Synanthropic Plants as an Underestimated Source of Bioactive Phytochemicals: A Case of Galeopsis bifida (Lamiaceae)

Hemp nettle (Galeopsis bifida Boenn.) is a synanthropic species of the Lamiaceae family that is widely distributed across Europe, Asia, and Siberia. Galeopsis bifida is deeply embedded in the ethnomedical tradition of Asian healers; however, this plant is still poorly characterized, both chemically and pharmacologically. To study Siberian populations of G. bifida, we used high-performance liquid chromatography with photodiode array and electrospray triple quadrupole mass detection for metabolic profiling. Ninety compounds were identified, including iridoid glycosides, phenylethanoid glycosides, hydroxycinnamates, and flavone glycosides, most of which were identified in G. bifida for the first time, while some phenolics were found to have potential chemotaxonomic significance in the Lamiaceae family and Galeopsis genus. An unequal quantitative distribution of the selected metabolites was observed within separate organs of the G. bifida plant, characterized by high accumulation of most compounds within the aerial part of the plant (leaves, flowers). Analysis of the content of specific chosen compounds within the leaves of different populations of G. bifida from Eastern Siberia revealed the existence of two chemical types based on metabolic specifics: the southern type accumulates flavone glucuronides, while the northern type tends to accumulate high levels of phenylpropanoids and acylated flavone glucosides. The first study of the bioactivity of G. bifida extract demonstrated that the herb has low toxicity in acute experiments and expresses antioxidant potential against free radicals in the form of DPPH˙, ABTS˙+, and superoxide radical, as well as high ferric reducing antioxidant power, oxygen radical absorbance capacity, and protective action in the carotene bleaching assay. In general, our results suggest the herb of G. bifida as a new, prospective synanthropic plant for medical application.


Introduction
Intensive use of natural landscapes and decreased areas of natural vegetation lead to the process of synanthropization, which has acquired the scale of anthropogenic evolution [1]. In a broad sense, synathropization refers to the process of adaptation of organisms to habitats in places dramatically transformed by humans, up to settlements and human dwellings. In connection with anthropogenic transformation, synanthropic species occupy an increasingly prominent place in the structure of biological diversity, which is especially important for the vast territories of Siberia and Asia [2]. Speaking of their practical importance, synanthropic species, as a rule, are not considered to be economically valuable due to the instability of their raw material base. However, these species are characterized by high reproductive energy as well as wide ranging ecological adaptability, which make

Compound Found in Galeopsis Species of Subgenus
Regarding the territory of Siberia, the most abundant synanthropic species is G. bifida Boenn. (hemp nettle) [32,33], which, despite its large biological reserves, has no economic value, although it is often used in various traditional medical systems of the Siberian peoples and some Asian countries. In Tibetan medicine, as well as its local branch in the form of Buryat traditional medicine, the herb G. bifida is widely used under various names ('jib rtsi, pri yang ky, zhim thig le) in the form of decoctions, rinses, and irrigation solutions, as well as applied in the treatment of various oral diseases (stomatitis, caries), gastrointestinal tract disorders (gastritis, gastroenteritis, ulcers, and inflammation of the esophagus, stomach, and intestines), kidney disorders (inflammation, cystitis), inflammation of the lungs and female genital organs, and eye diseases (conjunctivitis) [34,35]. In the medicine of the peoples of the Far East, infusions of G. bifida in vodka have been used in the treatment of oncological diseases of the stomach, sore throat, and epilepsy, as well as to increase food bitterness to stimulate appetite [36]. Additionally, leaf applications of G. bifida have been used to treat lichen, panaritium, and other skin wounds [36]. In the territory of Siberia, a decoction of the plant has been used as an expectorant to treat pulmonary tuberculosis and other respiratory infections, while a milk infusion was used for chronic rhinitis [37]. In the medical practice of the nomadic peoples of the North, a tincture of G. bifida herb was used to treat liver diseases [38]. In Kyrgyzstan, G. bifida tincture was recommended as an antihypertensive agent [39]. It should also be noted that young leaves of G. bifida were used earlier in the Baikal region, and are still used there today in food as a salad plant [40].
An ambiguous opinion exists regarding the toxicity of G. bifida, as well as Galeopsis species in general. Scientific reports [41] indicate that there is a possibility of temporary paralysis of the limbs when eating oil from the seeds of some species of Galeopsis (G. bifida, G. ladanum, G. speciosa, and G. tetrahit). A case of oil poisoning by seeds of G. segetum (also known as G. cannabina) has been described, which resulted in nausea, a feeling of heaviness in the lower extremities, and pain in the hands and in the region of the sacrum [42]. A scientific study of this phenomenon has not been carried out; therefore, the question of the reliability of this information remains open. However, a more recent study investigating the chemical causes of a pathological condition known as koturnism, which is caused by consumption of the meat of some species of quail that feed on G. ladanum [43], did not observe any toxic manifestations with regards to G. ladanum extract or stachydrin, which is its component.
Currently, despite satisfactory raw material reserves, G. bifida does not have any practical application, for example, as an official medicinal plant. The reason for this is lack of knowledge regarding the plant's chemical composition, as well as the lack of information on the pharmacological effects of extraction preparations from it. The known data on the metabolites of G. bifida indicate the presence of iridoids [7,8], flavonoids [17], fatty acids [22][23][24][25], acylglycerols [26], and essential oils [27].
In the current study, we performed a qualitative chromatographic analysis of G. bifida using high-performance liquid chromatography with photodiode array and electrospray triple quadrupole mass detection (HPLC-PAD-ESI-tQ-MS). Additionally, we performed quantification of selected metabolites within the different organs of G. bifida and natural populations, as well as investigations into the acute toxicity and antioxidant properties of the plant using various biological in vitro assays.

Metabolites of Galeopsis bifida: LC-MS Profile and Chemotaxonomic Significance
The existing data regarding the metabolites of Galeopsis species indicate the presence of a wide group of compounds with various polarities and chromatographic behaviors (Table 1). Therefore, prior to studying the LC-MS profile of G. bifida extract, we separated the total probe using a solid-phase extraction (SPE) technique on a polyamide cartridge in order to avoid missing any low content or trace compounds. Elution of the preconditioned cartridge using water, ethanol, and alkalized ethanol allowed the isolatation of a hydrophilic fraction of iridoids and two less polar fractions of phenylethanoid glycosides/neutral flavone glycosides and acidic/acylated flavone glycosides. Further analysis of the three eluates by HPLC-PAD-ESI-tQ-MS assay (Figure 1) demonstrated the good separation of a total of ninety compounds, identified by their retention times and ultraviolet (UV) and mass spectrometric data via comparison with reference standards and known literature information ( Table 2).
2.1.6. Chemotaxonomic Significance of G. bifida Metabolites As a result of the chromatographic research of G. bifida, approximaetly 100 metabolites of various chemical groups were identified. When choosing compounds that may have chemotaxonomic significance, special attention was paid to the iridoid glycosides, phenylethanoid glycosides, phenylpropanoids, and flavone glycosides.
Earlier attempts have been made to use iridoid glycosides as marker compounds within the genus Galeopsis and the Lamiaceae family [7][8][9]. The use of individual iridoids for the chemical division of the genus Galeopsis into the subgenera Ladanum and Galeopsis (Tetrahit) was unsuccessful. The assumption that harpagide 8-O-acetate, reptoside, and ajugoside are characteristic only of the species of subgenus Ladanum [7] is not supported by our data. In view of the fact that harpagide and its 8-O-acetate are more widespread in the Lamiaceae family, especially in tribe Stachydeae in genus Betonica and Stachys [44], the known conclusions about the applicability of specific iridoid glycosides for taxonomic purposes should be revised.
Phenylethanoid glycosides are widespread in the Lamiaceae family, especially in genera closely related to Galeopsis, such as Ballota [61], Lamium [62], Phlomis [63], and Stachys [64]. A high occurrence of verbascoside and isoverbascoside is characteristic of many species of Lamiaceae [75], especially the species belonging to the subfamily Stachyoideae [76]. The same principles apply to chlorogenic acids, which are found in most species of the Lamiaceae genus [76]. These features of the chemical composition of G. bifida, as well as the genus Galeopsis, indicate a low value of phenylethanoid glycosides and phenylpropanoids in terms of chemotaxonomic purposes.
It should be noted that there is a clear differentiation between the chemical characteristics of the species belonging to different subgenera: the presence of 5,7,8-trihydroxyflavones has not been established in species of the subgenus Galeopsis (Tetrahit) [17]; this finding is congruent with our research on G. bifida. In previous studies, two flavones have been isolated from G. ladanum, specifically ladanein (5,6-dihydroxy-7,4 -dimethoxyflavone) and ladanetin (5,6,4 -trihydroxy-7-methoxyflavone), the structural type of which does not correspond to the typical structure for the subgenus Ladanum [18]. Tomas-Barberan et al., following a chromatographic study of the genus Galeopsis, demonstrated that flavones with a hydroxy group at the C-6 position are not characteristic of the subgenus Ladanum [77]. Following this report [18], the presence of ladanein in the Lamiaceae family was also revealed in the tribes Ocimeae (Ocimum [78], Orthosiphon [79], Lavandula [80], Plectranthus [81]), Marrubieae (Ballota [82], and Marrubium [83]) and Nepetoideae (Rosmarinus [84], Salvia [85], and Thymus [77]), however, never in the tribe Galeopsis. A similar situation was observed with the flavone ladanetin, which is characteristic of the genus Dracocephalum [86], but not of Galeopsis. Considering the above, the presence of ladanein and ladanetin in G. ladanum and the genus Galeopsis remains doubtful.

Distribution of Selected Metabolites in G. bifida Organs
Studying the distribution of metabolites in the organs of medicinal plants is important for correctly selecting the part of the plant that contains the highest concentration of bioactive compounds. Due to the fact that G. bifida is harvested as a full-plant (including roots), it became necessary to determine the levels of individual compounds in the different parts of the plant. To do that, we used a quantitative HPLC-MS assay, which made it possible to determine the content of 18 compounds in the leaves, flowers, stems, and roots of G. bifida (Table 3)  The quantification data demonstrated an uneven distribution of the compounds within the various organs of G. bifida. Two dominant iridoid glycosides, harpagide 8-O-acetate and harpagide, were found in higher levels in the leaves (25.69 and 11.35 mg/g dry plant weight) and stems (18.53 and 9.37 mg/g dry plant weight), in contrast to the flowers and roots, which contained total iridoid glycosides content levels of 15.55 and 2.12 mg/g, respectively. The highest total phenylethanoid glycoside content was found in the leaves (61.38 mg/g) of G. bifida, followed by the flowers (50.06 mg/g), stems (18.47 mg/g), and roots (7.32 mg/g). The main compound was verbascoside, which amounted to 21.56, 18.98, 5.32, and 2.63 mg/g of the dry weight of the leaves, flowers, stems, and roots, respectively. Above all, attention should be drawn to the high levels of isoverbascoside (2.08-14.88 mg/g) and lavandulifolioside (1.57-16.37 mg/g) in the organs of G. bifida.
Caffeoylquinic acid showed higher concentrations in the leaves (47.52 mg/g in total), dominated by 5-O-caffeoylquinic acid (45.20 mg/g). Meanwhile, the total level of caffeoylquinic acid in the other organs of G. bifida was 16.01 mg/g in the flowers, 9.21 mg/g in the stems, and 2.90 mg/g in the roots. Flavone glycosides comprised a large group of compounds with high content in the leaves (80.56 mg/g in total) and flowers (53.48 mg/g in total), with the predominant luteolin 7-O-glucuronide contributing 29.73 and 39.63 mg/g of the leaf and flower dry weights, respectively. Apigenin 7-O-glucuronide was the second highest-level flavonoid in the leaves (19.32 mg/g) and scutellarein 7-O-glucuronide was also in high levels in the flowers (5.22 mg/g). The content of non-acylated flavone glycosides was greater than acylated derivatives in all organs of G. bifida. These results make obvious the fact that the roots are poor in metabolites and, thus, the above-ground parts of G. bifida (leaves, flowers, and stems) should be recommended for use in medical applications.
Due to the absence of information regarding the metabolite content of Galeopsis species, we compared the results of our study with known data concerning the metabolite content of other Lamiaceous species. Háznagy-Radnai et al. studied the total content of iridoid glycosides in ten Stachys species collected in Bulgaria, and found that the levels reached 15.2 mg/g in S. officinalis leaves, 16.8 mg/g in S. officinalis flowers, and 14.7 mg/g in S. recta roots [87]. The level of harpagide and harpagide 8-O-acetate in Leonurus species from central regions of Russia were found to be 0.11-0.37 and 0.10-0.37 mg/g in Leonurus quinquelobatus herb, respectively, and 0.06 and 0.04 mg/g in Leonurus cardiaca herb, respectively [88]. Despite iridoid accumulation, some Leonurus species have been characterized by high phenylethanoid glycoside content, varying from 3.09 mg/g in L. quinquelobatus to 26.17 mg/g in L. cardiaca, and high verbascoside levels in six Siberian Leonurus species, ranging from 0.89-3.66 mg/g [89]. Flavonoids, as the most studied group of Lamiaceae phenolics, were at highest levels in the family and selected species. For example, the levels of flavonoids in herbs of Stachys byzantine-11.1 mg/g, Salvia officinalis-5.12 mg/g, Mentha suaveolens-3.9 mg/g [90], Mentha piperita-30.2-63.2 mg/g [91], Panzerina lanata-29.3 mg/g [92], Thymus baicalensis-18.4 mg/g, Thymus sibiricus-26.5 mg/g [93], Nepeta glutinosa-7.3-10.2 mg/g, Ziziphora pamiroalaica-8.3-10.1 mg/g [94], and Dracocephalum palmatum-10.5-35.4 mg/g [95] are known. Undoubtedly, the herb of G. bifida is a good source of iridoids and phenolic compounds that contain a comparable or greater level of phytocomponents.

Two Siberian Chemotypes of G. bifida
Investigations into geographical variations of the chemical profiles of plants allowed us to understand the level of stability of metabolic pathways of selected species, as well as the power of climatic influence on the plant populations. This is particularly relevant for species with a wide area of distribution, such as the common hemp-nettle, which is located across the whole of Eurasia. In this study, we analyzed eight Siberian populations of G. bifida located at the southern (Buryatia Republic, populations P1-P4) and northern (Sakha Yakutia Republic, populations P5-P8) borders of the Russian area (Figure 2a). Moreover, it should be pointed out that we used the leaf samples only because they contained the highest content of metabolites. The results of HPLC-PAD-ESI-tQ-MS profiling of G. bifida extracts demonstrated the stability of qualitative metabolite patterns in all samples analyzed; however, the quantification data indicated various levels of the selected compounds (Table 4). Considering the features of metabolite accumulation in G. bifida leaves, the following conclusions can be drawn: The specificity of iridoid glycoside accumulation in the southern populations of G. bifida lies in Moreover, it should be pointed out that we used the leaf samples only because they contained the highest content of metabolites. The results of HPLC-PAD-ESI-tQ-MS profiling of G. bifida extracts demonstrated the stability of qualitative metabolite patterns in all samples analyzed; however, the quantification data indicated various levels of the selected compounds (Table 4). Considering the features of metabolite accumulation in G. bifida leaves, the following conclusions can be drawn: Table 4. Content of selected compounds in leaves of G. bifida from eight Siberian populations (P1-P8).  The specificity of iridoid glycoside accumulation in the southern populations of G. bifida lies in the fact that southern and northern populations have similar content levels of harpagide and its O-acetate (8.57-18.33 mg/g vs. 9.35-14.53 mg/g, respectively), while northern populations have a much greater content of harpagide 8-O-acetate in contrast to southern populations (<0.10-4.14 mg/g vs. 24.52-31.82 mg/g, respectively).
The level of phenylethanoid glycosides in northern populations (61.16-69.79 mg/g) was higher than in southern populations (6.21-12.73 mg/g), while the selected compounds of isoverbascoside and leonoside B were trace components in southern populations, in contrast to northern populations (12.76-18.67 and 1.57-1.93 mg/g, respectively).
Caffeoylquinic acids were detected in all populations, however, their concentrations varied from 35.02 to 44.90 mg/g and from 5. The results of the principal component analysis (PCA) confirmed the division of the studied G. bifida population into two types: type I or southern type, located on the left side of the diagram, and type II or northern type, located on the right side of the diagram (Figure 2b). The total scores plot of PCA in a two-component model amounted to 89.3% of the total variability. These results indicate the existence of at least two chemotypes of G. bifida in the Siberian region: the southern chemotype, with the predominance of non-acylated flavone glucuronides, and the northern chemotype, with a high content of acetylated iridoids, phenylethanoid glycosides, caffeoylquinic acids, and acylated flavone glycosides. Obviously, additional studies need to be performed to ensure the correctness of this theory.
In the debate regarding possible reasons for chemical variation between the southern and northern populations of G. bifida, climatic differences between the Buryatia Republic, with a warm humid continental climate, and the Sakha Yakutia Republic, located in the subarctic area of Siberia, should be mentioned. The cold climate of the northern territories promotes the accumulation of compounds such as acetylated iridoids, phenylethanoid glycosides, caffeoylquinic acids, and acylated flavone glycosides in G. bifida plants, while warm climates result in high concentrations of the non-acetylated iridoid glycosides and non-acylated flavone glucuronides. This early comparative data regarding the chemical composition of plants collected from the various regions of Siberia demonstrate greater storage ability in the northern plant populations for flavonoids [53,56,96], simple phenolics [97], ellagitannins [98], iridoids [99], caffeoylquinic acids [100], sesquiterpenes [101], and coumarins [102].

Bioactivity of G. bifida Extracts: Acute Toxicity and Antioxidant Potential
Existing data concerning the possible toxicity of Galeopsis extracts [15,16] inspired us to determine the acute toxicity of G. bifida methanol extracts (GBME) prior to proceeding with other pharmacological experiments. Intraperitoneal administration of GBME from the southern population, P3, and northern population, P7, in doses of 1-3000 mg/kg, did not cause the death of experimental animals (mice) during a week. According to our data, this shows that GBME is a plant extract with low toxicity.

Extract
The power of GBME prepared from the southern populations, P1-P4, was lower than the power of GBME prepared from the northern populations, P5-P8. This phenomenon is obviously caused by the higher content of phenolics in the extracts from P5-P8. The data regression analysis of "antioxidant activity-compound content" relationships confirmed these findings via high values of the regression coefficients (r 2 ) of the linear equations (>0.5) ( Table 6). These conclusions are reinforced by the known pharmacological data relating to Galeopsis plants reporting the low toxicity of G. ladanum extract [29] and the good antioxidant potential of G. speciosa extract in the DPPH assay (IC 50 2.85-4.00 µg/mL), phosphomolybdenum assay, and linoleic acid peroxidation assay (64.5%) [30]. It can be said for Lamiaceous plants as a whole that their extracts are safe and effective antioxidants, such as those that have traditionally been used, such as skullcaps [65], motherworts, sages, dead-nettles [30], lemon balm, peppermints [103], and thymes [104]. Hemp nettle is a good addition to the list of known medicinal plants with potential as bioactive.

Plant Material and Chemicals
Samples of Galeopsis bifida were collected in the eight Siberian regions in the flowering stage on the same day (20.VI.2019) ( Table 7)

Plant Extracts Preparation
The extract of G. bifida herb for the qualitative analysis was prepared from the total aerial part (leaves, flowers, and stems) of the P5 sample and 100 g of the dry powdered herb was extracted by 60% ethanol with sonication (60 min, 50 • C, ultrasound power 100 W, frequency 35 kHz) for that purpose. Liquide extract was filtered through the filter paper and concentrated in vacuo until dryness. The yield of the dry extract from G. bifida herb was 19% from dry plant weight. The dry extracts of leaves from the samples P1-P8 for the quantitative analysis and study of biological activity were produced using the same technology with the yields 28% (P1), 24% (P2), 25% (P3), 29% (P4), 33% (P5), 35% (P6), 31% (P7), and 30% (P8) of dry plant weight.

Polyamide Solid-Phase Extraction
The separation of the extract of G. bifida herb before qualitative chromatographic analysis was realized with solid-phase extraction (SPE) on the polyamide cartridges Chromabond (Polyamide 6; 6 mL, 1000 mg; Sorbent Technologies, Inc., Norcross, GA, USA) preconditioned with methanol (50 mL) and water (70 mL). The dry extract (100 mg) was dissolved in 25% methanol (10 mL), centrifuged (6000× g, 15 min), and the supernatant volume reached 10 mL in the volumetric flask (10 mL; solution A). An aliquote of solution A (5 mL) was mixed with 100 µL of trifloroside solution (internal standard-1; 2 mg/mL in 20% methanol), 100 µL of scopoletin 7-O-neohespridoside solution (internal standard-2; 2.5 mg/mL in 40% methanol), and 50 µL of 3,5-di-O-feruloylquinic acid solution (internal standard-3; 1 mg/mL in 40% methanol), and the mixture was passed through preconditioned polyamide SPE-cartridge eluted with water (40 mL; eluate I), 85% methanol (50 mL; eluate II), and 0.45% NH 3 in methanol (50 mL; eluate III). Eluates I, II, and II were concentrated in vacuo, dissolved in 1 mL of methanol, and stored at 4 • C before chromatographic analysis (Section 3.4). The injection volume was 1 µL and the elution flow 100 µL/min. The UV-Vis spectra were registered in the spectral range of 200-600 nm. Mass spectrometric detection was performed both in negative and positive ESI mode and the temperature levels of ESI interface, desolvation line, and heat block were 300 • C, 250 • C, and 400 • C, respectively, and the flow of nebulizing gas (N 2 ), heating gas (air), and collision-induced dissociation gas (Ar) were 3 L/min, 10 L/min, and 0.3 mL/min, respectively. The mass spectra were registered as 3 kV source voltage and collision energy +15-+25 eV in the positive mode and −15-35 eV in the negative mode by the scanning range of m/z 50-2000. LabSolution's workstation software with the inner LC-MS library was used to managing the LC-MS system. The final identification of metabolites was done after an integrated analysis of retention time, ultraviolet, and mass spectra with the reference samples and/or literature data.

Metabolite Quantification
Quantification of compounds in G. bifida extracts was realized in chromatographic conditions (mode III), as described above (Section 3.4) and HPLC-MS data (full scan MS, peak area) were used for calculation. Eighteen metabolites were quantified and seventeen solutions To build reference standard calibration curves, the stock solutions were diluted with methanol (1-100 µg/mL), chromatographed, and MS peak area data were used to plot "concentration-peak area" graphs. The validation criteria (correlation coefficients, r 2 ; standard deviation, S YX ; limits of detection, LOD; limits of quantification, LOQ; and linear ranges) were calculated as described previously [107] ( Table S4). All analyses were carried out five times, and the data were expressed as mean value ± standard deviation (S.D.). For the analysis of G. bifida plant samples (leaves, flowers, stems, and roots), pulverized material (200 mg) was extracted with 60% ethanol (5 mL) twice by sonication (20 min, 50 • C, ultrasound power 100 W, frequency 35 kHz), followed by centrifugation (6000× g, 20 min) and filtering (0.22-µm PTFE syringe filter) to the volumetric flask (10 mL). The samples of G. bifida extracts were prepared the same way using 50 mg of dry material.

Acute Toxicity
Experiments were performed on adult male C57BL/6 mice (body weight range 80-100 g; 6-8 weeks of age) obtained from the 'Pushchino' Laboratory Animal Breeding House (Moscow, Russia). Animals were housed at 22 • C under a 12/12 light/dark cycle, with free access to food and water. Acute toxicity experiments (LD 50 ) was determined using recommendations of the Guidelines for Preclinical Drug Trials [108] after oral administration of G. bifida extracts (samples P3 and P7) by gavage at the doses of 1 (8 animals), 10 (8 animals), 100 (10 animals), 1000 (10 animals), and 3000 (10 animals) mg/kg in a volume 10 mL/kg. The animals were continually observed for a week and there were no clinical signs of toxicity or mortality in the experimental groups. The experimental procedure was authorized by the Institute of General and Experimental Biology's Ethical Committee (protocol No LM-0324, 27.01.2012) before starting the study and was conducted under the internationally accepted principles for laboratory animal use and care.

Antioxidant Activity
Microplate spectrophotometric assays were used to study the scavenging activity of G. bifida extracts against the 2,2-diphenyl-1-picrylhydrazyl radical and the 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) cation radical, as described earlier [53,95] and the superoxide radicals scavenging capacity was determined using pyrogallol auto-oxidation assay [109]. Ferric reducing antioxidant power was determined by spectrophotometrical assay and used the reduction of the Fe 3+ -2,4,6-tri(2-pyridyl)-1,3,5-triazine complex to the Fe 2+ at low pH [110]. The fluorimetric method of peroxyl radical generation by thermal decomposition of 2,2 -azobis(2-amidino-propane) dihydrochloride was used to measure the oxygen radical absorbance capacity assay [111] and peroxide-radical-induced destruction of the β-carotene was used in the spectrophotometric carotene-bleaching assay [112]. Trolox, as a reference standard (1-100 µg/mL in methanol), was used for the expression of the values of antioxidant parameters as µmol Trolox-equivalents/g of dry weight. All the analyses were carried out five times and the data were expressed as mean value ± standard deviation (SD).

Statistical and Multivariate 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.) of some replicates. The linear regression analysis and generation of calibration graphs were conducted using Advanced Grapher 2.2 (Alentum Software Inc., Ramat-Gan, Israel). Principal component analysis based on a data matrix (18 markers × 8 samples) was performed using Graphs 2.0 utility for Microsoft Excel (Komi NTc URO RAN, Syktyvkar, Russia) to generate an overview for group clustering.

Conclusions
Galeopsis bifida is a ruderal synanthropic species found throughout most of Eurasia. Early ethnopharmacological information has not been scientifically confirmed in the modern world; therefore, the use of this species is not widespread. In the course of this study, it was shown that G. bifida is characterized by the ability to accumulate phenolic compounds of different classes. In particular, the composition of G. bifida phenylpropanoids was established for the first time and it was shown that these compounds are represented by caffeoylquinic acids, as well as phenylethanoid glycosides. Flavonoids of this plant species consist of flavones in the form of p-coumaroyl glucosides and glucuronides. Of the 90 identified compounds, 82 were found in G. bifida for the first time. The finding of organ specificity among the accumulation of phenolic compounds in G. bifida indicates a greater practical significance of the aerial part of this species due to the ability of leaves and flowers to accumulate individual compounds. In the course of the study of the Siberian populations of G. bifida, the existence of two chemotypes characterized by geographical confinement was shown. This phenomenon can be important when choosing locations to collect plant materials from regarding specific parameters of their chemical composition. For the first time, a study of the pharmacological properties of G. bifida was carried out and it was found that its extracts can be considered as low-toxic antioxidant agents.
Considering the early ethnopharmacological information on the use of G. bifida, as well as data on its chemical composition, it can be assumed that recommendations for use of this species in the treatment of liver and stomach diseases, as well as many other illnesses, are due to its high content of compounds with antioxidant and anti-inflammatory activity, such as verbascoside, 3-O-caffeoylquinic acid, luteolin, and apigenin glycosides. In this regard, we conclude that the synanthropic plant species G. bifida is not just a weedy and unimportant plant, but instead, has great potential as a medicinal species and, thus, research into this species should be continued.

Supplementary Materials:
The following are available online at http://www.mdpi.com/2223-7747/9/11/1555/s1, Table S1: Ultraviolet spectral patterns of compounds found in Galeopsis bifida, Table S2: Content of selected compounds in extracts of G. bifida from eight Siberian populations, Table S3: Reference standards used for the qualitative and quantitative analysis by HPLC-PAD-ESI-tQ-MS, Table S4: Regression equations, correlation coefficients, standard deviation, limits of detection, limits of quantification and linear ranges for 17 reference standards used in HPLC-MS quantification.
Funding: This research was funded by Ministry of Education and Science of the Russian Federation, grant number AAAA-A17-117011810037-0.