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Multivariate analysis
of the phytochemical composition
and antioxidant properties
in twenty‑ve accessions
across three Achillea species
Mostafa Farajpour
1*, Mohsen Ebrahimi
2*, Mohammad Sadat‑Hosseini
3,
Dhia Falih Al‑Fekaiki
4 & Amin Baghizadeh
5
This study explored the chemical composition, antioxidant activity, and total phenol content of aerial
parts from 25 accessions of three Achillea species (Achillea wilhelmsii C. Koch, Achillea vermicularis
Trin., and Achillea tenuifolia Lam.). The plants were collected from various natural habitats across Iran,
encompassing regions such as Central, Western, Southern, Northern, Western, and Northwestern
parts of the country. Subsequently, they were grown together under eld conditions. The study
revealed signicant variation in essential oil yields among accessions of A. wilhelmsii, ranging from
0.01 to 0.107%, A. vermicularis with a range of 0.075 to 1.5%, and A. tenuifolia showing a variation of
0.1 to 2%. The study utilized Gas Chromatography–Mass Spectrometry (GC–MS) analysis, revealing
75, 49, and 75 compounds in the essential oils of A. wilhelmsii, A. tenuifolia, and A. vermicularis,
respectively. Major components included camphor, 1,8‑cineole, anethole, α‑pinene, and phytol in A.
wilhelmsii, 1,8‑cineole, camphor, levo‑carvone, and δ‑terpinene in A. vermicularis, and β‑cubebene,
elixene, β‑sesquiphellandrene, 1,8‑cineole, camphor, and δ‑terpinene in A. tenuifolia. The essential
oil compositions of A. wilhelmsii and A. vermicularis were predominantly characterized by oxygenated
monoterpenes, whereas that of A. tenuifolia was characterized by sesquiterpenes. Cluster analysis
grouped accessions into three clusters, with A. tenuifolia forming a distinct group. Principal
Component Analysis (PCA) triplot (62.21% of total variance) conrmed these results and provided
insights into compound contributions. Furthermore, total phenolic content and antioxidant activity of
the accessions of three species were assessed over 2 years. A. tenuifolia exhibited the highest levels in
both categories, with statistically signicant linear regression between antioxidant activity and total
phenol content for A. tenuifolia and A. wilhelmsii. These ndings emphasize signicant phytochemical
diversity within Achillea species, positioning them as promising natural sources of antioxidants.
Further exploration and selection of specic accessions within each species are crucial for unlocking
their medicinal potential and supporting cultivation and conservation eorts.
Keywords A. tenuifolia, A. vermicularis, A. wilhelmsii, Total phenol
Medicinal plants have been demonstrated to play an important role in human health and cultures. Substantial
research over the past few decades has identied a wide variety of valuable phytochemicals present among
dierent species1. Herbal medicines contribute signicantly to elds ranging from nutrition to cosmetics to
pharmaceuticals2. Traditional medical practices involving herbal remedies have been employed for millennia
OPEN
1Crop and Horticultural Science Research Department, Mazandaran Agricultural and Natural Resources
Research and Education Center, Agricultural Research, Education and Extension Organization (AREEO), Sari,
Iran. 2Department of Agronomy and Plant Breeding, College of Abourihan, University of Tehran, Tehran,
Iran. 3Department of Horticultural Science, Faculty of Agriculture, University of Jiroft, Jiroft, Iran. 4Department of
Food Sciences, Agriculture College, Basrah University, 61004 Basrah, Iraq. 5Department of Biotechnology, Institute
of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology,
Kerman, Iran. *email: m.farajpour@areeo.ac.ir; mebrahimi@ut.ac.ir
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globally for maintaining wellness and managing illness, as seen traditionally in regions such as China, India, Cen-
tral and South America, and Africa3. Currently, herbal therapies still represent the primary healthcare approach
for approximately 85% of people worldwide4. Frequently used medicinal plants in the Mediterranean basin par-
ticularly include species from families like Lamiaceae, Asteraceae, and Apiaceae. Phytochemical analyses have
pinpointed compounds within herbal extracts, essential oils, and fruit juices possessing therapeutic properties5.
An estimated over 50,000 medicinal plant types exist globally, serving as a rich source for drug nding eorts6.
Conventional medical systems have long relied on medicinal plants to support health and wellbeing7, and they
continue playing important roles in modern medicine and pharmacology8.
e Achillea genus, an esteemed repository of medicinal attributes within the Asteraceae family, boasts a
diverse collection of over 100 wild species9. Its global utilization extends to proven ecacies in treating various
ailments, ranging from gastrointestinal disorders and inammation to wound healing and diuretic applications10.
Of particular signicance is yarrow, an ancient medicinal plant, prompting meticulous consideration of raw
material quality during collection and processing. Essential for subsequent chemophenetic investigations is the
analysis of the chemical composition of specialized metabolites, attributing Yarrow’s pharmacological eec-
tiveness to compounds such as caeoylquinic acids, avonoids, and sesquiterpene lactones, contributing to its
multifunctional biological activity11,12.
Achillea species exhibit a spectrum of eects, including immunosuppressive, anti-inammatory, and anti-
oxidant properties13. Further, these plants demonstrate noteworthy wound-healing and antimicrobial eects
against various bacteria, along with antitumor eects on dierent cell lines. e breadth of their eects extends
to anti-arrhythmic, anti-thrombotic, vasorelaxant, anti-hyperlipidemic, anti-hypertensive, hepatoprotective,
and gastroprotective actions, as well as endocrine eects like anti-diabetic, estrogenic, and anti-spermatogenic
properties13. A wealth of ethnopharmacological characteristics associated with Achillea has been documented,
covering an extensive range of medicinal uses, and the essential oils and extracts have been scrutinized, revealing
a diverse array of phytochemicals contributing to therapeutic properties14.
Crucial in safeguarding lipids from oxidation and oering numerous health benets is the role of natural
antioxidants15. ese compounds, instrumental in preventing chronic diseases by mitigating oxidative dam-
age caused by reactive oxygen species (ROS), have gained recent popularity as functional and nutraceutical
ingredients, providing natural alternatives to synthetic antioxidants in the food industry16. Synthesized through
shikimate and phenylpropanoid metabolic pathways in plants, phenolic compounds, including avonoids and
phenolcarbonic acids, stand out for their pharmacological activity in yarrow16,17. e diverse properties of Achil-
lea plant extracts contribute to their antioxidant potential.
Over the past three decades, signicant research has delved into essential oils from Achillea species, uncover-
ing predominant monoterpene compounds like 1,8-cineole, camphor, borneol, α- and β-pinenes9,18–20.
Medicinal plants, recognized as a valuable resource for drug discovery, oer potential new compounds inspir-
ing scientic innovation21. Despite historically serving as the primary form of healthcare in developing nations,
only a fraction of Iran’s rich medicinal plant species, including Achillea, have been thoroughly studied for their
phytochemical components and antioxidant activity9.
Iran’s vast territory harbors a diverse array of medicinal and aromatic plants, including nineteen identied
Achillea species, seven of which are endemic22,23. Despite a wealth of published reports on these plant species,
there remains a dearth of information focusing specically on Achillea species in dierent regions9,18–20,24.
Achillea wilhelmsii C.Koch is a perennial herb belonging to the Asteraceae family. It grows to a height of
15–30 cm with herbaceous stems and white, petioless leaves. e plant owers from May to June, producing
yellowish-white blooms. It is native to Western Asia and naturally found in Iran. Locally, A. wilhelmsii is used
traditionally to treat abdominal pain, stomach ache, vomiting, leucorrhoea, dysmenorrhea, stomachache, dia-
betes, and obesity through decoction and infusion remedies25. e main bioactive compounds identied in this
plant are carvacrol, linalool, camphor, 1,8-cineole, borneol, and α-pinene26,27.
Achillea tenuifolia Lam is a native perennial herb that grows to a height of 20–90 cm, with elongated, narrow
leaves lacking petioles28. It is distributed across Western Asia, Eastern Europe, and the Mediterranean region. e
main compounds isolated from this plant are germacrene D, α-humulene and 1,8-cineole28. In Iranian traditional
medicine, A. tenuifolia is implicated as appetite enhancers29.
Achillea vermicularis Trin. is a perennial herb with several branched stems emerging from the base. It reaches
20–50 cm in height and owers from late spring through midsummer30. Traditionally, indigenous peoples have
prepared A. vermicularis remedies to cure cold, u and upset stomach31. Previous pharmacological studies have
demonstrated these species possesses antidiabetic, antispasmodic, antianxiety, anti-inammatory, analgesic,
and antibacterial properties26,32–34.
e climatic diversity in Iran presents an ideal environment for a rich germplasm of medicinal plants, with
exciting prospects for discovering unique species with valuable essential oil compositions. ese ndings hold
great promise for advancing human health and well-being through potential therapeutic applications of these
plants.
In light of the above, this study aims to bridge the existing gap by evaluating the phytochemical composi-
tion, antioxidant activity, and total phenol content of plant extracts from 25 accessions of three specic Achillea
species: A. vermicularis, A. wilhelmsii and A. tenuifolia. rough a comprehensive analysis, this research seeks
to contribute valuable insights into the potential phytochemical components, antioxidant properties, and total
phenolic content of these Achillea species, thereby enhancing our understanding of their medicinal properties.
is knowledge may pave the way for potential therapeutic applications and advancements in human health
and well-being.
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Material and methods
Plant materials
e study gathered seeds from 25 dierent accessions of three Achillea plant species (A. vermicularis, A. wil-
helmsii, and A. tenuifolia) in Iran. e voucher samples are stored at the Herbarium of the Research Institute of
Forests and Rangelands in Tehran (Table1). e plants were identied based on Flora Iranica35, Identication
was conrmed by Dr. Valiolah Mozafarian of the Research Institute of Forests and Rangelands in Iran.
Initially, the seeds were cultivated in a greenhouse and then transferred to the eld when they reached a height
of around 10 cm. e seedlings were grown using a randomized complete block design at the research farm of
the College of Abouraihan, University of Tehran. Each accession was planted in 1 m2 plots with sandy-loam
soil. e plants were harvested during the initial owering stage to assess their phytochemical components, total
phenol, and antioxidant activity.
e Pakdasht region, where the study was conducted, experiences distinctive seasonal characteristics. Sum-
mers are characterized by extremely hot temperatures, arid conditions, and clear skies, while winters are marked
by very cold temperatures, dry air, and mostly clear weather. roughout the year, temperatures typically range
from 1 to 38°C, with occasional instances of temperatures dropping below − 3°C or rising above 41°C. All
methods in the study were conducted in accordance with the applicable guidelines and regulations.
Extracting essential oils
One hundred grams of dried samples from the aerial parts of the plant were samples were ground into a ne
powder and then were subjected to hydrodistillation using a Clevenger apparatus for a duration of 3 h.
Phytochemicals composition of the essential oils
GC–MS analysis was performed using a Varian CP-3800 instrument equipped with a VF-5 capillary column
(30 m × 0.25 mm i.d., lm thickness 0.25 µm). Helium was used as the carrier gas at a ow rate of 1 mL/min,
and the temperature program was set at 60°C for 1 min, followed by an increase to 250°C at a rate of 3°C/min,
and held for 10 min. e injector and detector temperatures were maintained at 250°C and 280°C, respectively.
To identify the components of the essential oils, the retention index (RI) was utilized by subjecting n-alkanes
(C6–C24) to programmed temperature conditions. e resulting RI values were then compared to the internal
reference MS library (Wiley 7.0) and published data in the literature36.
Table 1. Geographical location of 25 Iranian Achillea sp accessions.
Achillea sp. Code Voucher numbers Province City
A. wilhelmsii
W1 8451 Isfahan Daran
W2 15796 Lorestan Kuhdasht
W3 17628 Qom Dastjerd
W4 19489 Kurdistan Baneh
W5 33976 Yazd Tab as
W6 34431 Hormozgan Bandar-Abbas
W7 35561 Mazandaran Polur
W8 39346 Qazvin Tarom Soa
A. vermicularis
V1 9687 Kurdistan Sanandaj
V2 9872 Kurdistan Baneh
V3 10342 Yazd Khatam
V4 19471 West Azerbaijan Mahabad
V5 19488 West Azerbaijan Mirabad
V6 22593 Kurdistan Saqqez
V7 23155 Zanjan Zanjan
V8 26032 Kurdistan Divandarreh
V9 35179 West Azerbaijan Khoy
V10 35181 West Azerbaijan Salmas
A. tenuifolia
T1 14234 West Azerbaijan Salmas
T2 14300 Kurdistan Divandarreh
T3 25948 Kurdistan Dehgolan
T4 25977 Kurdistan Saqqez
T5 35180 West Azerbaijan Mahabad
T6 39374 Qazvin Takestan
T7 34662 Kurdistan Sanandaj
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Extracting the plant extracts
e plant materials were dried at room temperature to remove moisture. Once fully dried, the plants were ground
into a ne powder using a mill. For extraction, 5 g of each powdered sample was accurately weighed and trans-
ferred to separate Erlenmeyer asks. To each ask, 50 mL of 80% methanol solvent was added. Extraction was
carried out using maceration, where the plant powder was soaked and agitated in the methanol. A magnetic stir
plate and orbital shaker set to 150 rpm were used to gently mix the samples at 25°C for 24 h. Aer maceration,
the mixtures were strained through lter paper to separate the extracts from insoluble residues. e ltered
extracts were concentrated by evaporating the methanol under reduced pressure using a rotary evaporator. en,
the pure extract was collected in a small container and stored at 4°C until total phenol and antioxidant activity
analyses. Prior to the analyses, the samples were dried and used immediately.
Total phenolic content (TPC)
One millilitre of diluted extract (0.1 g in 10 mL of distilled water) was combined with 1 ml of 6 M HCl and 5
mL of 75% methanol/water solution. e resulting mixture was subjected to shaking for 2 h at 90°C in a water
bath. Subsequently, the solution was diluted to a nal volume of 10 ml using distilled water. One milliliter of
this diluted solution was mixed with 5 ml of previously tenfold diluted Folin & Ciocalteau reagent and 15 ml of
sodium carbonate solution (7 g/100 mL). e resulting mixture was brought to a nal volume of 100 mL with
distilled water. e absorbance of the solution at 760 nm was measured using a spectrophotometer, comparing
it against a blank prepared using distilled water instead of the extract, which had undergone the same extraction
steps. e experiment was conducted in triplicate, and our methodology closely followed the approach described
by Çam etal.37, with the exception that we employed four dierent concentrations of gallic acid solution (1.0,
0.4, 1.6, and 2.2 mg per milliliter) in this study. Finally, the total phenolic content in the extract was quantied
and reported as milligrams of gallic acid per milliliter of the sample extract.
Antioxidant activity
e antioxidant activity of the extract was assessed following the methodology of Brand-Williams etal.38, with
minor modications. e experiment employed four dierent concentrations including 10, 100, 250, and 500
ppm of the extract (0.1, 1, 2.5, and 5 mg in 10 mL of distilled water, respectively). Subsequently, 0.1 ml of each
concentration was added to 3.9 ml of a 6 × 10–5 mol/L methanol DPPH solution. For the control sample, 0.1 ml
of methanol were mixed with 3.9 ml of the methanolic DPPH solution. e spectrophotometer was calibrated
using pure methanol as the zero reference. Aer an incubation period of 30 min, the absorbance of all samples
was measured at a wavelength of 515 nm.
Statistical analysis
e PCA analysis was conducted using version 9.1 of the Statistical Analysis Soware (SAS Institute, Cary, NC)
for Windows. A heat map clustering analysis was performed to visualize the similarity patterns among samples
based on their phytochemicals components values. Hierarchical clustering was applied using Euclidean distance
measure and the arithmetic mean method (UPGMA). e heat map displays the clustering of accessions on the
y-axis and phytochemicals components on the x-axis, with color intensity indicating the standardized value for
each trait in each accession. is analysis helped group accessions exhibiting similar response patterns. Also, a
correlation heat map was generated to examine relationships between phytochemicals components. Pairwise
correlation coecients between the components were computed and plotted in a color-coded matrix, with red
indicating positive correlation and blue representing negative correlation. e correlation coecient values
were depicted based on their absolute strengths. e results of the clustering, heat map correlation, and triplot
analyses were visualized as a colored heat map using MetaboAnalyst39. Additionally, the graphs were created
using Prism 9 (GraphPad).
Results and discussion
Essential oil yield
e essential oil yield of 25 accessions from three Achillea species (A. wilhelmsii, A. tenuifolia, and A. vermicula-
ris) was evaluated over two consecutive years. Figure1 presents the essential oil yields for each accession during
the rst and second year of cultivation. In the rst year, yields ranged from 0.01 to 1.2% whereas in the second
year yields were generally higher between 0.02 and 2%. Statistically signicant dierences were observed between
the 2years for all three species (p < 0.01). Yields increased for the majority of accessions in the second compared
to the rst year. Certain A. tenuifolia accessions such as T4 exhibited notably higher essential oil production in
the second year. Based on the ranges observed, A. tenuifolia accessions generally exhibited the highest essential
oil yields, followed by A. vermicularis, with A. wilhelmsii having the lowest yields. In the rst year, A. tenuifolia
accessions produced 0.1–1.04% oil, A. vermicularis yields varied from 0.12 to 1.2%, while A. wilhelmsii yields
were under 0.1%. Similarly, in the second year, A. tenuifolia accessions yielded 0.71–2%, A. vermicularis varied
from 0.075 to 1.45%, and A. wilhelmsii increased but remained low at 0.02–0.107%. e results demonstrate
considerable variation in the essential oil yields among the accessions and between the 2years. In general, it can
be observed that the essential oil yields tend to be higher in the second year compared to the rst year for all
three Achillea species. is nding suggests that the plants undergo certain physiological changes that positively
inuence essential oil production as they mature. One possible explanation for the increased essential oil yield
in the second year is the establishment and development of the plants during the rst year. As perennial plants,
the rst year is typically characterized by a longer growth period until owering, which occurred in August.
e extended growth period in the rst year may have prioritized vegetative growth over secondary metabo-
lite production, resulting in lower essential oil yields. In contrast, the second year exhibited a shorter growth
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cycle, with owering occurring in May. is shorter growth period likely allowed for increased accumulation
of phenolic antioxidants in the aerial parts by the time of owering, leading to higher essential oil yields. e
observed increase in essential oil yield in the second year highlights the importance of considering the stage of
plant maturity when studying essential oil production in perennial species. It suggests that the developmental
stage and growth cycle signicantly inuence the biosynthesis and accumulation of essential oil constituents.
e results demonstrate considerable variation in essential oil yields among accessions. For A. wilhelmsii, the
yields ranged from 0.01 to 0.107% in our study, whereas, the literature reports of 0.14–0.82%27,40,41. Similarly,
Rabbi Angouran30 observed 0.7% yield in A. vermicularis, comparable to our observed range of 0.075–1.5%
across accessions. For A. tenuifolia, Sedkon etal.28 reported a range of 0.16–1.59%, encompassing our observed
variation between accessions of 0.1–2%. Overall, the literature comparisons validate the substantial intra-specic
variability in oil yields observed among our Achillea accessions under uniform cultivation.
Essential oil compounds
A total of 75 compounds were identied in the A. wilhelmsii accessions, as presented in Table2. Among the
identied compounds, camphor was determined to be the predominant constituent in this species. e W5
accession exhibited the highest camphor content (31.48%), whereas the remaining seven accessions displayed
varying concentrations of this compound (Fig. S1a). e second most signicant compound in this species
was 1,8-cineole, with concentrations ranging from 4.31% to 18.82%. e W4 accession exhibited the highest
proportion of 1,8-cineole and also displayed another notable compound, anethole, at a concentration of 21.63%.
e A. wilhelmsii accessions contained α-pinene in quantities ranging from 1% to 6.7%. e W8 accession
displayed a noteworthy amount of piperitone (13.66%), which was only found in small amounts in the other
W1
W2
W3
W4
W5
W6
W7
W8
0.00
0.05
0.10
0.15
First year
Second year
V1
V2
V3
V4
V5
V6
V7
V8
V9
V10
0.0
0.5
1.0
1.5
2.0
T1
T2
T
3
T4
T5
T
6
T7
0.0
0.5
1.0
1.5
2.0
2.5
Essential oil yield (%)Essential oil yield (%)
Essential oil yield (%)
Figure1. Essential oil yield of 25 accessions of the three studied Achillea sp.
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Name RI W1 W2 W3 W4 W5 W6 W7 W8
2-Methylbutyl acetate 894 0.36 0.48 0.22 0 0.49 0.17 0 0
Santolina triene 932 0 0.18 0 0.1 0 0 0 0
α-Pinene 977 6.65 6.71 1.47 2.39 6.69 1.51 0.98 1.51
Camphene 1003 1.42 1.62 0.55 3.26 4.86 0.55 0.57 1.18
β-phellandrene 1042 3.15 1.85 4.04 3.77 1.1 4.19 0.85 1.39
β-Pinene 1048 1.2 0.6 0.24 0.96 1.26 0.23 0.34 0.44
β.-Myrcene 1071 0.34 0.3 0 0.3 0.17 0 0.61 0.53
Perillen 1109 0.54 0.34 0.19 0 0.48 0.18 0 0.16
4-Carene 1115 0.56 1.5 0.41 0.22 0.83 0.42 0 0.14
m-Cymene 1128 1.22 0.68 0.41 0.13 0.6 0.42 0 0.21
1,8-cineole 1140 15.42 5.74 6.07 18.84 5.29 6.46 4.31 9.47
δ-Terpinene 1186 0.5 1.9 0.28 2.43 0.11 0.29 2.43 0.38
cis-Sabinenehydrate 1204 0.59 4.23 0.94 1.49 0.17 0.93 1.13 2.21
Artemesia alcohol 1223 0 0 0 0.13 0 0 0.42 0
α-campholenal 1238 0.4 0.15 0.11 0 0 0.16 0 0
Linalool 1256 7.18 2.59 2.3 0.8 5.4 2.35 1.08 2.46
Butanoic acid,2-methyl-, 2-methylbutyl 1261 1.25 0.72 0.38 0.48 0.89 0.43 0.64 0.26
2-Methylbutyl isovalerate 1270 0.49 0.7 0.7 0 0.35 0.8 0 0.19
ujone 1284 0 4.91 1.22 0 0 1.22 0 0
Chrysanthenone 1292 0 0.42 5.47 4.3 0 5.63 5.64 10.68
α.-Campholenal 1300 0.76 0.51 0 0.1 0 0 0 0
trans-Pinocarveol 1323 0.4 1.59 0.97 0.52 0.29 1 0.67 1.17
Camphor 1333 9.45 17.7 5.88 11.2 31.48 6.45 9.99 14.03
3,9-Epoxy-1-p-menthene 1347 3.22 2.65 1.79 0.25 0.79 1.9 0 0
Pinocarvone 1359 1.04 1.72 2.19 1.01 1.02 2.23 1.6 1.15
Borneol 1374 2.64 6.8 1 1.69 1.21 1.06 2.63 2.23
Pinocamphone 1381 0.34 0 0.17 0 0 0.17 0 0
p-Menth-1-en-4-ol 1388 0.43 5.36 0.72 0.57 0.21 0.73 0.71 0.65
α-Terpineol 1411 2.43 1.53 1.84 0.99 0.85 1.78 1.26 1.69
trans-Piperitol 1432 0 0.3 3.79 0 0 3.74 0 2.53
trans-Carveol 1448 0.21 1.66 0.33 0 0 0.3 0 0.29
Isogeraniol 1464 0.48 0.21 0.36 0 0.19 0.29 0 0
Piperitone 1499 0 0 0 1.98 0 0 0 13.66
verbenyl acetate 1503 0.47 0 1.37 0.99 0.12 1.36 0.87 0
p-Mentha-1,8-dien-3-one, (+)- 1521 0 0 0.13 0.26 0 0.27 0.41 0.51
α.-Cyclogeraniol acetate 1532 0 2.86 0.63 2.23 0 0.63 0 0
(. + /-.)-Lavandulol, acetate 1540 0 0 4.99 0 0 5.08 0 0
Anethole 1545 1.45 3.93 1.32 21.63 15.27 1.33 8.77 0.56
p-ymol 1553 0.86 0.34 0.2 0 1 0.19 0 0
cis-CarvylAcetate 1607 0.94 0.31 1.31 0 0 1.19 0 0
γ-Terpineol 1641 0.24 2.05 0.17 0 0.16 0.18 0 0.32
Geranyl acetate 1668 0.39 0 0 0 0 0 1.07 0.46
5-Isopropenyl-2-methylcyclopent-1 1689 0 0.65 1.76 1.86 0 1.32 2.61 4.35
Caryophyllene 1721 2.38 1.25 1.5 0.15 0.52 1.59 0.39 0
β-cubebene 1798 1.65 0.51 2.1 3.29 1.24 2.11 3.2 3.42
Elixene 1817 2.43 1.03 2.07 0.29 1.08 1.94 0.63 0.53
α.-Farnesene 1828 0 0 0.33 0.15 0 0.19 0 0
β-Cedrene 1836 0.34 0.16 4.11 0 0 4.38 0 0
Hotrienol 1858 0 0 0.21 0 0 0.21 0.48 0
Longipinocarvone 1912 0.65 0.7 0.65 0 0.43 0.58 1.11 0
Spathulenol 1917 0.27 0 0.43 0.29 0.47 1.21 0.58
Caryophyllene oxide 1924 1.73 0.79 2.42 0.17 0.54 2.56 0.61 0
trans-Nerolidol 1958 6 0 0.93 2 0.19 0.94 0.52 0
Agarospirol 1967 0 0 0 0.15 0 0 1.08 0
Spathulenol 1971 0.25 0 0 0 0 0 0.95 0.2
Tetracyclo[6.3.2.0(2,5).0(1,8)]tridecan-9-ol, 4,4-dimethyl- 1978 0.92 1 1.94 0 0.31 1.82 0 0
Continued
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accessions. All accessions demonstrated similar levels of phytol compounds. Chrysanthenone component was
also detected in the essential oil of this species.
Based on the observed variations in compound number and concentration, it can be inferred that there is
considerable phytochemical diversity within this species across dierent regions of the country. e principal
compounds identied in the essential oil of the aerial parts of A. wilhelmsii in this study were camphor, 1,8-cin-
eole, anethole, α-pinene, and phytol.
Previous investigations have reported similar compounds, such as camphor, 1,8-cineole, and α-pinene, as well
as dierent compounds, including carvacrol, linalool, and borneol26,27,42. e previous reports and the ndings
of this research suggested that camphor and 1,8-cineole are the principal constituents of the essential oil in this
species. Nonetheless, dierent studies have reported dierent major compounds for this plant. ese disparities
may be attributed to variations in physiology, environment, geography, genetics, and plant material diversity43. In
addition, Saeidi etal.27 conducted a study to analyze the essential oil composition of twenty A. wilhelmsii acces-
sions collected from their natural habitats across southwest Iran. e researchers identied several components,
including chrysanthenone, trans-carveol, linalool, neoiso-dihydrocarveol acetate, camphor, lifolone, 1,8-cineole,
borneol, α-pinene, trans-piperitol, (E)-caryophyllene, (E)-nerolidol, and lavandulyl acetate, which were present
abundantly in the essential oil of A. wilhelmsii populations. Many of these components were also detected in
the accessions studied in the present research. However, certain components such as neoiso-dihydrocarveol
acetate, lifolone, and lavandulyl acetate were exclusively identied in the previous study, whereas components
like anethole and phytol were specically identied in the present study. One possible explanation for these
dierences is that the accessions in the present study were cultivated in specic locations, while Saeidi etal.27
collected accessions from their natural habitats. Also, in the present study, a broader range of locations across
the country was covered, which may explain some of the dierences observed compared to the previous study.
A total of 75 compounds were identied in A. vermicularis, similar to A. wilhelmsii, as presented in Table3.
Among these compounds, the composition of 1,8-cineole was recognized as the most signicant in this species.
e V2 accession exhibited the highest percentage of 1,8-cineole at 26.22% (Fig. S1b). All accessions, except
V7, contained varying percentages of this compound. e second most important composition was camphor,
with a range of 0% to 28%, and the highest percentage was found in the V6 accession. is accession also exhib-
ited a prominent compound of this species, levo-carvone, at a concentration of 15.38%. e species displayed
δ-terpinene in concentrations ranging from 0 to 10%. Several unique compounds were found in the accessions of
this species. For instance, V1 contained 10% pinocarvone compound, V7 contained 17.16% cyclohexadecanolide
compound, and V9 contained 40.54% pulegone compound. All accessions demonstrated similar percentages of
mehp composition. Based on the observed changes in each composition and their respective ranges, it can be
inferred that there is signicant phytochemical diversity within this species.
Name RI W1 W2 W3 W4 W5 W6 W7 W8
tau-Cadinol 1981 0 0 0 0.18 0 0 1.82 0.42
14-Methyloxacyclotetradecan-2-one 1986 2.03 1.04 0 0 0.68 0 1.07 0.31
β.-Eudesmol 1989 0 0.6 4.74 0.47 0.22 5.14 1.69 0.58
Acetic acid, 1-methyl-3-(2,6,6-trimethylcyclohex-1-enyl)propyl ester 1994 0 0 1.29 0.1 0 1.63 1 0.6
Humulane-1,6-dien-3-ol 2007 0.51 0.23 2.93 0.2 0 3.13 3.74 0.45
9-Ethylbicyclo(3.3.1)nonan-9-ol 2015 0 0 4.15 0.19 0 4.31 0 0.31
Camazulene 2029 0 0 0.4 0.09 0 0.33 0.52 0
Cyclohexylidenecyclohexane 2044 0.12 0 0.39 0.11 0 0.29 0.78 0.34
Andrographolide 2056 0.26 0 0.15 0 0.42 0.15 0.2 0.19
Farnesol, acetate 2065 0.74 0.2 0 0 0 0.34 1.68 0
Cyclohexadecanolide 2071 2.14 1.11 2.09 2.6 1.64 1.84 7.43 4.47
Phthalicacid, methyl octyl ester 2114 0.38 0.22 0 0.27 0.23 0.28 0.61 0.32
Octadec-9-enoic acid 2122 0.29 0.18 0.29 0.34 0.19 0.21 0.96 0.52
Phytol 2131 3.69 2.18 5.66 2.66 2.83 4.75 4.74 3.24
β.-Cholestanol acetate 2149 0.57 0.14 0.53 0.31 0.44 0.42 0.76 0.29
Tetratriacontane 2167 0.23 0 0.27 0.17 0 0.16 0.31 0.31
Eicosane 2212 0.15 0 0.16 0.14 0 0.13 0.5 1.02
Mehp 2224 2.99 0.78 2.31 1.13 1.94 1.88 1.28 2.12
Hexatriacontane 2273 0 0 0 0 0 0 0.43 0
Monoterpenes hydrocarbons – 31 21.42 13.66 32.4 21.39 14.3 10.09 15.41
Oxygenated monoterpenes – 35.66 61.03 36.5 49.99 59.4 37 39.57 58.89
Sesquiterpenes – 6.8 2.95 10.11 3.88 2.84 10.2 4.22 3.95
Oxygenated sesquiterpenes – 9.81 2.32 13.39 1.56 3.62 14.5 15.62 2.83
Table 2. Chemical composition of essential oils (%) of eight A. wilhelmsii accessions. e values in the table
are percentages of a given constituent in the total oil. e data were sorted based on the retention index (RI) of
the components.
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Name RI V1 V2 V3 V4 V5 V6 V7 V8 V9 V10
2-Methylbutyl acetate 894 0 0 0.42 0 0.17 0 0 0 0 0
Santolina trien 931 0 0.2 0 0.13 0.12 0 0 0 0 1.23
α-Pinene 977 4.64 3.08 1.49 2.6 2.02 4.23 0 3.85 0.28 5.13
Camphene 1003 0.81 2.54 0.22 0.71 2.72 2.46 0 2.43 0.11 1.3
β-phellandrene 1042 0.49 4.84 3.24 1.4 3.03 3.6 0 4.82 0.41 4.73
β-Pinene 1049 7.89 1.53 0.32 3.23 0.59 1.02 0 0.91 0.61 1.34
β.-Myrcene 1072 0.47 0.39 0.13 0.51 0.31 0.41 0 0.54 0.2 0.51
Yomogi alcohol 1083 0 0.34 0 0.27 0.27 0 0 0 0.16 0
4-Carene 1115 0.11 0.44 0.63 0.47 0.54 0.16 0 1.17 0 0.38
m-Cymene 1128 0 0.16 0.39 0.12 0.15 0.28 0 0.34 0 0.37
1,8-cineole 1140 8.42 26.22 8.1 15.19 16.7 22.11 0 23.61 7.97 23.57
δ-Terpinene 1186 0.22 9.71 0.24 8.93 10.1 0.74 0 1.93 0 0.48
cis-Sabinenehydrate 1204 1.03 2.97 0.8 5.69 3.59 1.01 0 1.98 0 1.56
Artemesia alcohol 1223 0 1.81 0 0.65 0.44 0.09 0 0 0 0
( +)-4-Carene 1232 0 0.21 0 0.16 0.17 0 0 0.48 0.12 0
Linalool 1256 6.25 1.14 0.76 2.63 1.49 0.69 0.55 1.25 0 0.67
Butanoic acid, 1261 0 0.4 0 0.32 0.69 0.32 0 0.67 0 0.77
ujone 1266 1.01 0 2.05 0 0.39 0 0 0 0 0
Chrysanthenone 1292 1 3.11 0 0.26 5.66 0.54 0.64 3.55 0 0.61
trans-Pinocarveol 1323 0.43 0.54 22.73 0.43 0.61 0.67 2.68 1.31 0 1.35
Camphor 1332 2.44 13.44 1.93 5.82 4.14 28.08 0 8.82 0 6.81
9-Ethylbicycl(3.3.1)nonan-9-o 1340 0 0 0 0 0 0 0 0 20.65 0
Pinocarvone 1359 10.5 0.78 2.45 0.54 1.02 0.76 0 2.86 0 2.07
Borneol 1375 2.62 2.07 1.49 1.2 3.31 1.34 1.51 9.83 9.84 1.96
p-Menth-1-en-4-ol 1388 0.46 0 0.48 1.71 1.54 0.56 0 4.29 0 0.78
α.-ujenal 1393 0 1.58 0.35 0 0 0.24 0 0 0 0
Artemisia ketone 1400 0 0.09 0 0 0.19 0.1 0 0.57 0 0
α-Terpineol 1412 6.41 0.14 0.78 6.54 0.99 2.3 1.19 2.89 0 2.66
trans-Piperitol 1432 0.21 0 0 0 0 1.16 0 0.25 0 1.21
trans-Carveol 1448 0.36 2.19 0 0.3 0.12 0.14 0 0.89 0 0
Artemisia ketone 1472 0 0 0 0.25 0.46 0.21 0 0 0 0
Pulegone 1477 0 0 0 0 0 0 0 0 40.54 0
Levo-carvone 1484 0 0 0.18 0 0.27 15.38 0 0.55 0 0
Piperitone 1499 0.41 0.32 0 1.11 0.81 0 0.61 0 1.01 21.23
verbenyl acetate 1503 0.33 0 0 0 1.28 0 0 0 0 0
α-Citral 1520 0.15 0.2 0 0 0.35 0 0 0 0.17 0
L-bornyl acetate 1542 0.8 0.21 0 0 21.22 0 0 0.73 0.15 0
Anethole 1546 0.28 3.06 37.63 1.1 0 1.8 1.58 0.28 0.28 3.24
cis-CarvylAcetate 1608 0 0.26 0 0 0.12 0 0 0.24 0 0
Pulespenone 1616 0 0 0 0 0 0 0 0 4.73 0
5-Allyl-2-methoxyphenol 1635 0 0.13 0 0 0 0.11 0.48 0.36 0 0.31
Aglaiene 1665 0 0.16 0 0 0.17 0 0 0 0 0
Geranyl acetate 1668 0.75 0 0 0.6 0 0.49 0 0.41 0 0
Methyl eugenol 1698 0.22 0.58 0 0.14 0.39 0.28 1.19 0.44 0 0.31
Caryophyllene 1721 2.12 0 0.42 2.17 0 0 0 0 0 0
α.-Himachalene 1790 0.38 0 0 0.43 0 0 0.59 0 0 0
β-cubebene 1798 1.65 2.44 0.15 4.21 3.14 1.18 7.9 4.33 0.35 1.67
Elixene 1817 0.39 0.39 1.34 0.62 0.59 0.28 1.01 0.59 0 0.24
γ-Cadinene 1839 0.61 0 0.33 0.5 0 0 0 0 0 0
δ.-Cadinene 1846 0.14 0 0 0.15 0 0 0 0 0 0
Hotrienol 1858 0.3 0 0 0.19 0 0 1.04 0 0 0
8-Hydroxylinalool 1879 0.32 0 0.17 0 0 0 1.3 0.63 0 0.28
β.-Terpineol 1886 0.43 0 0 0.16 0 0 1.4 0.62 0 0
Longipinocarvone 1912 0.63 0 0.71 0.6 0 0 0 0 0 0
Spathulenol 1917 0.81 0.21 0 0.68 0.63 0.22 3.27 0.48 0.12 0.79
Caryophyllene oxide 1924 1.37 0 0.91 1.39 0.21 0 1.47 0 0 0
Continued
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In a study by Rabbi-Angourani30, the main compositions of the essential oil of A. vermicularis were found to
be camphor, bornel acetate, and 1 and 8-cineole. is nding aligns with a report on A. vermicularis growth in
Turkey, which identied camphor and 15-hexadecanolide as the major components of the essential oil44. Previous
studies conducted on A. vermicularis from Iran also reported 1,8-cineole, camphor, and germacrene D as the
main components45,46. In another study by Rezaei etal.47, the major constituents of the essential oil were identied
as camphor, borneol, and terpinen-4-ol. In the present study, the most important components identied in the
essential oil of the aerial parts of A. vermicularis were 1,8-cineole, camphor, δ-terpinene, anethole, borneol, and
trans-pinocarveol. ese ndings are consistent with previous studies conducted in Turkey, which also reported
camphor and 1,8-cineole as the most important compounds in this species48.
e conrmation of these ndings across dierent regions and years suggests that these compounds are
consistently produced under various environmental conditions, although the reported percentages may vary.
e essential oil of A. tenuifolia species yielded a total of 49 identied compounds, as outlined in Table4. It
is worth noting that the number of compounds obtained in A. tenuifolia was signicantly lower compared to the
other two species investigated. e most prominent compound observed in A. tenuifolia was β-cubebene, which
was present in all accessions. Among the accessions, T4 exhibited the highest percentage (50.23%) of β-cubebene,
while T3 had the lowest percentage (29.79%) (Fig. S1c). Another notable compound in A. tenuifolia was elixene,
with a composition range of 5.95 to 8.91%. e T6 accession displayed the highest percentage (13.88%) of elixene,
along with β-sesquiphellandrene. Additionally, two compounds, 1,8-cineole and camphor, were identied as
major compounds in A. tenuifolia. ese compounds were also found in the other two species, A. vermicularis and
A. wilhelmsii, where they constituted the primary compounds. In contrast to the other two species, A. tenuifolia
accessions did not exhibit a unique compound, which could be attributed to the close proximity of the sample
collection sites or a lower diversity of chemical compounds in this species. Notably, all accessions of A. tenuifolia
displayed similar percentages of mehp composition. e key essential compounds identied in the aerial parts of
A. tenuifolia in this study were β-cubebene, elixene, β-sesquiphellandrene, 1,8-cineole, camphor, and δ-terpinene.
Previous studies have shed light on the signicant chemical compounds of this plant. A study conducted on
dierent parts of the plant reported that ower compounds included limonene and α-cadinol, leaf compounds
included limonene, α-pinene, caryophyllene oxide, α-gurjunene, bornyl acetate, and δ-cadinene, while stem
compounds included limonene, α-pinene, and spathulenol49. Aghjani etal.50 identied camphor and borneol as
the primary chemical compounds in the owers of this plant. e compounds contribute to the diverse biological
activities of the essential oil and methanol extract of Achillea species, including antioxidant and antimicrobial
properties44.
e major components in the essential oil of the three studied Achillea species are presented in Table5. Among
the species, A. tenuifolia exhibited signicantly higher amounts of β-cubebene and elixene compared to the
other two species. Interestingly, all three species had similar levels of α-pinene. In terms of specic compounds,
Name RI V1 V2 V3 V4 V5 V6 V7 V8 V9 V10
α.-Santalene 1950 0.61 0.18 0.14 0.68 0.4 0 2.71 0 0 0.43
trans-Nerolidol 1959 3.42 0 0 0.52 0 0 5.86 0 0 0
Agarospirol 1968 2.23 0 0 2.38 0 0 0.82 0 0 0.45
tau-Cadinol 1981 4.67 0.24 0.87 3.65 0.19 0.21 1.38 0.39 0 0.23
β.-Eudesmol 1989 4.82 0.45 0.12 3.24 0.47 0.22 3.85 1.02 0 0.59
Acetic acid, trimethylcyclohex-1 1994 0 0.19 0 0.43 0.21 0.12 1.88 0.3 0 0.19
Humulane-1,6-dien-3-ol 2007 5.73 0.21 0 4.34 0.31 0.33 2.52 0.38 0 0.43
Heptadecane 2013 0 0.13 0.1 0.43 0.11 0.14 1.24 0 0 0
Cyclohexylidenecyclohexane 2044 0.52 0 0.05 0.66 0 0 2.28 0.42 0 0.23
Cyclohexadecanolide 2071 0.84 1.48 1.21 1.18 0.72 1.77 17.16 2.03 0 1.9
Eicosane 2084 0.09 0 0 0.1 0 0 0.31 0 0 0
Phthalicacid, methyl octyl ester 2114 0 0.24 0.26 0.11 0.18 0.24 0.86 0 0 0
Octadec-9-enoic acid 2121 0 0.24 0.18 0.13 0.15 0.19 1.31 0 0 0.11
Phytol 2131 1.22 1.01 2.21 2.38 1 0.71 9.81 0.53 0 0.42
β.-Cholestanol acetate 2149 0.14 0.21 0.21 0.14 0 0 1.21 0 0 0
Hexatriacontane 2167 0.11 0.14 0.25 0.13 0 0 1.02 0.2 0 0
Tetratriacontane 2212 0.12 0 0.12 0.19 0 0 0.79 0.2 0 0
Mehp 2224 0.49 1.52 0.79 0.84 0.98 1.86 1.47 1.4 0.38 2.95
Hexatriacontane 2273 0 0 0.07 0.09 0 0 0.5 0 0 0
Monoterpenes hydrocarbons – 14.63 22.9 6.27 18.14 19.6 12.62 0 16.13 1.73 15.1
Oxygenated monoterpenes – 36.71 35 71.8 29.64 49.08 56.27 14.17 43.42 56.72 45.82
Sesquiterpenes – 5.9 3.17 2.38 8.76 4.3 1.46 12.21 4.92 0.35 2.34
Oxygenated sesquiterpenes – 24.46 1.42 2.61 17.48 2.15 1.21 21.55 3.01 0.12 2.68
Table 3. Chemical composition of essential oils (%) of ten A. vermicularis accessions. e values in the table
are percentages of a given constituent in the total oil. e data were sorted based on the retention index (RI) of
the components.
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Table 4. Chemical composition of essential oils (%) of seven A. tenuifolia accessions. e values in the table
are percentages of a given constituent in the total oil. e data were sorted based on the retention index (RI) of
the components.
Name RI T1 T2 T3 T4 T5 T6 T7
α-Pinene 977 1.6 2.55 3.18 4.98 2.27 2.38 4.7
Camphene 1003 0.36 0.39 0.67 0 0.46 0 0.65
β-phellandrene 1042 0.3 0.79 2.45 1.38 1.07 1.16 1.08
β-Pinene 1048 0.19 0.37 0.51 0 0 0 0.54
Yomogi alcohol 1083 0.54 0 0 0 0 0 0.54
m-Cymene 1128 1.12 1.28 1.53 2.07 1.82 0.86 1.7
1,8-cineole 1140 1.45 4.75 5.23 3.04 6.7 5.39 5.31
δ-Terpinene 1186 5.93 3.54 2.31 1.62 2.35 2.69 4.99
cis-Sabinenehydrate 1204 0.51 0.57 1.25 0.94 1.31 0 0.29
Artemesia alcohol 1223 0.81 1.5 0.51 0 0 0.69 2.19
Linalool 1256 0.33 0.36 0.85 0 0.51 0 0.27
Chrysanthenone 1291 0.91 2.3 3.85 0 2.7 0 0
trans-Pinocarveol 1325 0.44 1.56 0.88 0 1.65 0 0.63
Camphor 1333 2.33 3.32 5.52 3.96 3.73 2.16 2.95
3,9-Epoxy-1-p-menthene 1347 0.96 2 1.84 1.82 1.32 0.97 1.45
Pinocarvone 1359 0 0.41 0.68 0 0 0 0.42
Borneol 1375 0 0.3 0.46 0 0 0 1.04
p-Menth-1-en-4-ol 1388 0.4 0.57 0.95 1.18 1.19 0 0.4
α.-Terpineol 1411 0.48 1.49 1.01 0 1.55 1.05 1.12
trans-Piperitol 1433 0.78 3.82 1.17 0 2.92 1.2 0.58
Piperitone 1499 0 1.32 0.56 0 0 0.59 0.38
verbenyl acetate 1503 2.04 0 1.24 0 0.84 0 0
α.-Cyclogeraniol acetate 1531 0 0 1.66 0 0 0.92 0
Anethole 1546 1.85 0.8 1.14 2.1 2.2 1.39 1.71
Bicyclohexyl 1595 0 0.2 0 0 0 0 0.34
Copaene 1665 0.7 0.5 0.44 0 0 0 0.57
β.-Elemen 1683 0.64 0.49 0.41 0 0.58 0.89 0
Caryophyllene 1721 1.82 1.03 0.93 4.76 0.71 0.99 1.92
(Z)-.β.-Farnesene 1763 2.2 3.11 2.23 0 2.23 0.57 1.79
β-cubebene 1798 44.89 35.13 29.79 50.23 42.88 36.75 35.99
α.-Farnesene 1811 0 0.83 1.23 1.03 0 0 0.55
Elixene 1817 5.95 7.51 7.13 7.18 6.07 8.91 7.19
δ.-Cadinene 1846 0.75 0.65 0.2 0 0 0 0.86
β-Sesquiphellandrene 1851 0 0.81 1.91 6.87 4.38 13.88 0.8
Spathulenol 1917 3.51 3.13 3.3 1.91 2.1 3.68 2.71
Caryophyllene oxide 1924 0.75 0 0 0 0 0 0.59
Ent-Spathulenol 1926 0 0.81 0.92 0 0 0 0
α.-Santalene 1950 0.29 0.53 0.64 0 0 0 0.85
trans-Nerolidol 1959 0.23 0.49 0.58 0 0 0.61 0.41
Spathulenol 1973 0.77 0.96 1.03 0 0.64 1.03 0.84
tau-Cadinol 1981 0 0.6 0.58 1.52 0 0.69 0
α.-Cadinol 1989 3.39 0.94 1.81 0.95 0.88 1.05 1.66
Acetic acid, 1-methyl-3-(2,6,6-trimethylcyclohex-1-enyl)propyl ester 1994 0.66 0.45 0.47 0 0 0.73 0.35
Humulane-1,6-dien-3-ol 2007 1.42 2.35 2.3 1.66 2.22 4.06 1.35
Cyclohexylidenecyclohexane 2044 0.72 0.36 0.37 0 0 0 0.59
Enanthone 2068 0 0 0.52 0 1.02 0 1.24
Cyclohexadecanolide 2071 0.77 0.96 0.47 0 0 0 0
Phytol 2132 0 0 0 0 0 1.51 0.49
Mehp 2224 1.26 0.86 0.77 0.82 1.32 1.27 1.53
Monoterpenes hydrocarbons – 11.49 13.67 15.88 13.09 14.67 12.48 19.51
Oxygenated monoterpenes – 10.88 18.32 20.07 8.18 18.6 7.08 11.98
Sesquiterpenes – 57.49 50.91 45.19 70.07 56.85 61.99 50.88
Oxygenated sesquiterpenes – 10.07 9.28 10.52 6.04 5.84 11.12 7.56
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A. wilhelmsii displayed higher values of camphor, anethole, phytol, and trans-nerolidol compared to the other
two species. On the other hand, the amount of 1,8-cineole in A. vermicularis was approximately double that of
A. wilhelmsii, and the amount in A. wilhelmsii was approximately double that of A. tenuifolia. e essential oil
components present in four classes (Tables2–4). e results showed signicant variations in the composition of
essential oils among the dierent Achillea species and their classes. In A. wilhelmsii, the oxygenated monoter-
penes are the dominant class, ranging from 35.66 to 61.03% across the eight accessions. e monoterpene
hydrocarbons and sesquiterpenes are also present in notable amounts, but in lower proportions compared to
the oxygenated monoterpenes. e A. vermicularis samples exhibit a more diverse essential oil prole. e oxy-
genated monoterpenes are still a signicant component, ranging from 14.17 to 71.8%. In contrast, the essential
oil composition of A. tenuifolia was dominated by sesquiterpenes, which account for 45.19–70.07% of the total
essential oil components across the seven accessions.
Multivariate analysis
Cluster analysis was conducted using the key components of essential oils from the studied accessions,
namely β-cubebene, elixene, borneol, camphor, 1,8-cineole, α-pinene, δ-terpinene, phytol, anethole,
β-sesquiphellandrene, trans-pinocarveol, and trans-nerolidol. e analysis resulted in the classication of the
accessions into three main groups (Fig.2). e rst group comprised seven accessions of A. tenuifolia. Based on
the results, the accessions of this species were distinguished from other accessions primarily due to signicantly
higher levels of β-cubebene and elixene components in their essential oils. Within this group, accession T6 was
separated from other A. tenuifolia accessions due to its high level of β-sesquiphellandrene in its essential oil. e
second and third groups were formed by the accessions of A. wilhelmsii and A. vermicularis. e cluster analysis
did not dierentiate between the accessions of these two species, indicating a similarity in their essential oil
compositions. However, accession V3 did not belong to the second and third groups due to its elevated levels of
anethole and trans-pinocarveol components in its essential oil.
e results of principal component analysis (PCA) revealed that the rst three principal components (PCs)
accounted for 62.21% of the total variance (Table6). PC1, which explained 28.78% of the variance, exhibited a
signicant positive correlation with β-cubebene, elixene, and β-sesquiphellandrene, and a signicant negative
Table 5. Mean values of the major essential oil components in the three Achillea sp.
Component A. wilhelmsii A. vermicularis A. tenuifolia
Phytol 3.7 1.9 0.3
Elixene 1.25 0.54 7.13
α-pinene 3.5 2.73 3.1
Anethole 6.8 4.9 1.6
Camphor 13.3 7.15 3.42
1,8-cineole 8.95 15.2 4.55
δ-terpinene 1.4 3.23 3.35
β-cubebene - 2.71 39.4
trans-nerolidol 1.32 1.03 0.33
trans-pinocarveol 0.82 3.1 0.74
β-sesquiphellandrene - - 4.1
Figure2. Heat map clustering of the 25 accessions of three studied Achillea sp based on the eight measured
minerals. e color scales represent the values were normalized by Z-score ((value-mean value)/standard error)
for each character.
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correlation with borneol and camphor. PC2, explaining approximately 18.82% of the variance, showed positive
correlations with 1,8-cineole, α-pinene, and δ-terpinene, while displaying negative correlations with phytol
and anethole. Additionally, PC3 explained 14.61% of the total variation among the study accessions and was
positively correlated with trans-pinocarveol, while negatively correlated with trans-nerolidol. e PCA triplot
conrmed the clustering results, as the accessions of A. tenuifolia were closely grouped together (Fig.3). Also,
some accessions from A. wilhelmsii and V3 were found to be distant from other accessions of the same species,
as well as from A. vermicularis accessions.
Cluster analysis and PCA have played a pivotal role in advancing our understanding of the chemical com-
positions of essential oils derived from dierent Achillea species. Yener51 employed PCA to successfully identify
A. nobilis subsp. neilreichii as distinct in terms of its composition, while Turkmenoglu44 utilized PCA to group
species based on their chemotypes. ese studies exemplify the eectiveness of PCA in discerning unique chemi-
cal proles within the Achillea genus. Similarly, Sadyrbekov52 employed cluster analysis to categorize species
according to their chemical compositions, further underscoring the signicance of these analytical techniques
in comprehending the diverse essential oil compositions found in Achillea species.
e correlation coecients among the top essential oil components of the accessions of three Achillea species,
including A. wilhelmsii, A. vermicularis, and A. tenuifolia, were presented as heat map correlation (Fig.4). e
Table 6. PCA based on the eight minerals of 25 Achillea sp. accessions.
Label Minerals
Principal components
PC1 PC2 PC3
1 β-cubebene 0.95 0.05 0.05
2 Elixene 0.95 − 0.02 0.05
3 β-sesquiphellandrene 0.73 0.00 0.03
4Borneol − 0.52 0.27 − 0.09
5Camphor − 0.47 0.41 0.06
6 1,8-cineole − 0.52 0.59 0.13
7 α-pinene − 0.09 0.51 − 0.03
8 δ-terpinene 0.20 0.46 0.10
9Phytol − 0.37 − 0.70 − 0.44
10 Anethole − 0.30 − 0.43 0.76
11 Trans-pinocarveol − 0.19 − 0.53 0.75
12 Trans-nerolidol − 0.18 − 0.51 − 0.61
– Eigenvalue 3.45 2.25 1.75
– % of variance 28.78 18.82 14.61
–Cumulative% 28.78 47.60 62.21
Figure3. PCA triplot based on the three rst PC of the 25 accessions of three studied Achillea sp.
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results indicated the strength and direction of the relationships between the components. β-sesquiphellandrene
exhibited a moderate positive correlation with β-cubebene, elixene, and trans-pinocarveol. β-cubebene displayed
a strong positive correlation with elixene and a moderate positive correlation with trans-pinocarveol. It also had
weak negative correlations with α-pinene, camphor, 1,8-cineole, borneol, anethole, phytol, and trans-nerolidol.
Elixene showed a strong positive correlation with trans-pinocarveol. It also had weak negative correlations with
α-pinene, camphor, 1,8-cineole, borneol, anethole, phytol, and trans-nerolidol. Anethole showed a strong posi-
tive correlation with trans-pinocarveol and a weak positive correlation with phytol. Phytol showed a moderate
positive correlation with trans-nerolidol.
ese correlation coecients provide insights into the relationships between the essential oil components
and can be used to understand the composition and characteristics of the dierent Achillea species accessions.
Antioxidant activity and total phenol content
In this study, the total phenol content and antioxidant activity of the samples were assessed over a period of
2years.
e analysis of A. wilhelmsii species revealed that the W4 and W7 accessions exhibited higher levels of total
phenol content in comparison to other accessions in the initial year (Fig.5). ese two accessions were the sole
ones that displayed elevated phenol levels in the rst year compared to the second year, while the remaining
accessions demonstrated higher phenol levels during the second year relative to the rst. A paired t-test was
employed to compare the 2 years in terms of this characteristic, which revealed no statistically signicant dier-
ence between the two periods. Furthermore, the W4 (IC50 = 278.32) and W7 (IC50 = 243.21) accessions exhibited
greater antioxidant activity in the rst year when compared to other samples (Table7), whereas in the second
year, the W3 accession (IC50 = 203.23) displayed the highest antioxidant activity. With the exception of the W7
accession, all accessions demonstrated higher antioxidant activity in the second year compared to the rst.
Regression analysis for each accession conducted for each year demonstrated statistically signicant models
(p < 0.01). Moreover, the coecient of determination (R-squared) exceeded 0.93 in the majority of models,
indicating a high degree of accuracy for the models.
Although there was no signicant dierence observed in total phenol content across the 2-year period,
there was a noteworthy dierence in antioxidant activity (p < 0.05). Figure6 illustrates the relationship between
antioxidant activity and total phenol content. Linear regression analysis for these two variables was statistically
signicant, and the coecient of determination was relatively high for both years within this species. While
phenolic compounds are widely recognized as the principal bioactive compounds associated with antioxidants16,
it should be noted that total phenol content does not encompass the entirety of antioxidants53.
e analysis of A. vermicularis species revealed that the V2 accession exhibited a signicantly higher total
phenol content in the second year compared to other accessions. Conversely, the V6 accession displayed the
highest phenol levels in the rst year. Apart from these two accessions, there were no signicant dierences in
phenol content among the accessions over the 2-year period. Notably, the V2 and V6 accessions, characterized by
higher phenol levels, also demonstrated superior antioxidant activity compared to other samples. In the second
year, all accessions, except for V6, exhibited higher antioxidant activity relative to the rst year. e regression
Figure4. Heat map correlation among the major essential oil composition of 25 accessions of three studied
Achillea sp.
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analysis results for each accession in each year established the statistical signicance of the obtained models
(p < 0.01), with the exception of the model associated with the V6 accession in the second year. Furthermore, the
coecient of determination yielded high values in most cases, indicating a strong model accuracy. Although there
was no signicant variation in total phenol content throughout the 2-year period, antioxidant activity displayed a
signicant dierence (p < 0.01). Linear regression analysis conrmed the signicance of this relationship for the
two variables in the rst year, supporting the linearity of the model. However, in the second year, the relationship
between the variables was found to be non-linear.
e analysis of A. tenuifolia species revealed that the highest total phenol contents in the rst and second
years were obtained from T6 and T3 accessions, respectively. e range of variation in total phenol content was
low in the rst year but increased in the second year. e t-test analysis indicated a signicant dierence between
the 2 years for both total phenol content and antioxidant activity (p < 0.01). All accessions demonstrated higher
antioxidant activity in the second year compared to the rst year. e results of regression analysis for each
accession in each year revealed the statistical signicance of the obtained models (p < 0.01). Additionally, the
coecient of determination exhibited high values in most cases. e results of linear regression analysis revealed
a linear relationship between antioxidant activity and total phenol content in both years.
e results demonstrated that among the examined species, A. tenuifolia displayed the highest level of anti-
oxidant activity. However, there was a relatively comparable level of antioxidant activity observed across the
studied species. Additionally, A. tenuifolia exhibited the highest total phenol content.
e year factor had a signicant eect on the antioxidant activity of all three studied Achillea species, while it
was only statistically signicant for total phenol content in A. tenuifolia. As the plants are perennial, in the rst
year of establishment, the growth period until owering was longer compared to the second year. In the rst
year, the plants owered in August, while in the second year owering occurred in May. e extended growth
W1
W2
W3
W4
W5
W6
W7
W8
0.00
0.05
0.10
0.15
0.20
First year
Second year
Total phenolic (mg GA/ml)
V1
V2
V3
V4
V5
V6
V7
V8
V9
V10
0.0
0.1
0.2
0.3
0.4
Total phenolic (mg GA/ml)
T1
T2
T3
T4
T5
T6
T7
0.00
0.05
0.10
0.15
0.20
Total phenolic (mg GA/ml)
Figure5. Total phenol contents of 25 accessions of the three studied Achillea sp.
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period in the rst year likely diverted more resources towards vegetative growth rather than secondary metabo-
lite production54. In contrast, the shorter growth cycle in the second year allowed for increased accumulation
of phenolic antioxidants in the aerial parts by the time of owering in May. is may explain the higher anti-
oxidant activity levels observed in all three species during the second year. Meanwhile, the year eect on total
phenol content was only signicant for A. tenuifolia possibly due to greater sensitivity or capacity for phenolic
accumulation in this species.
e results of present study are consistent with a study conducted by Polatoglu etal.48, which reported signi-
cant DPPH scavenging activity in the essential oils of A. tenuifolia and A. vermicularis. Several Achillea species,
such as A. vermicularis, A. wilhelmsii, and A. tenuifolia, have been identied as possessing noteworthy antioxidant
activity and exhibiting high total phenolic content45,55–57. ese properties can be attributed to the presence of
bioactive compounds, including phenolics and essential oils, in these species56,58. However, it should be noted
that the antioxidant activity of A. tenuifolia’s root extracts does not necessarily correlate with their total phenol
content57. Also, Al-Ogaili etal.59 revealed that Iraqi A. tenuifolia contains high levels of polyphenols, indicating
its potential as a source of antioxidants. ese ndings underscore the potential of Achillea species, including
A. vermicularis, A. wilhelmsii, and A. tenuifolia, as natural sources of antioxidants with promising applica-
tions in the pharmaceutical and medical elds. Also, signicant antioxidant properties and total phenol content
were observed in Achillea species collected from their original site56. ese plants exhibited higher antioxidant
properties and total phenol content compared to our study, which may be attributed to dierent factors such as
elevation, region, and organs used60–62.
Conclusion
To highlight the novel ndings of this study, the results revealed substantial dierences in the essential oil proles
and antioxidant potentials among accessions from three Achillea species (A. wilhelmsii, A. tenuifolia, and A.
vermicularis) when cultivated under uniform eld conditions. Notably, evaluating multiple accessions together
for the rst time demonstrated considerable intraspecic chemical diversity and phenolic variations between
genotypes of the three species that had not been previously reported. e dominant compounds diered between
the species, with camphor being predominant in A. wilhelmsii, 1,8-cineole in A. vermicularis, and β-cubebene
and elixene in A. tenuifolia. However, certain compounds, such as 1,8-cineole and camphor, were consistently
found across all species. Cluster analysis grouped the accessions into three main clusters, with A. tenuifolia
accessions forming a distinct group characterized by higher levels of β-cubebene and elixene. Additionally, the
Table 7. Antioxidant activities of 25 accessions of three studied Achillea sp.
Accessions
Year1 Ye a r 2
IC50 b a R2IC50 b a R2
W1 333.37 0.14 − 0.5 0.93 252.44 0.121 18.75 0.96
W2 360.91 0.11 10.3 0.96 259.34 0.086 20.33 0.78
W3 343.07 0.13 5.4 0.98 203.23 0.178 9.79 0.94
W4 278.32 0.13 − 0.36 0.99 258.26 0.11 11.1 0.99
W5 387.38 0.17 − 5.58 0.98 320.02 0.182 2.8 0.95
W6 360.71 0.16 − 3.34 0.96 282.63 0.13 15 0.99
W7 243.21 0.15 − 12 0.98 332.12 0.164 8.6 0.99
W8 413.33 0.16 − 1.35 0.99 269.23 0.167 2.8 0.98
V1 373.33 0.15 − 6 0.99 260.77 0.13 16.1 0.97
V2 361.18 0.135 1.24 0.95 212.76 0.17 13.83 0.94
V3 367.46 0.13 2.23 0.94 248.07 0.15 12.79 0.94
V4 369.51 0.124 4.18 0.99 274.17 0.12 17.1 0.97
V5 362.19 0.169 − 11.21 0.99 244.38 0.13 18.23 0.88
V6 290.47 0.148 7.01 0.99 402.50 0.1 9.75 0.61
V7 356.58 0.152 − 4.2 0.98 309.00 0.1 19.1 0.82
V8 390.27 0.146 − 6.98 0.99 255.39 0.18 4.03 1
V9 362.20 0.127 4 0.98 230.00 0.13 20.1 0.8
V10 347.73 0.11 11.75 0.99 260.87 0.16 8.26 0.99
T1 352.96 0.142 − 0.12 0.99 309 0.1 19.1 0.82
T2 345.99 0.142 0.87 0.98 244.92 0.13 18.16 0.89
T3 326.92 0.13 7.5 0.99 216.25 0.16 15.4 0.95
T4 385.95 0.121 3.3 0.98 290 0.1 21 0.84
T5 361.18 0.135 1.24 0.95 282.09 0.11 18.97 0.90
T6 255.60 0.157 9.87 0.99 243.18 0.17 8.66 0.98
T7 364.03 0.149 − 4.24 0.98 225.16 0.19 7.22 0.97
t-value 3.83* (indicating that the antioxidant activity of the 2 years of the
study diered signicantly at a signicance level of 0.05.)
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study assessed the total phenolic content and antioxidant activity of Achillea species over a 2-year period. Among
the examined species, A. tenuifolia exhibited the highest levels of total phenol content and antioxidant activity.
However, there was a relatively comparable level of antioxidant activity observed across the studied species.
Furthermore, linear regression analysis revealed a positive relationship between antioxidant activity and total
phenol content in both years for A. tenuifolia and A. wilhelmsii. ese ndings emphasize the phytochemi-
cal diversity within Achillea species and highlight the inuence of genetic and environmental factors on their
essential oil compositions and antioxidant properties. Moreover, the study underscores the potential of Achillea
species as a reliable source of antioxidants for use in the food and pharmaceutical industries. Further research,
including the selection of specic accessions within each species, can provide deeper insights into their chemical
composition and medicinal potential.
Data availability
All data are within the manuscript.
Received: 14 February 2024; Accepted: 21 May 2024
0.00 0.05 0.10 0.15
200
250
300
350
400
450
Total phenolic content (mg GA/ml)
IC50
Y= -1131.7x + 392.76
R
2
= 0.87
0.00 0.02 0.04 0.06
0.08
150
200
250
300
350
Total phenolic content (mg GA/ml)
IC50
Y= -3620.4x + 407.46
R
2
= 0.92
0.00 0.05 0.10 0.15
250
300
350
400
Total phenolic content (mg GA/ml)
IC50
Y= -807.74x + 384.87
R
2
= 0.96
0.00.1 0.
20
.3
0
100
200
300
400
500
Total phenolic content (mg GA/ml)
IC50
Y= -405.04x + 292.48
R
2
= 0.28
0.00 0.01 0.02 0.03 0.04
200
250
300
350
400
450
Total phenolic content (mg GA/ml)
IC50
Y= -4576.5x + 450.73
R
2
= 0.80
0.00 0.05 0.10
0.15
200
250
300
350
Total phenolic content (mg GA/ml)
IC50
Y= -846.88x + 325.51
R
2
= 0.97
First year Second year
A. wilhelmsii
A.
vermicularis
A. tenuifolia
Figure6. e relationships between antioxidant activity and total phenol content of three Achillea species over
2 years.
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Author contributions
M.E. and M.F. conceived and designed the research. M.F. conducted experiments and wrote the manuscript.
M.S.H., and D.F.A.L. conducted experiments. A.B., M.E. and M.S.H. elaborated on the results and discussion,
while doing a critical reading of the manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Correspondence and requests for materials should be addressed to M.F.orM.E.
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