Dracocephalum palmatum S. and Dracocephalum ruyschiana L. Originating from Yakutia: A High ‐ Resolution Mass Spectrometric Approach for the Comprehensive Characterization of Phenolic Compounds

: Dracocephalum palmatum S. and Dracocephalum ruyschiana L. contain a large number of tar ‐ get analytes, which are biologically active compounds. High performance liquid chromatography (HPLC) in combination with an ion trap (tandem mass spectrometry) was used to identify target analytes in extracts of D. palmatum S. and D. ruyschiana L. originating from Yakutia. The results of initial studies revealed the presence of 114 compounds, of which 92 were identified for the first time in the genus Dracocephalum . New identified metabolites belonged to 17 classes, including 16 phe ‐ nolic acids and their conjugates, 18 flavones, 5 flavonols, 2 flavan ‐ 3 ‐ ols, 1 flavanone, 2 stilbenes, 10 anthocyanins, 1 condensed tannin, 2 lignans, 6 carotenoids, 3 oxylipins, 2 amino acids, 3 sceletium alkaloids, 3 carboxylic acids, 8 fatty acids, 1 sterol, and 3 terpenes, along with 6 miscellaneous com ‐ pounds. It was shown that extracts of D. palmatum are richer in the spectrum of polyphenolic com ‐ pounds compared with extracts of D. ruyschiana , according to a study of the presence of these com ‐ pounds in extracts, based on the results of mass spectrometric studies.


Introduction
The genus Dracocephalum L. (family Lamiaceae) is represented on the territory of the Republic of Sakha (Yakutia) by five species-Dracocephalum jacutense Peschkova, D. nutans L., D. palmatum Stephan, D. ruyschiana L., and D. stellerianum Hiltebr [1]. These are A total of 23 compounds (phenylpropanoids, coumarins, flavonoids, and triterpenes) were isolated from a crude alcoholic extract of the aerial parts of Dracocephalum palmatum in studies by Olennikov et al. (2013) [4]. A research by Kim et al. (2020) aimed to evaluate the tumor suppressive effect of D. palmatum extract in diffuse large B cell lymphoma (DLBCL) and its underlying mechanism. The effect of D. palmatum extracts on several DLBCL cell lines significantly reduced cell viability and increased apoptosis and, at the same time, did not affect the survival of normal cells in vitro and in vivo. These studies indicate that the cytotoxic effect may be specific to cancer cells [5]. Lee et al. (2020) studied the anticancer potential of dried leaves of D. palmatum Stephan using human prostate cancer PC-3 cells. The results showed that the use of D. palmatum extract induces apoptosis and has intracellular ROS (reactive oxygen species)-independent antitumor effects on prostate cancer cells associated with increased expression of superoxide dismutase (SOD2) [6].
The habitat of Dracocephalum ruyschiana L. extends far to the north; its growth was noted in the Lena and Vilyui river basins, in grass, larch, birch, and mixed forests and meadow steppes. This species has erect stems 20-55 cm high, sparsely shortly pubescent at the nodes and in the upper part, with shortened vegetative shoots in the leaf axils. Dracocephalum ruyschiana forms continuous "carpet" populations in the Amga River valley in the conditions of Central Yakutia (N 60°31′09.0″ E 131°26′26.7″) ( Figure 2). Kakasy et al. (2006) identified the composition of D. ruyschiana L. extracts using HPLC and GC-MS with particular emphasis on their flavonoids, aliphatic, aromatic carboxylic acids, and sugars. GC-MS analysis identified and quantified as the main components monosaccharides, sugar alcohols, disaccharides, and trisaccharides, 33 components in total [7].
A review by Zeng et al. (2010) is devoted to the study of the chemical compositions of plants of the genus Dracocephalum L. Since the 1970s, 246 compounds, including terpenoids, steroids, flavonoids, alkaloids, lignans, phenols, and coumarins, have been identified from the genus Dracocephalum. As can be seen, terpenoids are the dominant constituents within the genus Dracocephalum [8]. Five new flavone tetraglycosides, 5 new benzyl alcohol glycosides, and 19 known compounds were isolated from the extract of the aerial parts of D. ruyschiana. D. ruyschiana L. (Lamiaceae) is a traditional medicinal plant in Mongolia [9].
In this work, we used an HPLC-MS/MS-ion trap to carry out a phytochemical study involving a detailed metabolomic and comparative analysis of D. palmatum and D. ruyschiana extracts. Aboveground, phytomass of D. palmatum was collected during expedition work on the territory of the Pole of Cold Oymyakon during the period of seed ripening (from 15 to 25 July 2019). Phytomass of D. ruyschiana was collected on the territory of the river Amga, Yakutia, in June 2019.

Results
Extracts of D. palmatum S. and D. ruyschiana L. were analyzed by an HPLC-MS/MS ion trap to better interpret the diversity of available phytochemicals. All of them have a rich bioactive composition. 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. A total of 114 compounds were successfully characterized in extracts of D. palmatum and D. ruyschiana based on their accurate MS and fragment ions by searching online databases and the reported literature.

Tetrahydroxyflavones
The flavone luteolin (compound 5) has already been characterized as a component of Eucalyptus [16], and Triticum aestivum [17]. The flavone luteolin was found in extracts of D. palmatum and D. ruyschiana. The CID spectrum in positive ion modes of luteolin from extracts of D. palmatum is shown in Figure 4. The [M + H] + ion produced two fragment ions at m/z 152 and m/z 237 ( Figure 4). It was identified in the bibliography in extracts of Eucalyptus [16], and Triticum aestivum [17].

Tetrahydroxyflavone
The flavonol kaempferol (compound 31) has already been characterized as a component of potato leaves [37], and rapeseed petals [38]. Flavonol kaempferol was found in extracts of D. palmatum and D. ruyschiana.

Hexahydroxyflavone
The hexahydroxyflavone ampelopsin (compound 34) has already been characterized as a component of Impatiens glandulifera Royle [39]. It was identified in extracts of D. palmatum. The CID spectrum in positive ion modes of ampelopsin from extracts of D. palmatum is shown in Figure 8.

Dihydroflavonols
The dihydroflavonols dihydrokaempferol (compound 32) and dihydroquercetin (compound 33) have already been characterized as a component of strawberry [40] and Solanum tuberosum [41]. The flavonols dihydrokaempferol and dihydroquercetin were Intens.   Figure 9). The fragment ion with m/z 269 yields two daughter ions at m/z 267 and m/z 183. This compound was identified in the bibliography in extracts from of strawberry [40] and Solanum tuberosum [41].

Materials
Aboveground, phytomass of D. palmatum S. was collected during expedition work on the territory of the Pole of Cold Oymyakon during the period of seed ripening (from 15 to 25 July 2019). Phytomass of D. ruyschiana L. was collected on the territory of the river Amga, Yakutia, in June 2019. The identification of the species was carried out by E. G. Nikolin, PhD (IBPK SB RAS). All samples were morphologically authenticated according to the current standard of Pharmacopoeia of the Eurasian Economic Union [44]. Herbariums of plants are kept in the collection of the educational and scientific laboratory "Molecular Genetic and Cellular Technologies" of the Institute of Natural Sciences of North-Eastern Federal University (Yakutsk, Republic of Sakha (Yakutia), Russian Federation).

Chemicals and Reagents
HPLC-grade acetonitrile was purchased from Fisher Scientific (Southborough, UK), and MS-grade formic acid was from Sigma-Aldrich (Steinheim, Germany). Ultrapure water was prepared from a Siemens Ultra Clear (Siemens Water Technologies, Munich, Germany), and all other chemicals were analytical grade.

Fractional Maceration
Fractional maceration technique was applied to obtain highly concentrated extracts [45]. From 500 g of the sample, 10 g of leaves was randomly selected for maceration. The total amount of the extractant (ethyl alcohol of reagent grade) was divided into three parts and consistently infused to the grains with the first, second, and third parts. A solid-solvent ratio was 1:20. The infusion of each part of the extractant lasted 7 days at room temperature.

Mass Spectrometry
MS analysis was performed on an ion trap, amaZon SL (Bruker Daltonics, Bremen, Germany), equipped with an ESI source in negative and positive ion modes. 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. A four-stage ion separation mode (MS/MS mode) was implemented. An ion trap was used in the scan range m/z 100-1.700 for MS and MS/MS. All experiments were repeated three times. A four-stage ion separation mode (MS/MS mode) was implemented.

Conclusions
The extracts of D. palmatum S. and D. ruyschiana L. contain a large number of polyphenolic complexes, which are biologically active compounds. For the most complete and safe extraction, the method of maceration with MeOH was used. To identify target analytes in extracts, HPLC was used in combination with an ion trap. The results of the preliminary study showed the presence of 114 compounds corresponding to the genus Dracocephalum, of which 92 were identified for the first time in the genus Dracocephalum L.