Avance 600 nmr spectrometer

The morphological characteristics of the endophytic fungi isolated from E. exserta were observed using a CX31 Digital Fluorescence Microscope (Mshot, Guangzhou, China). High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) was carried out on a Q-TOF mass spectrometer from Bruker maXis with an ESI interface (Bruker, Fremont, CA, USA). Nuclear magnetic resonance (NMR; ¹ H and ¹³ C) spectra were recorded on a Bruker Avance-600 NMR spectrometer (¹ H at 600 MHz and ¹³ C at 151 MHz) (Bruker, Fremont, CA, USA). Semi-preparative HPLC separation was performed on a Lumtech instrument equipped with an HPLC K-501 pump and a K-2501 UV detector using a Wondasil 38020-41 C₁₈ column (250 mm × 21.2 mm, 5 μm, Welch Materials Inc.). Water used for experiments was purified with an ultrapure water machine (Exceed-Cb-10, Aike, Chengdu, China). MeOH was of HPLC grade, and all the other reagents were of analytical grade. All solvents used for HPLC were filtered through a 0.45 μm nylon membrane before use. Silica gel (60–100, 100–200 and 200–300 mesh, Qingdao Marine Chemical Inc.) and Sephadex LH-20 (GE Healthcare) were used for separation and isolation. Precoated silica gel GF-254 plates (Qingdao Marine Chemical Inc.) were used for analytical TLC. Spots were visualized under UV light (254 or 356 nm).

Plasma samples prepared from fasted blood at weeks 0 and 12 were prepared for NMR spectroscopy by mixing 400 µL plasma with 200 µL of D2O. All NMR measurements were performed on a Bruker Avance 600 NMR Spectrometer (Bruker BioSpin, Rheinstetten, Germany) with 1H operating frequency of 600.13 MHz and equipped with a 5 mm TXI inverse probe. The Carr–Purcell–Meiboom–Gill (CPMG; cpmgpr1d, Bruker pulse sequence) pulse sequence with water suppression was applied for the acquisition of 1H NMR spectra of plasma samples at 310 K. The total spin–spin relaxation delay was 100 ms, the spin-echo delay was 1 ms, and the relaxation delay between pulses was 3 s. The spectra were acquired by 64 scans, 32k data points, a spectral width of 17.34 ppm, and an acquisition time of 1.57 s.
Spectra were imported to Chenomx NMR suite v 8.0 (Chenomx Inc., Edmonton, AB, Canada) and were automatically phased and manually baseline corrected. Referencing of the ppm scale was based on the format singlet at 8.5 ppm, and identification of metabolites was based on the build in database. After integration, concentrations were calculated relative to the blood glucose levels measured enzymatically.

NMR spectra were captured on an Avance-600 NMR spectrometer (Bruker, 57 Karlsruhe, Germany) at room temperature. High-resolution electrospray ionization mass spectrometry (HRESIMS) spectra data were recorded on a 6500 series quadrupole-time-of-flight (Q-TOF) mass spectrometer (Agilent, Santa Clara, CA). Mass spectrometry also recorded on LCMS 8050 (Shimadzu, Tokyo, Japan). High-performance liquid chromatography (HPLC) analysis was performed on a 1260 Infinity LC system (Agilent, Santa Clara, CA), and the column used was a 250 mm × 4.6 mm i.d., 5 µm, ZORBAX Eclipse XDB (Agilent, Santa Clara, CA). Semipreparative HPLC was performed on a 1260 series system (Agilent), and the column used was a 250 mm × 9.4 mm i.d., 5 μm, ZORBAX Eclipse XDB (Agilent). Column chromatography was performed using silica gel (100−200 mesh) (Qingdao Ocean Chemical Co. Ltd., Qingdao, China) and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden). All chemical reagents were purchased from a chemical reagent company (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) and used without further purification.

Media samples were prepared for NMR analyses based on protocols as described by Beckonert and colleagues [24 (link)]. Briefly, 300 μl media samples were mixed 1:1 with NMR phosphate buffer (50% v/v D20 (GOSS Scientific, UK, 0.01% v/v sodium 3-(trimethylsilyl) propionic acid 2,2,3,3-d4 ([TSP)], pH 7.4) and centrifuged at 12,470 × g for 5 minutes. Some 550 μl of sample was transferred into 5-mm NMR tubes (Bruker, Germany) and all samples from both studies were run on a Bruker Avance 600 NMR Spectrometer with TXI probe head (Bruker), using XWIN-NMR software (Bruker Biospin, Germany). ¹H NMR data were acquired by applying a standard one-dimensional (1D) pulse programme for 128 scans (after eight dummy scans), that included water irradiation during the recycle delay, set at 2 seconds (s). The pulse sequence was set to: recycle delay-90°-t-90°-tm-90°-ACQ, whereby 90° pulse length was set to between 16.5 μs, t (short delay) = 2 s, tm (mixing time) = 100 ms and ACQ (acquisition period) = at 2.73 s per scan. Spectral data underwent baseline correction, internal reference (TSP) peak calibration and phasing, using an in-house MATLAB algorithm (version R2012b, Mathworks Inc, USA) and Topspin 3.1 software (Bruker BioSpin, Germany). Water regions and HEPES buffer peaks were removed followed by to automatic spectral alignment and probabilistic quotient normalization [25 (link)] in MATLAB.

A Bruker Avance 600 NMR spectrometer with a 5 mm inverse probe head at a ¹H resonance frequency of 600.13 MHz was used to perform a number of NMR experiments at a temperature of 298 K. One-dimensional proton, 2D phase-sensitive total correlation spectroscopy (TOCSY)³⁸ , nuclear Overhauser effect spectroscopy (NOESY)³⁹ and natural abundance ${}^1$ H- ${}^{13}$ C and ${}^1$ H- ${}^{15}$ N heteronuclear single quantum coherence spectroscopy ( ${}^1$ H- ${}^{13}$ C-HSQC) and ${}^1$ H- ${}^{15}$ N-HSQC⁴⁰ spectra were acquired, with relaxation delays of 2.5 s, 2.0 s, 1.5 s, 1.0 s and 2.0 s, respectively, and acquisition times of 3.4 s, 340 ms, 280 ms, 170 ms and 100 ms, respectively. Mixing times of 60 ms and 200 ms were used for the TOCSY and NOESY, respectively.
The ¹H spectral widths were 6.0 kHz, 7.2 kHz, 6.0 kHz and 9.6 kHz, and the spectra were acquired with 4, 16, 32 and 64 transients for each of the 1024, 2048, 256 and 128 t1 increments for the TOCSY, NOESY, ${}^1$ H- ${}^{13}$ C-HSQC and ${}^1$ H- ${}^{15}$ N-HSQC, respectively. The ¹³C spectral width was 21.1 kHz and the ¹⁵N spectral width was 10.9 kHz. All two-dimensional spectra were processed with the Bruker TopSpin program, version 4.0.6 (Bruker BioSpin, Germany), using the sine squared window function, and the ¹H signal of TSP was used as the chemical shift reference.

The 300 μL liquid sample was directly mixed with 100 μL DMSO-d6 for ¹³C NMR determination. The spectra were obtained on a Bruker AVANCE 600 NMR spectrometer using 12 μs pulse width (90°). The relaxation delay was 6 s. To achieve a sufficient signal-to-noise ratio, inverse-gated proton decoupling method was applied. The spectra were taken at 150 MHz with 800–1200 scans accumulated.

Optical rotations were measured on an Autopol III automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). UV spectra were collected on a Cary 300 spectrometer (VARIAN, Palo Alto, CA, USA). IR spectra were collected on a Nicolet Nexus 470 FT-IR spectrometer (Thermo Scientific, Waltham, MA, USA). CD spectra were collected on a J-810 spectropolarimeter (Jasco Corporation, Tokyo, Japan). ¹H and ¹³C-NMR spectra were collected on a Bruker Avance-600 NMR spectrometer (Bruker Corporation, Billerica, MA, USA). HRESIMS spectra were collected on a Waters Xevo G2 Q-TOF spectrometer (Waters, Milford, MA, USA). HPLC analysis was performed on an Agilent 1260 series (Agilent Technologies, Santa Clara, CA, USA) with a C₁₈ RP-column (Eclipse XDBC₁₈, 150 × 4.6 mm, 5 μm, Agilent Technologies, Santa Clara, CA, USA). Semi-preparative HPLC was performed on a SSI 23201 system (Scientific Systems Inc., State College, PA, USA) with a YMC-Pack ODS-A column (250 × 10 mm, 5 μm, YMC CO., LTD. Shimogyo-ku, Kyoto, Japan). MPLC was performed on a LC3000 series (Beijing Tong Heng Innovation Technology, Beijing, China) with a Claricep^TM Flash i-series C₁₈ cartridge (20–35 μm, 40 g, Bonna-Agela, Wilmington, DE, USA). Size exclusion chromatography was carried out using a Sephadex LH-20 (GE Healthcare, Chicago, IL, USA) column.