LAUSR

Silene nutans L. (syn. S. brachypoda Rouy, Cucubalus nutans Lam., Otites nutans Opiz) is a boreal Euro-Siberian species of the family Caryophyllaceae that is broadly distributed in northern and middle Europe, European Russia, Caucasus, and western and eastern Siberia [1]. Early reports of the chemical composition of S. nutans included ecdysteroids, which were most interesting for research [2–9], and triterpenoids [10], flavonoids [11], and essential oil [12]. Siberian populations of S. nutans have not been previously studied. Therefore, the chemical composition of S. nutans growing in Buryatia Republic was studied during continuing studies of plant sources of ecdysteroids and phenolic compounds in the genus Silene in the flora of Baikal region. S. nutans (whole plants) was collected during flowering in the vicinity of Mandrik station (Pribaikalsky District, Republic of Buryatia, Russia; Jul. 12, 2017; 52°4′16.47′′ N, 107°27′26.64′′ E; h = 672 m above sea level). Raw material specimens are preserved in the herbarium of the IGEB SB RAS (No. Ca/ae-17/07-14/0132). The species was identified by Dr. T. A. Aseeva (IGEB SB RAS). Raw material was air-dried in the shade to ≤5% moisture and milled to particle size 1–2 mm. Column chromatography (CC) used polyamide, Al2O3, normal (SiO2) and reversed-phase silica gel (RP-SiO2), and Sephadex LH-20 (Sigma-Aldrich, St. Louis, MO, USA). Preparative HPLC used a Summit liquid chromatograph (Dionex, Sunnyvale, CA, USA) with a LiChrospher RP-18 column (250 × 10 mm, ∅ 10 μm; Supelco, Bellefonte, PA, USA); mobile phase H2O (A) and MeCN (B) at flow rate (ν) 1 mL/min; and column temperature 35°C. Spectrophotometric studies used an SF-2000 spectrophotometer (OKB Spectr, St. Petersburg, Russia). Mass spectrometric analysis used an LCMS-8050 TQ mass spectrometer (Shimadzu, Columbia, MD, USA) with electrospray ionization (ESI) in charged-ion mode with capillary potential +29 V (ecdysteroids) and –10 V (flavonoids, phenylpropanoids); field potential 3.0 kV; ionization source temperature 250°C; and molecular-mass scan range (m/z) 100–1900 amu. NMR spectra were recorded on a VXR 500S NMR spectrometer (Varian, Palo Alto, CA, USA). Plant raw material was quantitatively analyzed using a microcolumn HPLC-UV. For this, raw material (40 mg) was placed into an Eppendorf tube (2 mL), treated with EtOH (70%, 1 mL), irradiated with ultrasound (50 kHz, 40 min, 35°C), and centrifuged (6,000 g, 20 min). The obtained extract was filtered through a 0.45-μm membrane filter and used for analysis (1 μL). HPLC-UV conditions: Milichrom A-02 microcolumn liquid chromatograph (EcoNova, Novosibirsk, Russia) ProntoSIL-120-5-C18AQ column (2 × 75 mm, ∅ 5 μm; Metrohm AG, Herisau, Switzerland); mobile phase 0.2 M LiClO4 in 0.006 M HClO4 (A) and MeCN (B) in gradient mode (%B): 0–10 min 12–35%, 10–15 min 35–70%; ν 150 μL/min; column temperature 35°C; and UV detector at λ 245 and 330 nm. Calibration curves were plotted using solutions (in DMSO–MeOH, 1:9) of commercial reference standards such as 20-hydroxyecdysone (≥95%, OOO Fitopanatseya, Moscow, Russia), saponarin (≥98%), schaftoside (≥90%), isoorientin (≥99%), vitexin-2′′-O-rhamnoside (≥99%), vitexin (≥99%), isovitexin (≥99%, Extrasynthese, Lyone, France), isoscoparin (≥98%), 5-O-feruloylquinic acid (≥95%, Wuhan ChemFaces Biochemical Co., Ltd., Wuhan, Hubei, PRC), isovitexin-2′′-O-rhamnoside (≥90%, Sigma-Aldrich), 6-O-feruloylglucose (≥98%, Synthose, Ontario, Canada), ferulic acid-4-O-glucoside (≥98%, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and 3-O-p-coumaroylquinic acid (≥98%, Carbosynth, Compton, UK). Ecdysteroids were analyzed using 20-hydroxyecdysone; orientin-2′′-O-glucoside, orientin; 1-O-feruloylglucose, 6-O-feruloylglucose; and 4-Oand 5-O-p-coumaroylquinic acids, 3-O-p-coumaroylquinic acid [13]. Results were given as means of three parallel determinations (± standard deviation, SD). Statistical analysis used Statistica 10 software (Dell, Round Rock, TX, USA).