Ascend 700 mhz spectrometer

The (+)-usnic acid was isolated from the extracts of marine fungus, Mycosphaerella sp., which was collected from a marine sediment at Donghae-si, Korea. The fungus was cultivated in 6 L of potato dextrose broth (PDB) dissolved in seawater in 27 °C, at 140 rpm shaking incubator for 7 days. The broth was extracted with ethyl acetate and yielded 4.01 g extract. The crude extract was fractionated into 3 fractions with a silica gel column chromatography using CH₂Cl₂ and MeOH as solvent. Fractions were further purified by C18 HPLC (Phenomenex luna C18 column, 250 mm × 10 mm, 5 μm) using 65% CH₃CN in H₂O to yield 6.8 mg (+)-usnic acid. NMR spectra were measured using a Bruker Ascend 700 MHz spectrometer. Electrospray ionization source (ESI) mass data was obtained using Agilent Technologies 6120 quadrupole mass spectrometer coupled with Agilent Technologies 1260 series HPLC. The optical rotation of (+)-usnic acid was measured in chloroform using a 10 mm path length cell on a Digital Polarimeter P-2000, Jasco Inc.

Persicaria senticosa was collected from Seo-myeon, Chuncheon-si, Gangwon-do, Korea, in 2016 (GPS: N 37° 55′ 30.1^″, E 127° 37′ 57.0^″, altitude: 434 m). Voucher specimens (G071) were authenticated by Dr. Chun Whan Choi. Persicaria senticosa were deposited at the herbarium of Biocenter, Gyeonggido Business & Science Accelerator, Suwon, South Korea (Fig. S3). The 99.9% methanol extract of thirty-four Polygonum was obtained from the Korea Plant Extract Bank at the Korea Research Institute of Bioscience and Biotechnology (Daejeon, Korea). ¹H and ¹³C NMR experiments were performed on a Bruker Ascend 700 MHz spectrometer with tetramethylsilane (TMS). LC-ESI-MS was obtained on a Triple TOF 5600+ instrument (AB SCIX, USA) and HRESI-MS on a LTQ Orbitrap XL instrument (Thermo, USA). Thin-layer chromatography (TLC) was conducted on Silica gel 60 F₂₅₄ (Merck, Germany) and Silica gel 60 RP-18 F_254S (Merck, Germany) plates. Column chromatography(CC) was performed using Silica gel 60 (70~230 mesh, Merck, Germany), ODS-A (12 nm S-7 μm, YMC GEL, Japan), and preparative HPLC was performed on LC-8A (Shimadzu, Japan).

The NMR spectra were recorded with a Bruker Ascend 700 MHz spectrometer in solvent system pyridine-d₅—methanol-d₄, 9:1 (v/v) with a sample concentration of 10 mg ml⁻¹.
One-dimensional ¹H spectra were collected using standard parameters.
Two-dimensional ¹H spectra were measured in the phase-sensitive mode with a spectral width of 7716 Hz. The DQF-COSY spectrum was acquired in a 6080 × 512 matrix with 32 accumulations per increment and was processed in a 4 K×2 K matrix. The TOCSY spectrum was acquired with a mix time of 110 ms in a 2048 × 512 matrix with 32 accumulations per increment in a 2 K × 1 K matrix. The ROESY spectrum was acquired with a mix time of 300 ms in a 2048 × 512 matrix with 64 accumulations per increment in a 2 K × 1 K matrix. HSQC and HMBC experiments were performed with pulse field gradients. The HSQC spectrum was acquired in the phase-sensitive mode. The spectral windows for ¹H and ¹³C axes were 7716 Hz and 35211 Hz, respectively. The data were collected in a 1024×256 matrix and processed in a 2 K × 1 K matrix. The HMBC spectrum was acquired in absolute value mode. The spectral windows for ¹H and ¹³C axes were 7716 Hz and 39063 Hz, respectively. The data were collected in a 2048×256 matrix and processed in a 2 K × 1 K matrix.

NMR spectra of all cell extracts and their corresponding culture media were recorded at 298 K on Bruker Ascend 700 MHz spectrometer equipped with a cryogenic probe. ¹H NMR spectrum of each sample was acquired using one dimensional Nuclear Over Hauser Effect Spectroscopy (NOESY). The NOESY experiment was chosen due to its better sensitivity and more efficiency in water suppression compared to water presaturation experiment. The acquisition time for each ¹H NMR spectrum was 3.53 min, consisting of 64 scans with a width of 12 ppm. Additional support for identification was obtained using two-dimensional (2D) NMR such as J-resolved (JRES), homonuclear spectroscopy (COSY) and heteronuclear multiple bond coherence (HMBC).