500 mhz nmr spectrometer

All the compounds were recorded by a 500 MHz Bruker NMR spectrometer (Bruker Daltonics Inc., Bremen, Germany) with ¹H, ¹³C and DEPT135 NMR in CDCl₃ solution with TMS as internal standard for protons. The 2D NOSEY experiment was carried out in the 500 MHz Bruker NMR spectrometer with 32 of the scans number. Chemical shift values are mentioned in δ (ppm) and coupling constants (J) in Hz. MS (ESI) was measured on an ESI-Thermo Fisher LTQ Fleet instrument spectrometer (Thermo Fisher Scientific Inc., Waltham, MA)The reaction progress was supervised by thin-layer chromatography (TLC), spots were observed under UV light (254 nm and 365 nm) and colorized with 5% phosphomolybdic acid. Silica gels (300-400 mesh, Qingdao Marine Chemical Ltd., Qingdao, China) were used for column chromatography. Waters 1525 series equipped with a PDA detector (Waters Corporation, Milford, MA) was used for analytical HPLC, and YMS-pack ODS-A column (250 mm × 10 mm, 5 µm) was used for semipreparation. All reagents were purchased from commercial sources, and purifications and desiccations of available solvents were accomplished by standard techniques prior to use. Positive control drug CA-4 was purchased from Sigma, and isoCA-4 was obtained refer to the previous report [37 (link)]. The purity of all compounds was confirmed to be greater than 95% through analytical HPLC.

The NMR data were obtained using a 500 MHz Bruker NMR spectrometer (Zurich, Switzerland) at 25 °C. MSP1-1 and MSP1-2 (20 mg each) were dissolved in D₂O solvent and lyophilized. The dried polysaccharides (20 mg) were transferred to a standard 5 mm probe after dissolution in 500 μL D₂O solvent. The operating frequencies of ¹H NMR and ¹³C NMR were 500 and 125 MHz, respectively. The acquisition times were set to 16 times for ¹H NMR spectra, 1024 times for ¹³C NMR spectra, 12 times for ¹H/¹H correlation spectroscopy (COSY), 12 times for heteronuclear single quantum correlated spectroscopy (HSQC), 32 times for nuclear overhauser effect spectroscopy (NOESY), and 64 times for heteronuclear multiple bond correlation (HMBC) spectra. MestReNova software (Version: 9.1.0-14011; Mestrelab Reserch, Santiago de Compostela, Spain) was used to process the NMR data of the polysaccharide samples.

Different compounds were isolated following the method of Chaudhuri, et al. [9 (link)]. Briefly, the S. pinnata stem bark was cut into small pieces, dried, ground into powder, and extracted with 70 % methanol and water. The lyophilized extract was re-extracted successively with hexane, chloroform, ethyl acetate and water. All of the fractions were concentrated in reduced pressure, and the ethyl acetate fraction was further purified through silica gel column chromatography. Dichloromethane and methanol elution yielded four compounds namely SPE1, SPE2, SPE3 and SPE4. Structures of the bioactive compounds were analysed using different spectroscopic methods such as EIMS (JEOL JMS-700, Germany), FT-IR spectra recorded in KBr (Perkin Elmer, USA) and different nuclear magnetic resonance (NMR) experiments including ¹H and ¹³C with a Bruker-500 MHz NMR Spectrometer (Germany).