Unity inova 500 mhz spectrometer

Linoleic acid hydroperoxide (LHP) was enzymatically synthesized by the reaction of linoleic acid with soybean lipoxygenase, according to the method that was described by Ohkawa et al. [48 (link)], with some modifications. Briefly, 50 mg of linoleic acid was dissolved in 0.1 M borate buffer (pH 9). Subsequently, 15 munit/mg soybean lipoxygenase was added, followed by incubation under saturated oxygen conditions at 4 °C for 2 h. After incubation, 2.5 g of NaCl was added to stop the reaction, and the hydroperoxides were extracted with anhydrous diethyl ether. To dry the ether extract, anhydrous sodium sulfate was used to remove any water, and the extract was filtered. The filtrate was evaporated to dryness in a rotary evaporator, resuspended in MeOH, and stored at −80°C. The maximal absorption at 234 nm was used to calculate the final concentration of LHP by applying Beer’s law while using ε = 23,000 as the molar extinction coefficient. The LHP structure was determined on the basis of its mass (Micromass Quattro LC) and nuclear magnetic resonance (NMR, Varian Unity INOVA 500 MHz spectrometer).

The chemical structure of the extracted ALG was confirmed by Fourier Transform Infrared (FTIR) spectroscopy and nuclear magnetic resonance (NMR) and was compared with a commercial sample. For ¹H NMR analysis, an ALG sample (10 mg/mL) was dissolved in D₂O and analyzed on a Varian UNITY Inova 500 MHz spectrometer (Varian, Palo Alto, CA, USA).

The soursop kombucha freeze-dried samples (10 mg) were mixed with 0.375 ml of methanol-D4 and 0.375 ml of KH₂PO₄ buffer in D₂O (pH 6.0) containing 1% TSP as internal standard for relative quantification of the identified metabolites. The mixture was vortexed for 1 min and sonicated at 30°C for 15 min in an ultra-sonicator (Branson, United States). The solution was centrifuged at 13,000 rpm for 10 min, and 600 μl of supernatant was transferred to a nuclear magnetic resonance tube for proton nuclear magnetic resonance (¹H-NMR) analysis (Muhialdin et al., 2020b (link)). Spectra were recorded at 25°C with frequency of 500 MHz on a Varian Unity INOVA 500 MHz Spectrometer (Varian Inc., CA). Each sample was subjected to 64 scans and recorded with an acquisition time of 193 s, with a pulse width of 10 ppm and a relaxation delay of 1 s. The spectra were automatically phased and bucketed using Chenomx software, with standard bins of δ 0.05 ranging from region δ 0.50 to 10.00. The analysis was required to remove residual methanol region (δ 3.28–3.33) and water region (δ 4.70–4.96). Two-dimensional ¹H–¹H J-resolved and was employed to identify metabolites. Partial least square analysis (PLS) and principal component analysis (PCA) were performed using SIMCA-P software (Umetrics AB, Umeå, Sweden).

Melting points were determined in an IA9200 Electrothermal apparatus equipped with a digital thermometer in glass capillary tubes and are uncorrected. Infrared spectra were recorded on a Perkin Elmer 281 FTIR spectrometer in KBr disks or NaCl crystal windows. Elemental analyses for C, H, N, and S were within ±0.4% of theoretical values and were performed on a Carlo Erba Elemental Analyzer Mod. 1108 apparatus. ¹H NMR spectra were recorded on a Varian Unity Inova 500 MHz spectrometer in DMSO-d₆ solution. Chemical shifts are given in δ values (ppm), using tetramethylsilane (TMS) as the internal standard; coupling constants (J) are given in Hz. Signal multiplicities are characterized as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad). All the synthesized compounds were tested for purity on TLC (aluminum sheet coated with silica gel 60 F254, Merck, Kenilworth, NJ, USA) and visualized by UV (λ = 254 and 366 nm). Purification of synthesized compounds by column chromatography was performed using silica gel 60 (Merck, Kenilworth, NJ, USA). All chemicals and solvents were reagent grade and were purchased from commercial vendors.