INTRODUCTION

Research interest in the Silene L. genus (family Caryophyllaceae Juss.) is due to the large number of species (more than 700 species) [1], (according to Plant list—487 species) [2], rich composition of ecdysteroids and flavonoids. Earlier, on the basis of chromatographic analysis of seeds, we had identified more than 100 new sources of ecdysteroids of the genera Silene L., Lychnis L., Petrocoptis A. Braun, Sagina L., Saponaria L. et al. [3–5].

Currently, flavonoids of more than 230 species of various Caryophyllaceae genera [6, 7], about 35 species of the Silene genus and 3 species of Lychnis have been studied. It has been shown that many of them are rich producers of flavonoids [6–10]. The most commonly synthesized polyphenols of the C6-C3-C6 series in species of the Silene genus are C-mono- and diglycosides of flavones apigenin, luteolin, etc. The study of a number of Silene species [6, 7] showed that all the studied plants contain vicenins, isovitexin, orientin, homoorientin and their 8-α, 6-α, and 6-β isomers, isosaponarin, vitexin. Thus, in 12 species of the Silene genus, including S. graminifolia Otth, S. jenisseensis Willd, S. chlorantha Willd, vicenins 1, 2, 3 were found [10]. Shaftoside was first discovered in S. schafta S.G. Gmel. ex Hohen [11]. Active studies of secondary metabolites of currently Silene species—S. nutans, S. italica, S. aprica, S. sibirica, S. repens, S. samojedorum, S. dioica, S. graefferi [12–17] have led to the discovery of a large number of flavonoid components. The well-known C–monoglycosides of flavones—isoorientin, isovitexin, isoscoparin, vitexin, and isovitexin glycosides, etc.; C-diglycosides—shaftoside, isoshaftoside, vicenin-2, vicenin-3, carlinoside, lucenin-2; O-glycosides—cosmosiin, cinaroside; C,O-glycosides—saponarin, isovitexin-2"-O-arabinoside, isovitexin-6"-O-arabinoside, etc. In addition, new flavones—selenerepin from S. repens [14], silenesides A, B, C, D, E, F, and G from S. aprica, S. samojedorum, S. jeniseensis, S. italica, S. dioica, their structures have been established [15, 16]. Chemically, S. italica and S. nutans have been studied to a greater extent [13, 18, 19].

Various types of biological activity of flavone C-glycosides have been identified, such as vicenin-1, which in combination with trigoneoside Ib is recommended for the treatment of autoimmune diseases [20]. Vicenin-2 exhibits antioxidant, anti-inflammatory, antitumor and hepatoprotective properties, in addition, antidiabetic activity [21]. Orientin successfully slows down the aging process, and also has antioxidant, antiviral, antibacterial, anti-inflammatory, cardioprotective, radiation protective effects [22]. Vitexin exhibits powerful hypotensive, antiinflammatory and antispasmodic (nonspecific) properties [23]. The biological activities of some Silene species are known. Thus, antimicrobial activity of more than 30 species of the genus Silene has been established [24, 25]. V. Darmograi et al. have developed methods for the treatment of purulent wounds based on Silene extracts [26]. Studies of the activity of extracts, as well as the amount of flavonoids isolated from S. chalcedonica and some Silene species introduced in the Siberian Botanical Garden of Tomsk State University (SibBS TSU), have shown the manifestation of hemorheological, gastroprotective, anti-inflammatory, cerebroprotective, analgesic, actoprotective, antitumor effects [27–31]. The data obtained so far indicate the prospects of identifying new sources of pharmacologically valuable flavonoids among the species of the Caryophyllaceae family.

The purpose of this work is to study the composition and content of flavonoids of 12 Silene species introduced in the south of Western Siberia.

EXPERIMENTAL

Plant raw materials. The objects of research are plants of the Silene genus, the seeds of which were obtained from botanical gardens in Western Europe (Germany, Poland, France, Belarus and other countries), successfully introduced into culture in SibBG TSU: Silene caramanica Boiss., S. chalcedonica (L.) E.H.L. Krause, S. colpophylla Wrigley, S. graminifolia Otth., S. frivaldszkyana Hampe., S. linicola C.C. Gmelin., S. nemoralis W.K. (Wald. et. Kit.) Pers., S. jenisseensis, S. paradoxa L., S. roemeri Friv., S. sendtneri Boiss., S. viridiflora L. Sp. Pl. Some of them (S. frivaldszkyana, S. roemeri, S. sendtneri, etc.) are endemic to the countries of the Balkan peninsula. The species was determined by the researcher of SibBS TSU Ivanova N.A. and professor of the Botany Department of Biological Institute of Tomsk State University (BIO TSU) A.I. Pyak. Vouchers are stored in the P.N. Krylov Herbarium, TSU.

Quantitative determination of flavonoids. Preparation of extracts: air-dry raw materials weighing 1 g was extracted five times with 70% ethyl alcohol in a water bath at a temperature of 55°C. The obtained extracts were combined and concentrated using a rotary evaporator (IKA RV 10, Germany) at heating up to 50°C. The resulting solutions were centrifuged.

The analysis of flavonoids in concentrated extracts was performed by high-performance liquid chromatography (HPLC) on a liquid chromatograph “Shimadzu LC-20AD” (Japan), a diode-matrix detector, a chromatographic column Perfect Sil Target ODS—3; 4.6 × 250 mm, the grain size of the sorbent is 5 microns. Eluent A: a mixture of acetonitrile, isopropyl alcohol (5 : 2 v/v), eluent B: 0.1% trifluoroacetic acid. Analysis time—60 min. The elution rate is 1 mL/min. Elution mode: low pressure gradient; gradient program: 0–40 min 15–35% eluent A, 40–60 min 35% eluent A. The sample volume is 5 µL. Analytical wavelength for registration of flavonoids λmax = 272 and 330 nm.

Phenolic compounds were identified using standards (Sigma-Aldrich, purity ≥95.0%). Identification of signals on chromatograms was carried out by comparing the retention times of the components of extracts and reference samples, as well as absorption maxima in UV spectra. The flavonoid content was calculated from the peak areas of the sample and the corresponding standards using a calibration curve constructed using the LC Postrun Calibration Curve software. The analysis was carried out in three repetitions, statistical calculations were carried out in Microsoft Excel, 2007. The data is presented in the form of arithmetic mean and standard error.

Isolation of flavonoids. Air-dry raw materials S. chalcedonica (200 g). extracted fivefold with 70% ethyl alcohol solution. The resulting extract was filtered and concentrated under vacuum at a temperature of 55°C. The concentrated extract was diluted with water in a ratio of 1 : 2 (v : v) and re-filtered. Then the filtrate was cleaned from lipophilic substances three times with n-hexane. The control of the content of BAS in fractions was carried out by the HPLC method. The extraction of the amount of secondary metabolites from the purified filtrate was carried out by repeated extraction with n-butanol. The yield of the butanol fraction in dry form was 21.2% of the dry raw material. When concentrating the butanol fraction dissolved in 80% ethyl alcohol, a yellow-brown precipitate fell out, the yield of which was 1.26% of the raw material). Recrystallization of the precipitate using 70 and 95% ethyl alcohol allowed to obtain a pure compound, the content of which was 81% of the sum of the peaks of flavonoids or 1.02% of the raw materials).

Identification of a dedicated connection. The structure of the compound was determined by HPLC, UV spectroscopy, mass spectrometry, 1H and 13C NMR spectroscopy. Flavonoids of other types are obtained by spontaneous precipitation from concentrated ethanol extracts at a temperature of 8°C and identified by HPLC by comparison with standards and isolated flavonoid.

Mass spectrometric analysis was performed on a 6500 QTRAP mass spectrometer (SCIEX, USA) in the electrospray ionization mode (ESI) in the region of positive and negative ionization. The solutions were infused with a syringe embedded in a mass spectrometer at a rate of 5 µL/min. Molecular ions were scanned in Q1 mode at a speed of 200 Da/s in the mass range of 500–700 Da. Parameters of the mass spectrometer operation: curtain gas pressure (CUR) = 20 psi, spray gas (GS1) and desiccant gas (GS2) 15 psi each, desiccant gas temperature (TEM) = 100°C, declustering potential (DP) = 80 V, inlet potential (EP) = 10 V, the voltage at the ion source (IS) = 5500 V (positive ionization) and 4500 V (negative ionization). The fragmentation mass spectrum was analyzed in the Enhanced Product Ion (EPI) mode. The selected molecular ions were fragmented with a gradual increase in the values of fragmentation energy (CE) from 5 to 30 V, the mass spectrum was measured in the range from 50 to 600 Da. NMR spectra of 1H and 13C were recorded on a Bruker AV = 400 NMR spectrometer, solvent—DMSO-d6.

RESULTS AND DISCUSSION

According to experimental data, the studied species of the Silene genera have been successfully introduced into SibBS TSU. Due to the fact that flavones were discovered for the first time in most of the studied species and it was possible to isolate one of them, its structure was established, which is a convincing proof of its presence in the studied species.

Identification of Flavonoids

The compound isolated from S. chalcedonica was identified by comparing the spectral characteristics (UV, MS, NMR 1H and 13C) (electronic application) with those described in the literature. In the remaining species, flavonoids were identified by comparing HPLC data with standards. The observed short-wave (270 nm) and long-wave (336 nm) absorption maxima (Fig. 1) are characteristic of the chromophore system of flavones.

Fig. 1.
figure 1

HPLC chromatogram/UV compounds isolated from S. chalcedonica.

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Thus, based on the spectral and chromatographic characteristics obtained by us (UV, MS, NMR, 1H, and 13C, HPLC), the major component of the sum of S. chalcedonica flavonoids was identified as 8-α-Larabinopyranosyl-6-β-D-glucopyranosyl apigenin—shaftoside.

It is shown that the studied 12 Silene species are rich sources of flavonoids, 15–18 compounds were identified in them, especially the C-glycosides of the flavones apigenin and luteolin (Table 1). In addition, a number of unidentified compounds have been discovered, which, according to the characteristic absorption spectra of 270 and 330 nm, are attributed to flavonoids. Identification of flavonoids based on HPLC data indicates that chromatographic profiles of flavonoids of Silene extracts (Table 1) differ. In Silene species 2–12, a more diverse flavonoid profile was found than in S. chalcedonica, in which shaftoside, neovitexin and isovitexin were found within the existing standards.

Table 1. Composition of flavonoid extracts of the studied Silene species
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1, Silene chalcedonica; 2, S. frivaldszkyana; 3, S. roemeri; 4, S. sendtneri; 5, S. viridiflora; 6, S. paradoxa; 7, S. colpophylla; 8, S. nemoralis; 9, S. graminifolia; 10, S. linicola; 11, S. jenisseensis; 12, S. caramanica. FL 1—FL 19 are unidentified flavonoids.

Almost 50 years after the first discovery of shaftoside in the genus Silene—S. schafta [11], new sources of this C-diglycosyl flavone were identified in the species of the genus Silene—S. repens, S. sibirica, S. italica, S. dioica, S. aprica, S. samojedorum, S. nutans [12–16, 18, 19]. In this work, the presence of shaftoside was established for the first time in all 12 Silene species studied—S. sendtneri, S. roemeri, S. viridiflora, S. paradoxa, S. nemoralis, S. frivaldszkyana, S. graminifolia, S. jenisseensis, S. linicola, S. caramanica, S. colpophylla, S. chalcedonica (Table 2).

Table 2. The content of identified flavonoids in some Silene species (flowering phase, % per a.s.s.)
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It is a major component in the amounts of flavonoids of most of the studied species. Its share in the sum of all flavonoids of the species S. chalcedonica, S. caramanica, S. paradoxa, S. nemoralis (flowering phase) is 81–86%, and in S. roemeri, S. graminifolia, S. viridiflora, S. colpophylla, S. frivaldszkyana 50–76%. Analysis of the distribution of another C-diglycoside of flavone—vicenin-2, which was previously found in S. chlorantha, S. graminifolia, S. jenissensis, S. italica, S. nutans, S. wolgensis, S. saxatilis [6, 13, 18], S. repens, and S. sibirica [14, 19], showed that it was found in all the studied species, with the exception of S. chalcedonica, which was previously assigned to the genus Lychnis L. Vicenin-2 was discovered for the first time in 9 species, its presence in S. jenisseensis, S. graminifolia was reported earlier [6].

Of the C-monoglycosides of flavones, vitexin was detected for the first time in S.sendtneri, S. roemeri, S. paradoxa, S. frivaldszkyana, S. linicola, it was not detected in S. graminifolia and S. jenisseensis, whereas its presence was previously reported [6] and isovitexin in the latter [6, 15]. Neovitexin was detected for the first time in all species, with the exception of S. linicola, S. caramanica, and S. jenisseensis. It is known that neovitexin is less common, it was found earlier in species of the family Caryophyllaceae—S. chalcedonica [32] and Myosoton aquaticum (L.) Moench [7]. Orientin was first discovered in S. sendtneri, S. roemeri, S. viridiflora, S. paradoxa, S. nemoralis, S. frivaldszkyana, S. colpophylla, in addition, its presence is confirmed in S. graminifolia and S. jenisseensis [6]. In extract S. roemeri and S. chalcedonica found isovitexin, in S. paradoxa—cinaroside.

The total content of flavonoids (identified and unidentified, but having absorption maxima characteristic of the chromophore system of flavones) in the aboveground part of the studied species is quite high, the highest is characterized by S. nemoralis (6.75%), high—S. sendtneri (5.15%), S. caramanica (5.06%), S. frivaldszkyana (4.75%), S. paradoxa (4.28%), S. chalcedonica (3.35%). The highest content of vicenin-2 is characteristic of S. linicola and S. graminifolia; shaftoside—S. nemoralis, S. caramanica, S. sendtneri, S. frivaldszkyana, S. paradoxa, S. chalcedonica; orientin—S. linicola; neovitexin— S. sendtneri (Table 3).

Table 3. Dynamics of flavonoid content in the aboveground part of Silene species during the growing season, % per a.s.s.
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An assumption is made about the dynamic process of flavonoid synthesis based on different levels in different phases of vegetation, as well as the fact that individual flavonoids were not detected in all periods of development. In this regard, the study of the flavonoids accumulation dynamics during the growing process has been undertaken using the example of 4 endemic species that are most successfully adapted to the conditions of Western Siberia. It is established that there is a change in the composition of flavonoids during the growing season. Thus, in a number of species—S. nemoralis, S. graminifolia, S. linicola, orientin was detected in the flowering phase (Table. 2), whereas in S. roemeri—only at the beginning of vegetation and budding, and in S. colpophylla—during the formation of reproductive organs (Table 3). The greatest variety of flavonoids is found in plants flowering and fruiting phases. It should be noted that throughout the growing season, shaftoside is present in the aboveground part of all species, similarly to vicenin-2—in S. roemeri and S. sendtneri; neovitexin—in S. frivaldszkyana and S. sendtneri, whereas in S. colpophylla—only in the budding and flowering phases. Vitexin in minor amounts was found in all phases of development in S. frivaldszkyana, whereas in S. sendtneri—only in the flowering phase. The presence of vicenin-2 in S. frivaldszkyana was established only during the formation of buds and flowers, and in the flowering phase much more. In all likelihood, there is a species specificity in the need for the presence of individual flavonoids in certain phases of development, depending on the biology of the species.

It should be noted that the phases of maximum accumulation of C-diglycosides in the aboveground part of S. roemeri are the beginning of vegetation, S. frivaldszkyana is flowering. In S. colpophylla, the highest levels of shaftoside were determined at the beginning of vegetation, and vicenin-2—in flowering, whereas in S. sendtneri shaftoside—during flowering, and vicenin-2—at the end of the growing season.

It is likely that the importance of individual components of synthesized flavonoids in the development of perennial Silene species in certain periods increases or weakens. Thus, the proportion of vicenin-2 in S. roemeri (Table 3) in the total S. roemeri the total amount of flavonoids varies from the maximum at the beginning of the growing season (8.9%) to the minimum during bud formation (3.3%), while this indicator of shaftoside is quite high and varies during the growing season from 47.1 to 74.1%, reaching a maximum during bud formation.

The proportion of vicenin-2 in the total amount of flavonoids was insignificant during the formation of buds and flowers of S. frivaldszkyana (0.9 and 2.7%), shaftoside—the highest at the beginning of the growing season and in budding 92.7 and 89.6%, respectively, despite the fact that the highest content of shaftoside in S. frivaldszkyana is noted in the flowering phase—3.63%, the share of it decreased to 76.4% during this period.

Probably, due to the fact that shaftoside is a major component throughout the growing season, the nature of changes in the content of the entire amount of flavonoids is observed similar to shaftoside—in S. sendtneri the maximum of both indicators was noted in flowering (5.15 and 3.84%), S. roemeri—at the beginning of the growing season (5.18 and 3.30%, respectively). Thus, it is possible to collect plant raw materials purposefully in the phases of maximum accumulation of individual flavonoids and their amounts.

Comparison with the literature data showed that in the well-known producers of S. jenissensis and S. graminifolia flavones, in which the presence of vitexin, isovitexin, vicenins 1,2,3,orientin, and isoorientin was previously detected, we first identified shaftoside, and in the latter—neovitexin. In all other species, all flavones have been studied for the first time. Perhaps these differences in the composition of flavonoids are explained by the influence of the place of plant growth or by a limited set of standards at the disposal of researchers.

CONCLUSIONS

In the studied Silene species, well-known and characteristic flavonoids of the genus were identified for the first time. Shaftoside and neovitexin were first discovered in S. graminifolia S. jenissensis. In 9 studied species of the genus Silene, vicenin-2 was detected for the first time. Shaftoside was found for the first time in all the studied introducents. The structure of shaftoside isolated from S. chalcedonica was established by physicochemical methods—UV, MS, NMR 1H, and 13C.

Neovitexin was detected for the first time in the species S. chalcedonica, S. frivaldszkyana, S. roemeri, S. sendtneri, S. viridiflora, S. paradoxa, S. colpophylla, S. nemoralis, S. graminifolia; vitexin—S. chalcedonica, S. frivaldszkyana, S. roemeri, S. paradoxa, S. linicola

The producers of orientin are S. linicola, S. graminifolia, S. roemeri, S. colpophylla, S. nemoralis, S. colpophylla and S. jenisseensis. In addition to common flavonoids, species—specific compounds were also found in the extracts—cinaroside in S. paradoxa, isovitexin S. roemeri and S. chalcedonica.

The peculiarities of changes in the composition of flavonoids during the vegetation of plants have been studied. It is shown that shaftoside is present in all species, and vicenin-2, neovitexin and vitexin—in a number of species throughout the growing process. Orientin was found in separate phases of development, mainly during the period of active plant growth and the development of reproductive organs.

The data obtained allow us to recommend the studied Silene species as sources of valuable flavonoids with antioxidant, anti-inflammatory, antitumor, gastroprotective, analgesic and antidiabetic effects.