Caucasian Dragonheads: Phenolic Compounds, Polysaccharides, and Bioactivity of Dracocephalum austriacum and Dracocephalum botryoides

Dracocephalum botryoides Steven and Dracocephalum austriacum L. are unexplored species of the Dracocephalum genus (Lamiaceae family) with a distribution in the Caucasus, where they are used in folk medicine and local cuisine. There are no data on the chemical composition of these Dracocephalum species. In this study, the application of a liquid chromatography-mass spectrometry technique for the metabolite profiling of methanol extracts from herbs and roots of D. austriacum and D. botryoides resulted in the identification of 50 compounds, including benzoic acid derivatives, phenylpropanoids, flavonoids and lignans. Water-soluble polysaccharides of the herbs and roots of D. austriacum and D. botryoides were isolated and characterized as mostly pectins with additive arabinogalactan-protein complexes and starch-like compounds. The antioxidant potential of the studied extracts of Dracocephalum and selected phenolics and water-soluble polysaccharides were investigated via radical-scavenging and ferrous (II) ion chelating assays. This paper demonstrates that herbs and roots of D. austriacum and D. botryoides are rich sources of metabolites and could be valuable plants for new biologically active products. To the best of our knowledge, this is the first study of whole plant metabolites and their antioxidant activity in D. austriacum and D. botryoides.


Introduction
For thousands of years, medicinal plants have been a valuable source of therapeutic agents, and they remain an important basis for the discovery of modern medicines [1]. The practice of herbal medicine builds on indigenous knowledge of the use of native plants for both the prevention and treatment of diseases. Local people have developed effective methods for identifying, collecting, using, and conserving medicinal plants and their habitats [2]. The implementation of ethnopharmacological and phytochemical studies on the study of plants from the local flora is an important task in the search for promising medicinal raw materials [3].
The Caucasus is a floristically diverse region and is of interest to modern researchers due to its extensive thickets of medicinal plants and the widespread use of local plant resources as medicinal plant raw materials [4]. Plants of the Lamiaceae family, and the Dracocephalum genus (dragonhead) in particular, are among the most used in folk medicine in the Caucasus [5,6]. There are 74 species in the Dracocephalum genus [7], with about 30 species of economic significance and used as medicinal plants [8]. This genus is of interest to researchers due to its wide range of biological activities, such as anti-inflammatory [9,10], 30 species of economic significance and used as medicinal plants [8]. This genus is of interest to researchers due to its wide range of biological activities, such as anti-inflammatory [9,10], antibacterial [11], antioxidant [12], etc. The genus Dracocephalum is represented in the Caucasus, in particular Azerbaijan, by six species, among which Dracocephalum botryoides Steven and Dracocephalum austriacum L. are of special interest as they have not been investigated ( Figure 1) [13]. Dracocephalum botryoides is a perennial plant with numerous simple and branched pubescent stems, 10-20 cm in height; ovate-rounded leaves, 1-1.5 cm long and 0.8-1.2 cm wide; and purple flowers on short pedicels with false whorls. It grows on rocky slopes, near streams and on rocks in the alpine belt, at an altitude of 2500-3600 m. Dracocephalum austriacum is a perennial plant with single or several pubescent stems, 20-60 cm in height; linear or lanceolate leaves, 2-3 cm long and 1-2.5 mm wide; and dark purple flowers on short pedicels with false whorls. It grows on limestone and rocky slopes, on steppe and subalpine meadows up to 2400 m [14]. Freshly harvested D. austriacum and D. botryoides are applied as a spice in Azerbaijani cuisine, and the fresh herb of D. botryoides is used in vegetable salads. These Dracocephalum species are used in the preparation of the famous Azerbaijani dish kükü, which is made by some local people from eggs, fresh herbs, butter, and milk [15]. Moreover, one of the features of the first dishes of Azerbaijani cuisine is their use for medicinal purposes. Thus, xəmiraşı flour soup, with the addition of dragonhead, was used in Azerbaijani villages in ancient times to treat diseases of the respiratory and gastrointestinal tracts [16]. Furthermore, an herb infusion of D. botryoides was applied in liver diseases, gastritis, and ulcers, while an herb infusion of D. austriacum was employed as an anti-inflammatory and wound healing remedy in Azerbaijani folk medicine [17].
There is information about the essential oils for both Dracocephalum species [18] and germacrone for D. botryoides [19]. However, the chemical data known about the Dracocephalum genus makes it possible to characterize some species of this genus as known and valuable sources of bioactive components. Earlier phytochemical studies of the genus Dracocephalum reported the presence of flavonoids, terpenoids, alkaloids, lignans, coumarins, polysaccharides, and cyanogenic glycosides [9,20]. An increased interest in phenolic Freshly harvested D. austriacum and D. botryoides are applied as a spice in Azerbaijani cuisine, and the fresh herb of D. botryoides is used in vegetable salads. These Dracocephalum species are used in the preparation of the famous Azerbaijani dish kükü, which is made by some local people from eggs, fresh herbs, butter, and milk [15]. Moreover, one of the features of the first dishes of Azerbaijani cuisine is their use for medicinal purposes. Thus, terest to researchers d tory [9,10] There is informa germacrone for D. bot alum genus makes it valuable sources of flour soup, with the addition of dragonhead, was used in Azerbaijani villages in ancient times to treat diseases of the respiratory and gastrointestinal tracts [16]. Furthermore, an herb infusion of D. botryoides was applied in liver diseases, gastritis, and ulcers, while an herb infusion of D. austriacum was employed as an anti-inflammatory and wound healing remedy in Azerbaijani folk medicine [17].
There is information about the essential oils for both Dracocephalum species [18] and germacrone for D. botryoides [19]. However, the chemical data known about the Dracocephalum genus makes it possible to characterize some species of this genus as known and valuable sources of bioactive components. Earlier phytochemical studies of the genus Dracocephalum reported the presence of flavonoids, terpenoids, alkaloids, lignans, coumarins, polysaccharides, and cyanogenic glycosides [9,20]. An increased interest in phenolic compounds was noted when analysing chemical information on various Dracocephalum species, which can be explained by their good antioxidant activity [12,[21][22][23].
As part of the ongoing work involving the metabolomic study of the Dracocephalum genus [24][25][26][27], we realized the first analysis of herb and root extracts of D. austriacum and D. botryoides using high-performance liquid chromatography with diode array and electrospray triple quadrupole mass detection (HPLC-PDA-ESI-QQQ-MS). Water-soluble polysaccharides (WSPS) of the herb and roots of D. austriacum and D. botryoides were also investigated. As most of the metabolites found in D. austriacum and D. botryoides were phenolic compounds, the antioxidant potential of the studied extracts of Dracocephalum, as well as WSPS and selected phenolic compounds, was studied using five in vitro models. To the best of our knowledge, this is the first study of whole plants metabolites and their antioxidant activity for D. austriacum and D. botryoides.

LC-MS Profiles of Herb and Root Extracts of Two Dracocephalum Species
Chromatographic profiles of herbs and roots of D. botryoides and D. austriacum were realized by high-performance liquid chromatography with photodiode array and electrospray ionization mass spectrometric detection (HPLC-PDA-ESI-QQQ-MS). Compounds of both Dracocephalum species were identified after a precise interpretation of the chromatographic and spectral data (using retention times and ultraviolet-visible spectra/mass spectral patterns, respectively) in comparison with reference standards and literature data. The obtained HPLC-PDA-ESI-QQQ-MS chromatograms of herb and root extracts from D. austriacum and D. botryoides revealed the presence of 50 compounds with interpretable data (Figures 2 and 3), details of which are shown in Table 1.  Table 1. IS-3′,4′-di-O-acetyl-cis-khellactone (5 μg/mL).  Table 1. IS-3 ,4 -di-O-acetyl-cis-khellactone (5 µg/mL).  Table 1. IS-3′,4′-di-O-acetyl-cis-khellactone (5 μg/mL).

Benzoic Acid Derivatives, Phenylpropanoids and Lignans
Two derivatives of benzoic acid {4-hydroxybenzoic acid 4-O-glucoside (8), 4-hydroxybenzoic acid O-hexoside-O-malonyl ester (16)} were found in the herb of D. botryoides. 4-Hydroxybenzoic acid 4-O-glucoside (8) was identified using the reference standard. The mass spectrometric analysis of compound 16 demonstrated the loss of malonyl (86 a.m.u.) and hexose fragments (162 a.m.u.), and the remaining fragment with m/z 299 was related to the 4-hydroxybenzoic acid O-hexoside moiety. The assumed structure of compound 16 was found to be 4-hydroxybenzoic acid O-hexoside-O-malonyl ester. Compound 8 has not been found previously in the Dracocephalum genus but has been detected in Nandina domestica before [28].
Seventeen compounds were determined as phenylpropanoids, separated into danshensu Trace amounts of danshensu O-hexoside (1) was revealed in the herb of D. austriacum, while danshensu O-acetyl ester was detected in the roots of D. botryoides. The presence of danshensu and its derivatives in Dracocephalum species has been revealed for the first time; previously, danshensu was detected in the genus Salvia [29] and Orthosiphon [30].
In Schizotenuin A (46) was identified using the reference standard and was revealed in the herb of D. austriacum in trace amounts. Benzofuran lignan nepetamultin A (50) was identified in the herbs and roots of D. austriacum and D. botryoides by comparison with the reference standard. Compounds 48 and 49 had UV spectra typical of caffeic acid derivatives (λ max 290 and 321 nm), and their mass spectra showed the primary loss of the particle with m/z 162, related to hexose, and the remaining fragment, with m/z 743, was related to nepetamultin A and the loss of the particle with m/z 212 due to the loss of hydroxyhydrocafeic acid. The supposed structures of 49 and 50 were found to be nepetamultin A O-hexosides. Compounds 46 and 50 have not previously been found in the Dracocephalum genus, although schizotenuin A has been detected in Lycopus lucidus [37] and Schizonepeta tenuifolia [38], while nepetamultin A has been revealed in Nepeta multifida [39].
As a result of the chromatographic research of D. austriacum and D. botryoides, 50 metabolites of various chemical groups were identified. This study demonstrates that the whole plant of both Dracocephalum species are characterized by a specific accumulation of phenolic metabolites. The highest content of phenolic compounds was typically found in the herb of both Dracocephalum species, while the roots were characterized by the lowest phenolic diversity. botryoides had a similar chemical profile, with a predominance of the phenylpropanoids rosmarinic acid and lithospermic acid B. This may affect the biological properties of these species, particularly the antioxidant activity of dragonhead extracts, since the activity of diverse phenolic groups is known to be variable [59][60][61]. Table 1. Retention times (t), ultraviolet (UV), and mass spectral (ESI-MS) data of compounds 1-50 were found in leaves and roots of D. austriacum and D. botryoides, in addition to their content (mg/g of dry plant weight, in brackets S.D.).  (1) identified compounds after comparison of UV, mass-spectral data, and retention time with reference standards; (2) putatively annotated compounds after comparison of UV and mass-spectral data with literature data [62]. b traces-<LOQ (limit of quantification).

Water-Soluble Polysaccharides of Herb and Roots of Two Dracocephalum Species
The fractions of WSPS were obtained from the herb and root samples of D. austriacum and D. botryoides by hot (90 • C) water extraction, followed by triple ethanol precipitation, deproteinization, ion-exchange resin and polyamide purification. The pure WSPS gave 0.6-1.2% yield (of dry plant weight) and were completely soluble in hot water ( Table 2) [64]. The presence of α-linkages and aldopyranoses were confirmed by the strong absorption bands at 831-840 cm −1 and 886 cm −1 in the anomeric region of the FTIR spectra, respectively. The FTIR spectral properties of WSPS from roots of D. austriacum and D. botryoides were generally close to that of the herb samples, except in the 900-1200 cm −1 region, and the anomeric region showed shapes specific for the starch-like polymers. The spectra of WSPS in the ultraviolet region had shoulders at 270-280 nm and 320-331 nm, indicating the presence of phenolic fragments ( Figure 5). The pure polysaccharides (like apple pectin) did not have maxima in the UV spectrum. The shortwave band was most likely the secondary benzoic band (B-band) caused by the p-hydroxy-phenyl fragments, while the longwave band was a K-band, owing to the n ⟶ π* transitions of the p-coumaroyl-like fragments. The ionization of phenolic functional groups by alkaline additives (NaOH) resulted in the appearance of additional maxima at 275-290, 310-339, 369-373 and 394-395 nm, typical for the simple phenolic compounds in alkaline media. The WSPS of herbs and roots of D. austriacum and D. botryoides, therefore, had a complex structure consisting of at least three fragments of carbohydrate, protein, and phenolic nature. The polysaccharides were mostly pectins with additive arabinogalactan-protein complexes and starch-like compounds, linked with phenolic fragments of benzoic acids, benzoic aldehydes and hydroxycinnamates.  Table 3. Phenolic compounds released after alkaline hydrolysis of water-soluble polysaccharides of herbs and roots of D. austriacum and D. botryoides.

D. botryoides Roots
Benzoic acid The WSPS of herbs and roots of D. austriacum and D. botryoides, therefore, had a complex structure consisting of at least three fragments of carbohydrate, protein, and phenolic nature. The polysaccharides were mostly pectins with additive arabinogalactanprotein complexes and starch-like compounds, linked with phenolic fragments of benzoic acids, benzoic aldehydes and hydroxycinnamates.
Dragonhead polysaccharides are still poorly researched; currently, there are only data relating to polysaccharides of the herb of D. palmatum, which are pectin, arabinogalactans, and a starch mixture [24], and information about polysaccharides of D. moldavica [20]. Dracocephalum membership in the subtribe Nepetinae, tribe Mentheae, which is included in the subfamily Nepetoideae of the Lamiaceae family, demonstrates the taxonomical proximity to Agastache, Cedronella, Nepeta, Glechoma, Hymenocrater and Meehania species [65]. Even so, polysaccharides have not been studied in the above genera. The closest known data about carbohydrate polymers belongs to the Menthinae and Salviinae subtribes, where galacturonans, glucans and arabinogalactans were found as components of Lycopus [66], Mentha [67,68], Origanum [69], Prunella [70], Rosmarinus [71], Salvia [72] and Thymus [73], indicating the prevalence of these polymers in the Mentheae tribe that Dracocephalum represents. The high phenolic content of polysaccharides of D. austriacum and D. botryoides seems unusual for Lamiaceae plants but is common for many other plants, showing the presence of cross-connected pectin-phenol and hemicellulose-lignin complexes [74]. Some of polyphenol linked polysaccharides are bioactive polymers with antioxidant, antidiabetic, and antitumor properties [75].

Antioxidant Activity of Extracts of D. austriacum and D. botryoides, Selected Phenolics and Water-Soluble Polysaccharides
Phenols are widely known as protective compounds against free radicals and toxic metals [76][77][78], so we decided to study the antioxidant potential of extracts of D. austriacum and D. botryoides as possible radical scavengers and metal chelators. Five well-known antioxidant assays were used to study the scavenging properties against 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH • ), 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) cation radicals (ABTS •+ ), hydroxyl radicals and superoxide anion radicals, as well as the ferrous (II) ion chelating ability [79]. Seven selected compounds representing various phenolic groups detected in D. austriacum and D. botryoides were analysed, as well as WSPS. Trolox was used as a reference substance (Table 4). Table 4. Bioactivity of extracts of D. austriacum and D. botryoides, selected phenolics and water-soluble polysaccharides (WSPS) in five antioxidant assays a .
Object The studied extracts of Dracocephalum demonstrated good scavenging effects against synthetic free radicals, such as DPPH • and ABTS •+ . The herb extract of D. botryoides was the most active, with IC 50 values of 28.63 and 25.20 µg/mL for DPPH • and ABTS •+ radicals, respectively, while roots of this species showed the lowest IC 50 value among the extracts of Dracocephalum (40.67 and 36.14 µg/mL, respectively). Individual phenylpropanoids rosmarinic acid and lithospermic acid B showed superior scavenging activity against the DPPH • radical, with IC 50 values of 6.22 and 9.08 µg/mL, respectively, while the flavonoids eriodictyol 7-O-glucoside and luteolin 7-O-glucoside demonstrated maximal inhibition of the ABTS •+ radical (IC 50  Previous studies have been conducted on the antioxidant activity of other Dracocephalum species. The water-soluble extract of D. moldavica revealed DPPH • radical scav-enging activity, with an IC 50 of 445.90 µg/mL, and superoxide anion radical scavenging activity (IC 50 467.20 µg/mL) [21]. The methanol extract of D. kotschyi inhibited DPPH • , with IC 50 values of 60.69 and 51.54 µg/mL for two-year and six-year-old samples, respectively [12]. The IC 50 values for the DPPH • , ABTS •+ and OH • radical-scavenging activities for the extract of D. rupestre were 50.01, 43.62 and 28.59 µg/mL, respectively [80], and the methanol extract of shoots of D. polychaetum had an IC 50 value of 5600 µg/mL in the DPPH • inhibiting assay [50].
The data obtained in five in vitro assays demonstrated a good efficacy for the herb extract of D. botryoides as an antioxidant agent. This trend is not surprising at all; the presence of strong antioxidants, such as phenylpropanoids and flavonoids in the herb extract of D. botryoides have led to such results. The antioxidant activity of herb and root extracts of D. botryoides and D. austriacum, as well as their water-soluble polysaccharides, have been shown here for the first time.

Conclusions
In this paper, the whole plants of D. austriacum and D. botryoides were shown to be natural accumulators of diverse metabolites of a phenolic and non-phenolic nature. The metabolites of these plant species were investigated for the first time using HPLC-PDA-ESI-QQQ-MS, and the water-soluble polysaccharides of roots of D. austriacum and D. botryoides were studied. The dragonhead polysaccharides were mainly found to be pectins with additive arabinogalactan-protein complexes and starch-like compounds, linked with phenolic fragments of benzoic acids, benzoic aldehydes and hydroxycinnamates. The phenolic diversity in herbs and roots of D. austriacum and D. botryoides implies the presence of antioxidant properties, which were confirmed by five in vitro assays (DPPH • , ABTS •+ , OH • , O 2 •− radical-scavenging activity and Fe 2+ -chelating activity). Thus, the information presented highlights the potential of the herbs and roots of D. austriacum and D. botryoides for possible future plant remedies or sources of new functional products.
The extracts of herbs and roots of D. austriacum and D. botryoides for the antioxidant study were prepared from the ground samples (50 g), treated by the 70% methanol (1 L) in an ultrasonic bath (30 min, 45 • C, ultrasound power 100 W, frequency 35 kHz), filtered, reduced in a vacuum until dryness and stored (−20 • C) before analysis. The yields of the extracts from D. austriacum were 23.4% for the herb sample, 19.8% for the root sample. The yields of the extracts from D. botryoides were 21.7% for the herb sample and 18.1% for the root sample. The injection volume was 1 µL and the flow rate was 120 µL/min. The UV-Vis spectra were recorded by an SPD-M20A photodiode detector (spectral range 200-600 nm) equipped with a post-column derivatization reactor. The used temperature levels were 300 • C in the ESI interface, 250 • C in the desolvation line, and 400 • C in the heat block. The flow values were 3 L/min for the nebulizing gas (N 2 ), 10 L/min for the heating gas (air), and 0.3 mL/min for the collision-induced dissociation gas (Ar). The source voltage of mass spectra was 3 kV and the collision energy −10-30 eV (negative ionization) by the scanning range of m/z 80-1900. The LC-MS system was managed by LabSolution's workstation software equipped with the inner LC-MS library. The integrated analysis of retention time, ultraviolet and mass spectra data after comparison with the reference standards and literature data was used for the identification of metabolites [62].

HPLC-PDA-ESI-QQQ-MS Metabolite Quantification
To quantify 50 metabolites of herbs and roots of D. austriacum and D. botryoides, the HPLC-PDA-ESI-QQQ-MS conditions were used (Section 4.3). Totally, 34 reference standards were separately weighed (10 mg) and dissolved in the methanol-DMSO mixture (1:1) in volumetric flasks (10 mL) preparing the stock solution (1000 µg/mL) used for the calibration curve building. The calibration solution (1, 10, 25, 50, 100 µg/mL) chromatographed in known HPLC-PDA-ESI-QQQ-MS conditions and mass spectral data was used to create 'concentration-mass spectrometric peak area' correlation. The principal validation criteria including correlation coefficients (r 2 ), standard deviation (S YX ), limits of detection (LOD), limits of quantification (LOQ), and linear ranges were found using the known method [81] ( Table S2). Five HPLC runs were sufficient for the quantitative analyses and the results were expressed as mean value ± standard deviation (S.D.).

Polysaccharides Extraction and Analysis
The samples of dried milled herb and roots of D. austriacum and D. botryoides (100 g) were added to distilled water (1.0 L), heated on a boiled water bath (1 h) and after cooling to room temperature water extracts were filtered under reduced pressure and concentrated down in vacuo to 200 mL. The concentrated residues were mixed with 95% ethanol (1:5) and after 2 h the precipitates were centrifuged at 3000× g. The crude polysaccharide fractions were redissolved in 200 mL of water. The Sevag method [82] was applied for deproteination and was followed by dialysis for 48 h against distilled water using dialysis tubes with an MW-cut off of 2 kDa (Sigma-Aldrich, St. Louis, MO, USA). The non-dialysed parts were loaded on to a KU-2-8 cation-exchange resin column (H + -form, 200 g; Closed Joint-Stock Company Tokem, Kemerovo, Russia) which were eluted with 2 L of distilled water. Eluates were concentrated in vacuo up to 200 mL and then liophylized. The resulting water-soluble polysaccharides were whitish powders.
For estimation of total carbohydrate content modified anthrone-H 2 SO 4 spectrophotometric assay with glucose as a standard compound was used [83]. To evaluate content of uronic acids 3,5-dimethylphenol method was applied calculated as galacturonic acid [84]. The Bradford method using Coomassie G250 was employed for determination of protein content [85]. Phenolic content was analysed using the Folin-Ciocalteu method [86]. Reactions of water-soluble polysaccharides solutions with iodine, resorcinol, Yariv and Fehling's reagents were carried out accordingly [87][88][89]. The IR spectra were registered in a spectral range of 4000-600 cm −1 using a FT-801 Fourier-transform infrared spectrometer (Simex, Novosibirsk, Russia) coupled with a single reflection ATR device. Alkaline hydrolysis procedure and HPLC-DAD-MS analysis conditions of the released products were as described by us earlier [90].

Statistical Analysis
Statistical analyses were performed by one-way analysis of variance, and the significance of the mean difference was determined by Duncan's multiple range test. Differences at p < 0.05 were considered statistically significant. The results are presented as mean values ± standard deviations (S.D.). The linear regression analysis and generation of calibration graphs were conducted using Advanced Grapher 2.2 (Alentum Software Inc., Ramat-Gan, Israel).

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/plants11162126/s1. Table S1: Reference standards used for the qualitative and quantitative analysis by HPLC-PDA-ESI-QQQ-MS assays. Table S2: Regression equations, correlation coefficients, standard deviation, limits of detection, limits of quantification and linear ranges for 34 reference standards.