Hypoxis corms produce a thick black leachate when soaked in water. The leachate was found to inhibit sproiting of the corm. The leachate was concentrated and and a H-NMR profile was obtained. Annotation of compounds was obtained using Chenomx and published data. To confirm the compounds, the samples were spiked with pure standards.
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The 250 ml leachate was dried in a SpeedVacTM vacuum concentrator (ThermoFisher Scientific, Waltham, USA) for 24 h, with a 50 mg dried sample weighed out into 2 ml Eppendorf tubes for extraction and analysis. Thereafter, 0.75 ml methanol-d4 (CH3OH-d4) and potassium dihydrogen phosphate (KH2PO4), buffered in deuterium water (D2O) (pH 6.0), containing 0.1% (w/w) Trimethylsilylpropionic acid sodium salt, were added to samples. The mixtures were vortexed at room temperature for 1 minute, ultra-sonicated for 20 minutes and then centrifuged for another 20 minutes at 10 000 rpm. The supernatant from each tube was transferred to a 5 mm nuclear magnetic resonance (NMR) tube (Norell, Sigma-Aldrich, St. Louis, USA) for analysis. The NMR spectral data were obtained using a 600 MHz 1H-NMR spectrometer (Varian Inc., Cal-ifornia, USA), with 32 scans recorded. The human metabolome , and other refer-ences were used for the annotation of compounds existing in the leachate. To confirm the annotated compounds, samples were spiked with pure standards of succinate, lactose, propylene glycol, gallic acid and choline with subsequent NMR analysis. For this analysis, 100 mg of leachate was extracted with 0.75 ml methanol-d4 (CH3OH-d4) and potassium dihydrogen phosphate (KH2PO4), buffered in deuterium water (D2O) (pH 6.0), containing 0.1% (w/w) TSP (Trimethylsilylpropionic acid sodium salt), and 500 µL of the extract then added to the NMR tube. The standards of lactose (0.684 mg/mL), choline (0.1 mg/mL), succinate (190.240 mg/mL) and propylene glycol (0.2 mL/mL) were prepared in 0.75 ml methanol-d4 (CH3OH-d4) and potassium dihy-drogen phosphate (KH2PO4), buffered in deuterium water (D2O) (pH 6.0), containing 0.1% (w/w) TSP (Trimethylsilylpropionic acid sodium salt), and 500 µL of each stand-ard solution added to the NMR tube containing the leachate extract. The final volume per NMR tube was therefore 1 mL. An increase in the peaks of the annotated com-pounds was considered as confirmation of the presence of the compounds in the leachate.