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Comparative Study of Chemical Composition and Cholinesterase Inhibition Potential of Essential Oils Isolated from Artemisia Plants from Croatia

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28 September 2023

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30 September 2023

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Abstract
The essential oil (EO) of Artemisia plants contains a large number of bioactive compounds that are widely used. The aim of this study was to analyse the chemical composition of EOs of six Arte-misia plants collected in Croatia and to test their cholinesterase inhibitory potential. GC-MS analysis of EO of A. absinthium showed that the dominant compounds are cis-sabinyl acetate and cis-epoxy-ocimene; in EO of A. abrotanum it is borneol; in EO of A. annua it is artemisia ketone, camphor, and 1,8-cineole; in EO of A. arborescens it is camphor and chamazulene; in EO of A. verlotiorum it is cis-thujone, 1,8-cineole, and trans-thujone; in EO of A. vulgaris, it is trans-thujone and trans-epoxy-ocimene. EO of the five studied Artemisia species from Croatia is rich in mono-terpenoid compounds (1,8-cineole, artemisia ketone, cis-thujone, trans-thujone, cis-epoxy-ocimene, camphor, borneol, and cis-sabinyl acetate). EO of A. arborescens is also rich in chamazulene. The results also showed that the tested EOs have moderate cholinesterase inhibi-tion potential, especially the EOs of A. annua, A. vulgaris, and A. abrotanum. This is the first anal-ysis of the chemical composition of the EOs of four Artemisia plants and the first analysis of cho-linesterase potential for plants collected in Croatia.
Keywords: 
Subject: Biology and Life Sciences  -   Life Sciences

1. Introduction

The genus Artemisia (family Asteraceae) includes a large number of species distributed in Europe, Asia, Africa and North America. Plants of the genus Artemisia are aromatic and are widely used in traditional medicine for their medicinal properties [1,2]. The genus Artemisia is of particular interest because in 2015 the Nobel Prize was awarded for the detection of the sesquiterpene lacone artemisinin in it and its antimalarial activity was demonstrated. The main constituents of Artemisia plants are mainly specific sesquiterpene lactones, essential oil, flavonoids, coumarins and phenolic acids [3]. The essential oil of these plants contains a large number of bioactive chemical compounds, which are widely used in the chemical industry as well as in medicine, cosmetics and food industry. The components of these oils show antifungal, antibacterial, and antiparasitic effects [4]. They also stimulate appetite, improve digestion by stimulating bile secretion, stimulate the liver, eliminate indigestion and flatulence. The species of this genus are used in modern medicine for their apoptosis-inducing, antitumor, and antiplasmodial effects, as well as for the treatment of viral infections [5]. It is used in the form of tea, extracts, and spirits, and the flower buds are dried and ground into powder. are used as a spice.
Essential oils (EOs) are secondary plant metabolites, characteristic ingredients of medicinal and aromatic plants. They are used in various industries and fields, from pharmaceuticals and cosmetics to food and aromatherapy [6]. Recently, the scientific community is paying more and more attention to the use of substances isolated from nature and their use in the therapy of pathological conditions.
Alzheimer's disease (AD) is a progressive senile dementia that primarily affects the elderly. The decline in cognitive abilities is due to a deficiency of acetylcholine in the patient's brain tissue. This leads to an impairment of the patient's quality of life. There are two main forms of cholinesterase in the mammalian brain: Acetylcholinesterase (AChE) and Butyrylcholinesterase (BChE), both of which have the ability to degrade acetylcholine and butyrylcholine, respectively. AChE is found in the synaptic cleft (soluble form) and in synaptic membranes (membrane-bound form), whereas BChE is mainly associated with glial cells [7]. AChE is the major enzyme that hydrolyzes acetylcholine into choline and acetate. For this reason, inhibition of AChE is the mainstay of treatment for AD. Since existing inhibitors of these enzymes are associated with undesirable side effects, there is a constant need to research and invent new cholinesterase inhibitors isolated from nature [8].
The aim of this work was the isolation and identification of the chemical composition of EOs from six samples of plant species of the genus Artemisia, originating from the territory of Croatia: A. absinthum; A. abrotanum; A. annua; A. arborescens; A. verlotiorum; A. vulgaris. The isolated EOS were also evaluated for their ability to inhibit cholinesterase, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), to draw conclusions about the potential of the essential oils of these plants on these two enzymes, which are important in the treatment of AD. To our knowledge, this is the first report of the chemical composition of four of the six plants studied (A. abrotanum; A. annua; A. arborescens; A. verlotiorum) collected in Croatia, and the first test of the anticholinesterase potential of the EOs of Artemisia plants from Croatia.

2. Materials and Methods

2.1. Chemicals

Acetylcholinesterase (AChE, from Electrophorus electricus – electric eel, type V-S), Acetylthiocholine iodide (ATChI), Butyrylcholinesterase (BChE, from equine serum), Butyrylthiocholine iodide (BTChI), and 5,5-dithiobis (2-nitrobenzoicacid) (DTNB, Ellman’s reagent), were purchased from Sigma-Aldrich GmbH (Steinheim, Germany); Ethanol was purchased from Kemika, Zagreb, Croatia;

2.2. Plant material

Plant parts of six different species of the genus Artemisia were collected immediately after full flowering at different locations in Croatia, Table 1. Collection and identification of plant material was performed by botanist Prof. Mirko Ruscic. The voucher specimens of the plant material were deposited in the Herbarium of the Department of Biology, Faculty of Natural Sciences, University of Split (AABS_2020, AABR_2021, AANN_2020, AABR_2020, AVER _2020, AVUL_2020).

2.3. Isolation of The Essential Oil

The EOSs of six different Artemisia plants were isolated from previously dried plant material by hydrodistillation in a Clevenger apparatus using according to the method previously described by Bektasevic et al. [9]. The isolated essential oils were filled into vials, dried over anhydrous Na2SO4 and stored at 4 °C until analysis.

2.4. Identification and Quantification of the Chemical Constituents of the Essential Oil by GC-MS

Separation and analysis of the essential oils from Artemisia plants was performed by GC-MS using a gas chromatograph (gas chromatograph model 8890 equipped with an automatic liquid injector model 7693A), and a tandem mass spectrometer (MS) model 7000D GC/TQ (Agilent Inc., Santa Clara, CA, USA). Chromatographic separation was performed on the nonpolar HP-5MS column (30 m × 0.25 mm × 0.25 µm, Agilent Inc.). Helium was used as the carrier gas at a flow rate of 1.0 mL min, the sample injection volume was 1 µL, and the split ratio was 1:50. Analyses were performed using MS full scan (33-350 m/z). The ion source temperature was set at 230 °C, the interface temperature was set at 250 °C, and the ionization energy was 70 eV. The column temperature programme was set at 70 °C for the first 2 min and then heated to 200 °C at 3 °C/min and kept isothermal for 18 min. The analysis was performed twice, and the results are presented as the mean of the obtained results.
The compounds of the essential oils were identified by comparing their retention indices with the series of n-hydrocarbons (C8-C40) analyzed under the same conditions as the essential oil. Individual components were identified by comparing their mass spectra to library entries from two commercial databases, Wiley 7 MS library (Wiley, NY, USA) and NIST02 (Gaithersburg, MD, USA), and by comparing their mass spectra and retention indices to published data [10]. The relative proportions of oil components (%) were calculated based on the peak areas on the chromatography column. Retention indices (RI) were calculated based on the retention times of series of alkanes and using the equation of van den Dool and Kratz [11].

2.5. Cholinesterase Inhibitory Assay

Cholinesterase inhibitory activity was determined against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) at a concentration of 1 mg/mL using an ELISA microplate reader according to the Ellman method [12]. The method was based on the reaction of Ellman's reagent (DTNB) and thiocholine, yielding a yellow colored product. Enzyme activity was measured according to the method previously described by Bektasevic et al. [9] and lasted 6 min with three replicates each time. The percentage of AChE / BChE enzyme inhibition by essential oils or extracts was calculated according to the following formula:
% inhibition of AChE / BChE = {[(Ae − Abe) − (Au − Abu)]/(Ae − Abe)} × 100;
Ae—absorbance of enzyme without an inhibitor, Abe—absorbance of a blank for enzyme without a substrate, Au—absorbance of enzyme with an inhibitor, Abu—absorbance of blank for enzyme without an inhibitor

3. Results and Discussion

In this work, the chemical composition and cholinesterase inhibition potential of the essential oils (EOs) of six Artemisia plants (A. absinthum; A. abrotanum; A. annua; A. arborescens; A. verlotiorum; A. vulgaris) collected in Croatia were studied.

3.1. Phytochemical Profile

EOs of six species of the genus Artemisia collected immediately after full flowering in Croatia were isolated from dried plant material by hydrodistillation and analysed by coupled gas chromatography-mass spectrometry system (GC-MS).
The chemical composition of the essential oils is given in Table 2, while the GC-MS total ion chromatograms are shown in Figure 1. The compounds in the Table 2 are grouped by compound class and by ascending retention index (RI).
The EOs of the studied Artemisia species in the dry plant material from which they were isolated ranged from 0.2% (A. vulgaris) to 1.6% (A. absinthium). The essential oil of A. absinthium was reddish-brown, the oil of A. arborescens was dark blue, while all other studied oils were yellow. The most abundant compounds in the EO of A. absinthium are the monoterpenoids cis-sabinyl acetate (38.5%) and cis-epoxy-ocimene (28.8%). All other components of this EO have a proportion of less than 5%. The monoterpenoids are present in this EO in a high proportion of 78.1% (w/w). This is followed by other compounds (7.4%), sesquiterpenes (3.7%), monoterpenes (3.1%), and sesquiterpenoids (2.1%).
According to Orav et al. [13], four chemotypes characteristic of A. absinthium growing in Europe were found: sabinene- and myrcene-rich oil, α- and ß-thujone-rich oil, epoxy-ocimene-rich oil, and (E)-sabinyl acetate-rich oil. Some mixed chemotypes were also found. According to this classification, the oil isolated from the plant collected in Croatia belongs to the mixed chemotype (epoxy-ocimene-rich oil and (E)-sabinyl acetate-rich oil).
The EO of this plant species collected in Croatia (it is not specified where, full flowering, dried and powdered) was previously analyzed by Juteau et al. [14]. Analysis of this oil revealed ß-thujone (26.0%), (Z)-6,7-epoxyocymene (9.0%), linalool (5.9%), and sabinene (5.5%) as the main constituents. All other constituents of this oil were present in amounts less than 4.5%.
Analysis of EO of this plant collected in the southern part of neighboring Serbia (Bela Palanka and Nis, above ground and previously dried) showed that the main components are ß-thujone (19.8 and 63.4%), cis-ß-epoxy-ocimene (10.7 and 0.0%), trans-sabinyl acetate (8.8 and 0.0%), sabinene (8.1 and 10.8%), and linalyl-3-methylbutanoate (7.5 and 4.5%) [15]. The composition of the essential oil of A. absinthium collected in the northwestern Italian Alps, Piedmont (full bloom, air-dried) revealed cis-epoxyocimene (24.8%), trans-chrysanthenyl acetate (21.6%), and camphor (17.1%) as the main constituents [16].
The main constituent of the EO of A. abrotanum is the monoterpene alcohol borneol (48.0%). Camphor (9.5%), camphene (7.0%), sabinene (5.2%), and chrysanthenone (4.7%) are also present in significant proportions. Other identified constituents of this EO account for less than 4%. The predominant compound class in this oil is monoterpenoids (74.4%). This is followed by monoterpenes (16.7%) and other compounds (0.8%).
There are many different chemotypes of A. abrotanum from different geographical locations ((+)-piperitone chemotype, trans-sabinyl acetate/α-terpineol chemotype, 1,8-cineole/α-thujene/α-pinene chemotype, eucalyptol chemotype, davanol/davanone/hydroxydavanon chemotype) [17]. The EO of the A. abrotanum from Croatia is particularly rich in borneol, and we could conclude that it is a borneol chemotype. This is not the case with any other oil of this plant.
To date, not one analysis of EO of this plant species collected in Croatia has been performed. Two analyzes of EO of this plant has been performed in neighboring countries, Austria and Italy. The results of the Austrian EO analysis (plant from the Botanical Garden of the University of Veterinary Medicine Vienna, Austria, in full bloom) showed that the most abundant components of this EO are the derivative davanone (22.5%) and 4-methyl-pent-2-enolide (15.7%) [18]. The EO composition of A. abrotanum from the northwestern Italian Alps, Piedmont (full bloom, air-dried) revealed 1,8-cineole (34.7%), bisabolol oxide (18.4%) and ascaridol (16.0%) as the predominant components [16].
To date, not one analysis of the EO of this plant species collected in Croatia has been performed. Two analyzes of EO of this plant were performed in the neighboring countries, Austria and Italy. The results of the Austrian EO analysis (plant from Botanical Garden of the University of Veterinary Medicine Vienna, Austria, full bloom) showed that the most abundant components of this EO were the derivative davanone (22.5%) and 4-methyl-pent-2-enolide (15.7%) [18]. EO composition of A. abrotanum from the northwestern Italian Alps, Piedmont (full bloom, air-dried) revealed 1,8-cineole (34.7%), bisabolol oxide (18.4%) and ascaridole (16.0%) as predominant constituents [16].
The monoterpenoids artemisia ketone (22.3%), camphor (22.0%), and 1,8-cineole (16.2%) were identified as the dominant constituents of EO from A. annua. Caryophyllene oxide (5.3%) and artemisia alcohol (3.2%) were also identified with lower proportions. All other constituents of this EO were present in minor proportions. The predominant compound class in this EO was monoterpenes (74.0%). This was followed by monoterpenes (8.6%), sesquiterpenoids (8.2%), sesquiterpenes (5.6%) and other compounds (1.9%).
Depending on the variety, the dominant compounds of EO, isolated from A. annua, were artemisia ketone and camphor, camphor and 1,8-cineole, 𝛼-pinene and pinocarvone, artemisia ketone and 1,8-cineole, and a chemotype with phenolic compounds [19]. According to the chemical composition, the EO isolated from A. annua collected in Croatia, belongs to the artemisia ketone/camphor/1,8-cineole chemotype.
To date, not one analysis of EO of A. annua collected in Croatia has been performed. A few analyzes have been performed in neighboring countries. The EO of the cultivated plant collected in spring in Bosnia and Herzegovina (Kiseljak, near Sarajevo) and previously dried contains a high percentage of artemisia ketone (30.7%) and artemisia alcohol (6.5%) [20]. The analysis of this plant species cultivated near Sarajevo, Bosnia and Herzegovina (air-dried and hydrodistillated), contains artemisia ketone (28.3%) and camphor (16.9%) as the main components [19], while the analysis of A. annua harvested after flowering period from the natural habitat, air-dried and hydrodestilated after one year of storage revealed selina-3,11-dien-6α-ol (9.6%), cis-thujopsenoic acid (7.0%), caryophyllene oxide (7.0%) and alloaromadendrene epoxide (4.7%) as the main constituents [21]. The most abundant volatile compounds of A. annua EO from Serbia were artemisia ketone (25.4 %) and trans-caryophyllene (10.2 %), followed by 1,8-cineole, camphor, germacrene D and β-selinene [22] . Ickovski et al. [23] identified artemisia ketone (55.8%) and α-pinene (12.7%) as main components components of A. annua collected near Nis, Serbia (fresh aerial parts). Radulovic et al. [24] were also performed an analysis of A. annua EO from Serbia (Nis) (air-dried) and identified artemisia ketone (35.7%), α-pinene (16.5%) and 1,8-cineole (5.5%) as the most abundant components while The analysis of this EO collected in Belgrade, Serbia (aerial and air-dried), contains pinocarvone (29.40%), artemisia ketone (19.19%), caryophyllene oxide (5.93%), and 1,8-cineole (4.72%) as the most abundant constituents [25]. The flowering aerial parts of A. annua collected from the banks of the Arno River in Pisa (Italy) in late September 2015 and previously air-dried contained artemisia ketone (22.1%), 1,8-cineole (18.8%), and camphor (16.9%) as main constituents [26]. The essential oil of plants collected in Sesto Fiorentino, Italy, at the full flowering stage (fresh plant material) contained numerous constituents, of which the most important were germacrene D (21.2%), camphor (17.6%), (E)-β-farnesene (10.2%), (E)-β-caryophyllene (9%), and bicyclogermacrene (4.2%) [27]. The composition of EO of A. nnua collected in the northwestern Italian Alps, Piedmont (full flower, air-dried), revealed 1,8-cineole (34.7%), α-pinene (19.6%), bisabolol oxide (18.4%), ascaridole (16.0%), and camphor (15.5%) as the main constituents [16]. The chemical composition of the EO of 85 individuals of A. annua cultivated in Budaörs, near Budapest, Hungary, (fresh plant material) showed that the main constituents were artemisia ketone (33–75%) and artemisia alcohol (15–56%) [28].
The monoterpenoid camphor (39.5%) and the bicyclic unsaturated hydrocarbon, the sesquiterpene camazulene (33.9%), were identified as the major constituents of EO isolated from A. arborescens. Terpinen-4-ol (3.2%), camphene (2.4%), and ß-myrcene (2.1%) occur in lower proportions, while the other constituents of this oil occur in proportions of less than 2%. The dominant class of compounds in this oil are monoterpenoids (45.5%) and sesquiterpenoids (35.7%). They are followed by monoterpenes (8.8%), other compounds (2.7%) and sesquiterpenes (1.6%).
Different chemotypes have been identified for the essential oils of A. arborescens: a ß-thujone/camphor chemotype (Sardinia, Italy, around Usellus) and Morocco; a chamazu-lene/camphor chemotype (northwestern United States and in southern parts of Italy, Calabria, Sicily, and the Aeolian Islands); and a ß-thujone/chamazulene chemotype (Liguria (Sacco), Sicily, Sardinia, and Algeria) [29]. According to this classification, the EO isolated from the plant collected in Croatia belongs to the chamazulene/camphor chemotype.
To date, not a single analysis of the essential oil of this plant species collected in Croatia has been performed. Several analyzes of the oil of this plant have been carried out in neighboring countries. The analysis of EO of this plant (above-ground biomass of plant, blossom stage) collected from two sites in Italy (Capo Zafferano and Termini Imerese) revealed that the most abundant constituents of the EO are chamazulene (43.12 and 36.83%), ß-thujone (19.57 and 19.89%), and camphor (8.78 and 8.68%). The results of GC-MS analysis of this plant collected in Italy in three locations (Sicily, Calabria and the Aeolian Islands, Lipari) (fresh plant material, leaves; at vegetative phase; EO isolated by microwave assisted hydrodistillation) showed that the most abundant components of this EO are camphor (21.4, 39.5 and 20.1%), camazulene (37.6 27.1 and 34.6%) [31]. The EO of A. arborescens from Sardinia, Italy, isolated from plant material collected at three developmental stages of the plant (from vegetative state to postflowering), belongs to the ß-thujone / chamazulene chemotype. The most abundant constituents of this EO were chamazulene (51.5; 34.2 and 25.6%), ß-thujone (38.8; 33.8 and 53.2%) and germacrene D (3.2; 5.4 and 4.3%) [29]. The EOs of the aerial parts of several A. arborescens populations (flowering stage) collected from different sites in Sicily (Petru, Diga, Felice) were analyzed by GC–FID and GC–MS systems. β-Thujone (20.5–55.9%), chamazulene (15.2–49.4%), camphor (1.3–8.4%) and germacrene D (2.8–3.4%) were identified as the most abundant compounds of these oils [32]. The analysis of EO, isolated from the fresh plant material of this plant collected in the vegetative stage (January) in the northwestern part of Sicily, Italy, showed that the most abundant constituents of this oil (steam distillation) are ß-thujone (45.04%), chamazulene (22.71%), and camphor (6.78%) [33]. GC- MS analysis of this EO oil collected in Montenegro (Budva and Stari Ulcinj island) (aerial parts, air-dried) showed that the most abundant constituents were α thujone (0.0 and 28.59%), camphor (6.44 and 39.46%) and camphene (7.08 and 2.35%) [25].
The monoterpenoids cis-thujone (46.3%), 1,8-cineole (10.9%), and trans-thujone (9.0%) were identified as the predominant constituents of the EO of A. verlotiorum. Caryophyllene oxide (6.0%) and ß-caryophyllene (5.8%) were presented in slightly lower proportions. Other compounds of this oil were identified in amounts of less than 2.5%. The dominant class of compounds in this EO was monoterpenoids (75.2%). This was followed by sesquiterpenes (8.8%) and sesquiterpenoids (8.8%), as well as monoterpenes (3.8%) and other compounds (2.0%). As for the chemical composition, the analyzed essential oil from Croatia belongs to the thujone/1,8-cineole chemotype.
So far, not one analysis of EO from this plant has been performed on a plant collected in Croatia, but several EO analyzes have been performed on plant material collected in neighboring countries. Seasonal variations in the chemical composition of the oil isolated from this plant collected during the year in Pisa Province, Italy (aerial parts, air-dried, showed that the most abundant constituents of this oil were 1,8-cineole (12.8–32.2%), germacrene D (3.8–18.1%), α-thujone (2.3–8.0%), ß-thujone (8.3–14.7%), ß-caryophyllene (1.8–10.6%), borneol (3.3–9.9%), camphor (3.6–8.3%), and myrcene (0.4–11.2%) [34]. The composition of the EO of A. verlotiorum from the northwestern Italian Alps, Piedmont (full bloom, air-dried) revealed caryophyllene oxide (21.4%), borneol (17.6%), camphor (11.2%), 1,8-cineole (10.6%), and spathulenol (9.2%) as the main components [16].
The monoterpenoids trans-thujone (40.3%) and cis-epoxy-ocimene (15.5%) were identified as dominant constituents of A. vulgaris EO. The EO also contains cis-thujone (5.6%), toreiol (3.7%), davanone (3.2%), 1,8-cineole (3.2%), and other compounds in lesser amounts. The predominant compound class in this EO was monoterpenoids (69.4%). Followed by sesquiterpenoids (12.1%), monoterpenes (10.4%), sesquiterpenes (4.4%) and other compounds (2.7%). Four different chemotypes of EO from A. vulgaris were found: One with the coexistence of ar-curcumene and α-zingiberene; two characterized by the presence or absence of thujone and santolinatriene; and a fourth characterized by the presence of crysanthenyl acetate (40%) [35]. Accordingly, the Croatian EO of A. vulgaris belongs to the thujone chemotype.
GC-MS analysis of the EO of this plant collected in Dalmatia, Croatia, (aerial plant material, air-dried) revealed that the most abundant constituents of this oil at the of full flowering (August) were ß-thujone (20.8%), α-pinene (15.1%), 1,8-cineole (11.7%), camphor (8.7%), and α-thujone (8.5%) trans-chrysanthenyl acetate (18.5%), 1,8-cineole (15.2%), and α-phellandrene (12.9%) [36]. Chemical analysis of the essential oil of this plant, collected in the area of Niš, Serbia, at the time of full flowering, showed that the dominant compounds in the oil of the aerial part of the plant (isolated directly after drying and after one year of storage) are 1,8-cineole (28.9%), sabinene (13.7%) and ß-thujone (13.5%) [15]. The composition of the EO of A. vulgaris collected from north-west Italian Alps, Piedmont (full bloom, air-dried) revealed camphor (47.7%) as dominant compounds. In this EO, camphene (9.1%), verbenone (8.6%) and trans-verbenol (7.0%) were also identified as components contained in larger propositions [16].
The chemical composition of EOs of the studied plant species of the genus Artemisia (A. absinthium, A. abrotanum, A. annua, A. verlotiorum, A. vulgaris) revealed that the studied EOs are dominated by monoterpenoid components: 1,8-cineole, artemisia ketone, cis-thujone, trans-thujone, cis-epoxyocimene, camphor, borneol, cis-sabinyl acetate. In one plant species (A. arborescens), the azulene derivative chamazulene occurs as a major compound. It is a blue-violet azulene derivative biosynthesized from the sesquiterpene matricin

3.2. Cholinesterase inhibition potential of Artemisia essential oils from Croatia

The ability of EOs from Artemisia plants collected in Croatia to inhibit the enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) was tested by the Ellman method [12]. The concentration of tested EOs in the solutions was 1 mg/mL, while the concentration of EOs in the reaction systems was 45.45 µg/mL. The results are shown in Table 3.
The essential oils isolated from Artemisia from Croatia show moderate ability to inhibit the enzyme AChE (29.7 – 55.2%). Among these oils, the oil of A. annua shows the best inhibitory effect, while the EO of A. absinthium shows the weakest effect on this enzyme at the tested stock solution concentration of 1 mg/mL. As expected, the inhibition of BChE by these EOs shows a slightly weaker activity compared to the inhibition of AChE, with the exception of EO from A. absinthium. The results obtained were compared with those of the known good inhibitors of these enzymes, huperzine A and galantamine, Table 3.
To the best of our knowledge, we report here the first results on cholinesterase inhibitory activity of selected Artemisia plants collected in Croatia. Only one study was conducted on the antiAChE potential of the EOs of the tested Artemisia species collected in the areas of neighboring countries. The flowering aerial parts of A. annua collected in late September in Pisa (Italy) along the Arno riverbank showed an AChE inhibition potential IC50=472.4 mg/L [25].
Numerous researchers have evaluated the pure compounds included in the essential oil composition for their ability to inhibit AChE. Less pure compounds have been tested for their BuChE inhibition [37]. Despite major differences in methodology, the results of these tests showed that monoterpenoids are the most potent inhibitors of these enzymes. Among them, 1,8-cineole and camphor, which are present in greater proportions in the essential oils of Artemisia plants, are quite potent inhibitors, especially of AChE. It can be concluded that these are the components of the oil that can be attributed with the ability to inhibit AChE. 1,8-Cineole has also been shown to be a good BChE inhibitor. At the same time, synergistic or antagonistic effects must also be taken into account, so it is difficult to say with absolute certainty which constituents of a mixture of compounds are responsible for the biological effect [37].
Few more tests on the inhibitory potential of EOs from Artemisia plants on AChE/BChE were performed: A. absinthium collected in Pakistan [38] and Algeria [39] and A. annua (flowers) from China [40].

4. Conclusions

The chemical composition of EOs of the studied plant species of the genus Artemisia (A. absinthium, A. abrotanum, A. annua, A. verlotiorum, A. vulgaris) revealed that the studied EOs are dominated by monoterpenoid components: 1,8-cineole, artemisia ketone, cis-thujone, trans-thujone, cis-epoxyocimene, camphor, borneol, cis-sabinyl acetate. In one plant species (A. arborescens), the azulene derivative chamazulene occurs as a major compound.
Artemisia essential oils isolated from Croatia showed moderate ability to inhibit the enzyme AChE. Among these oils, the oil of A. annua showed the best inhibitory activity compared to the known ChE inhibitors galantamine and huperzine A. EO isolated from A. vulgaris and A. abrotanum also showed significant inhibitory activity, especially on AChE. Inhibition of BChE by these EOs shows a slightly weaker activity compared to the inhibition of AChE, with the exception of EO from A. absinthium.
This is the first analysis of the chemical composition of the EOs of four Artemisia plants studied and the first analysis of cholinesterase potential for Artemisia plants collected in Croatia.

Author Contributions

Conceptualization, OP and MB; methodology, OP, IC, AS; validation, OP, IC, AS; formal analysis, OP, AS; investigation, OP, AS; resources, OP, MB; data curation, OP; writing—original draft preparation, OP; MB; writing—review and editing, OP; MB; visualization, OP, MB; supervision, OP; project administration, OP; funding acquisition, OP; MB. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Wright, C.W. Artemisia, 1st ed.; CRC Press: London, United Kingdom, 2001. [Google Scholar]
  2. Bora, K.S.; Sharma, A. The Genus Artemisia, a comprehensive review. Pharm Biol 2011, 49, 101–109. [Google Scholar] [CrossRef]
  3. Ekiert, H.; Klimek-Szczykutowicz, M.; Rzepiela, A.; Klin, P.; Szopa, A. Artemisia Species with High Biological Values as a Potential Source of Medicinal and Cosmetic Raw Materials. Molecules 2022, 27, 6427. [Google Scholar] [CrossRef]
  4. Kaul, V.K.; Nigam, S.S.; Banerjee, A.K. Insecticidal activity of some essential oils. Indian J. Pharm. 1978, 40, 22–26. [Google Scholar]
  5. Taleghani, A.; Emami, S.A.; Tayarani-Najaran, Z. Artemisia: a promising plant for the treatment of cancer. Bioorg Med Chem. 2020, 28, 115180. [Google Scholar] [CrossRef]
  6. Bolouri, P.; Salami, R.; Kouhi, S.; Kordi, S.; Lajayer, B. A.; Hadian, J.; Astatkie, T. Applications of Essential Oils and Plant Extracts in Different Industries. Molecules 2022, 27, 8999–9016. [Google Scholar] [CrossRef]
  7. Giacobini, E. Do cholinesterase inhibitors have disease-modifying effects in Alzheimer's disease? CNS Drugs 2001, 15, 85–91. [Google Scholar] [CrossRef]
  8. Briggs, R.; Kennelly, S. P.; O’Neill, D. Drug treatments in Alzheimer’s disease. Clin. Med. 2016, 16, 247–253. [Google Scholar] [CrossRef]
  9. Bektasevic, M.; Jurin, M.; Roje, M.; Politeo, O. Phytochemical Profile, Antioxidant Activity and Cholinesterase Inhibition Potential of Essential Oil and Extracts of Teucrium montanum from Bosnia and Herzegovina. Separations 2023, 10, 421. [Google Scholar] [CrossRef]
  10. Adams, R.P. Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy; 4.1 ed.; Allured Publishing Corporation: Carol Stream, IL, USA, 2017. [Google Scholar]
  11. Dool, H.V.D.; Kratz, P.D. A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography. J. Chromatogr. A 1963, 11, 463–471. [Google Scholar] [CrossRef] [PubMed]
  12. Ellman, G. L.; Courtney, K. D.; Andres Jr., V.; Featherstone, R. M. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 1961, 7, 88–95. [Google Scholar] [CrossRef]
  13. Orav, A.; Raal, A.; Arak, E.; Müürisepp, M.; Kailas, T. Composition of the essential oil of Artemisia absinthium L. of different geographical origin. Proc. Estonian Acad. Sci. Chem. 2006, 55, 155–165. [Google Scholar]
  14. Juteau, F.; Jerkovic, I.; Masotti, V.; Milos, M.; Mastelic, J.; Besseire, J. M.; Viano, J. Composition and Antimicrobial Activity of the Essential Oil of Artemisia absinthium from Croatia and France. Planta Med. 2003, 69, 158–161. [Google Scholar] [CrossRef] [PubMed]
  15. Blagojevic, P.; Radulovic, N.; Palic, R.; Stojanovic, G. Chemical Composition of the Essential Oils of Serbian Wild-Growing Artemisia absinthium and Artemisia vulgaris. J. Agric. Food Chem. 2006, 54, 4780–4789. [Google Scholar] [CrossRef] [PubMed]
  16. Mucciarelli, M.; Caramiello, R.; Maffei, M. Essential Oils from Some Artemisia Species Growing Spontaneously in North-West Italy. Flavour Frag. J. 1995, 10, 25–32. [Google Scholar] [CrossRef]
  17. Saunoriūtė, S.; Ragažinskienė, O.; Ivanauskas, L.; Marksa, M. Essential oil composition of Artemisia abrotanum L. during different vegetation stages in Lithuania. Chemija 2020, 31, 52–56. [Google Scholar]
  18. Obistioiu, D.; Cristina, R. T.; Schmerold, I.; Chizzola, R.; Stolze, K.; Nichita, I.; Chiurciu, V. Chemical characterization by GC-MS and in vitro activity against Candida albicans of volatile fractions prepared from Artemisia dracunculus, Artemisia abrotanum, Artemisia absinthium and Artemisia vulgaris. Chem. Cent. J. 2014, 8, 6. [Google Scholar] [CrossRef]
  19. Vidic, D.; Copra-Janicijevic, A.; Milos, M.; Maksimovic, M. Effects of different methods of isolation on volatile composition of Artemisia annua L. Int. J. Anal. Chem 2018, 1–6. [Google Scholar] [CrossRef]
  20. Cavar, S.; Maksimovic, M.; Vidic, D.; Paric, A. Chemical composition and antioxidant and antimicrobial activity of essential oil of Artemisia annua L. from Bosnia. Ind. Crops Prod. 2012, 37, 479–485. [Google Scholar] [CrossRef]
  21. Vidic, D.; Cavar Zeljkovic, S.; Dizdar, M.; Maksimovic, M. Essential oil composition and antioxidant activity of four Asteraceae species from Bosnia. J. Essent. Oil Res. 2016, 28, 445–457. [Google Scholar] [CrossRef]
  22. Acimovic, M.; Stankovic Jeremic, J.; Todosijevic, M.; Kiprovski, B.; Vidovic, S.; Vladic, J.; Pezo, L. Comparative Study of the Essential Oil and Hydrosol Composition of Sweet Wormwood (Artemisia annua L.) from Serbia. Chem. Biodivers 2022, 19, 202100954. [Google Scholar] [CrossRef]
  23. Ickovski, J. D.; Stepic, K. D.; Stojanovic, G. S. Composition of essential oils and headspace constituents of Artemisia annua L. and A. scoparia Waldst. et Kit. J. Serb. Chem. Soc. 2020, 85, 1565–1575. [Google Scholar] [CrossRef]
  24. Radulovic, N. S.; Randjelovic, P. J.; Stojanovic, N. M.; Blagojevic, P. D.; Stojanovic-Radic, Z. Z.; Ilic, I. R.; Djordjevic, V. B. Toxic essential oils. Part II: Chemical, toxicological, pharmacological and microbiological profiles of Artemisia annua L. volatiles. Food Chem. Toxicol. 2013, 58, 37–49. [Google Scholar] [CrossRef]
  25. Janackovic, P.; Rajcevic, N.; Gavrilovic, M.; Novakovic, J.; Giwelib, A.; Stesevic, D.; Marin, P. D. Essential oil composition of five Artemisia (Compositae) species in regards to chemophenetics. Biochem. Syst. Ecol. 2019, 87, 103960. [Google Scholar] [CrossRef]
  26. Bedini, S.; Flaminib, G.; Cosci, F.; Ascrizzi, R.; Echeverria, M. C.; Guidi, L.; Landi, M.; Lucchi, A.; Conti, B. Artemisia spp. essential oils against the disease-carrying blowfly Calliphora vomitoria. Parasites & Vectors 2017, 10, 80–90. [Google Scholar] [CrossRef]
  27. Bilia, A. R.; Flaminib, G.; Morgennic, F.; Isacchia, B.; Vincieria, F. F. GC MS Analysis of the Volatile Constituents of Essential Oil and Aromatic Waters of Artemisia annua L. at Different Developmental Stages. Nat. Prod. Commun. 2008, 3, 2075–2078. [Google Scholar] [CrossRef]
  28. Héthelyi, E. B.; Cseko, I. B.; Grósz, M.; Márk, G.; Palinkás, J. J. Chemical Composition of the Artemisia annua Essential Oils from Hungary. J. Essent. Oil Res. 1995, 7, 45–48. [Google Scholar] [CrossRef]
  29. Ornano, L.; Venditti, A.; Ballero, M.; Sanna, C.; Quassinti, L.; Bramucci, M.; Lupidi, G.; Papa, F.; Vittori, S.; Maggi, F.; Bianco, A. Chemopreventive and antioxidant activity of the chamazulene-rich essential oil obtained from Artemisia arborescens L. growing on the Isle of La Maddalena, Sardinia, Italy. Chem. Biodivers. 2013, 10, 1464–1474. [Google Scholar] [CrossRef]
  30. Said, M. E.-A.; Militello, M.; Saia, S.; Settanni, L.; Aleo, A.; Mammina, C.; Bombarda, I.; Vanloot, P.; Roussel, C.; Dupuy, N. Artemisia arborescens Essential Oil Composition, Enantiomeric Distribution, and Antimicrobial Activity from Different Wild Populations from the Mediterranean Area. Chem. Biodivers. 2016, 13, 1095–1102. [Google Scholar] [CrossRef]
  31. Lo Presti, M.; Crupi, M. L.; Zellner, B. d’A.; Dugo, G.; Mondello, L.; Dugo, P.; Ragusa, S. Characterization of Artemisia arborescens L. (Asteraceae) leaf-derived essential oil from Southern Italy. J. Essent. Oil Res. 2007, 218–224. [Google Scholar] [CrossRef]
  32. Militello, M.; Carrubba, A.; Blázquez, M. A. Artemisia arborescens L.: essential oil composition and effects of plant growth stage in some genotypes from Sicily. J. Essent. Oil Res. 2012, 24, 229–235. [Google Scholar] [CrossRef]
  33. Militello, M.; Settanni, L.; Aleo, A.; Mammina, C.; Moschetti, G.; Giammanco, G. M.; Amparo Blàzquez, M.; Carrubba, A. Chemical composition and antibacterial potential of Artemisia arborescens L. essential oil. Curr. Microbiol. 2011, 62, 1274–1281. [Google Scholar] [CrossRef] [PubMed]
  34. Chericoni, S.; Flamini. G.; Campeol, E.; Cioni, P. L.; Morelli, I. GC-MS analysis of the essential oil from the aerial parts of Artemisia verlotiorum: Variability during the year. Biochem. Syst. Ecol. 2004, 32, 423–429. [Google Scholar] [CrossRef]
  35. Williams, J. D.; Campbell, M. A.; Jaskolka, M. C.; Xie, T. Artemisia vulgaris L. Chemotypes. Am. J. Plant Sci., 2013, 4, 1265–1269. [Google Scholar] [CrossRef]
  36. Jerkovic, I.; Mastelic, J.; Milos, M.; Juteau, F.; Masotti, V.; Viano, J. Chemical variability of Artemisia vulgaris L. essential oils originated from the Mediterranean area of France and Croatia. Flavour Fragr. J. 2003, 18, 436–440. [Google Scholar] [CrossRef]
  37. Burcul, F.; Blazevic, I.; Radan, M.; Politeo, O. Terpenes, Phenylpropanoids, Sulfur and Other Essential Oil Constituents as Inhibitors of Cholinesterases. Curr. Med. Chem. 2020, 27, 4297–4343. [Google Scholar] [CrossRef]
  38. Khan, F. A.; Khan, N. M.; Ahmad, S.; Nasruddin; Aziz, R.; Ullah, I.; Almehmadi, M.; Allahyani, M.; Alsaiari, A. A.; Aljuaid, A. Phytochemical Profiling, Antioxidant, Antimicrobial and Cholinesterase Inhibitory Effects of Essential Oils Isolated from the Leaves of Artemisia scoparia and Artemisia absinthium. Pharmaceuticals 2022, 12, 1221. [Google Scholar] [CrossRef] [PubMed]
  39. Orhan, I. E.; Belhattab, R.; S¸enol, F. S.; Gülpinar, A. R.; Hosbas, S.; Kartal, M. Profiling of cholinesterase inhibitory and antioxidant activities of Artemisia absinthium, A. herba-alba, A. fragrans, Marrubium vulgare, M. astranicum, Origanum vulgare subsp. glandulossum and essential oil analysis of two Artemisia species. Ind. Crop. Prod. 2010, 32, 566–571. [Google Scholar] [CrossRef]
  40. Wang, B.; Yang, F. Sun, Q.; Yang, Z.; Zhu, L. Chemical Composition and Antiacetylcholinesterase Activity of Flower Essential Oils of Artemisia annua at Different Flowering Stage. Iran. J. Pharm. Res. 2011, 10, 265–271. [Google Scholar]
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Figure 1. GC-MS total ion chromatograms of Artemisia essential oils (AABS-A. absinthum; AABR-A. abrotanum; AANN-A. Annua; AARB-A. arborescens; AVER-A. verlotiorum; AVUL-A. vulgaris).
Figure 1. GC-MS total ion chromatograms of Artemisia essential oils (AABS-A. absinthum; AABR-A. abrotanum; AANN-A. Annua; AARB-A. arborescens; AVER-A. verlotiorum; AVUL-A. vulgaris).
Preprints 86472 g001
Table 1. Location, coordinates and year of collection Artemisia plants.
Table 1. Location, coordinates and year of collection Artemisia plants.
Species Species code Locality/Year Coordinates
Geogr. latitude (N)
Geogr. longitude (E)
Artemisia absinthium L. AABS Sinj, Croatia / 2020 43°43ʹ27.29ʹʹ
16°40ʹ28.29ʹʹ
Artemisia abrotanum L. AABR Vrgorac, Croatia / 2021 43°12ʹ36.63ʹʹ
17°24ʹ4.83ʹʹ
Artemisia annua L. AANN Split, Croatia /2020 43°31ʹ34.24ʹʹ
16°28ʹ2.95ʹʹ
Artemisia arborescens (Vaill.) L. AARB Split, Croatia / 2020 43°30ʹ30.5ʹʹ
16°25ʹ17.76ʹʹ
Artemisia verlotiorum Lamotte AVER Zagreb, Croatia /2020 45°48ʹ59.08ʹʹ
15°55ʹ55.59ʹʹ
Artemisia vulgaris L. AVUL Sinj, Croatia / 2020 43°43ʹ27.29ʹʹ
16°40ʹ28.29ʹʹ
Table 2. The chemical composition of Artemisia essential oils from Croatia.
Table 2. The chemical composition of Artemisia essential oils from Croatia.
AABS AABR AANN AARB AVER AVUL
% EO (w/w) 0.5 1.6 0.6 1.1 0.3 0.2
Compounds RI
ß-thujene 928 1.9 - 0.9 - - -
α-pinene 937 0.4 1.7 1.2 1.6 0.5 1.2
camphene 952 - 7.0 2.9 2.4 - 0.5
ß-pinene 976 - - 1.3 - 1.4 2.3
sabinene 979 - 5.2 0.9 - 0.2 1.8
2-pentylfuran 989 - 0.9 - - - -
ß-myrcene 993 - - - 2.1 - -
α-phellandrene 1006 - - - - - 0.7
α-terpinene 1018 - - 0.4 0.8 - 0.2
p-cymene 1027 0.8 1.9 0.4 0.5 1.2 2.9
limonene 1031 - - - - 0.2 0.4
γ-terpinene 1061 - - 0.6 1.4 0.3 0.4
Monoterpenes 3.1 16.7 8.6 8.8 3.8 10.4
yomogi alcohol 1000 - - 1.2 - - -
1,8-cineole 1034 1.0 3.0 16.2 - 10.9 3.2
artemisia ketone 1065 - - 22.3 - - -
cis-sabinene hydrate 1069 - - 0.3 1.5 0.3 0.3
artemisia alcohol 1086 - - 3.2 - - -
linalool 1100 0.8 - 1.5 - - -
trans-sabinene hydrate 1102 - - - - 0.3 -
trans-3-caren-2-ol 1103 - - - - 0.5 -
cis-thujone 1107 3.1 - - - 46.3 5.6
trans-thujone 1118 0.9 - - - 9.0 40.3
cis-p-menth-2-en-1-ol 1122 - 0.9 0.3 - 0.4 -
chrysanthenone 1127 - 4.7 0.3 - - -
cis-epoxy-ocimene 1136 28.8 - - - - 15.5
ß-pinone 1139 - 0.8 - - -
trans-p-menth-2-en-1-ol 1141 - 0.8 0.6 - 0.5 -
trans-sabinol 1142 0.8 - 0.6 - - 0.2
trans-epoxyocimene 1143 1.1 - - - - -
camphor 1146 - 9.5 22.0 39.5 0.7 2.7
ß-pinene oxide 1160 - - 1.2 - 0.5 -
pinocarvone 1165 - 0.9 0.6 - 0.4 -
borneol 1167 - 48.0 0.3 0.5 0.3 0.7
lavandulol 1169 - - 0.4
terpinen-4-ol 1178 0.8 - 1.2 3.2 0.7 0.5
trans-p-mentha-1(7),8-dien-2-ol 1188 0.5 - - - - -
α-terpineol 1191 - - 0.5 0.4 - -
myrtenol 1196 - - - - 0.3 0.4
myrtenal 1202 - 0.7 0.8 - - -
trans-piperitol 1210 - 1.0 - - - -
trans-carveol 1220 - - - - 2.3 -
neral 1229 - - - - 0.3 -
carvotanacetone 1245 - - - - 0.7 -
cis-chrysanthenyl acetate 1264 1.8 - - - - -
perilla aldehyde 1275 - - - - 0.5 -
isobornyl acetate 1287 - 3.6 - - - -
thymol 1293 - 0.5 - - - -
perilla alcohol 1297 - - 0.2 - 0.3 -
cis-sabinyl acetate 1299 38.5 - - - - -
Monoterpenoids 78.1 74.4 74.0 45.5 75.2 69.4
α-copaene 1377 - - 1.1 - - 0.3
ß-bourbonene 1385 - - - - - 0.2
ß-caryophyllene 1419 1.3 - 1.3 0.5 5.8 1.3
α-humulene 1454 - - - - 0.9 0.1
γ-muurolene 1477 - - 0.5 - - 0.3
γ-himachalene 1480 0.4 - - - - -
α-amorphene 1484 0.5 - - - - -
germacrene D 1485 - - 0.4 1.1 1.1 -
ß-selinene 1486 1.5 - 2.3 - 0.3 1.2
α-selinene 1496 - - - - 0.3 -
δ-cadinene 1524 - - - - 0.4 -
Sesquiterpenes 3.7 0.0 5.6 1.6 8.8 4.4
spathulenol 1577 - - 0.2 - 1.5 -
caryophyllene oxide 1582 1.3 - 5.3 - 6.0 2.5
davanone 1589 - - - - - 3.2
humulene epoxide 1608 - - 0.3 - 0.4 1.1
α-copaen-4-ol 1611 - - 0.3 - - 0.3
10-epi-γ-eudesmol 1623 - - 0.7 - - -
longifolenaldehyde 1629 - - 0.6 1.8 - 0.8
torreyol 1655 - - - - 0.5 3.7
cubenol 1656 - - 0.2 - - -
ß-bisabolol 1671 - - 0.3 - - -
eudesma-4,15(7)-dien-1ß-ol 1685 - - 0.3 0.4 0.2
chamazulene 1728 0.8 - - 33.9 - 0.2
Sesquiterpenoids 2.1 0.0 8.2 35.7 8.8 12.3
hexanal 800 - - - - - 0.3
trans-2-hexen-1-ol 853 - - - - 0.9 0.8
1-octen-3-ol 980 - - - - 0.8
phenylacetaldehyde 1046 - 0.8 - - - 0.5
benzyl isovalerate 1388 - - 1.6 - - -
eugenol 1359 - - 0.3 - 0.3
2-ethyl-4-methyl-1,3-pentadienyl benzene*# 1515 2.9 - - 0.8 - 0.2
2-ethyl-4-methyl-1,3-pentadienyl benzene*# 1616 4.5 - - 1.9 - 0.6
hexadecanoic acid 1960 - - - - - 0.3
Other Compounds 7.4 0.8 1.9 2.7 2.0 2.7
TOTAL 94.4 91.9 98.3 94.3 98.6 99.2
AABS-A. absinthum; AABR_A. abrotanum; AANN_A. Annua; AARB-A. arborescens; AVER-A. verlotiorum; AVUL-A. vulgaris; * correct isomer is not identified; # identification performed only on the basis of MS and confirmed on the basis of the identification previously performed by Juteau et al. [14]; “-“- not identified. The presented results are given as the mean of two ranalyses.
Table 3. Cholinesterase inhibition potential of Artemisia essential oils.
Table 3. Cholinesterase inhibition potential of Artemisia essential oils.
Inhibition % AABS* AABR* AANN* AARB* AVER* AVUL* huperzineA& galantamine#
AChE 29.7 49.6 55.2 41.1 34.3 54.4 90.7 78.60
BChE 33.8 47.0 35.8 33.5 31.4 23.0 58.8 40.9
Tested concentrations *1 mg/mL; &0,1 mg/mL; #5 μg/mL.
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