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Article

Chemical and Biological Characterization of Metabolites from Silene viridiflora Using Mass Spectrometric and Cell-Based Assays

by
Nilufar Z. Mamadalieva
1,2,3,4,*,
Alexey Koval
4,
Maksud M. Dusmuratov
5,
Hidayat Hussain
6 and
Vladimir L. Katanaev
4,*
1
Institute of the Chemistry of Plant Substances, Uzbekistan Academy of Sciences, Mirzo Ulugbek Str. 77, Tashkent 100170, Uzbekistan
2
Tashkent Institute of Irrigation and Agricultural Mechanization Engineers, National Research University, Kori Niyazov Str. 39, Tashkent 100000, Uzbekistan
3
Department of Pharmacy and Chemistry, Faculty of Medicine, Alfraganus University, Tashkent 100190, Uzbekistan
4
Translational Research Center in Oncohaematology, Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland
5
Department of Organic Synthesis, Faculty of Industrial Pharmacy, Tashkent Pharmaceutical Institute, Oybek Str. 45, Tashkent 100015, Uzbekistan
6
International Joint Laboratory of Medicinal Food Development and Health Products Creation, Biological Engineering Technology Innovation Center of Shandong Province, Heze Branch of Qilu University of Technology (Shandong Academy of Sciences), Heze 274000, China
*
Authors to whom correspondence should be addressed.
Biomolecules 2024, 14(10), 1285; https://doi.org/10.3390/biom14101285
Submission received: 7 August 2024 / Revised: 30 September 2024 / Accepted: 9 October 2024 / Published: 11 October 2024

Abstract

:
A comprehensive metabolite profiling of the medicinal plant Silene viridiflora using an UHPLC-ESI-MS/MS method is described for the first time. A total of 71 compounds were identified and annotated, the most common of which were flavonoids, triterpene glycosides, and ecdysteroids. The three major compounds schaftoside, 26-hydroxyecdysone, and silviridoside can be chosen as the markers for the assessment of the quality of S. viridiflora preparations. The methanol extract and a variety of metabolites identified in S. viridiflora were screened for their cytotoxic and Wnt pathway-inhibiting activities against triple-negative breast cancer (TNBC), the deadliest form of cancer in women. 2-Deoxy-20-hydroxyecdysone with submicromolar IC50 was identified as a result. The structure–activity relationship derived from the data from the in vitro proliferation assay showed that the hydroxyl group present at position C-2 of steroid core reduces the ecdysteroids’ cytotoxicity against cancer cells.

1. Introduction

Caryophyllaceae is a large family of flowering plants commonly known as the pink family or carnation family. It includes around 80 genera and more than 2600 species, distributed worldwide. Species within this family offer a diverse range of uses across ornamental, medicinal, culinary, ecological, and cultural contexts, making it an important group of plants with practical significance [1]. Silene L. is a large genus of plants from the Caryophyllaceae family with more than 700 species of annual, biennial, and perennial plants. Young shoots and the leaves of some Silene species are used as medicine and food in European countries. Silene viridiflora, a member of the Caryophyllaceae family, is native to Eurasia and widely distributed in Crimea–Siberia and Mediterranean countries (France, Italy, and the Balkans) [2]. It is a perennial and grows primarily in temperate biomes. The plant has simple, broad leaves and capsule fruits. Individuals can grow to 50–80 cm. The aerial parts of S. viridiflora contain ecdysteroids (up to 1.5% dry basis), triterpene glycosides, lipids, neutral substances, carbohydrates, and microelements [3]. Biological activities, such as tonic, immunomodulator, actoprotector, adaptogen, antioxidant, and enzyme inhibitory activities, have been reported for the extracts, ecdysteroids and mixtures of ecdysteroids isolated from the S. viridiflora [3,4,5,6]. Ecdysteroids and triterpene glycosides are regarded as a common phytochemical feature of S. viridiflora [3,7,8]. Nevertheless, no data are present on the whole metabolite composition in the aerial parts of this species. Therefore, it was relevant to conduct a phytochemical analysis of S. viridiflora in order to understand the chemistry for the potential health benefits of this plant.
In the present work, the ultra-high performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UHPLC-ESI-MS) was applied to investigate the metabolite composition of the methanolic extract obtained from the aerial parts of S. viridiflora. In addition, the methanol extract and a variety of metabolites identified in S. viridiflora were screened for their potential to influence Wnt signaling and proliferation in triple-negative breast cancer (TNBC) cells. As TNBC is the deadliest form of gynecological cancer and is the target of massive attempts to develop natural-product-based anticancer compounds [9,10,11], such information is notably essential to obtain a deeper insight into the bioactive molecules in S. viridiflora and their mechanisms of action. A structure–activity relationship (SAR) study then determined the main structural features required to inhibit TNBC cells for the tested set of ecdysteroids.

2. Materials and Methods

2.1. Chemicals and Reagents

Methanol, acetonitrile, water and formic acid were purchased from Merck (Darmastadt, Germany). Ultrapure water and ammonium formate (≥99%, MS eluent additive) were obtained from Sigma Aldrich (Steinheim, Germany). Docetaxel was purchased from LC Laboratories (Woburn, MA, USA). The reference compounds quinic acid, p-coumaric acid, ferulic acid, oleanolic acid, isovitexin-7-O-glucoside, and rutin were purchased from Sigma-Aldrich (Milan, Italy) and Merck KGaA (Darmstadt, Germany). 20-Hydroxyecdysone, 2-deoxy-20-hydroxyecdysone, and shaftoside, obtained from the Institute of the Chemistry of Plant Substances (Tashkent, Uzbekistan), were used in this study with purities > 99%. Cell culture media (DMEM), supplements, dimethylsulfoxide (DMSO) were obtained from Gibco (Waltham, MA, USA). Renilla luciferase and XtremeGENE 9 reagent were purchased from Addgene (Cambridge, MA, USA) and Roche Holding (Bazel, Switzerland), respectively.

2.2. Plant Material

The aerial parts (flowers, leaves, and stems) of S. viridiflora were collected from the botanical field of the Institute of the Chemistry of Plant Substances (Tashkent, Uzbekistan). The taxonomic authentication was accomplished by Dr. A. Nigmatullaev of the Department of Herbal Plants of the ICPS. The voucher specimen of the plant was deposited in the departmental herbarium under the code 2017/087. The plant material was air-dried and powdered before use.

2.3. Preparation of Extract for Bioassays

The aerial parts of S. viridiflora were washed gently and left to dry naturally at room temperature. The dried plant material was ground to a fine powder with a Waring blender. After grinding, 50–100 g of the plant material was extracted with 200–500 mL of methanol. The extraction process by maceration was carried out by immersing the plant material in methanol for one day. The solvent was evaporated in a rotary vacuum evaporator at 40 °C. The obtained extract was then kept in a refrigerator until further use.

2.4. Preparation of Extract for UHPLC-MS Measurements

A total of 10 mg of powdered plant material was accurately weighed and placed into a tube with a cover, and 5 mL methanol solvent was added. After 15 min of ultrasonication at 50 °C, the extract was centrifuged at 14,000 g for 10 min to remove debris. After centrifugation, the supernatant was transferred to vials (1 mL) and filtered by a poly-tetraflouroethylene filter with a pore size 0.45 μm. This solution has been used for the UHPLC-MS measurements.

2.5. UHPLC-QTOF-MS/MS Analysis

Metabolites present in the methanol extract of S. viridiflora were identified using an advanced analytical technique, UHPLC-QTOF-MS/MS. The extract was injected into an Acquity-UPLC (Waters Inc., Milford, MA, USA) and separated on a Nucleoshell RP18 (150 mm × 2 mm × 2.7 µm; Macherey & Nagel, Düren, Germany) at 40 °C. The sample volume injected was set at 5 μL. The ESI mass spectra were acquired in positive and negative ion electrospray ionization mode by scanning over the m/z range 100–1200. The mobile phase consisted of 0.3 mM ammonium formate with 0.7 mM formic acid in water (A) and acetonitrile (B). The column flow was set at 0.3 mL/min, the autosampler temperature was 4 °C. Data interpretation was carried out using Sciex PeakView 2.1 software and ACD/MS Fragmenter (ACD/Lab, Toronto, ON, Canada). The putative known and unknown compounds were annotated by the Human Metabolome Database, MassBank Spectral Database, METLIN Metabolomics Database, as well as by comparison with standard compounds.

2.6. Cell Cultures

The cytotoxic activity of the samples was screened against the human triple-negative breast cancer BT-20 (HTB-19), HCC1395 (ATCC®CRL-2324), MDA-MB-231 (CRM-HTB-26), and HEK293 (CRL-1573) embryonic kidney cell lines. The cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 4.5 g/L glutamine and D-glucose, 10% fetal bovine serum (FBS), and a 1% penicillin/streptomycin mixture and incubated at 37 °C under a 5% CO2 humidified atmosphere. Cell passage and seeding performed after washing the adherent cells with PBS and detaching them using a Trypsin solution. The cells were detached and resuspended at 160,000 cells/mL and added into each well of a transparent 384-well plate in the final volume of 25 µL/well (4000 cells per well). The cells were maintained in DMEM containing 10% FBS at 37°C and 5% CO2 overnight. The next day, the 384-well plate was examined under an inverted microscope to identify cell seeding errors, growth characteristics, morphology and equal distribution. Then, the medium in each well was removed using a washer–dispenser (Biotek FX, Beersel, Belgium) and replaced by 40 μL of the fresh medium containing the indicated concentrations of compounds and incubated for 72 h.

2.7. Sample Preparation for Bioassays and Treatment of Cells

The 250 µg/mL concentrations of methanol extract and 20 mM concentrations of individual compounds were prepared as a stock solution in DMSO (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) prior to use. A complete medium was used to prepare this solution. The samples were further serially diluted in DMSO into eleven different concentrations and then added to the complete cell culture medium so as to attain the final concentrations ranging from 250 to 8.6 µg/mL (250, 178.6, 127.6, 91.1, 65.1, 46.5, 33.2, 23.7, 16.9, 12.1, and 8.6 µg/mL) for the extract and 20 to 1.45 µM (20, 15.4, 11.8, 9.1, 7.0, 5.4, 4.1, 3.2, 2.5, 1.9, and 1.5 µM) for the individual compounds in 384-well plates, which were added in four replicates. A 40 µL sample containing medium from each concentration was dispensed into each well. The concentration of the solvent, DMSO, was equal in all the wells and was fixed at 0.05% in the medium. As a negative control, cells were treated with DMEM containing DMSO. Docetaxel (concentration of 0.02 µM to 6.9 nM) is used as a positive control.

2.8. MTT Assay

The cytotoxicity of the samples was determined in triplicate using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay. The next day, the medium of the 384-well plates treated with samples was removed. Then, 25 µL of MTT solution (0.5 mg MTT in 1 mL PBS) was added to each well and incubated at 37 °C for 2 h. After incubation with MTT for 2 h, the solution was removed from the wells by the washer–dispenser, and the formed formazan crystals were then dissolved in 25 µL of DMSO. After a further 10 min of incubation at room temperature, the samples were mixed briefly, and the absorbance was detected at 510 nm with a Tecan Infinite 200 Pro Reader (Tecan Group Ltd., Männedorf, Switzerland). The cell viability rate (%) was calculated by the following formula:
Cell viability rate (%) = ((OD of treated cells − OD of media (blank)/(OD of control cells − OD of media (blank)) × 100%

2.9. TOPFlash Assay

The TOPFlash assay was performed as described [12,13]. TNBC BT-20 cell line stably transfected with TopFlash reporter plasmid was seeded at 450,000 cells/mL in a white opaque 384-well plate in the final volume of 25 μL. The cells were maintained and incubated at 37 °C with 5% CO2 overnight for attachment. Subsequently, they were transfected by a plasmid encoding Renilla luciferase under the CMV promoter using 12 μg/mL of DNA and 40 μL/mL XtremeGENE 9 reagent as described in the manufacturer’s protocol. The next day, the medium in each well was replaced with a 20 μL fresh medium containing Wnt3a (2.5 μg/mL) and compound dilution. Compound dilutions were prepared by serial dilution in DMSO and diluted with the amount of medium necessary to obtain their final concentrations indicated on the figures and tables and maintain a concentration of DMSO of 0.05% in all assay points. Wnt3a was added after 1 h of preincubation with compound dilution. After overnight incubation, the supernatant in each well was removed by the washer–dispenser, and the luciferase activity was measured as described [12,13]. Briefly, the culture medium was completely removed from all wells of the plate. Next, the luciferase activity of the firefly and Renilla luciferases was detected sequentially in individual wells of a 384-well plate through the injection of corresponding measurement solutions in Tecan Infinite 200 Pro multifunctional plate reader with an injection module [12].

2.10. Statistical Analysis

The experiments were carried out in four replicates. Continuous variables were presented as the mean ± SD. The IC50 was determined as the drug concentration which resulted in a 50% reduction in cell viability or the inhibition of biological activity. The level of significance was set at p < 0.05. The IC50 (inhibitory concentration which caused 50% inhibition) was estimated using the linear regression method of plots of the percent of cell viability against the concentration of the tested compounds using GraphPad Prism 8.0.1 software (San Diego, CA, USA).

3. Results and Discussion

3.1. UHPLC-HR-MS Profiling of Metabolites in a S. viridiflora Extract

As part of an ongoing study on secondary metabolites of the aerial parts of S. viridiflora metabolites [3,8,14,15,16], we performed, for the first time, qualitative chromatographic analyses of comprehensive compounds by means of UHPLC-ESI-MS/MS detection. UHPLC-MS/MS metabolite profiling of S. viridiflora revealed richness in diverse plant compounds. The base peak chromatograms of the methanol extract of S. viridiflora in both negative and positive ionization modes are displayed in Figure S1. The tentatively identified compounds are summarized in Table 1. Seventy-one compounds were tentatively identified based on the retention times, MS and MS/MS accurate masses, fragmentation patterns in MS/MS spectra, relative ion abundance, and comparison with reference standards and literature data. Compounds belonged to various classes, including flavonoid glycosides (19 compounds), triterpene glycosides (15), ecdysteroids (12), amino acids (10), phenolics (7), organic acids (3), sugar compounds (3), and polyols (2). In this study, all the compounds from S. viridiflora except metabolites 3133, 36, 40, 49, 5051, 58, and 65 [3,8,14,15,16] were annotated for the first time.

3.1.1. Flavonoids

Flavonoids were detected as the most abundant class, represented by 19 peaks belonging to flavanone, flavone, and flavonol subclasses, as C- and O-glycosides. In addition to the derivatives of apigenin, quercetin, and luteolin, a flavonone hesperidin was also detected in the extract. The annotated flavonoids are summarized in Table 1. Some researchers reported that the Silene species are rich sources of flavonoids, especially the C-glycosides of the flavones apigenin and luteolin [17]. The most common apigenin C-monoglycosides found in the studied Silene species are vitexin, isovitexin, and a number of their derivatives; luteolin derivatives, as well as orientin and others, are found slightly less frequently [18]. Chemical investigations show that most of the studied species contain vicenins, isovitexin, orientin, isoorientin and their 8-α, 6-α, and 6-β isomers, isosaponarin, and vitexin. The MS/MS analysis led to the successful identification of the aglycone moieties. MS/MS was performed to assist in structural elucidation, where the nature of sugars could be revealed from the elimination of the sugar residue, i.e., 162 amu (hexose, glucose) and 146 amu (rhamnose) or 132 amu (pentose, arabinose). Flavones are represented mainly by apigenin derivatives, which are considered the active principles of S. viridiflora. In this plant, apigenin flavones accumulate as C-glycosides 2324, 26, 2930, 34, 3839, and 42, except compound 26. Based on its fragmentation pattern and according to the literature, compound 26 was tentatively identified as cosmosiin (apigenin 7-O-glucoside). Quercetin O-glycosides were represented by five peaks (41, 43, 46, 47, 54), while peaks 25, 35, 37 and 45 represented luteolin glycosides. The major metabolite 29 was eluted at 7.14 min and has a [M − H] ion at m/z 563.1406 and the molecular formula C26H28O14. In the MS/MS spectra of this compound, the diagnostic ions at m/z 473.1054, 443.0950, and 383.0759 were produced by neutral losses of C3H6O3 (90Da), C4H8O4 (120Da), C2H4O2 (60Da), and +C4H8O4 (120Da), indicative of a flavonoid C-glycoside. By comparing the MS/MS spectra with the literature data [19], metabolite 29 was identified as schaftoside. The MS spectrum interpretation allows for the annotation of hesperidin signals with m/z 611.1980 [M + H]+ and 628.2221 [M + NH4]+ in peak 53. This flavonoid earlier was isolated from S. alba, S. conoidea, S. compacta, S. dichotoma, S. italica, S. supine, S. vulgaris, and S. schimperiana (Table 1) [20,21,22].
Table 1. Metabolites identified in Silene viridiflora using UHPLC-ESI-MS/MS in both negative and positive ionization modes.
Table 1. Metabolites identified in Silene viridiflora using UHPLC-ESI-MS/MS in both negative and positive ionization modes.
NRt (min)Tentatively Identified
Metabolites
Average m/zReference m/zError (ppm)Adduct TypeMS/MS FragmentsFormulaResourceReferenceClass
10.77Histidine156.0769156.07680.8[M + H]+ C6H9N3O2S. colorata, S. dioica[23,24]Amino acids
20.78Arginine175.1189175.1195−3.4[M + H]+ C6H14N4O2S. colorata, S. dioica[23,24]Amino acids
30.81Glutamine145.0622145.06192.1[M − H] C5H10N2O3S. colorata[23]Amino acids
147.0765147.07640.4[M + H]+
40.83Glutamic acid146.0452146.0459−4.7[M − H] C5H9NO4S. alba, S. colorata, S. dioica[23,24]Amino acids
50.84Glucaric acid209.0312209.03034.4[M − H] C6H10O8 Sugar compounds
60.85Pinitol195.0858195.0863−2.3[M + H]+ C7H14O6S. brahuica, S. ruscifolia[25,26]Polyols
7 *0.85Sucrose341.1069341.1084−4.3[M − H]387.1133 [M + HCOO]C12H22O11S. vulgaris, S. nutans, S. noctiflora, S. ruscifolia[26,27]Sugar compounds
343.1213343.1235−6.5[M + H]+365.1044 [M + Na]+
80.88Trehalose387.1130387.1144−3.6[M + HCOO]729.2293 [2M + HCOO]C12H22O11S. ruscifolia[26]Sugar compounds
360.1493360.1500−2.0[M + NH4]+
90.86Proline114.0549114.05490.2[M − H] C5H9NO2S. colorata[23]Amino acids
116.0709116.07062.8[M + H]+
100.86Norvaline118.0859118.0863−3.6[M + H]+ C5H11NO2 Amino acids
110.87Threonine120.0663120.06611.7[M + H]+ C4H9NO3S. colorata, S. dioica[23,24]Amino acids
12 *0.89Quinic acid191.0567191.056113.1[M − H] C7H12O6S. alba, S. conoidea, S. compacta, S. dichotoma, S. italica, S. supine, S. vulgaris[20,22] Polyols
193.07133193.07073.3[M + H]+
130.89Stachydrine144.1017144.1019−1.2[M + H]+ C7H13NO2 Amino acids
140.91Diglycolic acid133.0144133.01421.0[M − H] C4H6O5 Organic acid
150.93Tyrosine182.0803182.0812−4.8[M + NH4]+ C9H11NO3S. colorata[23]Amino acids
160.94Fumaric acid115.0036115.0037−0.5[M − H] C4H4O4 Organic acid
171.3Citric acid191.0193191.0197−2.1[M − H] C6H8O7S. vulgaris[28] Organic acid
186.34Tryptophan203.0815203.0826−5.4[M − H] C11H12N2O2 Amino acids
205.0978205.09723.1[M + H]+
196.47 *Ferulic acid193.0497193.0506−4.6[M − H] C10H10O4S. pratensis, S. schimperiana[21,29] Phenolics
206.51Salidroside299.1121299.1136−5.1[M − H]599.2322 [2M − H], 345.1189 [M + HCOO]C14H20O7 Phenolics
216.53p-Coumaric acid163.0395163.0401−3.4[M − H] C9H8O3S. alba, S. conoidea, S. compacta, S. dichotoma, S. italica, S. supine, S. vulgaris[20,22]Phenolics
165.0532165.05301.2[M + H]+147.0440 [M + H − H2O]+
226.69Chlorogenic acid353.0874353.0878−1.3[M − H] C16H18O9S. alba, S. dichotoma, S. italica, S. supine, S. vulgaris, S. albae, S. pendulae, S. compacta[20,22,30] Phenolics
355.1004355.10001.0[M + H]+372.1285 [M + NH4]+, 377.0836 [M + Na]+
236.71Isovitexin 7,2″-di-O-glucoside757.2169757.2186−2.3[M + H]+779.1987 [M + Na]+C33H40O20 Flavonoid glycoside
755.2010755.2040−4.0[M − H]
246.88Isosaponarin (isovitexin 4′-O-β-D-glucopyranoside)593.1499593.1512−2.1[M − H] C27H30O15S. armeria, S. bupleuroides, S. chlorifolia, S. compacta, S. cretacea, S. cubanensis, S. polaris[2,31] Flavonoid glycoside
256.89Luteolin 6-C-β-D-glucoside-8-C-α-L-arabinoside (carlinoside)579.1355579.13500.9[M − H]563.1368, 557.2928, 511.2869, 447.1487, 401.1426, 387.1638, 355.0651C26H28O15S. repens, S. sibirica[32,33] Flavonoid glycoside
266.92Cosmosiin (apigenin 7-O-glucoside)433.1128433.1129−0.4[M + H]+455.0948 [M + Na]+C21H20O10S. succulenta[34]Flavonoid glycoside
431.0981431.0984−0.7[M − H]
277.06Vanillic acid167.0345167.0350−3.0[M − H] C8H8O4S. schimperiana[21]Phenolics
287.12Caffeic acid181.0505181.05012.2[M + H]+163.0385C9H8O4S. dichotoma, S. italica, S. schimperiana, S. albae, S. pendulae[21,22,30] Phenolics
297.14Schaftoside (apigenin 6-C-β-D -glucoside-8-C-α-L-arabinoside)563.1406563.14010.9[M − H]511.2904, 473.1054, 443.0950, 431.1878, 383.0759C26H28O14S. aprica, S. repens, S. schafta, S. nemoralis, S. caramanica, S. sendtneri, S. frivaldszkyana, S. paradoxa, S. chalcedonica[17,32] Flavonoid glycoside
565.1538565.1552−2.6[M + H]+587.1359 [M + Na]+, 547.1468 [M + H − H2O]+
307.21Isovitexin 7-O-glucoside-2′’-O-rhamnoside739.2094739.20910.4[M − H] C33H40O19S. pratensis[35]Flavonoid glycoside
741.2219741.2236−2.4[M + H]+
31 *7.235,20,26-Trihydroxyecdysone (26-hydroxypolypodine B)511.2913511.29071.2[M − H]557.2967 [M + HCOO], 447.0890C27H44O9S. viridiflora[16]Ecdysteroids
327.302-Deoxy-5,20,26-trihydroxyecdysone519.2931519.29280.7[M + Na]+479.30023, 461.29126, 443.27814C27H44O8S. viridiflora[15]Ecdysteroids
337.3420-Hydroxyecdysone galactoside641.3543641.3537−0.9[M − H]687.3597 [M + HCOO], 613.2096, 563.1367, 461.1630C33H54O12S. brachuica, S. viridiflora[7]Ecdysteroids
347.40Vicenin 2 (apigenin-6,8-di-C-glucopyranoside)594.1589594.15850.7[M − H] C27H30O15S. boissieri, S. caramanica, S. chlorantha, S. colpophylla, S. commutata, S. cyri, S. foliosa, S. frivaldszkyana, S. graminifolia, S. jenissensis, S. italic, S. linicola, S. macrostyla, S. nemoralis, S. nutans, S. paradoxa, S. saxatilis, S. sendtneri, S. roemeri, S. wolgensis[2,36]Flavonoid glycoside
595.1638595.1658−3.3[M + H]+
357.43Orientin (luteolin-8-C-β-D-glucoside)447.0919447.0933−3.2[M − H] C21H20O11S. armeria, S. boissieri, S. bupleuroides, S. chlorantha, S. chlorifolia, S. commutata, S. compacta, S. cretacea, S. cubanensis, S. cyri, S. foliosa, S. graminifolia, S. jenissensis, S. italica, S. linicola, S. macrostyla, S. nutans, S. polaris, S. saxatilis, S. vulgaris, S. wolgensis[2]Flavonoid glycoside
449.1081449.10790.5[M + H]+
367.4520,26-Dihydroxyecdysone 495.2963495.29581.0[M − H]541.3018 [M + HCOO], 439.1798, 393.1740C27H44O8S. repens, S. viridiflora[17,37] Ecdysteroids
377.49Isoorientin (luteolin-6-C-β-D glucoside)447.0925447.0933−1.8[M − H] C21H20O11S. aprica, S. armeria, S. boissieri, S. bupleuroides, S. chlorantha, S. chlorifolia, S. commutata, S. compacta, S. cretacea, S. cubanensis, S. cyri, S. italic, S. littorea, S. foliosa, S. graminifolia, S. jenissensis, S. italica, S. macrostyla, S. nutans, S. polaris, S. saxatilis, S. viscariopsis, S. vulgaris, S. wolgensis[2]Flavonoid glycoside
449.1101449.10785.2[M + H]+471.0893 [M + Na]+
38 *7.51Saponarin (isovitexin 7-O-β-D-glucoside)593.1498593.1512−2.3[M − H] C27H30O15S. colorata, S. repens, S. nutans[1,38] Flavonoid glycoside
577.1530577.1552−3.9[M + H]+617.15063 [M + Na]+
397.52Vitexin-2″-O-rhamnoside (apigenin 8-C-β-D-glucoside-2″-O-rhamnoside)577.1563577.15571.0[M − H]503.1104, 471.0893, 413.0855C27H30O14S. nutans[38] Flavonoid glycoside
579.1704579.1708−0.7[M + H]+601.15365 [M + Na]+
407.57Integristerone A541.3018541.30131.0[M + HCOO]447.0884C27H44O8S. viridiflora[16]Ecdysteroids
519.2947519.29283.7[M + Na]+479.30194, 461.29297, 443.27985
41 *7.58Rutin (quercetin-3-O-rutinoside)609.1440609.1461−3.5[M − H] C27H30O16S. alba, S. conoidea, S. compacta, S. dichotoma, S. italica, S. supine, S. vulgaris, S. schimperiana[20,21,22]Flavonoid glycoside
611.1611611.16070.7[M + H]+
427.73Isovitexin (apigenin 6-C-β-D-glucopyranoside)431.0984431.09840.1[M − H] C21H20O10S. alba, S. aprica, S. armeria, S. boissieri, S. brachuica, S. bupleuroides, S. chlorantha, S. chlorifolia, S. commutata, S. compacta, S. cretacea, S. cubanensis, S. cyri, S. diclinis, S. dioica, S. foliosa, S. graminifolia, S. jenissensis, S. italica, S. macrostyla, S. multifida, S. nutans, S. polaris, S. repens, S. supina, S. turgida, S. wolgensis[2,18] Flavonoid glycoside
433.1144433.11293.3[M + H]+455.0926 [M + Na]+
437.74Quercetin-3-O-(6′’-O-malonyl)-β-glucoside551.1030551.1037−1.3[M + H]+515.3002, 392.2085, 279.1587C24H22O15 Flavonoid glycoside
447.7926-Hydroxyecdysone479.3014479.30091.1[M − H]1005.6133, 525.3069 [M + HCOO]C27H44O7S. repens[37]Ecdysteroids
457.80Diosmin (luteolin 4′-methyl ether-7-O-rutinoside)607.1992607.2000−1.3[M − H] C28H32O15S. succulent, S. schimperiana[21,34] Flavonoid glycoside
609.1842609.18144.6[M + H]+631.1658 [M + Na]+
467.82Hyperoside (quercetin-3-O-β-D-galactoside)463.0867463.0873−1.3[M − H] C21H20O12S. albae, S. pendulae, S. compacta[20,30] Flavonoid glycoside
477.83Narcissin (3-methylquercetin-3-O-rutinoside)623.1619623.16180.2[M − H] C28H32O16S. ruscifolia[26]Flavonoid glycoside
487.83Dihydroferulic acid195.0654195.0663−4.7[M − H] C10H12O4Gypsophila paniculata[39]Phenolics
497.842-Deoxy-5,20,26-trihydroxyecdysone495.2963495.29581.0[M − H]1037.6054, [2M + HCOO], 541.3018 [M + HCOO]C27H44O8S. viridiflora[15]Ecdysteroids
519.2918519.2934−3.1[M + Na]+479.2991, 461.2902, 443.2801
50 *7.9120-Hydroxyecdysone479.3014479.30091.1[M − H]525.3067 [M + HCOO]C27H44O7S. viridiflora[7]Ecdysteroids
481.3160481.3160−0.1[M + H]+503.2966 [M + Na]+, 463.3051 [M + H − H2O]+, 445.2938 [M + H − 2H2O]+, 427.2853 [M + H − 3H2O]+
517.932-Deoxyintegristerone A479.3014479.30091.1[M − H]525.3069 [M + HCOO], 441.1962, 369.0882C27H44O7S. otitis, S. italica ssp. nemoralis, S. viridiflora[16]Ecdysteroids
498.3402498.3425−4.6[M + NH4]+
528.09Vaccaroside B1296.62191296.6225−0.4[M + NH4]+1006.5222, 798.4625C60H94O29Vaccaria segetalis[40]Triterpenoids
538.12Hesperidin611.1980611.19760.7[M + H]+628.2221 [M + NH4]+C28H34O15S. alba, S. conoidea, S. compacta, S. dichotoma, S. italica, S. supine, S. vulgaris, S. schimperiana[20,21,22,23,24,25,26,27,28,29,30]Flavonoid glycoside
54 *8.34Quercitrin (quercetin 3-O-α-L-rhamnoside)449.1079449.10780.2[M + H]+ C21H20O11S. albae, S. pendulae[30]Flavonoid glycoside
558.52Armeroside E1131.52291131.52230.5[M − H]677.3533, 565.2568, 367.1237C54H84O25S. armeria[41]Triterpenoids
568.63Silenegallisaponin J1117.54361117.54310.5[M − H]707.2547, 649.3389, 581.2617, 509.3094C54H86O24S. gallica[42]Triterpenoids
578.68Sinocrassuloside II1131.52291131.52230.5[M − H]978.4760, 825.4244, 588.2588C54H84O25S. viscidula[43]Triterpenoids
58 *8.702-Deoxy-20-hydroxyecdysone509.3120509.31141.1[M + HCOO]463.3023 [M − H], 973.6228 [2M + HCOO]C27H44O6S. viridiflora[16]Ecdysteroids
465.3211465.3216−1.1[M + H]+487.3024 [M + Na]+, 447.3113 [M + H − H2O]+, 429.2997 [M + H − 2H2O]+, 411.2900 [M + H − 3H2O]+, 393.2787 [M + H − 4H2O]+, 355.2270, 331.2272, 287.2005
598.71Armeroside D1149.53351149.532910.5[M − H] C54H86O26S. armeria[41]Triterpenoids
608.73Makisterone C 553.3382553.33771.0[M + HCOO] C29H48O7 Ecdysteroids
507.3327507.33221.0[M − H]
618.89Armeroside G 1015.47551015.47500.5[M − H] C49H76O22S. armeria[41]Triterpenoids
628.97Armeroside F 987.4806987.48010.5[M − H] C48H76O21S. armeria, S. viscidula[41,43]Triterpenoids
639.403β,22α-Dihydroxyolean-12-en-23-al-28-oic acid 3-O-α-L-arabinopyranosyl-(1→3)-β-D-glucuronopyranoside793.4016793.40110.7[M − H] C41H62O15S. odontopetala[44]Triterpenoids
649.462-Deoxy-20-hydroxyecdysone-acetate505.3171505.31653−4.1[M − H] C29H46O7S. praemixta, S. wallichiana[45,46]Ecdysteroids
65 *10.12Silviridoside1115.53041115.52742.7[M − H]774.3528, 580.2618, 557.2574C54H84O24S. viridiflora[3]Triterpenoids
1117.54251117.5431−0.5[M + H]+1134.5691, 253.1077
6610.453-O-β-D-Glycuronopyranosyl-quillaic acid 28-O-hexapyranosyl-pentapyranosyl-xylopyranosyl ester1101.51231101.51180.5[M − H]749.3395, 573.2545, 550.2504, 311.1664C53H82O24 Triterpenoids
1120.55341120.5540−0.5[M + NH4]+971.4906, 680.4043, 571.2443
6710.553-O-[β-D-Glucopyranosyl-(1→2)-(β-D-xylopyranosyl-(1→3))-β-D-glucuronopyranosyl]-28-O-β-D-glucopyranosyl-3β-hydroxyolean-12-en-28-oic acid1087.53311087.53250.5[M − H]601.2628, 566.2651, 543.2607C53H84O23Beta vulgaris[47,48]Triterpenoids
1106.57421106.5747−0.5[M + NH4]+958.5082, 810.4453, 610.1826
6810.96Acetyl-23-hydroxyolean-12-en-28-oic acid-dipentosyl-hexosyl-deoxy-pentoside1085.55381085.55330.5[M − H]965.4340, 666.2781, 565.2729, 482.2128C54H86O22 Triterpenoids
1104.59491104.5955−0.5[M + NH4]+678.3821, 536.1646
6911.75Quillaic acid-3-O-β-D-glucuronopyranoside661.3593661.35880.8[M − H]535.3222, 279.1495, 185.0288C36H54O11S. vulgaris, Psammosilene tunicoides[49,50]Triterpenoids
680.4004680.4010−0.9[M + NH4]+536.1642
7013.303β,22α-Dihydroxyolean-12-en-23-al-28-oic acid 3-O-β-D-glucuronopyranoside707.3648707.36427−0.7[M + HCOO]661.3556, 330.1734, 311.1661C36H54O11S. odontopetala[44]Triterpenoids
7118.23Oleanolic acid455.3509455.3525−3.5[M − H] C30H48O3S. succulenta[51]Triterpenoids
*- Confirmed by comparison with corresponding reference standard.

3.1.2. Triterpenes

More than 70 triterpenes have been isolated from 12 Silene species to date [52]. The characteristic features of these triterpenes are an aldehyde or carboxyl group at C-23, a carboxyl group at C-28, secondary alcoholic functions at C-16, and rarely at C-11 [2]—the methodology that permitted the tentative detection and successful characterization of 15 triterpene glycosides. The triterpene glycosides in the methanol extract of S. viridiflora were tentatively annotated as glycosides of gypsogenic acid (peak 52), 16α-hydroxygypsogenic acid (55, 57, 59, 62), caulophyllogenin (56), 3,16α-dihydroxy-3,4-secoolean-4(24),12-dien-23,28-dioic acid (61), 3β,22α-dihydroxyolean-12-en-23-al-28-oic acid (63, 70), quillaic acid (65, 66, 69), 3β-hydroxyolean-12-en-28-oic acid (67, 71), and oleanane type triterpene (68). Monitoring these MS data, the sequential loss of hexoses (Gal, Man), GlcA and/or pentoses (Xyl, Ara) in the presence of Glc as the dominant sugar were characteristic for triterpene peaks. The position of the sugar attachment could not be confirmed by MS analysis, but based on the preceding literature was assumed to connect to either the C-3 and/or C-28 positions of aglycone. The glycosylation pattern of Silene could be qualified by high relative levels of 28-Rha and 28-Fuc in gypsogenin and quillaic acid, 28-Glc in 16α-hydroxygypsogenic acid, and glucuronic acid in these three sapogenins [53]. Four 16α-hydroxygypsogenic acid (3β,16α-dihydroxyolean-12-en-23,28-dioic acid)-containing triterpene glycosides, 55, 57, 59, and 62, showing fragment ions of 16α-hydroxygypsogenic, were unequivocally identified following the data reported in the literature [54,55]. In the negative ion mode of UHPLC-MS, the oleanane type triterpenoid saponins mostly afford major [M − H] ions [50].
Recently, the first major peak 65 in TIC of S. viridiflora was isolated and through HR-MS and 1D and 2D NMR elucidated as silviridoside (3-O-β-D-galacturonopyranosyl-quillaic acid 28-O-β-D-glucopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→3)]-β-D-fucopyranosyl ester) [3]. In this study, the second major peak 66 (Rt = 10.45 min) exhibited a parent ion [M − H] at m/z 1101.5223 with ion fragments at m/z 749.3395, 573.2545, 550.2504, and 311.1664. Under the positive ESI mode, the [M + NH4]+ adduct with m/z 1120.5534 was detected for this metabolite, with further fragmentation to produce mass fragments with m/z 971.4906 [M + H − Xyl]+, 680.4043, 571.2443. Thus, this compound could be assigned tentatively as 3-O-glycuronopyranosyl-quillaic acid 28-O-hexapyranosyl-pentapyranosyl-xylopyranosyl ester.

3.1.3. Ecdysteroids

In this study, twelve known ecdysteroids were identified in the methanol extract of aerial parts of S. viridiflora by UHPLC-ESI-MS/MS (Table 1). These ecdysteroids, namely 5,20,26-trihydroxyecdysone (26-hydroxypolypodine B) (peak 31), 2-deoxy-5,20,26-trihydroxyecdysone (32), 20-hydroxyecdysone galactoside (silenoside A or silenoside D) (33), 20,26-dihydroxyecdysone (36), integristerone A (40), 2-deoxy-5,20,26-trihydroxyecdysone (49), 20-hydroxyecdysone (50), 2-deoxyintegristerone A (51), and 2-deoxy-20-hydroxyecdysone (58), were previously isolated and characterized from S. viridiflora by CC and HPLC methods [8,14,15,16]. Also, three other ecdysteroids such as 26-hydroxyecdysone (44), makisterone C (60), and 2-deoxy-20-hydroxyecdysone-acetate (65) were annotated in S. viridiflora for the first time and were partially identified on the basis of their molecular weight and the fragmentation pattern. 26-Hydroxyecdysone (44, C27H44O7) was the major compound detected in S. viridiflora and was represented in the mass spectra with the signals at m/z 479.3014 [M − H], 525.3069 [M + HCOO] and 1005.6133 [2M + HCOO]. Another major metabolite 51 in the positive ion mode, with [M + NH4]+ at m/z 498.3402, demonstrated a fragmentation pattern typical for 2-deoxyintegristerone A. In the negative ion mode, the spectrum of metabolite 51 fragments at m/z 525.3069 [M + HCOO], 479.3014 [M − H], 441.1962, 369.0882 was observed. Based on the comparison of the stacked experimental and reference mass spectra, this peak was identified as 2-deoxyintegristerone A (51). Similarly, 2-deoxy-20-hydroxyecdysone was identified as peak 58 based on the signals at m/z 463.3023 [M − H] and m/z 509.3120 and m/z 973.6228 corresponding to its adduct ions [M + HCOO] and [2M + HCOO] in negative ion mode. In the positive ion mode, metabolite 58 was observable as a pattern of ionic adducts—m/z 465.3211 [M + H]+ and 487.3024 [M + Na]+. Moreover, this pattern was accompanied with several water molecules, yielding the signals at m/z 447.3113 [M + H − H2O]+, 429.2997 [M + H − 2H2O]+, 411.2900 [M + H − 3H2O]+, and 393.2787 [M + H − 4H2O]+ (Figures S2 and S3). Most previous studies reporting on the LC-MS/MS analysis of ecdysteroids were conducted in the positive ion mode at the low (<10 eV) collision energy, which yields mass spectra showing a prominent loss of H2O molecules from polyhydroxylated molecular ions, that is, [M+X − (H2O)n]+, where X=H, Na, or K [56]. The presence of integristerone A (40), 20-hydroxyecdysone (50), and 2-deoxy-20-hydroxyecdysone (58) was confirmed by comparison with the reference spectra.

3.2. Anticancer Activities

Recent investigations of the Silene genus shows that its species have anticancer properties, including extracts of S. firma, S. fortunei, S. succulenta [57]. The total hydro-methanolic extract and its fractions of S. succulenta aerial parts against different cancer cell lines revealed that the highest activity was against breast carcinoma cell lines. The n-hexane fraction was a highly effective fraction against breast carcinoma cell lines (MCF-7) with IC50 = 15.5 mg/mL. After a bio-guided fractionation of the n-hexane fraction, two major compounds, cyclic glycolipids, were isolated and found to inhibit the proliferation of the MCF-7 cells at the IC50 of 21.5 µM and 13.1 µM [57].
In recent years, there has been growing interest in the potential use of ecdysteroids for the treatment of breast cancer. Martins et al. [58,59] demonstrated that semisynthetic ecdysteroid derivatives were able to inhibit the ABCB1 transporter and restore doxorubicin resistance in mammalian cancer cells expressing the human ABCB1 transporter. Studies have shown that ecdysteroids can inhibit the growth of breast cancer cells in vitro and in vivo. For example, a study by Romaniuk-Drapała et al. [60] showed that 20-hydroxyecdysone (ecdysterone) and ajugasterone C were able to inhibit the growth of triple-negative breast cancer MDA-MB-231 cells. Shuvalov et al. [61] found that 20-hydroxyecdysone strongly induces autophagy in a group of breast cancer cells (MCF-7, MDA-MB-231, and MDA-MB-468 cells). They also showed that 20-hydroxyecdysone exhibits a synergistic effect in combination with doxorubicin and induces cell death. However, this effect was observed in a high concentration range of 250–750 µM.
Zibareva et al. [62] reported that an ecdysteroid-containing S. viridiflora extract has antitumor effects in vivo. According to these previous studies, the real intention was to explore the cytotoxic compounds from the S. viridiflora aerial parts against cancer cells. Several ecdysteroids were previously isolated and characterized from S. viridiflora by CC and HPLC methods [8,14,15,16]. In this study, comprehensive metabolite profiling of S. viridiflora was carried out using the UHPLC-ESI-MS/MS method. In particular, we identified nine compounds such as quinic acid (12), ferulic acid (19), p-coumaric acid (21), schaftoside (29), isovitexin-7-O-glucoside (38), rutin (41), 20-hydroxyecdysone (50), 2-deoxy-20-hydroxyecdysone (58) and oleanolic acid (71) in the methanol extract of S. viridiflora. Except for the ecdysteroids 50 and 58 [7], the rest of the compounds were identified for the first time in S. viridiflora. The cytotoxic activity of some natural ecdysteroids (7276) isolated from other species of Silene, Ajuga, and Serratula [2], along with their mechanism of action and structure–activity relationships was also evaluated. Furthermore, a methanol extract of S. viridiflora and compounds 12, 19, 21, 29, 38, 41, 50, 58 and 7176 was bioassayed by in vitro methods to determine the potential cytotoxic activity and for their ability to inhibit TNBC and healthy cells, which allowed a metabolite with the greatest inhibitory potential to be found (Table 2). The methanol extract showed significant cytotoxic effects towards BT-20, MDA-MB-231, HCC1395 and HEK293 cells at 10.1 ± 1.3, 10.4 ± 2.1, 9.27 ± 1.8 and >20 μg/mL with a viability of 50%. Among the tested samples, only 2-deoxy-20-hydroxyecdysone (58) was found to be the main inhibitor of cancer cells. This ecdysteroid exhibits strong cytotoxic activity against BT-20, HCC1395 and MDA-MB-231 cells, with an IC50 of 0.12 ± 0.006 μM, 0.21 ± 0.01 μM, and 0.53 ± 0.14 μM, respectively, while healthy HEK293 cells were inhibited at 0.27 ± 0.01 μM (Figure 1).
A structure–activity relationship (SAR) study then determined the main structural features required to inhibit TNBC cells for the tested ecdysteroids 50, 58, and 7276. The inhibitory activity data presented in Table 2 led us to generate an initial SAR model in order to investigate the cytotoxic effect of the hydroxyl group on the steroid core, as well as the type of substituents (acetate and lactone ring at 25-OH) on the activity profile that could be explored (Figure 2 and Figure 3). Turkesterone (75), which possesses a hydroxyl group at C11 in the C ring, suggested that 11-OH also decreased the ecdysteroids’ cytotoxic activity. In contrast, the effects of substitutions of 25-OH in the side chain with an acetate group and lactone ring exemplified in viticosterone E (74) and cyasterone (76), if present, remain unclear due to activity above the highest concentrations tested of available derivatives. The structure–activity relationship inspected from the in vitro assay for ecdysteroids showed that the hydroxyl group present at position C-20 of the steroid core increases the ecdysteroids’ cytotoxicity against cancer cells. However, the absence of the hydroxyl group at position C-2 of the steroid core leads to a higher increase in the cytotoxicity of ecdysteroids (58) against TNBC cells by at least two orders of magnitude (Figure 3).
Martins et al. [63] studied the SAR of the apolar dioxolane derivatives of 20-hydroxyecdysone. The results showed that the substituted dioxolane ring at the 2,3 position is far more important for strong activity than the one at positions 20,22. The ecdysteroid derivative monosubstituted at position 2,3, was the only ecdysteroid derivative that was able to exert a stronger activity than the ecdysteroids with the 2,3; 20,22 diacetonide group.
Breast cancer in general and TNBC in particular are highly dependent on the oncogenic Wnt signaling for growth and progression [64,65], and a number of small molecules inhibiting TNBC cell proliferation through the blocking of their Wnt signaling has been identified [66,67,68,69]. In this study, we used the TOPFlash assay to assess the inhibitory effects of the methanol extract and compounds 12, 19, 21, 29, 38, 41, 50, 58 and 71 of S. viridiflora on Wnt signaling. Since TNBC cells are known to be dependent on Wnt signaling, additional efforts were made to determine whether compounds derived from S. viridiflora could be active against this signaling cascade, although there was no reason to assume that this extract should have specific inhibitory activity against Wnt from the point of view of components or known activities. The TOPFlash assay is a versatile tool that can be used to study the Wnt signaling pathway in a variety of contexts, including drug screening, target validation, and mechanistic studies [70,71]. It is particularly useful for identifying inhibitors of the Wnt pathway, as these compounds will typically reduce Wnt-driven firefly luciferase activity in the assay. As a control of unspecific activity, e.g., cytotoxicity or the suppression of cell vitality and protein production, another luciferase, from Renilla reniformis, which is produced in a Wnt-independent manner and can be independently measured in the same wells, is used [72]. The obtained results showed that none of the tested samples of S. viridiflora inhibited Wnt signaling at a concentration of 20 µM (extract was tested at the 20 μg/mL concentration). Although 2-deoxy-20-hydroxyecdysone (58) has strong cytotoxic effect under the MTT test, the results suggested that it is not a Wnt signaling inhibitor and must act through another mechanism to prevent cancer cell proliferation.

4. Conclusions

In conclusion, this is the first comprehensive metabolite profiling of S. viridiflora using the UHPLC-ESI-MS/MS method. A total of 71 compounds were annotated and tentatively identified. The results have shown that flavonoids, triterpene glycosides, and ecdysteroids were the most abundant constituents in the species. The three major compounds schaftoside, 26-hydroxyecdysone, and silviridoside can be chosen as the markers for the assessment of the quality of S. viridiflora preparations. SAR studies for the ecdysteroids revealed that the absence of a hydroxyl group at C-2 in ring A and the presence of a hydroxyl group at C-20 on the side chain of ecdysteroids are important structural features for eliciting cytotoxic activity. These results suggest that the 2-deoxy-20-hydroxyecdysone can be employed/further developed as a chemotherapeutic drug candidate for the treatment of cancer. Future studies should include in vivo investigations to evaluate the effectiveness and safety of the compound.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biom14101285/s1, Figure S1: UHPLC-MS total ion chromatogram of the methanol extract obtained from S. viridiflora (A—negative ion mode, B—positive ion mode). Several peaks have been annotated in the chromatogram with the names of the metabolites shown in Table 1; Figure S2: (+) ESI-MS of 2-deoxy-20-hydroxyecdysone (58); Figure S3: Proposed MS/MS fragmentation of 2-deoxy-20-hydroxyecdysone (58) in the positive ion mode.

Author Contributions

N.Z.M.: Conceptualization, Methodology, Formal analysis, Investigation, Software, Data curation, Visualization, Writing—original draft, Writing—review and editing. A.K.: Methodology, Investigation, Software, Data curation, Writing—review and editing. M.M.D.: Methodology, Formal analysis, Investigation, Writing—original draft. H.H.: Investigation, Resources, Writing—review and editing. V.L.K.: Project administration, Resources, Funding acquisition, Writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Ministry of Higher Education, Science and Innovation of Uzbekistan, Swiss National Research Foundation (grant number IZSEZ0_216954) and Alexander von Humboldt Foundation (grant agreement ID: UZB 1157968 GF-E).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available upon request from the first author, N.Z.M.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Inhibitory effect at various (2–0.069 µM) concentrations of 2-deoxy-20-hydroxyecdysone (58) on cell lines BT-20 (A), HCC1395 (B), MDA-MB-231 (C), and HEK293 (D) (MTT test). Data are represented as an average ±SEM from N = 3 independent experiments, performed in duplicate for each concentration (n = 2). Statistical significance was assessed by one-way ANOVA with multiple comparisons and is shown as * p < 0.05, *** p < 0.001 (for each point within the indicated span).
Figure 1. Inhibitory effect at various (2–0.069 µM) concentrations of 2-deoxy-20-hydroxyecdysone (58) on cell lines BT-20 (A), HCC1395 (B), MDA-MB-231 (C), and HEK293 (D) (MTT test). Data are represented as an average ±SEM from N = 3 independent experiments, performed in duplicate for each concentration (n = 2). Statistical significance was assessed by one-way ANOVA with multiple comparisons and is shown as * p < 0.05, *** p < 0.001 (for each point within the indicated span).
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Figure 2. Structure of tested ecdysteroids.
Figure 2. Structure of tested ecdysteroids.
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Figure 3. Summary of the SARs for the anti-proliferative activities of tested ecdysteroids.
Figure 3. Summary of the SARs for the anti-proliferative activities of tested ecdysteroids.
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Table 2. In vitro cytotoxic activity of the methanol extract of S. viridiflora and compounds 12, 19, 21, 29, 38, 41, 50, 58 and 7176 against BT-20, HCC1395, MDA-MB-231, and HEK293 cell lines for exposure of 72 h (MTT test). The data are represented as an average ± SEM from N = 3 independent experiments, performed in duplicate for each concentration (n = 2).
Table 2. In vitro cytotoxic activity of the methanol extract of S. viridiflora and compounds 12, 19, 21, 29, 38, 41, 50, 58 and 7176 against BT-20, HCC1395, MDA-MB-231, and HEK293 cell lines for exposure of 72 h (MTT test). The data are represented as an average ± SEM from N = 3 independent experiments, performed in duplicate for each concentration (n = 2).
NSampleBT-20MDA-MB-231HCC1395HEK293
IC50, µM
12Quinic acid>20>20>20>20
19Ferulic acid>20>20>20>20
21p-Coumaric acid>20>20>20>20
29Schaftoside>20>20>20>20
38Isovitexin-7-O-glucoside>20>20>20>20
41Rutin>20>20>20>20
5020-Hydroxyecdysone>20>20>20>20
582-Deoxy-20-hydroxyecdysone0.12 ± 0.0060.53 ± 0.140.21 ± 0.010.27 ± 0.01
71Oleanolic acid>20>20>20>20
722-Deoxyecdysone>20>20>20>20
73Ecdysone>20>20>20>20
74Viticosterone E>20>20>20>20
75Turkesterone>20>20>20>20
76Cyasterone>20>20>20>20
MeOH extract (μg/mL)10.1 ± 1.310.4 ± 2.19.27 ± 1.8>20
Docetaxel (nM)4.4 ± 0.000817.7 ± 0.00296.5 ± 0.00074.47 ± 0.0013
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Mamadalieva, N.Z.; Koval, A.; Dusmuratov, M.M.; Hussain, H.; Katanaev, V.L. Chemical and Biological Characterization of Metabolites from Silene viridiflora Using Mass Spectrometric and Cell-Based Assays. Biomolecules 2024, 14, 1285. https://doi.org/10.3390/biom14101285

AMA Style

Mamadalieva NZ, Koval A, Dusmuratov MM, Hussain H, Katanaev VL. Chemical and Biological Characterization of Metabolites from Silene viridiflora Using Mass Spectrometric and Cell-Based Assays. Biomolecules. 2024; 14(10):1285. https://doi.org/10.3390/biom14101285

Chicago/Turabian Style

Mamadalieva, Nilufar Z., Alexey Koval, Maksud M. Dusmuratov, Hidayat Hussain, and Vladimir L. Katanaev. 2024. "Chemical and Biological Characterization of Metabolites from Silene viridiflora Using Mass Spectrometric and Cell-Based Assays" Biomolecules 14, no. 10: 1285. https://doi.org/10.3390/biom14101285

APA Style

Mamadalieva, N. Z., Koval, A., Dusmuratov, M. M., Hussain, H., & Katanaev, V. L. (2024). Chemical and Biological Characterization of Metabolites from Silene viridiflora Using Mass Spectrometric and Cell-Based Assays. Biomolecules, 14(10), 1285. https://doi.org/10.3390/biom14101285

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