Hydro rp

The targeted phenolic compounds present in seaweeds were quantified by Agilent 1200 series HPLC (Agilent Technologies, CA, USA) equipped with a photodiode array (PDA) detector according to our previously published protocol of Gu et al. [112 (link)] and Suleria, Barrow, and Dunshea [63 (link)]. The sample’s phenolic compounds were extracted by conventional and ultrasonication. Sample extracts were filtered by the 0.45 μm syringe filter (PVDF, Millipore, MA, USA). A Synergi Hydro-RP (250 × 4.6 mm i.d.) reversed-phase column with a particle size of 4 µm (Phenomenex, Lane Cove, NSW, Australia) was protected by a Phenomenex 4.0 × 2.0 mm i.d., C18 ODS guard column. The injection volume of the sample or standard was 25 μL. The mobile phase A and B were of water/acetic acid (98:2, v/v) and acetonitrile/water/acetic acid (50:50:2, v/v/v), respectively. The gradient profile was 90–10% B (0–20 min), 75–30% B (20–30 min), 65–35% B (30–40 min), 45–55% B (40–60 min), 90–10% B (60–61 min), 90–10% B (61–66 min). The flow rate was 0.8, and the column was operated at room temperature. The wavelengths of 280, 320, and 370 nm were simultaneously selected at the PDA detector. Empower Software (2010) was used for instrument control, data collection, and chromatographic processing.

Ultra-high performance liquid chromatography Orbitrap mass spectrometry (LC-MS) was performed using a C18 column (Hydro-RP, Phenomenex) with tributylamine as an ion-pairing agent, analyzed over a 25-minute run-time protocol in negative ion mode, covering an m/z range of 70–1000 Da on a Thermo Exactive Plus Orbitrap (ThermoFisher) [22 (link)] (Supplementary Materials). Base peak chromatograms are shown in Supplementary Figures 2–4. Following peak selection and rigorous quality control, peak annotation was carried out using an in-house library of 292 chemically confirmed metabolic intermediates from major human and P. falciparum metabolic pathways. Unidentified peaks were putatively annotated using Human Metabolome Database (HMDB; version 4.0) datasets [23 (link)] with confidence at an m/z tolerance of 10 ppm. We filtered the final dataset down to only include acids (based on selection of pKa < 7.0). The metabolomic data analysis pipeline is shown in a flow diagram (Supplementary Figure 1).

NMR spectra were collected by Bruker Avance DRX600 spectrometer (Bruker, Fällande, Switzerland) equipped with a 5 mm triple resonance (HCN) cold probe, using TMS as an internal standard. ESI-MS and HR-ESI-MS data were obtained from an Agilent 6210 LC/TOF-MS spectrometer (Agilent Technologies, Santa Clara, CA, USA). A suitable crystal was selected and measured on a Bruker APEX-II CCD diffractometer (Bruker, Fällande, Switzerland). The structure was solved with the ShelXT structure solution program using intrinsic phasing and refined with the ShelXL refinement package using least squares minimization [29 (link)]. A high performance liquid chromatography (HPLC) system, Essentia LC-16P apparatus (Shimadzu, Kyoto, Japan), equipped with a semi-preparative column (Phenomenex Hydro-RP, 250 mm × 10 mm, 4 µm, Torrance, CA, USA), was used to purify all compounds. Acetonitrile (Merck, Darmstadt, Germany) and H₂O used in HPLC system were chromatographic grade, and all other chemicals were analytical grade.

Optical rotations were measured on a Perkin-Elmer 343 polarimeter in MeOH. UV spectra were recorded on a Shimadzu UV-1601PC spectrometer in MeOH. ECD spectra were measured on a Chirascan-Plus CD Spectrometer in MeOH. ¹H and ¹³C NMR spectra were recorded in aceton-d₆ on a Bruker Avance-300, Avance-500 and Avance III-700 spectrometers (Bruker BioSpin GmbH) operating at 300 and 75, 500 and 125 MHz and 700 and 176 MHz, respectively. HRESIMS spectra were obtained on a Bruker maXis Impact II mass spectrometer (Bruker Daltonics GmbH). Low-pressure liquid column chromatography was performed using C₁₈-SiO₂ Gel ODS-A (12 nm, S—75 um, YMC Co., Ishikawa, Japan) and a Buchi B-688 Chromatography Pump on a Buchi glass column using Si gel KSK (50/100 μm, Imid Ltd., Russia). Plates precoated with Si gel (5–17 μm, 4.5 × 6.0 cm, Imid Ltd., Russia) and Si gel 60 RP-18 F254S (20 × 20 cm, Merck KGaA, Germany) were used for thin-layer chromatography. Preparative HPLC was carried out on a Shimadzu LC-20 (Shimadzu, Kyoto, Japan) and Agilent 1100 (Agilent Technologies, Santa Clara, CA, USA) chromatographs using a Shimadzu RID-20A and Agilent 1100 refractometers and YMC ODS-AM (YMC Co, 5 μm, 250 × 10 mm), Ultrasphere Si (5 μm, 250 × 4.6 mm), and Hydro-RP (Phenomenex, 4 μm, 250 × 10 mm) columns.

The concentrations of the targeted phenolic compounds in Vigna were determined using an Agilent 1200 series HPLC (Agilent Technologies, CA, USA) provided with a photodiode array (PDA) detector along with our previously published methods of Suleria, Barrow and Dunshea [20 (link)] and Wu et al. [21 (link)]. A Synergi Hydro-RP (250 × 4.6 mm i.d.) reversed-phase column with 4 µm (Phenomenex, Lane Cove, NSW, Australia) was protected by a Phenomenex 4.0 × 2.0 mm i.d., C18 ODS guard column. At the PDA detector, wavelengths of 280, 320, and 370 nm were chosen in parallel. In terms of instrument control, statistics collection, and chromatographic preparation, Empower Software (2010) was utilized.