Chemical Diversity of Artemisia rutifolia Essential Oil, Antimicrobial and Antiradical Activity

This paper presents the results of the study of the composition of the essential oil (EO) of Artemisia rutifolia by the GC/MS method as well as its antimicrobial and antiradical activities. According to the PCA-analysis, these EOs can be conditionally divided into “Tajik” and “Buryat-Mongol” chemotypes. The first chemotype is characterized by the prevalence of α- and β-thujone, and the second chemotype by the prevalence of 4-phenyl-2-butanone, camphor. The greatest antimicrobial activity of A. rutifolia EO was observed against Gram-positive bacteria and fungi. The EO showed high antiradical activity with an IC50 value of 17.55 μL/mL. The presented first data on the composition and activity of the EO of A. rutifolia of the Russian flora indicate the prospects of the species as a raw material for the pharmaceutical and cosmetic industry.


Introduction
Essential oils are a mixture of volatile flavor substances belonging to different classes of organic compounds (terpenes, their oxygenated derivatives, aromatic and aliphatic compounds). These compounds can pass through biological membranes to exert antioxidant, antimicrobial, antifungal, anti-inflammatory, antiviral, and other effects [1], making EOs widely used in the pharmaceutical and cosmetic industries and increasing the demand for new natural sources of EOs.
Plants of the Artemisia L. genus, which grow abundantly in arid and semi-arid regions of Asia, can serve as a reliable natural source of EOs. A promising species is Artemisia rutifolia Steph. ex Spreng. (family Asteraceae Bercht. Et J. Presl., section Absinthium (Mill.) D.-C.), which is a semi-shrub, up to 80 cm tall with strongly branched, woody perennial stems covered with brownish grey, cracked bark [2]. It grows in Afghanistan, Kazakhstan, Kyrgyzstan, Mongolia, Nepal, Pakistan, Russia (Western and Eastern Siberia), Tajikistan, and Western Asia [3], in mountain steppes, rocky slopes, and screes [4]. On the territory of Baikal Siberia, A. rutifolia is a relict species [5], the life expectancy of which can reach 80-90 years [6]. In Kyrgyzstan folk medicine, fresh leaves have been used for toothache, and a decoction for sore throat, heart, and stomach diseases [7]. The therapeutic value of the species exhibited is due to the variety of biologically active substances it contains.
This article is the first to investigate the chemical composition of the EO of A. rutifolia, growing in Buryatia (Russia), its antimicrobial and antiradical activities, and to conduct comparative chemometric analysis.
This article is the first to investigate the chemical composition of the EO of A. rutifolia, growing in Buryatia (Russia), its antimicrobial and antiradical activities, and to conduct comparative chemometric analysis.

Chemical Diversity of EOs
Comparative analysis of the obtained data and the literature review [13][14][15][16] (Appendix A) showed that the EO of plants growing in Buryatia was similar to the EOs of Mongolian plant populations in the content of the major components, but quite different from the EOs of plants from Tajikistan.
Samples from Mongolia and Buryatia (compared to those from Tajikistan) were characterized by a sufficiently high content of camphor, which is used in creams, ointments, and lotions to relieve pain, irritation, itching, and has antifungal and antibacterial properties [17].
Samples from Mongolia and Buryatia (compared to those from Tajikistan) were characterized by a sufficiently high content of camphor, which is used in creams, ointments, and lotions to relieve pain, irritation, itching, and has antifungal and antibacterial properties [17].
It should be noted that studies of isomeric thujones (α-and β-) were previously initiated because wormwood is widely used to flavor alcoholic beverages. The most famous alcoholic beverage, absinthe, is made from Artemisia absinthium.
Thujones are known to be the main constituents of the EOs of A. absinthium [18]. It has neurotoxicity manifested by hyperactivity, tremors, and tonic convulsions [19]. The effects of thujone on the human body are related to the inhibition of GABAA receptors, leading to dose-dependent excitation and convulsions, with (−)-α-thujone having a greater ability to induce convulsions than the (+)-β-isomer; it is more likely that the convulsive effect of thujone acts on a specific receptor system [20]. For this reason, isomeric thujones were long thought to be responsible for the manifestation of the so-called "wormwood epilepsy".
Modern studies show that it is the additional components (apart from the main oneethyl alcohol) of industrially produced absinthe that do not seem to have any harmful effects on health, leaving aside the effects of ethanol on the body. Absinthe has an exceptionally high alcohol content (>50% vol.). This can lead to serious health and social problems, but it is not unique to this drink. So-called "absinthism" cannot be clearly distinguished from chronic alcoholism [21].
The content of thujones in the EO of A. absinthium can vary within a wide range. On this basis, thujone and sabinyl acetate EOs of A. absinthium were distinguished [22]. Thujone-containing and thujone-free forms are also characteristic of other wormwood species (e.g., A. campestris [23], A. molinieri [24]).
On the other hand, the discovery of thujone-free forms of A. rutifolia growing in Buryatia is important for the creation of safer medicines, cosmetics, food supplements, and therapeutic foods based on them. In addition, it allows us to understand the influence of environmental conditions on thujone biosynthesis. The currently available amount of information on the composition of EOs of A. rutifolia does not allow us to draw detailed conclusions, but we note that the formation of chemotypes occurs under the influence of a long-term and relatively uniform action of certain climatic conditions. In the course of evolution, changes in the composition of enzymes occur by replacing one or more amino acids. If the modified enzyme produces a useful product for the plant, these changes are fixed in the genes [25].
At the biochemical level, mechanisms are formed to synthesize a specific set of enzymes that contribute to the production of EO components of one or another chemotype. The biosynthesis of thujones has been studied in detail for only a few species. It is known that the first monoterpene in this transformation chain is sabinene, whose formation is catalyzed by the enzyme sabinene synthase. Furthermore, isomeric thujones are formed from isomeric sabinols, probably also from (+)-sabinone [26].
The territories of Tajikistan, Mongolia, and Buryatia (Russia), where A. rutifolia grows, belong to the arid zone of Asia. The territories of Buryatia and Mongolia belong to the eastern (and Tajikistan- time, the area where the plants were collected in Tajikistan is on the border of the western sector: the interaction of various circulation processes leads to a strong variability in the moisture regimes (there is almost no precipitation in summer). However, the climate of a particular area was influenced by meso-and microclimatic factors in addition to the macroclimatic factors.
The area of plant collection in Mongolia is located in the Great Lakes basin, the mesoclimate of which is close to the semi-arid climate of Buryatia [27]. Thus, these places where the raw materials were collected can be ranked as follows (in the order of increasing the aridity of growing conditions of plants in summer) Buryatia → Mongolia → Tajikistan.
The increasing aridity of climatic conditions will likely lead to the biosynthesis of thujones. In addition, other sabinene derivatives, transand cis-sabinene hydrates, have been found in small amounts in the EOs of A. rutifolia growing in the territories of Buryatia and Mongolia; in Mongolian plants, sabinyl acetate was found. These compounds probably block thujone biosynthesis.

Antimicrobial Activity
The antimicrobial activity of A. rutifolia EO was experimentally determined using the disc diffusion method against Gram-positive bacteria (Streptococcus pyogenes, Staphylococcus aureus, Bacillus cereus), Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica), and fungi (Aspergillus niger, Candida albicans).
The antimicrobial activity of the samples was evaluated by the diameter of the growth inhibition zones of the test strains (mm). Each sample was tested in three replicates. The test results of the antimicrobial activity of the samples are shown in Table 2. Table 2. Antimicrobial activity of essential oil from the aerial part of Artemisia rutifolia against Gram-positive, Gram-negative bacteria, and fungi. The results indicate the greatest antimicrobial activity of A. rutifolia EO against Grampositive bacteria (Streptococcus pyogenes, Staphylococcus aureus, Bacillus cereus) and fungi (Aspergillus niger, Candida albicans), with pronounced activity against Aspergillus niger.

Streptococcus pyogenes
To a lesser extent, the growth inhibition of Gram-negative bacteria (Salmonella enterica subsp. enterica, Escherichia coli) was observed. Pseudomonas aeruginosa proved to be the most resistant to the EO: no growth inhibition was observed.
The greatest antimicrobial activity of A. rutifolia EO from Buryatia was observed against the Gram-positive bacteria and fungi, which is consistent with the literature data. For example, the minimum inhibitory activity (MIC) and minimum bactericidal concentration (MBC) of the EOs of A. rutifolia from Tajikistan were previously determined to be 10 mg/mL against E. coli ATCC 25922, and 5 mg/mL against MRSA NCTC 10442 [28].
EOs of A. rutifolia from Mongolia at a concentration of 150 mg/mL (or 3 µg/disc) inhibited the growth of S. enterica by 9.3 ± 0.76 mm, B. subtillus by 10.3 ± 0.58 mm, and S. aureus by 9.6 ± 1.5 mm, thus showed moderate antimicrobial activity [16]. The target for the antimicrobial action of the EO is probably the bacterial cell wall, which is known to be fundamentally different in structure in Gram-positive and Gram-negative bacteria. The cell wall of Gram-negative bacteria contains a strong lipid layer on its surface, with which the EOs lose their antimicrobial activity [29]. Therefore, the A. rutifolia EO is recommended for use as an antimicrobial agent against Gram-positive bacteria and fungi.

Antiradical Activity
In order to evaluate the possible antiradical potential of the EO of A. rutifolia, the DPPH test (2,2-diphenyl-1-picrylhydrazyl radical inhibition) was applied. To determine the antiradical properties of the EO, a kinetic curve was constructed using the IC 50 value (Figure 4).  According to the results of the test, it was found that the EO has high antiradical activity as the IC50 value was 17.55 μL/mL.
It is considered that the antioxidant potential of EOs is exhibited mainly due to the presence of oxygenated monoterpenes (especially of phenolic structure), while sesquiterpene hydrocarbons and their oxygenated derivatives have very low antioxidant activity [30]. The EOs from Tajikistan had a better antiradical potential (IC50 = 7.91 mg/mL) [28] compared to those from Buryatia (IC50 = 17.55 μL/mL) and the content of oxygenated monoterpenes was higher in the EOs of A. rutifolia from Tajikistan.
Previously, it has been shown that EOs exhibit much greater activity than their individual components, which may be due to the high percentage of major components, and synergism between the various components of the EO including minor ones [31]. For example, wormwood EOs, whose main components are camphor and 1,8-cineol, always show antiradical activity, while camphor and 1,8-cineol individually do not [32].
It has also been shown that cineol enrichment of the secondary oil fractions of bay laurel and the cube residue of rockrose enhances their antioxidant properties [1]. In the case of A. rutifolia, we believe that the EO of the plants from Buryatia has a higher antiradical activity due to the synergistic effect.

Plant Material Collection and EO Production
The  According to the results of the test, it was found that the EO has high antiradical activity as the IC 50 value was 17.55 µL/mL.
It is considered that the antioxidant potential of EOs is exhibited mainly due to the presence of oxygenated monoterpenes (especially of phenolic structure), while sesquiterpene hydrocarbons and their oxygenated derivatives have very low antioxidant activity [30]. The EOs from Tajikistan had a better antiradical potential (IC 50 = 7.91 mg/mL) [28] compared to those from Buryatia (IC 50 = 17.55 µL/mL) and the content of oxygenated monoterpenes was higher in the EOs of A. rutifolia from Tajikistan.
Previously, it has been shown that EOs exhibit much greater activity than their individual components, which may be due to the high percentage of major components, and synergism between the various components of the EO including minor ones [31]. For example, wormwood EOs, whose main components are camphor and 1,8-cineol, always show antiradical activity, while camphor and 1,8-cineol individually do not [32].
It has also been shown that cineol enrichment of the secondary oil fractions of bay laurel and the cube residue of rockrose enhances their antioxidant properties [1]. In the case of A. rutifolia, we believe that the EO of the plants from Buryatia has a higher antiradical activity due to the synergistic effect.

Plant Material Collection and EO Production
The aerial part of A. rutifolia, collected in 2022 in the Selenginsky District (Buryatia, Russia) during the vegetation period, was used as the object of study. The voucher specimens were identified by Dr. Oleg A. Anenkhonov and deposited at the Herbarium of Institute of General and Experimental Biology SB RAS (UUH 019695, 019696). Data on the sampling locations and EO yield are presented in Table 3 (compared to data from other studies). EOs were obtained by hydrodistillation from air-dry raw materials (aboveground part of plants, for 3 h) in the year of raw material collection, according to OFS.1.5.3.0010.15 "Determination of essential oil content in medicinal plant raw materials and herbal drugs" with a modified Clevenger apparatus.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis and Principal Component Analysis (PCA)
The component composition of the EOs was determined by gas chromatography-mass spectrometry (GC-MS) using an Agilent 6890 gas chromatograph (Agilent Technologies, USA) with an HP 5973N mass-selective detector (Hewlett-Packard, Palo Alto, CA, USA) and an HP-5MS capillary column (30 m × 0.25 mm × 0.2 µm; Hewlett-Packard), as previously described in [33].
The principal component analysis (PCA) method was applied to the contents of the EO components (Sirius software package ver. 6.0, Pattern Recognition Systems, a/s, Norway).

Antiradical Activity
The antiradical activity of the EOs was determined by the DPPH test (using a stable radical, 2,2-diphenyl-1-picrylhydrazyl). Briefly, a DPPH solution (0.006% in 95% ethanol) was added to the EO of A. rutifolia (25-1000 µL/mL in ethyl alcohol) and incubated for 30 min in the dark at room temperature. The antiradical activity was then determined spectrophotometrically on a ClarioStar Plus multimode plate reader at 517 nm.
The antiradical activity (in % inhibition) was calculated using the formula: where A 0 is the absorbance of the control sample, A 1 is the absorbance of the test sample. The IC 50 index was determined using regression analysis.

Antimicrobial Activity
The antimicrobial activity of the test samples was determined by the technique of diffusion in dense nutrient media. The inoculum was prepared by the direct suspension of the daily culture colonies of each test strain in a sterile isotonic solution to a density of 0.5 according to the McFarland turbidity standard, which approximately corresponds to a load of 1-2 × 10 8 CFU/mL. The resulting microbial suspension was applied evenly to the entire surface of the nutrient medium (agar) in three directions using a sterile cotton swab.
Mueller-Hinton agar was used as a nutrient medium for microorganisms with normal nutrient requirements, and Mueller-Hinton agar with the addition of 5% defibrinated blood was used for bacteria with complex nutrient requirements (Streptococcus pyogenes). After applying the microbial suspension, sterile paper discs were placed on the agar surface and 10 µL of the test samples was applied (one sample per disc). Factory paper discs with antimicrobial additives (norfloxacin for Gram-positive bacteria, ceftazidime for Gramnegative bacteria, fluconazole for fungi) were used as the positive controls.
Cultures were incubated at 37 • C (22 • C for molds and yeasts). The results were recorded after 24 h of incubation for bacteria and 48 h for mold and yeast. To determine the antimicrobial activity of the samples tested, the diameters of the microbial growth suppression zones around the disks were evaluated. Growth inhibition zones were measured to the nearest millimeter.

Conclusions
Thus, for the first time, the chemical composition and primary biological activities of the EOs of A. rutifolia collected in Buryatia were studied. The greatest antimicrobial activity of the EOs was noted with the Gram-positive bacteria (Streptococcus pyogenes, Staphylococcus aureus, Bacillus cereus) and fungi (Aspergillus niger, Candida albicans). In addition, it showed antiradical activity, and the IC 50 index was 17.55 µL/mL. The obtained preliminary results of the antimicrobial and antiradical activities allow us to consider that A. rutifolia is a promising raw material for the pharmaceutical and cosmetic industries; however, it is necessary to carry out further studies.
The variability of plants growing within the natural habitat greatly affects the composition of essential oils. Despite the variability in the composition, the volatile substances of plants that form essential oils are the most important chemical markers that are used to solve the issues of chemosystematic or the taxonomic assignments of plants. The analysis of our own and the literature data showed that the EOs of A. rutifolia can be conditionally divided into "Tajik" and "Buryat-Mongol" chemotypes. The first chemotype is characterized by the prevalence of αand β-thujone, and the second by the high content of 4-phenyl-2butanone and camphor. The composition is highly variable and greatly depends on the geographical confinement.  Data Availability Statement: All data generated or analyzed during this study are included in this published article.

Conflicts of Interest:
The authors declare no conflict of interest.