Synanthropic Plants as an Underestimated Source of Bioactive Phytochemicals: A Case of Galeopsis bifida (Lamiaceae)
Laboratory of Medical and Biological Research, Institute of General and Experimental Biology, Siberian Division, Russian Academy of Science, 6 Sakhyanovoy Street, Ulan-Ude 670047, Russia; olennikovdn@mail.ru; Tel.: +7-9021-600-627
Abstract
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.
1. 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 these species convenient for introduction into culture [3]. The study of prospective practical applications for synanthropic species will, in the future, solve the problem of their utilization and expand the range of useful plant species.
Among the various synanthropic species of Eurasia, the weed genus Galeopsis L. of the Lamiaceae family is currently represented by 14 species (10 of which grow in Eurasia) belonging to two subgenera, Galeopsis (Tetrahit) and Ladanum [4,5]. Botanically, Galeopsis species are annual pubescent grasses with ovoid or lanceolate petiolate leaves and sessile flowers in whorls in the leaf axils. These species are often found in crops, fields, garbage sites, and alongside roads. Information on the chemical composition of Galeopsis species indicates the presence of iridoids [6,7,8,9,10,11], diterpenes [12,13,14], triterpenes [13], benzoic acids [15], hydroxycinnamates [15,16], flavones [9,15,17,18,19,20,21], fatty acids [22,23,24,25], acylglycerols [26], and essential oils [27,28] (Table 1). Early pharmacological studies have indicated the presence of central nervous system (CNS) depressive [29], antioxidant [30], neuroprotective, and anticholinesterase activities [31] in extracts of the Galeopsis species.
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.
2. Results and Discussion
2.3. 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.
Owing to the high content of the phenolic compounds in G. bifida plant material and dry extracts (Table S2), especially compounds with expressed antioxidant potential, such as phenylethanoid glucosides [50], caffeoylquinic acids [100], and flavone glycosides [95], we studied the antioxidative properties of GBME from eight Siberian populations. Six assays were chosen for investigation, including cavenging capacity against free radicals of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) cation radical (ABTS), and superoxide radical, as well as ferric reducing antioxidant power, oxygen radical absorbance capacity, and carotene bleaching assay. Trolox was used as a reference substance (Table 5).
All studied GBMEs demonstrated high effectiveness as antioxidants in the six assays, with potential ranging from 286.6–632.4 μM Trolox-eq./g in scavenging of DPPH radicals, 293.4–693.0 μM Trolox-eq./g in scavenging of ABTS radicals, 182.4–363.7 μM Trolox-eq./g in scavenging of superoxide radicals, 103.9–361.2 μM Trolox-eq./g in ferric reducing antioxidant power, 253.0–631.0 μM Trolox-eq./g in oxygen radical absorbance capacity, and 298.3–734.8 μM Trolox-eq./g in carotene bleaching assay.
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 (r2) 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 (IC50 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.
3. Materials and Methods
3.1. 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). The species was authenticated by Dr. N.I. Kashchenko (IGEB SB RAS, Ulan-Ude, Russia) and Professor N.K. Chirikova (North-Eastern Federal University, Yakutsk, Russia). The plant material was dried in the ventilated heat oven at 40 °C within 3–4 days and stored at 3–4 °C before analysis. The reference compounds were purchased from ChemFaces (Wuhan, Hubei, PRC), Extrasynthese (Lyon, France), MedKoo Biosciences Inc. (Morrisville, NC, USA), Sigma-Aldrich (St. Louis, MO, USA), Toronto Research Chemicals (North York, ON, Canada), VILAR Corp. (Moscow, Russia), or isolated early from the various plants in our laboratory [55,57,100,105,106] (Table S3). Selected chemicals were from Sigma-Aldrich—acetonitrile for HPLC (Cat. No 34851, ≥99.9%); 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Cat. No A1888, ≥98%); 2,2′-azobis(2-methylpropionamidine) dihydrochloride (Cat. No 440914, ≥97%); β-carotene (Ct. No C4582, ≥95%); 2,2-diphenyl-1-picrylhydrazyl radical (Cat. No 281689, ≥97%); formic acid (Cat. No 33015, ≥98%); methanol (Cat. No. 322415, ≥99.8%); myoglobin (Cat. No M0630, ≥95%); pyrogallol (Cat. No P0381, ≥98%); 2,4,6-tri(2-pyridyl)-1,3,5-triazine (Cat. No 93285, ≥99%); and Trolox (Cat. No 238813, ≥97%).
3.2. 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.
3.3. 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% NH3 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).
3.4. High-Performance Liquid Chromatography with Photodiode Array Detection and Electrospray Ionization Triple Quadrupole Mass Spectrometric Detection (HPLC-PDA-ESI-tQ-MS)
Qualitative chromatographic analysis of metabolic profiles of G. bifida extracts was done by high-performance liquid chromatography with photodiode array detection and electrospray ionization triple quadrupole mass spectrometric detection (HPLC-PDA-ESI-tQ-MS) technique using a liquid chromatograph LC-20 Prominence coupled with photodiode array detector SPD-M30A (wavelength range 200–600 nm), and triple-quadrupole mass spectrometer LCMS 8050 (all Shimadzu, Columbia, MD, USA) and C18 columns. Two-eluent gradient elution was used for successful separation of compounds in three chromatographic modes: mode 1 (separation of SPE-polyamide eluate I)—column ProteCol™ C18 HPH125 (250 × 4.6 mm, Ø 5 μm; Trajan Scientific Australia Pty Ltd., Ringwood, Victoria, Australia); column temperature 25 °C; eluents A, 0.2% HCOOH in water; eluent B, MeCN; gradient program: 0–2 min 5–6% B, 2–9 min 6–11% B, 9–15 min 11–25% B, 15–20 min 25–55% B, 20–25 min 55–5% B; mode 2 (separation of SPE-polyamide eluate II)—column GLC Mastro (150 × 2.1 mm, Ø 3 μm; Shimadzu, Kyoto, Japan); column temperature 30 °C; eluents A, 0.5% HCOOH in water; eluent B, 0.5% HCOOH in MeCN; gradient program: 0–2 min 5–6% B, 2–9 min 6–11% B, 9–15 min 11–25% B, 15–20 min 25–55% B, 20–25 min 55–5% B; mode 3 (separation of SPE-polyamide eluate III and quantitative analysis of G. bifida organs and extracts)—column GLC Mastro (150 × 2.1 mm, Ø 3 μm; Shimadzu, Kyoto, Japan); column temperature 30 °C; eluents A, 0.5% HCOOH in water; eluent B, 0.5% HCOOH in MeCN; gradient program: 0–5 min 5–10% B, 5–10 min 10–15% B, 10–22 min 15–20% B, 22–28 min 20–34% B, 28–35 min 34–52% B, 35–40 min 52–80% B, 40–50 min 80–5% B. 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 (N2), 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.
3.6. 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 (LD50) 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.
3.7. 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 Fe3+-2,4,6-tri(2-pyridyl)-1,3,5-triazine complex to the Fe2+ 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).
3.8. 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.
4. 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.
Acknowledgments
The author acknowledges the Buryat Research Resource Center for the technical support in chromatographic and mass-spectrometric research and also the students of the North-Eastern Federal University (Yakutsk, Russia) and Buryat State University (Ulan-Ude, Russia) for helping to collect plant samples in the various Siberian regions.
Appendix Group
Supplementary Materials
The following are available online at https://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.
Conflicts of Interest
The author declares no conflict of interest. The funder had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
| Compound | Found in Galeopsis Species of Subgenus | |
|---|---|---|
| Galeopsis (Tetrahit) | Ladanum | |
| Iridoids | ||
| 6-Desoxyharpagide | G. pubescens [6] | |
| G. tetrahit [7] | ||
| Harpagide | G. bifida [8] | G. ladanum [9] |
| G. pubescens [9] | G. ladanum subsp. angustifolia [9] | |
| G. speciosa [9] | G. pyrenaica [8] | |
| G. tetrahit [9] | G. reuteri [8] | |
| G. segetum [9] | ||
| Harpagide 8-O-acetate | G. pubescens [8] | G. ladanum [8] |
| G. ladanum subsp. angustifolia [9] | ||
| G. pyrenaica [8] | ||
| G. segetum [8] | ||
| Galiridoside | G. bifida [8] | G. ladanum [8] |
| G. pubescens [8] | G. pyrenaica [8] | |
| G. speciosa [9] | G. segetum [8] | |
| G. tetrahit [10] | ||
| Gluroside | G. pubescens [6] | |
| G. tetrahit [6] | ||
| Reptoside | G. pubescens [7] | G. ladanum [7] |
| G. tetrahit [7] | G. ladanum subsp. angustifolia [7] | |
| G. pyrenaica [7] | ||
| G. segetum [7] | ||
| Ajugoside | G. bifida [7] | |
| G. tetrahit [7] | ||
| Antirrhinoside | G. segetum [7] | |
| Antirrhinoside 5-O-glucoside | G. segetum [7] | |
| Daunoside | G. pubescens [7] | |
| G. tetrahit [7] | ||
| 8-Epiloganin | G. ladanum subsp. angustifolia [11] | |
| Diterpenoids | ||
| Hispanolone | G. ladanum subsp. angustifolia [12] | |
| Galeopsin | G. ladanum subsp. angustifolia [12] | |
| Pregaleopsin | G. ladanum subsp. angustifolia [12] | |
| Galepsitrione | G. ladanum subsp. angustifolia [13] | |
| Galeolone | G. ladanum subsp. angustifolia [13] | |
| Galepsinolone | G. ladanum subsp. angustifolia [13] | |
| Hispanone | G. ladanum subsp. angustifolia [13] | |
| Galeuterone | G. reuteri [14] | |
| Pregaleuterone | G. reuteri [14] | |
| Triterpenoids | ||
| Hederagenin | G. ladanum subsp. angustifolia [13] | |
| Benzoic acids | ||
| 4-Hydroxybenzoic acid | G. segetum [15] | |
| Vanillic acid | G. segetum [15] | |
| Hydroxycinnamates | ||
| p-Coumaric acid | G. segetum [15] | |
| Caffeic acid | G. segetum [15] | |
| Ferulic acid | G. segetum [15] | |
| Martynoside | G. pubescens [16] | |
| Isomartynoside | G. pubescens [16] | |
| Flavones | ||
| 5,7,4′-Trisubstituted flavones | ||
| Apigenin | G. segetum [15] | |
| Apigenin 7-O-glucoside | G. bifida [17] | G. ladanum [17] |
| G. pubescens [17] | G. ladanum subsp. angustifolia [17] | |
| G. speciosa [9] | G. pyrenaica [17] | |
| G. tetrahit [17] | G. × wirtgenii [17] | |
| Apigenin 7-O-(6″-O-p-coumaroyl)-glucoside | G. bifida [17] | G. pyrenaica [17] |
| G. pubescens [17] | G. segetum [17] | |
| G. speciosa [9] | G. × wirtgenii [17] | |
| G. tetrahit [17] | ||
| Apigenin 7-O-glucuronide | G. bifida [17] | G. ladanum [17] |
| G. pubescens [17] | G. ladanum subsp. angustifolia [17] | |
| G. speciosa [9] | G. pyrenaica [17] | |
| G. tetrahit [17] | G. segetum [17] | |
| G. × wirtgenii [17] | ||
| 5,6,7,4′-Tetrasubstituted flavones | ||
| Scutellarein 7-O-glucuronide | G. pubescens [17] | |
| G. tetrahit [17] | ||
| Ladanetin | G. ladanum [18] | |
| Ladanein | G. ladanum [18] | |
| Salvigenin | G. ladanum subsp. angustifolia [13] | |
| 5,7,8,4′-Tetrasubstituted | ||
| Isoscutellarein 7-O-(2″-O-allosyl)-glucoside | G. ladanum [17] | |
| G. ladanum subsp. angustifolia [17] | ||
| G. pyrenaica [17] | ||
| G. segetum [17] | ||
| G. × wirtgenii [17] | ||
| Isoscutellarein 7-O-(2″-O-(6″’-O-acetyl)-allosyl)-glucoside | G. ladanum [17] | |
| G. ladanum subsp. angustifolia [17] | ||
| G. pyrenaica [17] | ||
| G. segetum [17] | ||
| G. × wirtgenii [17] | ||
| Isoscutellarein 7-O-(2″-O-(6″’-O-acetyl)-allosyl-6″-O-acetyl)- | G. ladanum [17] | |
| glucoside | G. ladanum subsp. angustifolia [17] | |
| G. pyrenaica [17] | ||
| G. segetum [17] | ||
| G. × wirtgenii [17] | ||
| Isoscutellarein 4′-methyl ester 7-O-(2″-O-(6″’-O-acetyl)-allosyl- 6″-O-acetyl)-glucoside | G. ladanum [19] | |
| Galangustin | G. ladanum subsp. angustifolia [20] | |
| 5,7,3′,4′-Tetrasubstituted | ||
| Luteolin | G. segetum [15] | |
| Luteolin 7-O-glucuronude | G. bifida [17] | G. ladanum [17] |
| G. pubescens [17] | G. ladanum subsp. angustifolia [17] | |
| G. speciosa [9] | G. pyrenaica [17] | |
| G. tetrahit [17] | G. segetum [17] | |
| G. × wirtgenii [17] | ||
| 5,7,8,3′,4′-Pentasubstituted flavones | ||
| 8-Hydroxychrysoeriol 7-O-(2″-O-allosyl)-glucoside | G. ladanum subsp. angustifolia [21] | |
| 8-Hydroxychrysoeriol 7-O-(2″-O-(6″’-O-acetyl)-allosyl)-glucoside | G. ladanum subsp. angustifolia [21] | |
| 8-Hydroxychrysoeriol 7-O-(2″-O-(6″’-O-acetyl)-allosyl- 6″-O-acetyl)-glucoside | G. ladanum subsp. angustifolia [21] | |
| Hypolaetin 7-O-(2″-O-allosyl)-glucoside | G. ladanum subsp. angustifolia [17] | |
| Hypolaetin 7-O-(2″-O-(6″’-O-acetyl)-allosyl)-glucoside | G. ladanum [17] | |
| G. ladanum subsp. angustifolia [17] | ||
| G. pyrenaica [17] | ||
| G. segetum [17] | ||
| G. × wirtgenii [17] | ||
| Hypolaetin 7-O-(2″-O-(6″’-O-acetyl)-allosyl- 6″-O-acetyl)-glucoside | G. ladanum [17] | |
| G. ladanum subsp. angustifolia [17] | ||
| G. segetum [17] | ||
| Hypolaetin 4′-methyl ester 7-O-(2″-O-allosyl)-glucoside | G. ladanum [17] | |
| G. ladanum subsp. angustifolia [17] | ||
| G. segetum [17] | ||
| Hypolaetin 4′-methyl ester 7-O-(2″-O-(6″’-O-acetyl)-allosyl)- | G. ladanum [17] | |
| glucoside | G. ladanum subsp. angustifolia [17] | |
| G. pyrenaica [17] | ||
| G. segetum [17] | ||
| Hypolaetin 4′-methyl ester 7-O-(2″-O-(6″’-O-acetyl)-allosyl- | G. ladanum [17] | |
| 6″-O-acetyl)-glucoside | G. ladanum subsp. angustifolia [17] | |
| G. pyrenaica [17] | ||
| G. segetum [17] | ||
| G. × wirtgenii [17] | ||
| Various | ||
| Fatty acids | G. bifida [22,23,24,25] | |
| Acylglycerols | G. bifida [26] | |
| Essential oil | G. bifida [27] | |
| G. pubescens [28] | ||
| G. tetrahit [28] | ||
| Compound | Content, mg/g of Dry Plant Weight ± S.D. | |||
|---|---|---|---|---|
| Leaves | Flowers | Stems | Roots | |
| Iridoid glycosides | ||||
| Harpagide | 11.35 ± 0.23 | 5.18 ± 0.11 | 9.37 ± 0.19 | 0.50 ± 0.01 |
| Harpagide 8-O-acetate | 25.69 ± 0.51 | 10.37 ± 0.20 | 18.53 ± 0.37 | 1.62 ± 0.03 |
| Phenylethanoid glycosides | ||||
| Verbascoside | 21.56 ± 0.51 | 18.98 ± 0.56 | 5.32 ± 0.14 | 2.63 ± 0.06 |
| Isoverbascoside | 14.88 ± 0.38 | 9.15 ± 0.23 | 2.15 ± 0.06 | 2.08 ± 0.05 |
| Lavandulifolioside | 10.21 ± 0.06 | 16.37 ± 0.40 | 8.79 ± 0.26 | 1.57 ± 0.04 |
| Leucosceptoside A | 9.37 ± 0.18 | 3.16 ± 0.06 | 1.75 ± 0.03 | 0.93 ± 0.02 |
| Leonoside A | 3.76 ± 0.07 | 1.58 ± 0.03 | 0.32 ± 0.01 | 0.11 ± 0.00 |
| Leonoside B | 1.60 ± 0.03 | 0.82 ± 0.02 | 0.14 ± 0.00 | <0.01 |
| Caffeoylquinic acids | ||||
| 1-O-Caffeoylquinic acid | 0.53 ± 0.01 | 0.18 ± 0.00 | <0.01 | <0.01 |
| 3-O-Caffeoylquinic acid | 0.92 ± 0.02 | 2.61 ± 0.06 | 0.94 ± 0.02 | <0.01 |
| 4-O-Caffeoylquinic acid | 0.86 ± 0.02 | 0.25 ± 0.00 | <0.01 | <0.01 |
| 5-O-Caffeoylquinic acid | 45.20 ± 1.31 | 12.97 ± 0.38 | 8.30 ± 0.25 | 2.90 ± 0.08 |
| Flavone glycosides | ||||
| Luteolin 7-O-glucuronide | 29.73 ± 0.59 | 39.63 ± 0.79 | 3.75 ± 0.07 | 0.15 ± 0.00 |
| Apigenin 7-O-glucuronide | 19.32 ± 0.37 | 1.93 ± 0.04 | 0.45 ± 0.01 | 0.14 ± 0.00 |
| 6-Hydroxyluteolin 7-O-glucuronide | 2.63 ± 0.05 | 3.84 ± 0.07 | 0.35 ± 0.00 | 0.10 ± 0.00 |
| Scutellarein 7-O-glucuronide | 4.16 ± 0.08 | 5.22 ± 0.10 | 0.26 ± 0.00 | 0.11 ± 0.00 |
| Luteolin 7-O-(6″-O-p-coumaroyl)-glucoside | 11.79 ± 0.23 | 1.84 ± 0.03 | 0.27 ± 0.00 | <0.01 |
| Apigenin 7-O-(6″-O-p-coumaroyl)-glucoside | 12.93 ± 0.25 | 1.02 ± 0.02 | 1.53 ± 0.03 | <0.01 |
| Total content | ||||
| Iridoid glucosides | 37.04 | 15.55 | 27.90 | 2.12 |
| Phenylethanoid glucosides | 61.38 | 50.06 | 18.47 | 7.32 |
| Caffeoylquinic acids | 47.51 | 16.01 | 9.21 | 2.90 |
| Flavone glycosides | 80.56 | 53.48 | 6.61 | 0.50 |
| Phenolic compounds | 189.45 | 119.55 | 34.29 | 10.72 |
| Compound | Content in Populations, mg/g of Dry Plant Weight ± S.D. (Variation Coefficient, %) | |||||||
|---|---|---|---|---|---|---|---|---|
| P1 (n = 21) a | P2 (n = 30) a | P3 (n = 34) a | P4 (n = 28) a | P5 (n = 18) a | P6 (n = 25) a | P7 (n = 20) a | P8 (n = 17) a | |
| Iridoid glycosides | ||||||||
| Harpagide | 8.57 ± 0.42 (4.9) | 10.36 ± 0.61 (5.9) | 14.69 ± 1.29 (8.8) | 18.33 ± 1.41 (7.7) | 2.95 ± 0.20 (6.8) | 4.14 ± 0.23 (5.6) | 2.16 ± 0.16 (7.4) | <0.01 |
| Harpagide 8-O-acetate | 14.53 ± 1.14 (7.9) | 10.69 ± 1.10 (10.3) | 11.82 ± 0.98 (8.3) | 9.35 ± 1.09 (11.7) | 24.52 ± 1.83 (7.5) | 27.18 ± 1.47 (5.4) | 31.82 ± 1.56 (4.9) | 27.53 ± 1.73 (6.3) |
| Phenylethanoid glycosides | ||||||||
| Verbascoside | 10.32 ± 0.64 (6.2) | 8.54 ± 0.79 (9.3) | 5.63 ± 0.47 (8.3) | 5.07 ± 0.52 (10.3) | 20.67 ± 1.01 (4.9) | 25.16 ± 1.24 (4.9) | 22.67 ± 1.79 (7.9) | 27.59 ± 2.23 (8.1) |
| Isoverbascoside | <0.01 | <0.01 | <0.01 | <0.01 | 15.02 ± 0.85 (5.7) | 12.76 ± 0.51 (4.0) | 17.73 ± 1.98 (11.2) | 18.67 ± 1.56 (8.4) |
| Lavandulifolioside | 1.53 ± 0.09 (5.9) | 0.94 ± 0.08 (8.5) | 0.27 ± 0.03 (11.1) | 0.59 ± 0.05 (8.5) | 10.86 ± 0.67 (6.2) | 9.82 ± 0.81 (8.2) | 11.67 ± 0.57 (4.9) | 10.33 ± 0.60 (5.8) |
| Leucosceptoside A | 0.56 ± 0.05 (8.9) | 0.42 ± 0.04 (9.5) | 0.31 ± 0.03 (9.7) | 0.12 ± 0.01 (8.3) | 9.95 ± 0.91 (9.2) | 10.53 ± 0.39 (3.7) | 8.64 ± 0.51 (5.9) | 9.37 ± 0.62 (6.6) |
| Leonoside A | 0.32 ± 0.03 (9.4) | 0.43 ± 0.03 (7.0) | <0.01 | <0.01 | 3.09 ± 0.33 (10.7) | 4.29 ± 0.27 (6.3) | 3.52 ± 0.15 (4.3) | 2.11 ± 0.10 (4.7) |
| Leonoside B | <0.01 | <0.01 | <0.01 | 1.72 ± 0.17 (9.9) | 1.57 ± 0.14 (8.9) | 1.43 ± 0.11 (7.7) | 1.93 ± 0.09 (4.7) | 1.72 ± 0.17 (9.9) |
| Caffeoylquinic acids | ||||||||
| 1-O-Caffeoylquinic acid | <0.01 | <0.01 | <0.01 | <0.01 | 0.62 ± 0.03 (4.8) | 0.27 ± 0.02 (7.4) | <0.01 | <0.01 |
| 3-O-Caffeoylquinic acid | 0.94 ± 0.08 (8.5) | 0.52 ± 0.05 (9.6) | 0.37 ± 0.03 (8.1) | <0.01 | 1.02 ± 0.05 (4.9) | 0.53 ± 0.03 (5.7) | 0.47 ± 0.03 (6.4) | <0.01 |
| 4-O-Caffeoylquinic acid | 0.22 ± 0.02 (9.1) | <0.01 | <0.01 | <0.01 | 0.73 ± 0.05 (6.8) | 0.56 ± 0.05 (8.9) | 0.18 ± 0.02 (11.1) | <0.01 |
| 5-O-Caffeoylquinic acid | 12.67 ± 1.06 (8.4) | 11.73 ± 1.45 (12.4) | 9.69 ± 0.56 (5.8) | 5.33 ± 0.55 (10.3) | 42.53 ± 1.65 (3.9) | 41.75 ± 3.95 (9.5) | 36.18 ± 4.23 (11.7) | 35.02 ± 2.94 (8.4) |
| Flavone glycosides | ||||||||
| Luteolin 7-O-glucuronide | 32.59 ± 1.89 (5.8) | 46.14 ± 2.26 (4.9) | 45.53 ± 3.82 (8.3) | 42.76 ± 3.12 (7.3) | 27.63 ± 2.32 (8.4) | 25.85 ± 1.47 (5.7) | 22.63 ± 2.47 (10.9) | 19.07 ± 0.95 (5.0) |
| Apigenin 7-O-glucuronide | 22.73 ± 1.45 (6.4) | 25.82 ± 1.49 (5.8) | 27.59 ± 1.71 (6.2) | 25.07 ± 1.43 (5.7) | 17.67 ± 2.19 (12.4) | 15.72 ± 1.07 (6.8) | 12.04 ± 1.01 (8.4) | 10.35 ± 0.51 (4.9) |
| 6-Hydroxyluteolin 7-O-glucuronide | 3.67 ± 0.21 (5.7) | 4.57 ± 0.18 (3.9) | 4.96 ± 0.35 (7.1) | 4.50 ± 0.35 (7.8) | 2.90 ± 0.22 (7.6) | 1.27 ± 0.10 (7.9) | 0.95 ± 0.06 (6.3) | 1.11 ± 0.10 (9.0) |
| Scutellarein 7-O-glucuronide | 2.75 ± 0.18 (6.5) | 3.57 ± 0.17 (4.8) | 4.18 ± 0.22 (5.3) | 4.09 ± 0.39 (9.5) | 3.84 ± 0.23 (6.0) | 2.04 ± 0.11 (5.3) | 1.57 ± 0.09 (5.7) | 2.47 ± 0.23 (9.3) |
| Luteolin 7-O-(6″-O-p-coumaroyl)-glucoside | 0.52 ± 0.05 (9.6) | 0.37 ± 0.03 (8.1) | 0.11 ± 0.01 (9.1) | <0.01 | 12.04 ± 0.89 (7.4) | 12.64 ± 0.87 (6.9) | 27.35 ± 2.19 (8.0) | 17.36 ± 1.02 (5.9) |
| Apigenin 7-O-(6″-O-p-coumaroyl)-glucoside | <0.01 | <0.01 | <0.01 | <0.01 | 14.07 ± 0.73 (5.2) | 17.53 ± 0.86 (4.9) | 29.11 ± 1.14 (3.9) | 20.63 ± 1.44 (7.0) |
| Total content | ||||||||
| Iridoid glucosides | 23.10 | 21.05 | 26.51 | 27.68 | 25.47 | 31.32 | 33.98 | 27.53 |
| Phenylethanoid glucosides | 12.73 | 10.33 | 6.21 | 7.50 | 61.16 | 63.99 | 66.16 | 69.79 |
| Saffeoylquinic acids | 13.83 | 12.25 | 10.06 | 5.33 | 44.90 | 43.11 | 36.83 | 35.02 |
| Non-acylated flavone glycosides | 61.74 | 80.05 | 82.26 | 76.42 | 52.04 | 44.88 | 37.19 | 33.00 |
| Acylated flavone glycosides | 0.52 | 0.37 | 0.11 | <0.01 | 26.11 | 30.17 | 56.46 | 37.99 |
| Flavone glycosides | 62.26 | 80.42 | 82.37 | 76.42 | 78.15 | 75.05 | 93.65 | 70.99 |
| Extract No | DPPH | ABTS | SSA | FRAP | ORAC | CBA |
|---|---|---|---|---|---|---|
| P1 | 286.6 ± 5.7 a | 293.4 ± 8.8 g | 182.4 ± 7.2 y | 103.9 ± 4.1 l | 253.0 ± 7.5 p | 298.3 ± 14.9 t |
| P2 | 347.1 ± 6.9 c | 326.2 ± 9.7 h | 193.6 ± 7.7 y | 115.2 ± 4.6 lm | 296.1 ± 8.9 p | 343.1 ± 17.1 u |
| P3 | 353.2 ± 8.9 c | 373.8 ± 11.2 i | 202.8 ± 8.1 y | 125.9 ± 5.0 m | 315.2 ± 9.4 q | 374.1 ± 18.7 v |
| P4 | 302.1 ± 6.0 b | 325.6 ± 9.7 h | 189.4 ± 7.5 y | 109.6 ± 4.4 l | 310.6 ± 9.3 pq | 357.2 ± 17.8 uv |
| P5 | 533.8 ± 10.6 d | 618.2 ± 18.5 j | 294.7 ± 11.8 z | 306.2 ± 12.2 n | 576.3 ± 17.2 r | 657.6 ± 32.8 w |
| P6 | 587.1 ± 11.7 e | 624.3 ± 18.7 j | 326.8 ± 12.9 z | 312.4 ± 12.4 n | 582.9 ± 17.4 r | 699.2 ± 34.9 w |
| P7 | 632.4 ± 12.5 f | 693.0 ± 19.5 k | 363.7 ± 14.5 ã | 329.1 ± 12.9 no | 631.0 ± 18.3 s | 734.8 ± 36.2 x |
| P8 | 604.4 ± 12.0 e | 646.2 ± 19.2 j | 318.2 ± 12.7 z | 361.2 ± 14.0 o | 596.7 ± 17.9 rs | 701.4 ± 35.0 wx |
| Compounds | DPPH | ABTS | SSA | FRAP | ORAC | CBA |
|---|---|---|---|---|---|---|
| Iridoid glucosides | 0.1726 | 0.1671 | 0.1726 | 0.0948 | 0.1488 | 0.1498 |
| Phenylethanoid glucosides | 0.9565 | 0.9437 | 0.9349 | 0.9828 | 0.9477 | 0.9540 |
| Caffeoylquinic acids | 0.8935 | 0.8880 | 0.8722 | 0.8960 | 0.8861 | 0.8954 |
| Non-acylated flavone glycosides | 0.6792 | 0.7288 | 0.7124 | 0.7862 | 0.7454 | 0.7352 |
| Acylated flavone glycosides | 0.9325 | 0.9237 | 0.9486 | 0.9109 | 0.9114 | 0.9102 |
| Flavone glycosides | 0.4458 | 0.5264 | 0.4333 | 0.5797 | 0.5868 | 0.5631 |
| Number | Collection Place | Population Area, km2 | Collection Date | Coordinates | Height (m a.s.l.) | Voucher Specimens No |
|---|---|---|---|---|---|---|
| P1 | Kizhinga, Kizhinginskii District, Republic Buryatia | 2.5 | 20.VI.2019 | 51°47′44.0″ N, 109°52′24.6″ E | 670 | BU/LAM-0619/59–114 |
| P2 | Babushkin, Kabanskii District, Republic Buryatia | 2.8 | 20.VI.2019 | 51°41′18.3″ N, 105°50′39.4″ E | 660 | BU/LAM-0619/63–127 |
| P3 | Tsakir, Zakamenskii District, Republic Buryatia | 1.7 | 20.VI.2019 | 50°24′54.7″ N, 103°34′42.0″ E | 1100 | BU/LAM-0619/76–139 |
| P4 | Tamir, Kyakhtinskii District, Republic Buryatia | 0.9 | 20.VI.2019 | 50°12′51.8″ N, 107°25′34.7″ E | 1150 | BU/LAM-0619/79–146 |
| P5 | Vilyuisk, Viluiskii Ulus, Republic Sakha (Yakutia) | 0.5 | 20.VI.2019 | 63°43′07.3″ N, 121°38′55.9″ E | 110 | YA/LAM-0619/269–418 |
| P6 | Yakutsk, Republic Sakha (Yakutia) | 0.4 | 20.VI.2019 | 62°00′51.1″ N, 129°38′06.6″ E | 100 | YA/LAM-0619/273–425 |
| P7 | Ust-Nera, Oymyakonskii Ulus, Republic Sakha (Yakutia) | 0.9 | 20.VI.2019 | 64°32′23.6″ N, 143°14′49.4″ E | 690 | YA/LAM-0619/293–453 |
| P8 | Verkhoyansk, Verkhoyanskii Ulus, Republic Sakha (Yakutia) | 0.2 | 20.VI.2019 | 67°27′53.3″ N, 133°24′37.0″ E | 400 | YA/LAM-0619/299–457 |