Secoiridoids from Dogwood (Cornus officinalis) Potentiate Progesterone Signaling

Optical rotation was measured on a PerkinElmer 241 polarimeter using a 100 mm cell. UV spectra were recorded on a Varian Cary 5000 spectrophotometer. IR spectra were acquired on a Thermo-Nicolet 6700 FT-IR equipped with a SMART iTR sampling accessory. ECD spectra were recorded on a JASCO J-815 spectrometer. NMR spectra were obtained on a Bruker 800 MHz AVANCE NMR spectrometer equipped with a 5 mm TXI room temperature inverse probe with xyz-axis pfg and Topspin version 1.3 operating software, and a Bruker 900 (226.2) MHz AVANCE NMR spectrometer equipped with a 5 mm TCI cryogenic inverse probe with z-axis pfg and TopSpin version 2.1 operating software, at the University of Illinois at Chicago Center for Structural Biology. Chemical shifts (δ) are given in ppm and coupling constants ( J ) are reported in Hz. 1 H and 13 C NMR chemical shifts were referenced to the CD 3 OD (δ H 3.31 ppm and δ C 49.0 ppm). High resolution mass spectra were obtained on a Bruker COMPACT ESIQTOF mass spectrometer at the University of Illinois at Chicago. HPLC was performed on a Shimadzu LC-20AB equipped with a SPD-20A UV/Vis detector and a Waters Prep LC4000 system equipped with a Water486 UV/Vis detector. All solvents were spectroscopic grade. Dried plant twigs and fruits of Cornus officinalis were provided by the UIC/NIH Center for Botanical Dietary Supplements (plant material code BC362 and BC282). Dried plant material (197.5 g) of Cornus officinalis was grinded and repeatedly extracted with MeOH (1L × 3) and CH 2 Cl 2 (1L × 3). The combined extracts (16.3 g) were successively partitioned between H 2 O and hexanes; the former fraction was sequentially repartitioned with EtOAc and n-BuOH (2.34 g). An aliquot of the n-BuOH layer (1.50 g) was fractionated using RP-C 18 preparative HPLC (10 mL/min, gradient of MeOH:H 2 O with 0.1% formic acid (FA) from 30:70 to 65:35 over 20 min and from 65:35 to 100:0 over 5 min, followed by an isocratic flow of 100% MeOH with 0.1% FA for 10 min), to afford fractions F1 to F16. F6, F8, F10, and F12 were rich with specialized metabolites. F6 and F8 were purified using RP-C 18 semi-preparative HPLC (2.0 mL/min, gradient of MeCN-H 2 O with 0.1% FA from 10:90 to 65:35 over 25 min and from 65:35 to 100:0 over 5 min, followed by an isocratic flow of 100% MeCN with 0.1% FA for 5 min) to afford secologanoside ( 3 ) and sweroside ( 5 ). F10 was purified using RP-C 18 analytical HPLC (0.8 mL/min, gradient of MeCN-H 2 O with 0.1% FA from 5:95 to 25:75 over 40 min, followed by an isocratic flow of 100% MeCN with 0.1% FA for 5 min) to afford secoxyloganin ( 4 ), (7 β )-7- O -methylmorroniside ( 7 ), (7 α )-7- O -methylmorroniside ( 6 ), and demethoxy-cornuside ( 1 ). F12 was purified using RP-C 18 semi-preparative HPLC (2.0 mL/min, gradient of MeCN-H 2 O with 0.1% FA from 1:9 to 1:0 over 30 min, followed by an isocratic flow of 100% MeCN with 0.1% FA for 5 min) to afford cornuside ( 2 ). An off-white amorphous powder; [α] D 20 −1.2 ( c 0.1, MeOH); UV (MeOH) λ max (log ε ) 275 (5.16) nm; ECD (MeOH) λ max (Δε) 227 (−7.5), 248 (+2.6) nm; IR (neat) ν max 3309, 2940, 2829, 1447, 1022 cm −1 ; 1 H NMR (900 MHz, CD 3 OD) and 13 C NMR (225 MHz, CD 3 OD), see Table 1 ; HRESIMS m/z 551.1395 [M + Na] + (calcd for C 23 H 28 NaO 14 , 551.1377). A solution of 1 (2.0 mg) in 3 N HCl (500 μL) was stirred at 90 °C for 1 h. After hydrolysis, the reaction mixture was concentrated, diluted with H 2 O, neutralized with 3N NH 4 OH, and dried. The residue was re-dissolved in 500 μL of a solution of L-cysteine methyl ester (50 mg/mL) and pyridine. After mixing thoroughly, the reaction mixture was incubated at 60 °C for 1 h. 500 μL of reagent consisting of phenyl isothiocyanate and pyridine was added and heated for another hour. The solution was filtered and injected into an UPLC-MS/MS system (0.5 mL/min, isocratic of MeCN-H 2 O at 15:85 with 0.1% FA) with C 18 UPLC column (Agilent, 2.1 × 50 mm). A peak corresponding to a hydrolysate sugar derivative was detected at 4.90 min. The same procedures were carried out with L- and D-glucose. The retention time and MS data of the detected peak in the test sample were compared with those of standard D- (t R = 4.98 min) and L-glucose (t R = 4.48 min), respectively. Cornuside ( 2 ) (A14599, Adooq Bioscience, Irvine, CA, USA), progesterone (P0130-25G, Sigma-Aldrich, St. Louis, MO, USA), D-glucose (G8270-100G, Sigma-Aldrich, St. Louis, MO, USA), and L-glucose (G5500-250MG, Sigma-Aldrich, St. Louis, MO, USA) were purchased from commercial sources. Ishikawa cells stably expressing PR-B were treated as described previously. 23 PRE/Luc activity assay were performed as previously described. 23 All data are presented as means ± the standard error of the mean (SEM) with n ≥3. Due to the large number of samples, screening data of extracts, partitioned layers, and HPLC fractions of C. officinalis were not statistically analyzed. All other data were analyzed by ANOVA followed by a Tukey’s or Dunnett’s post hoc test, with p < 0.05 considered significant. Analysis was performed using Prism version 7.0a.