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

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 Artemisia plants collected in Croatia and to test their cholinesterase inhibitory potential. GC–MS analysis of the 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 the EO of A. annua, they are artemisia ketone, camphor and 1,8-cineole; in the EO of A. arborescens, they are camphor and chamazulene; in the EO of A. verlotiorum, they are cis-thujone, 1,8-cineole and trans-thujone; and in the EO of A. vulgaris, they are trans-thujone and trans-epoxy-ocimene. The EO of the five studied Artemisia species from Croatia is rich in monoterpenoid compounds (1,8-cineole, artemisia ketone, cis-thujone, trans-thujone, cis-epoxy-ocimene, camphor, borneol and cis-sabinyl acetate). The EO of A. arborescens is also rich in chamazulene. The results also showed that the tested EOs have moderate cholinesterase inhibition potential, especially the EOs of A. annua, A. vulgaris and A. abrotanum. This is the first analysis of the chemical composition of the EOs of four Artemisia plants and the first analysis of cholinesterase potential for plants collected in Croatia.

1. Introduction

The genus Artemisia (family Asteraceae) includes a large number of species distributed in Europe, Asia, Africa and North America. The 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 discovery of the sesquiterpene lactone artemisinin, which was shown to have antimalarial activity. 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 the 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 and 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 when the flower buds are dried and ground into powder and 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, researchers have paid 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 mainly affects the elderly. The decline in cognitive abilities is due to a deficiency in 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 hydrolyses acetylcholine into choline and acetate. For this reason, the 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 to isolate and identify 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 and A. vulgaris. The isolated EOs were also evaluated for their ability to inhibit 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 and A. verlotiorum) collected in Croatia, and the first test of the anticholinesterase potential of the EOs of Artemisia plants from Croatia. Research of this type will contribute to new knowledge and possibly stimulate new research related to Artemisia plants. The final goal of this research is to find a pure substance capable of increasing acetylcholine levels in the brain, improving cognitive function and alleviating symptoms in patients with Alzheimer’s dementia.

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. Location, coordinates and year of collection of Artemisia plants.

Species	Species Code	Locality/Year	Coordinates
			Geogr. Latitude (N)
			Geogr. Longitude (E)
Artemisia absinthium L.	AABS	Sinj, Croatia/2020	43°43′27.29″
			16°40′28.29″
Artemisia abrotanum L.	AABR	Vrgorac, Croatia/2021	43°12′36.63″
			17°24′4.83″
Artemisia annua L.	AANN	Split, Croatia/2020	43°31′34.24″
			16°28′2.95″
Artemisia arborescens (Vaill.) L.	AARB	Split, Croatia/2020	43°30′30.5″
			16°25′17.76″
Artemisia verlotiorum Lamotte	AVER	Zagreb, Croatia/2020	45°48′59.08″
			15°55′55.59″
Artemisia vulgaris L.	AVUL	Sinj, Croatia/2020	43°43′27.29″
			16°40′28.29″

). The collection and identification of the plant material were 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 EOs of six different Artemisia plants were isolated from previously dried plant material by hydrodistillation in a Clevenger apparatus 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 essential oils from Artemisia plants were 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 at 250 °C and the ionisation energy at 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.

Essential oil compounds were identified by comparing their retention indices with the series of n-hydrocarbons (C₈–C₄₀) analysed under the same conditions as the essential oil. Individual components were identified by comparing their mass spectra with library entries from two commercial databases, Wiley 7 MS library (Wiley, New York, NY, USA) and NIST02 (Gaithersburg, MD, USA), and by comparing their mass spectra and retention indices with published data [10]. The relative proportions of oil components (%) were calculated based on the peak areas on the chromatography column. Retention indices (RIs) were calculated based on alkane series retention times and using the equation of van den Dool and Kratz [11].

2.5. Cholinesterase Inhibitory Assay

The inhibitory effect of cholinesterase was determined against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) at a concentration of 1 mg/mL using an ELISA microplate reader by the Ellman method [12]. The method is based on the reaction of Ellman’s reagent (DTNB) and thiocholine, yielding a yellow-coloured 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

where Ae—absorbance of enzyme without an inhibitor, Abe—absorbance of a blank for enzyme without a substrate, Au—absorbance of enzyme with an inhibitor and Abu—absorbance of blank for enzyme without an inhibitor.

To compare the results obtained in testing the ChE inhibitory effect of EOs, two known good inhibitors of these enzymes, huperzine A and galantamine, were also tested.

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 and A. vulgaris) collected in Croatia were studied.

3.1. Phytochemical Profile

The 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 a coupled gas chromatography–mass spectrometry system (GC–MS).

The chemical composition of the essential oils is given in

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
Compound	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
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
Monoterpene		3.1	15.8	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	-	-	-	-	-
Monoterpenoid		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	-
Sesquiterpene		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
Sesquiterpenoid		2.1	0.0	8.2	35.7	8.8	12.3
hexanal	800	-	-	-	-	-	0.3
trans-hex-2-en-1-ol	853	-	-	-	-	0.9	0.8
oct-1-en-3-ol	980	-	-	-	-	0.8
2-pentylfuran	989	-	0.9	-	-	-	-
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	1.7	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; RI, retention index (experimental); * 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. [13]; “-“, not identified. The presented results are given as the mean of two analyses.

, while the GC–MS total ion chromatograms are shown in Figure 1. The compounds in Table 2 are grouped by compound class and by ascending retention index (RI).

The EO content 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 were the monoterpenoids cis-sabinyl acetate (38.5%) and cis-epoxy-ocimene (28.8%). All other constituents of this EO were less than 5%. Monoterpenoids were present in this EO in a high proportion of 78.1% (w/w). They were followed by other compounds (7.4%), sesquiterpenes (3.7%), monoterpenes (3.1%) and sesquiterpenoids (2.1%).

According to Orav et al. [14], four chemotype 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 flowered, dried and powdered) was previously analysed by Juteau et al. [13]. The 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 the EO of this plant collected in the southern part of neighbouring Serbia (Bela Palanka and Nis, aboveground and previously dried) showed that the main components were 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, in Piedmont (full flower, 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 was the monoterpene alcohol borneol (48.0%). Camphor (9.5%), camphene (7.0%), sabinene (5.2%) and chrysanthenone (4.7%) were also present in significant proportions. Other identified constituents of this EO accounted for less than 4%. The predominant compound class in this oil was monoterpenoids (74.4%). This was followed by monoterpenes (15.8%) and other compounds (1.7%).

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/hydroxydavanone chemotype) [17]. The EO of the A. abrotanum from Croatia was particularly rich in borneol, and we could conclude that it is a borneol chemotype. This is not the case with any other oil from this plant.

To date, not one analysis of the EO of this plant species collected in Croatia has been performed. Two analyses of the EO of this plant has been performed in neighbouring 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 were 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 flowering, air-dried), revealed 1,8-cineole (34.7%), bisabolol oxide (18.4%) and ascaridol (16.0%) as the predominant components [16].

The monoterpenoids artemisia ketone (22.3%), camphor (22.0%) and 1,8-cineole (16.2%) were identified as the dominant constituents of the EO from A. annua. Caryophyllene oxide (5.3%) and artemisia alcohol (3.2%) were also identified at 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 the 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 the EO of A. annua collected in Croatia has been performed. A few analyses have been performed in neighbouring countries. The EO of the cultivated plant collected in spring in Bosnia and Herzegovina (Kiseljak, near Sarajevo) and previously dried contained a high percentage of artemisia ketone (30.7%) and artemisia alcohol (6.5%) [20]. An analysis of this plant species cultivated near Sarajevo, Bosnia and Herzegovina (air-dried and hydrodistillated), contained artemisia ketone (28.3%) and camphor (16.9%) as the main components [19], while analysis of A. annua harvested after the flowering period from the natural habitat, air-dried and hydrodistillated after 1 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 the main components of A. annua collected near Nis, Serbia (fresh aerial parts). Radulovic et al. [24] 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. An analysis of this EO collected in Belgrade, Serbia (aerial plant material, air-dried), contained 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 the 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 the EO of A. annua collected in the northwestern Italian Alps, Piedmont (full flowering, 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 sesquiterpene camazulene (33.9%) were identified as the major constituents of the EO isolated from A. arborescens. Terpinen-4-ol (3.2%), camphene (2.4%) and ß-myrcene (2.1%) occurred in lower proportions, while the other constituents of this oil occurred in proportions of less than 2%. The dominant class of compounds in this oil were monoterpenoids (45.5%) and sesquiterpenoids (35.7%). They were followed by monoterpenes (8.8%), other compounds (2.7%) and sesquiterpenes (1.6%).

For the essential oils of A. arborescens, different chemotypes have been identified: a ß-thujone/camphor chemotype (Sardinia, Italy, around Usellus and Morocco), a chamazulene/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 analyses of the oil of this plant have been performed in neighbouring countries. Analysis of the EO of this plant (aboveground biomass of the plant, flowering stage) collected in two locations in Italy (Capo Zafferano and Termini Imerese) showed that the most abundant constituents of the EO were chamazulene (43.12 and 36.83%), ß-thujone (19.57 and 19.89%) and camphor (8.78 and 8.68%) [30]. 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; in vegetative phase; EO isolated by microwave-assisted hydrodistillation) showed that the most abundant components of this EO were camphor (21.4, 39.5 and 20.1%) and 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), belonged 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 analysed 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 northwestern Sicily, Italy, showed that the most abundant constituents of this oil (steam distillation) were 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 detected in slightly lower proportions. Other compounds of this oil were detected in amounts of less than 2.5%. The predominant compound class 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 analysed essential oil of Croatia belongs to the thujone/1,8-cineole chemotype.

To date, not one analysis of the EO from this plant has been performed on a plant collected in Croatia, but several EO analyses have been performed on plant material collected in neighbouring 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 flowering, 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 contained cis-thujone (5.6%), torreiol (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 (12.3%), monoterpenes (10.4%), sesquiterpenes (4.4%) and other compounds (2.7%). Four different chemotypes of the EO from A. vulgaris were found: one with the coexistence of ar-curcumene and α-zingiberene, two characterised by the presence or absence of thujone and santolinatriene and a fourth characterised 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), showed that the most abundant constituents of this oil at full flowering (August) were ß-thujone (20.8%), α-pinene (15.1%), 1,8-cineole (11.7%), camphor (8.7%), α-thujone (8.5%), trans-chrysanthenyl acetate (18.5%), 1,8-cineole (15.2%) and α-phellandrene (12.9%) [36]. Chemical analysis of the EO 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 1 year of storage) were 1,8-cineole (28.9%), sabinene (13.7%) and ß-thujone (13.5%) [15]. The composition of the EO of A. vulgaris collected from the northwest Italian Alps, Piedmont (full flowering, air-dried), revealed camphor (47.7%) as the dominant compound. In this EO, camphene (9.1%), verbenone (8.6%) and trans-verbenol (7.0%) were also identified as constituents in major amounts [16].

The chemical composition of the 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 were dominated by monoterpenoid components: 1,8-cineole, artemisia ketone, cis-thujone, trans-thujone, cis-epoxyocimene, camphor, borneol and cis-sabinyl acetate. In one plant species (A. arborescens), the azulene derivative chamazulene occurred as a major compound. This is a blue-violet azulene derivative that is biosynthesised from the sesquiterpene matricin.

3.2. Cholinesterase Inhibition Potential of Artemisia Essential Oils from Croatia

The ability of the EOs from Artemisia plants collected in Croatia to inhibit the enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) was tested using the Ellman method [12]. The concentration of the tested EOs in solution was 1 mg/mL, while the concentration of EOs in the reaction systems was 45.45 µg/mL. This EO concentration is the most commonly used start concentration in tests of this type of biological activity since higher concentrations often lead to turbidity in the sample and can give false results. Since the results did not show significant activity, lower concentrations of samples were not tested. The results are shown in

Table 3. Cholinesterase inhibition potential of Artemisia essential oils.

Inhibition %	AABS *	AABR *	AANN *	AARB *	AVER *	AVUL *	Huperzine A &	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

AABS, A. absinthum; AABR, A. abrotanum; AANN, A. Annua; AARB, A. arborescens; AVER, A. verlotiorum; AVUL, A. vulgaris. Tested concentrations, * 1 mg/mL; ^& 0.1 mg/mL; ^# 5 μg/mL.

.

The essential oils isolated from Artemisia from Croatia showed a moderate ability to inhibit the enzyme AChE (29.7–55.2%). Among these oils, the oil of A. annua showed the best inhibitory effect, while the EO of A. absinthium showed 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 showed a slightly weaker activity compared with the inhibition of AChE, with the exception of the 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 the cholinesterase inhibitory activity of selected Artemisia plants collected in Croatia. Only one study was conducted on the anti-AChE potential of the EOs of the tested Artemisia species collected in the areas of neighbouring countries. The flowering aerial parts of A. annua collected in late September in Pisa (Italy) along the Arno riverbank showed AChE inhibition potential IC₅₀ = 472.4 mg/L [25].

Numerous researchers have evaluated the pure compounds contained in 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 were the most potent inhibitors of these enzymes. Among them, 1,8-cineole and camphor, which were present in greater proportions in the essential oils of Artemisia plants, were quite potent inhibitors, especially of AChE. It can be concluded that these are the components of the oil that can be attributed to 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 considered, so it is difficult to say with absolute certainty which constituents of a mixture of compounds are responsible for the biological effect [37].

Few other tests have been performed on the inhibitory potential of the EOs from Artemisia plants on AChE/BChE: A. absinthium collected in Pakistan [38] and Algeria [39] and A. annua (flowers) from China [40]. Research into natural products is very valuable since most of the existing inhibitors used to treat AD are of natural origin, such as galantamine and huperzine A. Of particular interest are lipophilic natural products that easily cross the blood–brain barrier. This is exactly the case with essential oils. The results obtained in this study showed that the tested oils of Artemisia plants have a moderate effect on the ability to inhibit these enzymes, especially A. annua, A. vulgaris and A. abrotanum. This is one more confirmation that the plants of this genus are of great biomedical importance.

4. Conclusions

The chemical composition of the 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 were dominated by monoterpenoid components: 1,8-cineole, artemisia ketone, cis-thujone, trans-thujone, cis-epoxyocimene, camphor, borneol and cis-sabinyl acetate. In one plant species (A. arborescens), the azulene derivative chamazulene occurred as a major compound. Artemisia essential oils isolated from Croatia showed a moderate ability to inhibit the enzyme AChE. Among these oils, the oil of A. annua showed the best inhibitory activity compared with the known ChE inhibitors galantamine and huperzine A. The EOs isolated from A. vulgaris and A. abrotanum also showed significant inhibitory activity, especially on AChE. The inhibition of BChE by these EOs showed somewhat weaker activity compared with the inhibition of AChE, with the exception of the EO from A. absinthium.

This is the first analysis of the chemical composition of the EOs from four Artemisia plants studied and the first analysis of the cholinesterase potential of Artemisia plants collected in Croatia.