Chemodiversity of Arctic Plant Dryas oxyodonta: LC-MS Profile and Antioxidant Activity

Dryas oxyodonta Yuz. is a perennial evergreen shrub from the Rosaceae family. D. oxyodonta thrives in subalpine and subarctic regions, as well as in highlands spanning from Central Asia to Siberia and Mongolia. Owing to a lack of information on its chemical composition, we conducted qualitative and quantitative chromatographic analyses on extracts from the leaves and flowers of D. oxyodonta sourced from various Siberian habitats. Employing high-performance liquid chromatography with photodiode-array detection and electrospray ionization triple-quadrupole mass spectrometric detection, we identified 40 compounds, encompassing gallotannins, hydroxycinnamates, procyanidins, catechins, flavonoids, and triterpenes. All Siberian populations of D. oxyodonta exhibited a notable abundance of phenolic compounds. Furthermore, we identified rare glycosides, such as sexangularetin and corniculatusin, as potential markers of the chemodiversity within the Dryas genus. Extracts from the flowers and leaves were effective scavengers of free radicals, including DPPH•, ABTS•+−, O2•−, and •OH radicals. Our findings unequivocally establish D. oxyodonta as a rich source of phenolic compounds with potent antioxidant activity, suggesting its potential utility in developing novel functional products.


Introduction
The Rosaceae family is horticulturally important, containing various economically significant fruiting and ornamental species [1].Chemotaxonomic investigations into Rosaceae have been conducted worldwide, examining botanical [2], genomic [3], and chemical perspectives [4].Despite extensive research, debates persist regarding the classification of certain genera within the Rosaceae family.One such controversial genus is Dryas, which belongs to the subfamily Dryadoideae [5], although it has historically been classified as a separate family (Dryadaceae), tribe (Dryadeae), or subtribe (Dryadinae) [6].Thriving predominantly in the cold regions of the Northern Hemisphere, particularly in subalpine and subarctic zones, the Dryas genus plays a significant role in the vegetation of high-mountain arctic and alpine tundras, often symbolizing these environments [7].The ability of Dryas to form dense alpine thickets is likely attributable to the structure of its fruit-an achene characterized by a long, persistent, and feathery shaft.Dryas has limited distribution in snowy conditions because achenes fall very close to the parent plant [8].
Dryas oxyodonta Juz. is a perennial evergreen shrub, forming cushion-shaped growths with creeping, branched stems reaching heights of up to 8 cm (Figure 1).Its leaves, simple and petiolate, have oblong blades measuring 1-3 cm in length.These leaves exhibit a two-toned appearance, with dark green tops and whitish, tomentose undersides adorned with blunt serrations along the edges.The solitary white flowers bloom on 3-6-centimeterlong peduncles [9].D. oxyodonta is distributed in subalpine and subarctic regions and in highlands stretching from Central Asia to Siberia and Mongolia [10].Among Siberian ethnic groups, particularly the Yakuts and Buryats, D. oxyodonta is used in traditional folk medicine for treating diarrhea and aiding digestion [11,12].
The similarity in chemical composition and the identification of patterns in the metabolomes of plant species from the same family offer insights into their chemodiversity.Chemical compounds aid in species identification and quality control of herbal medicinal products that are increasing in popularity [16].To perform chemodiversity studies, advanced analytical tools like high-performance liquid chromatography with photodiode-array detection and electrospray ionization triple-quadrupole mass spectrometric detection (HPLC-PDA-ESI-tQ-MS) are indispensable, serving as instrumental tools for fingerprinting plant extracts.
As part of our ongoing investigation into the metabolomes of Rosaceae family members [17][18][19], we conducted, for the first time, a comprehensive qualitative and quantitative chromatographic analysis of extracts from D. oxyodonta's leaves and flowers, collected from diverse Siberian habitats.Employing HPLC-PDA-ESI-tQ-MS, we evaluated these extracts for their chemical constituents and antioxidant potential.Additionally, we identified specific chemotaxonomic markers characteristic of the Dryas genus.
The similarity in chemical composition and the identification of patterns in the metabolomes of plant species from the same family offer insights into their chemodiversity.Chemical compounds aid in species identification and quality control of herbal medicinal products that are increasing in popularity [16].To perform chemodiversity studies, advanced analytical tools like high-performance liquid chromatography with photodiode-array detection and electrospray ionization triple-quadrupole mass spectrometric detection (HPLC-PDA-ESI-tQ-MS) are indispensable, serving as instrumental tools for fingerprinting plant extracts.
As part of our ongoing investigation into the metabolomes of Rosaceae family members [17][18][19], we conducted, for the first time, a comprehensive qualitative and quantitative chromatographic analysis of extracts from D. oxyodonta's leaves and flowers, collected from diverse Siberian habitats.Employing HPLC-PDA-ESI-tQ-MS, we evaluated these extracts for their chemical constituents and antioxidant potential.Additionally, we identified specific chemotaxonomic markers characteristic of the Dryas genus.

Metabolome of Dryas oxyodonta
The analysis of metabolites in D. oxyodonta extracts was conducted using the HPLC-PDA-ESI-tQ-MS methodology.This comprehensive analysis allowed us to identify 40 distinct compounds (Figures 2 and S1; Table 1).The identification process adhered to the recommended minimum reporting standards for chemical analysis, as outlined by the Chemical Analysis Working Group.Specifically, compounds were identified using reten-tion times, UV and MS spectra, and by comparison with standard compounds and the existing literature [20].Metabolite identification was performed at two levels, with nineteen compounds fully characterized at the first level and twenty-one provisionally annotated at the second level.
Plants 2024, 13, x FOR PEER REVIEW 3 of 15 recommended minimum reporting standards for chemical analysis, as outlined by the Chemical Analysis Working Group.Specifically, compounds were identified using retention times, UV and MS spectra, and by comparison with standard compounds and the existing literature [20].Metabolite identification was performed at two levels, with nineteen compounds fully characterized at the first level and twenty-one provisionally annotated at the second level.

Gallotannins, Hydroxycinnamates, Procyanidins, and Catechins
The analysis of D. oxyodonta revealed representatives from six compound groups, encompassing gallotannins, hydroxycinnamates, procyanidins, catechins, flavonoids, and triterpenes.Notably, two gallotannins were discerned, each at varying levels of identification.The presence of 1-O-galloyl glucose (3), also known as glucogallin, was confirmed by comparing retention times and spectral characteristics with a reference standard.Additionally, the nature of galloyl glucose (2) was elucidated by comparing UV and MS data with the existing literature, particularly focusing on the deprotonated ion and the loss of particles with m/z 152 (a gallic acid fragment) [22].
Previously, the flavonoids hyperoside, avicularin, and guaijaverin were identified in D. octopetala leaves [14].It is also worth noting that the aglycones quercetin, kaempferol, corniculatusin, and sexangularetin were previously discovered in the same plant [13].However, in the D. oxyodonta extract, we only identified flavonoid glycosides.Therefore, the compounds reynoutrin, trifolin, and juglanin were identified for the first time in the Dryas genus.

Triterpenes
Four triterpenes were discerned in the D. oxyodonta extract, including tormentic (35), corosolic (36), and ursolic (37) acids.Their identification was achieved by comparing their UV spectra, MS data, and t R with those of reference compounds.The ursolic acid isomer (39) was provisionally annotated by comparing its UV and MS spectra with data from the literature.Notably, triterpenoids have not been previously reported in species of the Dryas genus.
Thus, this study's metabolomic profile of the D. oxyodonta extract provides comprehensive insights into its chemical composition and unveils novel compounds within the Dryas genus.

Chemodiversity Significance of D. oxyodonta Metabolites for the Dryas Genus
We attempted to identify specific markers of chemodiversity for the Dryas genus to clarify its potential taxonomic position from a chemical perspective.Our investigation focused on several compound groups (gallotannins, hydroxycinnamates, procyanidins, catechins, triterpenes, and flavonoids), comparing them with existing data on D. octopetala and representatives from other subfamilies.
Gallotannins are widely distributed in representatives of the Rosaceae family [32].Gallotannins, in particular glucogallin, are known to participate in the biosynthesis of 1,2,3,4,6-pentagalloylglucose, a precursor to ellagitannins [33] frequently found in the Rosoideae subfamily [19,34,35].Notably, ellagitannins were not detected in D. oxyodonta despite the fact that the genus Dryas was previously included in the Rosoideae subfamily [36].Given the widespread distribution of gallotannins in the Rosaceae family, their presence in the Dryas genus does not serve as a unique chemodiversity marker.Moreover, the absence of 2-pyrone-4,6-dicarboxylic acid in D. oxyodonta extracts is noteworthy, because this compound is a chemotaxonomic marker of the Rosoideae subfamily and has been identified in numerous genera, such as Agrimonia, Filipendula, Fragaria, Geum, Potentilla, Rosa, Rubus, and Sanguisorba [37,38].
Hydroxycinnamates are common metabolites in the Rosaceae family [39][40][41].Specifically, caffeic, coumaric, and ferulic acid derivatives are prevalent within the Rosoideae subfamily [42][43][44][45].Although derivatives of hydroxycinnamic acids were identified in the D. oxyodonta extract, there are no records of their discovery in D. octopetala, likely owing to the lack of comprehensive knowledge about this species.Procyanidins and catechins, characteristic of the Dryadaceae subfamily, have also been found in other subfamilies, such as Rosoideae and Amygdaloideae, across genera like Agrimonia [46], Prunus [47], Malus [48], and Pyrus [49].Therefore, establishing a distinct chemodiversity pattern for procyanidins and catechins is challenging due to their ubiquitous presence in the plant metabolome.This situation mirrors the one observed with triterpenes.In D. oxyodonta, all detected triterpenes belonged to the ursane type, a common triterpenoid found in various members of the Rosaceae family [19,50,51].Consequently, this triterpenoid type cannot serve as a distinguishing criterion for chemodiversity within the Dryas genus.
The presence of derivatives of the flavonols quercetin and kaempferol is a common characteristic of the Rosaceae family, as validated by this study [52].Additionally, derivatives of the flavonols sexangularetin and corniculatusin are more specific to the Dryas genus, identified in D. oxyodonta in this study and previously in D. octopetala [14].Previously, the presence of sexangularetin derivatives has been observed in certain representatives of the Amygdaloideae (genera Crataegus, Sorbus, and Prunus) [1,53] and Rosoideae (Fragaria) subfamilies [54].Conversely, corniculatusin derivatives have only been documented within the Rosaceae family in the Dryadoideae subfamily, specifically in the Cowania, Purshia, and Dryas genera [14,55].Consequently, sexangularetin and corniculatusin glycosides are a phytochemical fingerprint for the Dryas genus, because these 8-methoxyflavonol derivatives have not been observed together in other species of the Rosaceae family.A similar occurrence of sexangularetin and corniculatusin in one botanical specimen has previously been reported for Lotus corniculatus (Fabaceae) [56,57].Thus, the phenolic fingerprint pro-files, specifically the flavonol glycosides of sexangularetin and corniculatusin, along with the presence of gallotannins and the absence of ellagitannins and 2-pyrone-4,6-dicarboxylic acid, could serve as markers in chemodiversity and potentially chemotaxonomy investigations within the Dryas genus.Regarding the question of whether the Dryas genus should be included in a separate subfamily, it is important to note that the metabolome of the studied representatives of this genus differs from existing data on the chemical composition patterns of members of other subfamilies within the Rosaceae family.This difference justifies the presence of a separate subfamily for Dryas.

Quantitative Analysis of Metabolites in Dryas oxyodonta Extracts
For the quantitative analysis of compounds using HPLC-PDA-ESI-tQ-MS, as well as for a comparative qualitative analysis of the components, samples of D. oxyodonta (flowers and leaves) were collected from three different regions of the high-mountain alpine tundra belt in Siberia: Sakha (1000 m), Buryatia (1900 m), and Altai (2300 m) (Table 2).These regions have a sharply continental climate, wide daily and annual temperature fluctuations, and moderate precipitation [58,59].Procyanidin B1 was identified as the primary compound in both flowers and leaves of D. oxyodonta collected from the Sakha region (55.11 and 73.84 mg/g, respectively).Additionally, both flowers and leaves of the same samples exhibited notably high levels of (−)-epicatechin (26.84 and 52.17 mg/g, respectively) and (+)-catechin (14.82 and 45.77 mg/g, respectively).Furthermore, the analysis revealed that the hyperoside content in D. oxyodonta flowers exceeded that in leaves by more than fivefold (43.27 vs. 8.21 mg/g, respectively).The primary compound in D. oxyodonta flowers from Buryatia was identified as hyperoside (58.33 mg/g), while (−)-epicatechin accumulated in the leaves (73.15 mg/g).High levels of procyanidin B1 and (+)-catechin were also observed in both flowers and leaves, similar to the samples from Sakha.However, the flowers from Altai had a different composition, with the flavonols avicularin (27.82 mg/g) and hyperoside (27.63 mg/g) being the predominant compounds.In the leaves from Altai and those from Buryatia, epicatechin was the dominant compound, with a concentration of 35.11 mg/g.However, similar to the samples from other regions, the flowers and leaves of specimens from Altai also exhibited high levels of procyanidin B1 and (+)-catechin.The overall flavonoid content was highest in the D. oxyodonta flowers from Altai samples (123.91 mg/g), and the maximum concentration of phenolic compounds was observed in the flowers from Buryatia (257.80 mg/g).
Notably, all of the studied samples contained potential marker compounds, namely, glycosides of sexangularetin and corniculatusin.Higher concentrations of glycosides were observed in the flowers, while the leaves contained lesser amounts or even trace quantities.This pattern of sexangularetin and corniculatusin derivatives' accumulation in flowers was previously described for L. corniculatus, where the authors proposed it as a taxonomic and ecological characteristic [56].However, quercetin and kaempferol unsubstituted at the 8 position accumulated maximally in L. corniculatus leaves, a trend that was not observed in the leaves of D. oxyodonta.Therefore, all populations of D. oxyodonta collected in Siberia exhibited a high concentration of phenolic compounds, indicating the presence of antioxidant activity.

Antioxidant Activity of D. oxyodonta Extracts
We conducted a comparative analysis of the antioxidant potential of D. oxyodonta extracts derived from flowers and leaves collected from various locations in Siberia (Table 3).The scavenging capacities of the analyzed extracts were assessed using four radical assays: DPPH •− , and • OH.D. oxyodonta extracts from flowers collected in Buryatia and Sakha exhibited the most pronounced antioxidant activity, while leaf extracts from the same locations showed less pronounced activity.These results align with expectations because the active extracts are characterized by high levels of potent antioxidants such as catechins, flavonoids, and procyanidins [60,61].In a previous study, the highest antioxidant activity among the examined species of Arctic plants was found in the D. octopetala extract [62].Consequently, herbal tea made from the vegetative parts of D. oxyodonta can be considered to be a valuable source of antioxidants, owing to its high phenolic compound contents.

Plant Material
Plant samples of Dryas oxyodonta (flowers and leaves) were collected from three different regions of Siberia: the Republic of Buryatia, Tunkinsky District ( ).Samples were collected at eight points in the alpine tundra (12-14 samples each).Fresh materials were dried in an IPLS-131 drying oven (Bestek Engineering LLC, Rostov-on-Don, Russia) under the following conditions: convection mode, 40 • C.After the samples reached a humidity of 9-12%, they were stored in an Edry D-450A auto-drying cabinet (Edry Co., Ltd., Taichung, Taiwan) before HPLC separation.

Extract Preparation
To prepare the D. oxyodonta extracts, 10 g of the ground plant materials (leaves and flowers) was treated twice with 100 mL of 70% methanol using an ultrasonic bath (Sapphire Ltd., Moscow, Russia) with the following sonication parameters: 30 min, 40 • C, frequency 35 kHz, and ultrasound power 100 W. The obtained liquid extracts were combined and centrifuged.The supernatants were filtered through cellulose filters and concentrated until dryness.The yields of the D. oxyodonta extracts were 4.3 g (Altai flower extract), 4.6 g (Altai leaf extract), 3.8 (Buryatia flower extract), 4.1 (Buryatia leaf extract), 4.4 g (Sakha flower extract), and 4.2 (Sakha leaf extract).The final dry extracts were conserved at 4 • C for subsequent usage in chromatographic experiments and antioxidant activity studies.

Liquid Chromatography-Mass Spectrometry Detection of Metabolites in D. oxyodonta Extracts
Fingerprinting of D. oxyodonta metabolites was carried out using liquid chromatographymass spectrometry (Table S1).LabSolutions LCMS software (ver.5.6) was applied to operate the LC-MS system [65].To identify metabolites, a set of chromatographic and spectral parameters (retention time and UV/MS spectra, respectively) were analyzed in comparison with data from reference compounds, data from the literature, and our own mass spectrometry library.For the preparation of the analyzed solution, D. oxyodonta extract (5 mg) was dissolved in methanol in a volumetric flask (5 mL) by shaking, followed by filtration through syringe filters with a pore size of 0.22 µm.

Statistical Analysis
Statistical analyses were carried out with the usage of one-way analysis of variance.Duncan's multiple range test was applied to find the significance of the mean differences.Differences were presumed to be statistically significant at p < 0.05.The results were provided as the mean ± S.D. Advanced Grapher 2.2 (Alentum Software, Inc., Ramat Gan, Israel) was applied for linear regression analysis, as well as for generating the calibration graphs.

Conclusions
The Dryas oxyodonta species thrives across extensive territories in the subalpine and subarctic zones of the Northern Hemisphere, creating valuable reserves.This study elucidates the chemical composition of this species, which has not been previously examined.In exploring the chemodiversity of D. oxyodonta, we established the marker role of certain rare flavonoids.The propensity of Dryas to accumulate phenolic compounds, alongside its associated high antioxidant activity, positions the studied species as a promising source of raw medicinal plant materials.
a b Identification levels: 1 identified compounds after comparison of UV and MS data and retention times with reference standards; 2 putatively annotated compounds after comparison of UV and MS data with data from the literature.

Table 2 .
Contents of compounds 1-40 in leaves and flowers of Dryas oxyodonta collected from three locations in Siberia, mg/100 g FW (±S.D.).
a Regression equation: y = a × x + b.