used as antiphlogistics, antispasmod- ics, hemostatics, stomachics and holagogues (Kastner et al., 1995; Ali et al., 2017). While the content of phytop- harmaceutically important compounds in plants also de- pends on the type of habitat and climatic conditions, it is presumed to be primarily genetically conditioned, and as such limited to specific taxa. Because of that, under- standing the genus systematics is not only of academic, but also of practical importance. Among the bioactive components in yarrow, essen- tial oils are the most important in therms of medicinal effects (Franz, 2007). The content of essential oils in dry above-ground plant parts is about 0.2-1 % (Nemeth, 2005). They include 6-19 % of chamazulene and more than 100 other components, among them monoterpenes and sesquiterpenes. The content and composition of es- sential oils are influenced by genetic, ontogenetic (Farha- di et al., 2020) and environmental factors (Stahl, 1952; Deufel, 1954; Radulovich et al., 2007). The differences are not only reflected in the essential oils, extracted from inflorescences, but also from the leaves (Judzentiene & Mockute, 2005). Differences in essential oil composi- tion are also known among taxa of different ploidy levels (Hofmann et al., 1992) and populations from different geographical provinces (Haziri et al., 2010). The most important groups of sesquiterpene lac- tones found in yarrow include azulenogenic and non- azulenogenic guanolides, guanolide-endo-peroxides, 3-oxy-guanolides, eudezmanolides, longipin, and ger- macrenes. The basic azulenogenic guanolide in yarrow is achillicin (Kastner et al., 1995). Below-ground plant parts are characterized by their ability to synthesize and accumulate alkamides with specific olefinic and acety- lenic patterns, which substitutes the synthesis of polia- cetylenic compounds, otherwise characteristic of the An- themideae (Greger and Hofer, 1989). Yar row, A. millefolium s.l., is one of the first docu- mented medicinal plants in Europe (Wagenitz, 1979). The drug Herba Milefolii is listed in the pharmacopoe- ias of many European countries. However, the European Pharmacopoeia (2004) explicitly mentions only Achillea millefolium L., a specific taxon from the Achillea millefo- lium agg. Many sources suggest there is no differentiation between individual taxa of the aggregat when collecting yarrow for use in folk medicine (Saukel & Länger, 1992). Moreover, it is known from literature that the hexaploid taxon A. millefolium s. str. usually does not contain proa- zulenes at all, although its content is the ground criterion for inclusion in pharmacopoeias (Dabrowska, 1972; Os- wiecimska, 1968, 1974). On the other hand, proazulenes are commonly found in di- and tetraploid species of the A. millefolium s.l. (Bugge, 1991; Adler et al., 1994). It is generally accepted that the diploid taxa A. asplenifolia and A. roseoalba do contain proazulenes, but A. setacea does not, despite also being diploid. Among tetraploid taxa, only A. collina produces proazulenes, but A. praten- sis and A. nobilis do not. Most sources also agree that the hexaploid A. millefolium and octoploid A. pannonica do not synthesize proazulenes. The aim of the present study was to extend the cur- rent knowledge on the phytochemical constituents in the A. millefolium agg. in Slovenia. The study included 41 yarrow populations from 41 locations all over Slovenia. The most abundant taxa were included: Achillea millefoli- um L., A. roseoalba Ehrend., A. collina (Wirtg.) Becker ex Rchb., A. distans Waldst. & Kit. ex Willd., A. pannonica Scheele, A. nobilis L. and A. pratensis Saukel & R. Länger. The goal was to estimate the content of essential oils in above-ground plant parts and test for the presence and content of proazulenes. Additionally, the study quanti- fied antioxidative activity of extracts from collected taxa as another property, important for use in folk medicine. 2 MATERIAL AND METHODS 2.1 COLLECTION AND PREPARATION OF PLANT MATERIAL Plant material was collected from 41 locations across Slovenia. All known basic ploidy levels of Achil- lea millefolium agg. were included. The taxon A. roseo- alba Ehrend., which grows in humid lowland meadows, is diploid (2n = 18). Taxa A. collina (Wirtg.) Becker ex Rchb., A. nobilis L. and A. pratensis Saukel & R. Länger are tetraploid (2n = 36), A. millefolium L. and A. distans Acta agriculturae Slovenica, 117/2 – 2021 3 Essential oil content, chamazulene content and antioxidative properties of Achillea millefolium agg. extracts from Slovenia Waldst. & Kit. ex Willd. are hexaploid (2n = 54) and A. panonica Scheele is octoploid (2n = 72). Plant material was collected and prepared in the same manner for all further analyses. 500 g to 2000 g of fresh above-ground plant material was collected at each site. The quantity particularly depended on the size and abundance of the plants of each taxon in a population. In taxa where plants are large, a few dozen plants were suf- ficient, but where they are smaller, a few hundred were collected. At each site, plants were harvested as close to each other as possible. Due to the large amount of mate- rial collected, it was impossible to ensure that it all came from the same individual. When different morphologi- cal variants were present at the same site, only plants of the same morphological type were collected. Additional specimens for morphological measurements were col- lected at each site and stored in a herbarium. The plants were cleaned of any foreign plant mate- rial, tied into small bundles, and hung in a dry, dark and airy space, where they dried at room temperature for two to three days. The upper parts of the air-dried plants with inflorescences, healthy green leaves and the attached parts of the stem were cut off, cut into approximately 10 cm long pieces and stored in paper bags. The dry plant material was stored at room temperature until further processing in a dark, dry room. 2.2 EXTRACTION OF ESSENTIAL OIL AND PROA- ZULENES Extraction of essential oils and proazulenes was performed in accordance with the 5 th edition of the Eu- ropean Pharmacopoeia (2004) using 20 g of cut drug, a 1000 ml round-bottomed flask and 500 ml of a mixture of 1 volume of water and 9 volumes of ethylene glycol as the distillation liquid. 0.2 ml of xylene in the graduated tube was added to takeup the essential oil. The destilation time was 2 hours. 2.3 ESTIMATION OF CHAMAZULENE CONTENT IN ESSENTIAL OIL The content of chamazulene in the essential oil was determined photometrically in accordance with the 5 th edition of the European Pharmacopoeia (2004). After distillation, the xylene with dissolved essential oil, and with as little distillation liquid as possible, was trans- ferred into a 50 ml volumetric flask. Photometric meas- urement of absorbance was performed on a Perkin Elmer spectrophotometer, Lambda 25 UV / VIS Spectrometer at 608 nm. 2.4 PREPARATION OF EXTRACTS FOR ANTIOXI- DATIVE PROPERTIES ESSAY Plant samples, prepared in step 2.1, were shredded and mixed by hand to homogenize. Approximately 50 g of each sample was prepared for grinding. The instruc- tion of the European Pharmacopoeia (2004) that the drug should not contain more than 5 % of stems with a diameter exceeding 3 mm, or more than 2 % of other foreign components, was followed. For extraction, 0.5 g of ground plant material was weighed and added to 5 ml of solvent in a glass centri- fuge. 80 % methanol (a mixture of methanol and dem- ineralized water in a volume ratio of 80 : 20) was used as solvent. Samples were stored in colorless bottles in the freezer at -18 °C. 2.5 MEASUREMENT OF ANTIOXIDATIVE PROP- ERTIES OF EXTRACTS Antioxidant activity cannot be measured directly, but the inhibitory effect of antioxidants in oxidation can, using a wide range of methods. Efficiency of oxidation can be determined by measuring any of the factors in the oxidation process – the substrate, the oxidant or the in- termediate and final products of oxidation (Antolovich et al., 2002). One commonly used method is based on the use of the stable free radical diphenyl picryl hydra- zyl (DPPH) (Molyneux, 2004; Y ordanov et al., 1997). The results of a DPPH tests were presented by the inhibition coefficient (IC), expressing DPPH inhibition in % and through TEAC (Trolox Equivalent Antioxidant Capac- ity) or antioxidant capacity in Trolox equivalents in TE units (Trolox Equivalent), i.e., in mM TE per 100 g of tested material. 2.6 STATISTICS Descriptive statistics and plot production was per- formed using Statistica, Data Analysis Software System (StatSoft Inc., USA). 3 RESULTS AND DISCUSSION 3.1 ESSENTIAL OIL AND CHAMAZULENE CON- TENT Measurement of essential oil content with steam distillation using the Clevenger apparatus showed no significant differences among taxa. Essential oil content Acta agriculturae Slovenica, 117/2 – 2021 4 Boris TURK et al. Figure 1: Essential oil volume in plant extracts by ploidy level and by taxon, expressed as ml per 20 g dry matter. Table 1: Descriptive statistics and statistical significance of differences in average essential oil volume in plant extract among ploidy levels, expressed ml per 20 g of dry matter. Table 2: Descriptive statistics and statistical significance of differences in average essential oil volume in plant extract among taxa, expressed in ml per 20 g of dry matter. Note: same letters in column Sig. denote cases with no statistically significant difference (Duncan test, p ≤ 0.05) Note: same letters in column Sig. denote cases with no statistically significant difference (Duncan test, p ≤ 0.05) Ploidy Average [ml] Sig. Min. [ml] Max. [ml] SD [ml] SE [ml] 2n = 72 0.155 a 0.155 0.155 2n = 36 0.169 a 0.055 0.555 0.112 0.020 2n = 54 0.220 a 0.125 0.415 0.132 0.066 2n = 45 0.245 a 0.245 0.245 2n 18 0.255 a 0.125 0.445 0.124 0.055 Taxon Average [ml] Sig. Min. [ml] Max. [ml] SD [ml] SE[ml] COLL 0.130 a 0.075 0.275 0.056 0.017 PANN 0.155 a 0.155 0.155 DIST 0.170 a 0.165 0.175 0.007 0.005 NOBI 0.188 a 0.155 0.215 0.031 0.018 PRAT 0.193 a 0.055 0.555 0.142 0.035 NOBIxMILL 0.245 a 0.245 0.245 ROSE 0.255 a 0.125 0.445 0.124 0.055 MILL 0.270 a 0.125 0.415 0.205 0.145 Acta agriculturae Slovenica, 117/2 – 2021 5 Essential oil content, chamazulene content and antioxidative properties of Achillea millefolium agg. extracts from Slovenia Figure 2: Chamazulene content in % of dry matter by ploidy level and by taxon. Table 3: Descriptive statistics and statistical significance of differences in average chamazulene content in % of dry matter among ploidy levels. Note: same letters in column Sig. denote cases with no statistically significant difference (Duncan test, p ≤ 0.05) was the lowest in A. collina, with 6.50 ml per kg of dry matter (s. d. 2.80 ml), followed by A. pannonica with 7.75 ml kg -1 , A. distans with 8.50 ml kg -1 (s. d. 0.35 ml), A. nobilis with 9.40 ml kg -1 (s. d. 1.55 ml), A. pratensis with 9.65 ml kg -1 (s. d. 7.1 ml), A. nobilis × A. millefolium with 12.25 ml kg -1 and A. roseoalba with 12.75 ml kg -1 (s. d. 6.20 ml). The highest essential oil content was estimated in A. millefolium, with 13.50 ml kg -1 of dry matter (s. d. 10.25 ml). Total essential oil content was consistent with exist- ing data (Gharibi et al., 2015), while maximum differ- ences between species were just approximately two-fold, much less than some other studies report. Orav et al. (2006) found nine-fold variance in essential oil yield in yarrow samples, and even twenty-seven-fold differences have been reported from Achillea samples from Iran (Ra- himmalek et al., 2009). Consistent essential oil content in our study may be explained by the fact that the present study only included species from the A. millefolium agg., whereas other studies also took into account some taxo- nomically less related species. In addition, care was taken to only use the inflorescences and uppermost leaves, with as little stems as possible, since some studies showed large differences in essential oil content between the two plant parts, e. g. 0.65 % (v/w) in flowers and 0.0125 % (v/w) in stems (Bocevska & Sovova, 2007). The oil yield in all our samples conformed to the European pharmacopoeia 5.0 (2004) standard which is not less than 0.2 %. Proazulenes, measured through chamazulene, were only present in A. roseoalba and A. collina. This is, to some extent, consistent with existing literature, suggest- ing only diploid and tetraploid taxa are proazulenogenic (Gherase et al., 2003; Nemeth et al., 2007; Konakchiev et al., 2005), although some researchers claim that azu- lenes can be found in all ploidy levels, albeit in different proportions (Kindlovits et al., 2012). However, the data on proazulene presence is quite contradictory (Nemeth, 2005). Even so, in our research, only the diploid A. ro- seoalba consistently contained chamazulene (with popu- lation differences ranging from 2.648 % of dry mass to 0.351  % of dry mass). No difference in chamazulene content was detected between white-flowering and pink- flowering diploid individuals from the same population. In contrast, chamazulene content in the tetraploid A. col- lina was less consistent, and not significantly different from other taxa, except A. roseoalba. Chamazulene con- tent found in different populations ranged from 0.171 % of dry mass to 0.003 % of dry mass. Ploidy Average [%] Sig. Min. [%] Max. [%] SD [%] SE [%] 2n = 54 0.001 a 0.0002 0.0014 0.0006 0.0003 2n = 45 0.001 a 0.0010 0.0010 2n = 72 0.003 a 0.0028 0.0028 2n = 36 0.020 a 0.0000 0.1706 0.0418 0.0076 2n = 18 0.164 b 0.0357 0.2742 0.1055 0.0472 Acta agriculturae Slovenica, 117/2 – 2021 6 Boris TURK et al. Table 4: Descriptive statistics and statistical significance of differences in average chamazulene content in % of dry matter among taxa. Table 5: Descriptive statistics and statistical significance of differences in average DPPH inhibition coefficients (IC) in % among ploidy levels. Figure 3: DPPH inhibition coefficient (IC) in % by ploidy level and by taxon. Taxon Average [%] Sig. Min. [%] Max. [%] SD [%] SE [%] PRAT 0.001 a 0.000 0.002 0.001 0.000 DIST 0.001 a 0.000 0.001 0.001 0.001 MILL 0.001 a 0.000 0.001 0.001 0.000 NOBIxMILL 0.001 a 0.001 0.001 NOBI 0.002 a 0.002 0.003 0.001 0.000 PANN 0.003 a 0.003 0.003 COLL 0.052 a 0.003 0.171 0.057 0.017 ROSE 0.164 b 0.036 0.274 0.106 0.047 Note: same letters in column Sig. denote cases with no statistically significant difference (Duncan test, p ≤ 0.05) Note: same letters in column Sig. denote cases with no statistically significant difference (Duncan test, p ≤ 0.05) Ploidy Average [%] Sig. Min. [%] Max. [%] SD [%] SE [%] 2n = 72 35.34 a 35.34 35.34 2n = 54 44.72 a 32.43 57.86 10.86 5.43 2n = 18 46.49 a 40.94 55.26 5.99 2.68 2n = 36 48.14 a 25.87 65.29 9.02 1.65 2n = 45 50.00 a 50.00 50.00 Acta agriculturae Slovenica, 117/2 – 2021 7 Essential oil content, chamazulene content and antioxidative properties of Achillea millefolium agg. extracts from Slovenia Table 6: Descriptive statistics and statistical significance of differences in average DPPH inhibition coefficients (IC) in % among taxa. Table 7: Descriptive statistics and statistical significance of differences in Trolox Equivalent Antioxidant Capacity (TEAC) in µM TE per 100 g of dry plant matter among ploidy levels. Table 8: Descriptive statistics and statistical significance of differences in Trolox equivalent antioxidant capacity (TEAC) in µM TE per 100 g of dry plant matter among taxa. Note: same letters in column Sig. denote cases with no statistically significant difference (Duncan test, p ≤ 0.05) Note: same letters in column Sig. denote cases with no statistically significant difference (Duncan test, p ≤ 0.05) Note: same letters in column Sig. denote cases with no statistically significant difference (Duncan test, p ≤ 0.05) Taxon Average [%] Sig. Min. [%] Max. [%] SD [%] SE [%] PANN 35.34 a 35.34 35.34 MILL 36.43 a 32.43 40.44 5.66 4.00 ROSE 46.49 a 40.94 55.26 5.99 2.68 COLL 46.91 a 34.83 65.29 8.52 2.57 PRAT 48.02 a 25.87 61.85 9.98 2.49 NOBI x MILL 50.00 a 50.00 50.00 DIST 53.02 a 48.17 57.86 6.86 4.85 NOBI 53.34 a 49.92 58.43 4.49 2.59 Ploidy Average [µM] Sig. Min. [µM] Max. [µM] SD [µM] SE [µM] 2n = 72 42.54 a 42.54 42.54 2n = 54 54.65 a 38.78 71.60 14.02 7.01 2n = 18 56.93 a 49.77 68.25 7.73 3.46 2n = 36 59.06 a 30.32 81.17 11.63 2.12 2n = 45 61.46 a 61.46 61.46 Taxon Average [µM] Sig. Min. [µM] Max. [µM] SD [µM] SE [µM] PANN 42.54 a 42.54 42.54 MILL 43.95 a 38.78 49.12 7.31 5.17 ROSE 56.93 a 49.77 68.25 7.73 3.46 COLL 57.47 a 41.88 81.17 11.00 3.32 PRAT 58.89 a 30.32 76.74 12.87 3.22 NOBIxMILL 61.46 a 61.46 61.46 DIST 65.34 a 59.09 71.60 8.84 6.25 NOBI 65.76 a 61.35 72.32 5.79 3.35 Acta agriculturae Slovenica, 117/2 – 2021 8 Boris TURK et al. 3.2 ANTIOXIDATIVE ACTIVITY OF THE EX- TRACTS The differences in antioxidative capacity were not statistically significant among extracts from plants with different ploidy levels and of different taxa. The results showed a large range of antioxidant efficacy in the sam- ples. The DPPH radical inhibition coefficient (IC) ranged from 25.87 % in a population of A. pratensis, to 65.29 % in a population of A. collina. The highest detected value of Trolox equivalent antioxidative capacity (TEAC) was more than twice as high as the lowest. The values ranged from 81.17 µM in an A. collina population to 30.32 µM in an A. pratensis population (both tetraploid). The dis- tribution of IC values was relatively continuous, with no obvious groupings. Based on the results, it can be as- sumed that specific antioxidative capacity is not associat- ed with a specific taxon or ploidy level. Since the amount of antioxidants, as well as proazulenes, as shown by Stahl (1952), can be affected by environmental conditions and stress, or can even be related to the plant communities in which yarrow grows (Michler & Arnold, 1999; Radušiene & Gudaityte, 2005), it might be worth exploring the cor- relation between environmental conditions, in which the sampled plants grew, and their antioxidative activity. 4 CONCLUSIONS Due to the importance of yarrow from the Achillea millefolium agg. in folk medicine and phytopharmaceu- ticals on one side, and great genotypical and phenotypi- cal plasticity of the aggregate on the other, distinguishing among individual taxa is crucial. It is known, for instance, that taxa with different ploidy levels exhibit different abil- ities for proazulenic compound synthesis. The influence of environmental conditions and stress at the growing site is also important (Gudaityte, 2008), although some authors did not find any correlation (Nemeth, 2007). No such evaluation of the most abundant taxa from the A. millefolium agg. has so far been done in Slovenia. The present study showed that the ability of proazu- lenic compound synthesis in Slovenian taxa greatly corre- sponds to the general patterns. The highest chamazulene content was found in the only diploid taxon included in the study, A. roseoalba. Although the differences in the content among individual populations were quite large, ranging from 2.65 % to 0.35 % of dry plant matter, it was the only taxon with consistent chamazulene presence. The only other taxon, where chamazulene was found, was the tetraploid A. collina. Here, chamazulene content never exceeded 0.17 % of dry plant matter. We can con- clude that only A. roseoalba, occurring predominantly in wet meadows and slightly acidic fens (Dunkel et. al., 2011; Saukel, 2008), is worth being collected as a source of chamazulene. There was a lot of variability in essential oil con- tent among samples. No significant differences among taxa or ploidy levels could be found, perhaps also due to the small number of samples. Still, it appears that when picking yarrow for its essential oils, all taxa are similarly suitable for collection. The composition of essential oils, which was not tested here, however, most probably dif- fers among taxa (Y ener et al., 2020). Similar conclusions were obtained from the assay of antioxidative properties. Antioxidative activity of the ex- tracts showed no significant differences among taxa, but variability within taxa was large. One can speculate that antioxidative capacity is not determined only genetically, but largely depends on environmental and stress condi- tions. 5 REFERENCES Adler, W ., Osvald, K., Fischer, R. (1994). Exkursionsflora von Ös- terreich. Bestimmungsbuch für alle in Österreich wildwach- senden sowie die wichstigsten kultivierten Gefäβpflanzen (Farnpflanzen und Samenpflanzen) mit Angaben über ihre Ökologie und Verbreitung. Stuttgart und Wien, Verlag Eu- gen Ulmer. 1180pp. Ali, S. I., Gopalakrishnan, B., & Venkatesalu, V. (2017). Phar- macognosy, phytochemistry and pharmacological proper- ties of Achillea millefolium L.: a review. Phytotherapy Re- search, 31(8), 1140-1161. https://doi.org/10.1002/ptr.5840 Antolovich, M., Prenzler, P. D., Patsalides, E., McDonald, S., & Robards, K. (2002). Methods for testing antioxidant ac- tivity. Analyst, 127(1), 183-198. https://doi.org/10.1039/ b009171p Bocevska, M., & Sovova, H. (2007). Supercritical CO2 extrac- tion of essential oil from yarrow. The Journal of Supercriti- cal Fluids, 40(3), 360-367. https://doi.org/10.1016/j.sup- flu.2006.07.014 Bugge, G. (1991). Untersuchungen der Sippen des Achillea- millefolium-Komplexes auf Azulengehalt und Ploidiegrad. Angewandte Botanik, 65(5-6), 331-339. Dabrowska, J. (1972). Obserwacje rozmieszczenia azulenowych i bezazulenowych form Achillea L. na Śląsku, na tle danych o rozmieszczeniu taksonów rodzaju Achillea L. na tym ob- szarze. Herba Polonica Journal, 18, 40-69. Deufel, J. (1954). Der Azulengehalt tetraploider Schafgarben. Pharmazie, 9(9), 756. Dunkel, F. G., Gregor, T., & Meierott, L. (2011). Achillea roseo- alba–a long ignored relict in Germany. Feddes Repertorium, 122(3‐4), 268-274. https://doi.org/10.1002/fedr.201000005 Edreva, A., Vitkova, A., Dagnon, S., Konakchiev, A., Gesheva, E., & Bojilov, D. (2017). Field-cultivated medicinal plants of Achillea millefolium: a source of bioactive compounds. Genetics and Plant Physiology, 7(1-2), 22-33. Edreva, A., Vitkova, A., & Gesheva, E. (2019). Field-cultivated Acta agriculturae Slovenica, 117/2 – 2021 9 Essential oil content, chamazulene content and antioxidative properties of Achillea millefolium agg. extracts from Slovenia plants from Achillea millefolium: total flavonoid content, antiradical and antioxidant activities in stems and leaves, and ratio of plant parts. Genetics and Plant Physiology, 9(1- 2), 03-10. Ehrendorfer, F., & Guo, Y. P. (2006). Multidisciplinary studies on Achillea sensu lato (Compositae-Anthemideae): new data on systematics and phylogeography. Willdenowia, 69- 87. https://doi.org/10.3372/wi.36.36105 European pharmacopoeia 5.0. (2004). Strassbourg, Council of Europe, 2416pp. Farhadi, N., Babaei, K., Farsaraei, S., Moghaddam, M., & Pirba- louti, A. G. (2020). Changes in essential oil compositions, total phenol, flavonoids and antioxidant capacity of Achil- lea millefolium at different growth stages. Industrial Crops and Products, 152, 112570. https://doi.org/10.1016/j.ind- crop.2020.112570 Franz, C., Bauer, R., Carle, R., T edesco, D., T ubaro, A., & Zitterl- Eglseer, K. (2007). Study on the assessment of plants/herbs, plant/herb extracts and their naturally or synthetically pro- duced components as ‘additives’ for use in animal produc- tion. EFSA Supporting Publications, 4(4), 070828. https:// doi.org/10.2903/sp.efsa.2007.ZN-001 Gharibi, S., Tabatabaei, B. E. S., & Saeidi, G. (2015). Compari- son of essential oil composition, flavonoid content and an- tioxidant activity in eight Achillea species. Journal of Essen- tial Oil Bearing Plants, 18(6), 1382-1394. https://doi.org/10 .1080/0972060X.2014.981600 Gherase, F ., Spac, A., Dorneanu, V ., Stănescu, U., & Grigorescu, E. (2003). Pharmacognostic research of some species of Achillea. Note 1. Volatile oils analysis. Revista medico-chi- rurgicala a Societatii de Medici si Naturalisti din Iasi, 107(1), 188-191. Greger, H., & Hofer, O. (1989). Polyenoic acid piperideides and other alkamides from Achillea millefolium. Phytochem- istry, 28(9), 2363-2368. https://doi.org/10.1016/S0031- 9422(00)97985-8 Gudaityte, O. (2008). Evaluation of yarrow (Achillea L.) in- traspecific diversity and selection of samples by phyto- chemical properties and morphological characters. Sum- mary of doctoral dissertation, Vilnius, 5-22. Guo, Y . P ., Ehrendorfer, F., & Samuel, R. (2004). Phylogeny and systematics of Achillea (AsteraceaeAnthemideae) inferred from nrITS and plastid trnLF DNA sequences. Taxon, 53(3), 657-672. https://doi.org/10.2307/4135441 Haziri, A. I., Aliaga, N., Ismaili, M., Govori-Odai, S., Leci, O., Faiku, F., ... & Haziri, I. (2010). Secondary metabolites in essential oil of Achillea millefolium (L.) growing wild in east part of Kosova. American Journal of Biochemistry and Biotechnology, 6(1), 32-34. https://doi.org/10.3844/ajbb- sp.2010.32.34 Hofmann, L., Fritz, D., Nitz, S., Kollmannsberger, H., & Draw- ert, F. (1992). Essential oil composition of three polyploids in the Achillea millefolium ‘complex’ . Phytochemistry, 31(2), 537-542. https://doi.org/10.1016/0031-9422(92)90034-N Judzentiene, A., & Mockute, D. (2005). Composition of inflo- rescence and leaf essential oils of Achillea millefolium L. with white, pink and deep pink flowers growing wild in Vil- nius (Eastern Lithuania). Journal of Essential Oil Research, 17(6), 664-667. https://doi.org/10.1080/10412905.2005.969 9027 Kastner, U., Glasl, S., Jurenitch, J. (1995). Achillea millefolium - ein Gallentherapeutikum? Gedanken über den aktuellen Einsatz einer alten Arzneipflanze. Zeitschrift für Phytother- apie, 16, 34-36. Kindlovits, S., & Németh, É. (2012). Sources of variabil- ity of yarrow (Achillea spp.) essential oil. Acta alimentaria, 41(Supplement-1), 92-103. https://doi.org/10.1556/AAl- im.41.2012.Suppl.9 Konakchiev, A., Mikhova, B., Todorova, M., Najdenski, H., Tz- vetkova, I., Vitkova, A., & Duddeck, H. (2005). Composi- tion of the essential oil of Achillea asplenifolia Vent. from Bulgaria. Journal of Essential Oil Bearing Plants, 8(3), 318- 323. https://doi.org/10.1080/0972060X.2005.10643459 Michler, B., & Arnold, C. G. (1999). Predicting presence of proazulenes in the Achillea millefolium. Folia Geobotanica, 34(1), 143-161. https://doi.org/10.1007/BF02803081 Molyneux, P. (2004). The use of the stable free radical diphe- nylpicrylhydrazyl (DPPH) for estimating antioxidant activ- ity. Songklanakarin Journa of Science and Technoogyl, 26(2), 211-219. Nemeth, E. (2005). Essential oil composition of species in the genus Achillea. Journal of Essential Oil Research, 17(5), 501- 512. https://doi.org/10.1080/10412905.2005.9698978 Nemeth, E., Bernáth, J., Sárosi, S., & Rajhárt, P. (2007). Hazai cickafark (Achillea spp.) populációk drogminőségének vizs- gálata. Evaluation of the quality of drugs from Hungarian yarrow populations.) Kertgazdaság–Horticulture, 39(1), 53- 59. Orav, A., Arak, E., & Raal, A. (2006). Phytochemical analysis of the essential oil of Achillea millefolium L. from various Eu- ropean Countries. Natural Product Research, 20(12), 1082- 1088. https://doi.org/10.1080/14786410500510849 Oswiecimska, M. (1968). Achillea collina Becker-ein proazulen- haltiges Taxon von Achillea millefolium L. s. l. Planta Medi- ca, 16(2), 201-207. https://doi.org/10.1055/s-0028-1099900 Oswiecimska, M. (1974). Korrelation zwischen chromo- somenzahl und prochamazulenen in Achillea von Os- teuropa. Planta Medica, 25(04), 389-395. https://doi. org/10.1055/s-0028-1097960 Radulovic, N., Zlatković, B., Palic, R., & Stojanovic, G. (2007). Chemotaxonomic significance of the Bal- kan Achillea volatiles. Natural Product Commu- nications, 2(4), 1934578X0700200417. https://doi. org/10.1177/1934578X0700200417 Radušiene, J., & Gudaityte, O. (2005). Distribution of proazu- lenes in Achillea millefolium sl wild populations in relation to phytosociological dependence and morphological char- acters. Plant Genetic Resources, 3(2), 136-143. https://doi. org/10.1079/PGR200568 Rahimmalek, M., Tabatabaei, B. E. S., Etemadi, N., Goli, S. A. H., Arzani, A., & Zeinali, H. (2009). Essential oil variation among and within six Achillea species transferred from dif- ferent ecological regions in Iran to the field conditions. In- dustrial Crops and Products, 29(2-3), 348-355. https://doi. org/10.1016/j.indcrop.2008.07.001 Saukel, J. (2008). Achillea millefolium agg. In: Fischer, M. A., Oswald, K., & W agner, W . (2008). Exkursionsflora für Öster- Acta agriculturae Slovenica, 117/2 – 2021 10 Boris TURK et al. reich, Liechtenstein und Südtirol. 3., verb. Aufl. Biologiezen- trum d. Oberösterr. Landesmuseen, Linz. 1392pp. Saukel, J., Länger, R. (1992). Die Achillea millefolium - Gruppe (Asteraceae) in Mitteleuropa, 1. Problemstellung, Merk- malserhebung und Untersuchungsmaterial. Phyton (Horn, Austria), 31(2), 185-207. Stahl, E. (1952). Ist der Proazulengehalt der Schafgarbe (Achillea millefolium L.) genetisch oder umweltbedingt?. Pharmazie, 7, 863-868. Vitkova, A., Anchev, M., Goranova, V., Todorova, M., & Ko- nakchiev, A. (2005). Achillea millefolium (Asteraceae) in Bulgaria. Farmatsiya, 52, 60-63. Wagenitz, G. (1979). Compositae I: Allgemeiner Teil, Eupatori- um-Achillea. Illustrierte Flora von Mitteleuropa, 6, 133-136. Yener, I., Yilmaz, M. A., Olmez, O. T., Akdeniz, M., Tekin, F., Hasimi, N., ... & Ertas, A. (2020). A Detailed Biological and Chemical Investigation of Sixteen Achillea Species’ Essential Oils via Chemometric Approach. Chemistry & Biodiversity, 17(3), e1900484. https://doi.org/10.1002/cbdv.201900484 Yordanov, N. D., & Christova, A. G. (1997). Quantitative spec- trophotometric and EPR-determination of 1, 1-diphenyl- 2-picryl-hydrazyl (DPPH). Fresenius‘ Journal of Analyti- cal Chemistry, 358(5), 610-613. https://doi.org/10.1007/ s002160050478