Primary Determination of the Composition of Secondary Metabolites in the Wild and Introduced Artemisia martjanovii Krasch: Samples from Yakutia

: Artemisia martjanovii Krasch is a rare representative of the genus Artemisia in Siberia and the Far East. The phytochemical composition of this endangered species is essential for its potential use in medicine. We used tandem mass spectrometry and HPLC-MS/MS methods to describe the metabolome from the stem and leaf extracts of A. martjanovii from Yakutia. The metabolome proﬁle analysis of A. martjanovii grown in the Botanical Garden of the North-Eastern Federal University, Yakutsk, Russia, and the wild A. martjanovii from Khangalassky district, Republic of Sakha (Yakutia) differed signiﬁcantly both in the polyphenol composition and other compound classes. In total, we identiﬁed 104 bioactive constituents from stem and leaf extracts, 56 compounds from the polyphenol group, and 48 from other compound classes. Twenty-seven compounds classiﬁed as polyphenol groups, i.e., ﬂavones apigenin, trihydroxy(iso)ﬂavone, salvigenin, cirsiliol, cirsilineol, nevadensin, syringetin, gardenin B, thymonin, and chrysoeriol C -hexoside; ﬂavonols: taxifolin, tetrahydroxy-dimethoxyﬂavone-hexoside, etc.; and 26 compounds from other classes are being reported for the ﬁrst time in the genus Artemisia L.


Introduction
The genus Artemisia L., or wormwood, is one of the largest genera of the family Asteraceae Dumort (Compositae Giseke).It is distributed throughout the northern hemisphere, in the temperate zone of Eurasia, in North and South Africa, and in North America.
Approximately 180 species of the genus Artemisia have been recorded on the territory of Russia [1].In the flora of Yakutia, there are thirty-six species of the genus Artemisia, five of which are listed in the "Red Data Book of Yakutia [2].In the flora of Central Yakutia, there are 22 representatives of the genus Artemisia, of which four species are protected.Seventeen species have passed the introduction test in the conditions of Central Yakutia [3].
One of the rare representatives of the genus Artemisia in Siberia and the Far East is Artemisia martjanovii Krasch, ex Poljakov.It is naturally distributed in the south of the 2 of 28 Krasnoyarsk Territory, the north-eastern part of Khakassia, and Central Yakutia.It is listed in the Red Books of the Krasnoyarsk Territory with the status "2" as a vulnerable species, mainly because of its declining and fragmented populations.In Khakassia, however, it is listed with the status "3" as a rare species, and in Yakutia with the status "3d" as an extremely rare subspecies, a relic with a limited geographical area [4].
T. E. Leonova first discovered A. martjanovii on the territory of Yakutia in 1967 on the slopes of the banks of the Lena River near the village of Bulgunnyakhtakh.At present, scattered, limited populations of the species can be found along the left bank of the Lena River near the villages of Bulgunnyakhtakh, Elanka, and Tit-Ary on sandy and rocky slopes.Studies of the ontogenesis and the age spectrum of the coenopopulation of the species show that all age stages are represented near the village of Elanka.It is a perennial shrub with lignified branched stems and annual vegetative and flowering shoots up to 20-50 cm in total height (Figure 1).The plant is covered with thick glandular hairs, the leaves are bipinnately divided, and it has a paniculate inflorescence of spherical baskets with a diameter of 4-5 mm.
Krasnoyarsk Territory, the north-eastern part of Khakassia, and Central Yakutia.It is in the Red Books of the Krasnoyarsk Territory with the status "2" as a vulnerable sp mainly because of its declining and fragmented populations.In Khakassia, howeve listed with the status "3" as a rare species, and in Yakutia with the status "3d" as a tremely rare subspecies, a relic with a limited geographical area [4].
T. E. Leonova first discovered A. martjanovii on the territory of Yakutia in 1967 o slopes of the banks of the Lena River near the village of Bulgunnyakhtakh.At pr scattered, limited populations of the species can be found along the left bank of the River near the villages of Bulgunnyakhtakh, Elanka, and Tit-Ary on sandy and slopes.Studies of the ontogenesis and the age spectrum of the coenopopulation o species show that all age stages are represented near the village of Elanka.It is a pere shrub with lignified branched stems and annual vegetative and flowering shoots up 50 cm in total height (Figure 1).The plant is covered with thick glandular hairs, the l are bipinnately divided, and it has a paniculate inflorescence of spherical baskets w diameter of 4-5 mm.
Members of the genus Artemisia L. are popularly used for their medicinal prope Within the wormwood group, almost the entire range of terpene compounds is fou the Asteraceae family.The essential oils of the studied species of wormwood accum valuable constituents.Studies have shown the presence of coumarins in the aerial p A. martjanovii.Additionally, the essential oil of the aerial part of the plant contains m terpenoids and sesquiterpenoids (pinene, δ-karene, ո-cymol, linalool, borneol, and neol acetate).These essential oil constituents have been shown to have antibacteria antifungal properties.Members of the genus Artemisia L. are popularly used for their medicinal properties.Within the wormwood group, almost the entire range of terpene compounds is found in the Asteraceae family.The essential oils of the studied species of wormwood accumulate valuable constituents.Studies have shown the presence of coumarins in the aerial part of A. martjanovii.Additionally, the essential oil of the aerial part of the plant contains monoterpenoids and sesquiterpenoids (pinene, δ-karene, η-cymol, linalool, borneol, and borneol acetate).These essential oil constituents have been shown to have antibacterial and antifungal properties.
In general, studies of the phytochemical composition of representatives of the genus Artemisia are of great importance for determining their potential use in medicine, in the development of new drugs, and in other pharmaceutical industries.Thus, the aim of this work is to conduct a comparative analysis of the chemical composition of the above-ground phytomass (leaves, stems) of A. martjanovii collected both in controlled grown conditions in the Botanical Garden of the North-Eastern Federal University (NEFU) and in the wild growing conditions in the vicinity of the settlement Elanka, Khangalassky district of Yakutia (N 61 • 26 75 ; E 128 • 11 11 ) during an expedition in June 2022 (Figure 1C).

Materials
The object of this study was the aerial parts (leaves, stems) of A. martjanovii collected both under controlled conditions in the Botanical Garden of NEFU and under wild growing conditions near the settlement Elanka, Khangalassky district, Yakutia (N 61 • 26 75 ; E 128 • 11 11 ), Russia.Leaves and stems were collected during the growing season of plants in June 2022.A. martjanovii has been cultivated in the Botanical Garden of NEFU for 19 years.The plant was introduced into cultivation based on a sample collected in 2004 from a rocky slope on the bank of the Lena River near the village of Elanka.

Chemicals and Reagents
All chemicals used in this study were of analytical grade.High-performance liquid chromatography (HPLC)-grade acetonitrile was purchased from Fisher Scientific (Southborough, UK).Mass-spectrometry (MS)-grade formic acid was purchased from Sigma-Aldrich (Steinheim, Germany).Ultra-pure water was prepared by using a SIEMENS ULTRA clear (SIEMENS Water Technologies, Günzburg, Germany).

Extraction
The fractional maceration technique was applied to obtain highly concentrated extracts [5].Approximately 500 g of the aerial parts of A. martjanovii (wild and collected in the Botanical Garden) were randomly selected for maceration.The total amount of the extractant (ethyl alcohol) was divided into three parts, and the parts of the plant were consistently infused with the first, second, and third parts.The solid-to-solvent ratio was 1:15.The infusion of each part of the extractant was completed for 10 days at room temperature.

Liquid Chromatography
HPLC was performed using a Shimadzu LC-20 Prominence HPLC (Shimadzu, Kyoto, Japan) equipped with a UV sensor and a C18 silica reverse phase column (4.6 × 150 mm, particle size: 2.7 µm) to perform the separation of multicomponent mixtures.The gradient elution program with two mobile phases (A, deionized water; B, acetonitrile with formic acid 0.1% v/v) was as follows: 0-2 min, 0% B; 2-50 min, 0-100% B; control washing 50-60 min, 100% B. The entire HPLC analysis was performed with a UV-vis detector SPD-20A (Shimadzu, Kyoto, Japan) at a wavelength of 230 nm for identification compounds; the temperature was 40 • C, and the total flow rate was 0.25 mL min −1 .The injection volume was 10 µL.Additionally, liquid chromatography was combined with a mass spectrometric ion trap to identify compounds.

Mass Spectrometry
Mass spectrometry analysis was performed on an ion trap amaZon SL (BRUKER DALTONIKS, Bremen, Germany) equipped with an ESI source in negative ion mode.The optimized parameters were obtained as follows: ionization source temperature: 70 • C, gas flow: 4 L/min, nebulizer gas (atomizer): 7.3 psi, capillary voltage: 4500 V, end plate bend voltage: 1500 V, fragmentary: 280 V, collision energy: 60 eV.An ion trap was used in the scan range m/z 100-1.700for MS and MS/MS.The chemical constituents were identified by comparing their retention index, mass spectra, and MS fragmentation with an in-house, self-built database (Biotechnology, Bioengineering, and Food Systems Laboratory, Far Eastern Federal University, Russia).The in-house, self-built database are based on data from other spectroscopic techniques, such as nuclear magnetic resonance, ultraviolet spectroscopy, and MS, as well as data from the literature that are continuously updated and revised.The capture rate was one spectrum/s for MS and two spectrum/s for MS/MS.Data acquisition were controlled by Windows software for BRUKER DALTONIKS.All experiments were repeated three times.A four-stage ion separation mode (MS/MS mode) was implemented.The structural identification of each compound was carried out on the basis of their accurate mass and MS/MS fragmentation by HPLC-ESI-ion trap-MS/MS.

Results and Discussion
We compared the global metabolome profiles of stem and leaf extracts of A. martjanovii growing under controlled conditions in the Botanical Garden of NEFU and under wild growing conditions near the settlement of Elanka, Khangalassky district, Yakutia, Russia.

Mass Spectrometry
Mass spectrometry analysis was performed on an ion trap amaZon SL (BRUKER DALTONIKS, Bremen, Germany) equipped with an ESI source in negative ion mode.The optimized parameters were obtained as follows: ionization source temperature: 70 °C, gas flow: 4 L/min, nebulizer gas (atomizer): 7.3 psi, capillary voltage: 4500 V, end plate bend voltage: 1500 V, fragmentary: 280 V, collision energy: 60 eV.An ion trap was used in the scan range m/z 100-1.700for MS and MS/MS.The chemical constituents were identified by comparing their retention index, mass spectra, and MS fragmentation with an in-house, self-built database (Biotechnology, Bioengineering, and Food Systems Laboratory, Far Eastern Federal University, Russia).The in-house, self-built database are based on data from other spectroscopic techniques, such as nuclear magnetic resonance, ultraviolet spectroscopy, and MS, as well as data from the literature that are continuously updated and revised.The capture rate was one spectrum/s for MS and two spectrum/s for MS/MS.Data acquisition were controlled by Windows software for BRUKER DALTONIKS.All experiments were repeated three times.A four-stage ion separation mode (MS/MS mode) was implemented.The structural identification of each compound was carried out on the basis of their accurate mass and MS/MS fragmentation by HPLC-ESI-ion trap-MS/MS.

Results and Discussion
We compared the global metabolome profiles of stem and leaf extracts of A. martjanovii growing under controlled conditions in the Botanical Garden of NEFU and under wild growing conditions near the settlement of Elanka, Khangalassky district, Yakutia, Russia.
We identified 104 bioactive compounds from extracts of A. martjanovii (fifty-six chemical constituents from the polyphenol group and forty-eight chemical constituents from other compound classes).The chemical structures of some tentatively identified polyphenols are shown in Figures 2-4.All the identified polyphenols and compounds from other compound classes, along with molecular formulas and MS/MS data for A. martjanovii, are summarized in Appendix A (Table A1).Polyphenols are represented by the following compound classes: flavones, flavonols, flavan-3-ols, flavanones, phenolic acids, anthocyanins, lignans, coumarins, stilbenes, and chalcones (Table 1).For the first time in the genus Artemisia L., twenty-seven compounds from the polyphenol group and twenty-six compounds from other compound classes have been tentatively identified.Notably, we found flavones: apigenin, trihydroxy(iso)flavone, salvigenin, cirsiliol, cirsilineol, nevadensin, syringetin, gardenin B, thymonin, chrysoeriol C-hexoside; flavonols: taxifolin, tetrahydroxydimethoxyflavone-hexoside, isorhamnetin 3-O-(6″-O-rhamnosyl-hexoside); flavan-3-ol (epi)-catechin; flavanones eriodictyol, (S)-eriodictyol-6-C-β-D-glucopyranoside; stilbene resveratrol; coumarins umbelliferone, fraxetin, tomentin; lignan podophyllotoxin, etc. Constituents of other compound classes include amino acids, carboxylic acids, saturated fatty acids, naphthoquinones, sesquiterpenoids, omega-3 fatty acids, oxylipins, etc.  Below is the distribution of the bioactive compounds recorded in our study in A. martjanovii samples from the wild and the Botanical Garden of NEFU.Below is the distribution of the bioactive compounds recorded in our study in A. martjanovii samples from the wild and the Botanical Garden of NEFU.Below is the distribution of the bioactive compounds recorded in our study in A. martjanovii samples from the wild and the Botanical Garden of NEFU.
Figures 2-4 show examples of the decoding spectra (Collision-Induced Dissociation (CID) spectrum) of the ion chromatogram obtained using tandem mass spectrometry.The CID-spectrum in positive ion modes of artemisinin C from extracts of leaves of wild A. martjanovii is shown in Figure 2.
The  [6].The CID-spectrum in positive ion modes of L-tryptophan from extracts of stems of wild A. martjanovii is shown in Figure 3.
The [M + H] + ion produced one fragment ion at m/z 188.17 (Figure 3).The fragment ion with m/z 188.17 yields one daughter ion at m/z 146.19.The fragment ion with m/z 146.19 yields two daughter ions at m/z 144.19 and m/z 118.23.It has been identified in the bibliography in extracts from Huolisu Oral Liquid [7], Rosa acicularis [8], Camellia kucha [9], Euphorbia hirta [10], Hylocereus polyrhizus [11], and rapeseed flower petals [12].The CIDspectrum in positive ion modes of atractylenolide II from extracts of A. martjanovii from the Botanical Garden of NEFU are shown in Figure 4.The [M + H] + ion produced three fragment ions at m/z 187.28, m/z 145.26, and m/z 119.27 (Figure 4).The fragment ion with m/z 187.28 yields three daughter ions at m/z 145.24 and m/z 131.20.The fragment ion with m/z 145.24 yields one daughter ion at m/z 130.39.It has been identified in the bibliography in extracts of Codonopsis Radix, Atractylodes macrocephalae rhizoma [13], and the Chinese herbal formula Jian-Pi-Yi-Shen pill [14].A Venn diagram showing the similarities and differences in the presence of chemical constituents in wild and introduced A. martjanovii (stems and leaf extracts) from the Botanical Garden of NEFU is shown in Figure 6.A Venn diagram showing the similarities and differences in the presence of chemical constituents in wild and introduced A. martjanovii (stems and leaf extracts) from the Botanical Garden of NEFU is shown in Figure 6.From Table 3, it can be seen that a certain number of chemical compounds were commonly detected in the stem and leaf extracts of both wild and Botanical Garden-grown A. martjanovii.These are the following constituents: undecanedioic acid; deoxyartemisinin I; gardenin B; salvigenin; caffeic acid; artemisin; dihydroxy-trimethoxyflavone; casticin; hydroxy myristic acid; pseudosantonin; dihydroxy-dimethoxyflavone; centaureidin; eupatilin; artemetin; myristoleic acid; atractylenolide I; artemisinic acid; atractylenolide II; artemisinin C; cirsimaritin.
Thus, the analyzed samples of ethanol extracts of the above-ground phytomass of A. martjanovii, growing wild and cultivated in the Botanical Garden for 19 years, showed the presence of 50 common compounds of polyphenolic nature and other groups and also contained 27 different compounds.In extracts of plants growing in the wild, a larger number of compounds from other groups were identified (38) compared to plants cultivated under artificial conditions (34).However, a larger number of polyphenolic compounds (43) were identified in cultivated plants than in wild plants (39).
In this study, the qualitative variability of compounds in the phytochemical profile of extracts from above-ground phytomass of wild and cultivated A. martjanovii may be associated with differences in geographical location (rocky slope of the riverbank and flat steppe, respectively), soil type, agronomic practices (in the case of the plants grown in the Botanical Garden of NEFU), as well as natural and anthropogenic disturbances in wild nature and artificial cultivation.
Plant secondary metabolites are formed under the influence of many environmental factors and plant growth conditions.The diversity of chemicals in plants indicates their adaptation strategy to changing conditions, and it is specific to each individual species and may vary depending on intraspecific differentiation (population differences).In this regard, A. martjanovii samples collected from two different habitats showed intraspecific variability in the qualitative composition of polyphenolic compounds and other groups of compounds identified using tandem mass spectrometry and HPLC-MS/MS methods.
The presence of flavonoids (flavones, flavonols, and flavanones), caffeic acid, chlorogenic acid, and dicaffeoylquinic acid in wild and cultivated A. martjanovii shows that this rare plant can be a potential source of antioxidant substances.This can be achieved through introductory cultivation combined with qualitative and quantitative profiling (control) of the constancy of the phytochemical composition.This will reduce the burden on the dwindling wild populations of this rare plant species.The results that we detected stilbene (resveratrol), coumarin, dihydrochalcone, lignan, amino acids, polysaccharides, an abundance of sesquiterpenoids (including santonin, artemisin, etc.), sesquiterpenoid lactones (including artemisinin C and artemannuin B), omega-3 fatty acids, oxylipins, naphthoquinones, and alkaloids (sespendol)-make A. martjanovii a potential source of these biologically active substances.In the future, however, it will be worthwhile to investigate the quantitative composition of the identified secondary metabolites.Artemisia absinthium [6]; Propolis [21]; Jatropha [22]; Rosmarinus officinalis [27]; Juglans mandshurica [29];

Figure 4 .
Figure 4. CID-spectrum of atractylenolide II from extracts of introduced A. martjanovii growing in the Botanical Garden of NEFU, at m/z 233.24.

Figure 4 .
Figure 4. CID-spectrum of atractylenolide II from extracts of introduced A. martjanovii growing in the Botanical Garden of NEFU, at m/z 233.24.

Figure 4 .
Figure 4. CID-spectrum of atractylenolide II from extracts of introduced A. martjanovii growing in the Botanical Garden of NEFU, at m/z 233.24.

27 Figure 5 .Figure 5 .
Figure 5.A Venn diagram showing the similarities and differences in the presence of chemical constituents in wild A. martjanovii and introduced A. martjanovii from the Botanical Garden of NEFU.A Venn diagram showing the similarities and differences in the presence of chemical constituents in wild and introduced A. martjanovii (stems and leaf extracts) from the Bo-

Figure 5 .
Figure 5.A Venn diagram showing the similarities and differences in the presence of chemical constituents in wild A. martjanovii and introduced A. martjanovii from the Botanical Garden of NEFU.

Figure 6 .
Figure 6.A Venn diagram showing the similarities and differences in the presence of chemical constituents in stem and leaf extracts of wild and introduced A. martjanovii from the Botanical Garden of NEFU.Tables 2 and 3 below show the distribution of the bioactive compounds in the stem and leaf extracts of wild A. martjanovii plant samples collected from near the settlement Elanka, Khangalassky district of Yakutia (N 61°26′75″; E 128°11′11″) and from the Botanical Garden of NEFU.

Figure 6 .
Figure 6.A Venn diagram showing the similarities and differences in the presence of chemical constituents in stem and leaf extracts of wild and introduced A. martjanovii from the Botanical Garden of NEFU.Tables 2 and 3 below show the distribution of the bioactive compounds in the stem and leaf extracts of wild A. martjanovii plant samples collected from near the settlement Elanka, Khangalassky district of Yakutia (N 61 • 26 75 ; E 128 • 11 11 ) and from the Botanical Garden of NEFU.

Table 1 .
The distribution of the bioactive compounds in wild and introduced A. martjanovii from the Botanical Garden of NEFU. Figure 3. CID-spectrum of L-tryptophan from stems extracts of wild A. martjanovii, at m/z 205.21.CID-spectrum of artemisinin C from leaf extracts of wild A. martjanovii, at m/z 249.24.CID-spectrum of L-tryptophan from stems extracts of wild A. martjanovii, at m/z 205.21.

Table 1 .
The distribution of the bioactive compounds in wild and introduced A. martjanovii from the Botanical Garden of NEFU.

Table 1 .
The distribution of the bioactive compounds in wild and introduced A. martjanovii from the Botanical Garden of NEFU.

Table 2 .
The distribution of the constituents in the stem and leaf extracts of wild and introduced A. martjanovii from the Botanical Garden of NEFU.

Table 3 .
The distribution of the constituents in stem and leaf extracts of wild and introduced A. martjanovii from the Botanical Garden of NEFU.

Table A1 .
Compounds were identified from the ethanol extracts of A. martjanovii in positive and negative ionization modes by HPLC-ion trap-MS/MS.

Table A1 .
Cont.Chemical constituents identified for the first time in genus Artemisia L. *