The LEGE-NPL (natural products library) solid inventory is composed of crude extracts. Thus, freeze-dried biomass was extracted three times with MeOH, with a sonication step of 5 min in between extractions, and was filtered and concentrated at 30 °C, using a rotary evaporator. The yields of extraction are described in the Supplementary Material (Figure S1) . The extracts were then fractionated by reverse-phase HPLC in a Waters Alliance e2695 Separations Module instrument, coupled to a photodiode array detector (Waters 2998 PDA) and an automatic Waters Fraction Collector III (Waters, Mildford, MA, USA). Each crude was injected at 40 mg mL−1 (500 µL; 1 mL loop) and separated on an ACE 10 C8 column (50 ×10 mm, ACE, Reading, UK), using a H2O:MeCN gradient (Table 4 ). Hence, each cyanobacterial extract was chromatographed into eight fractions (4 mL final volume, named A–H) into 48-deep well plates (Riplate, Ritter, Schwabmünchen, Germany), which were then dried on a CentriVap Concentrator (LabConco, Kansas City, MO, USA). These fractions were solubilized in 500 µL of DMSO and transferred to 96-deep well microplates (Nest Scientific, Woodbridge Township, NJ, USA) and stored at −80 °C, thus forming the LEGE-NPL liquid library (mother plates).
Waters 2998 pda
Manufactured by Waters Corporation
Sourced in United States, Germany
The Waters 2998 PDA is a photodiode array (PDA) detector designed for high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC) systems. It provides full-spectrum UV-Visible detection, offering a range of wavelengths from 190 to 800 nm. The detector is capable of acquiring data at high sampling rates, enabling comprehensive analysis of chromatographic peaks.
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Lab products found in correlation
Waters fraction collector 3, by Waters Corporation (1 mentions)
Centrivap concentrator, by Labconco (1 mentions)
Lichrospher 100 rp 18, by Merck Group (1 mentions)
Waters 600e pump, by Waters Corporation (1 mentions)
Superspher 100 rp 18, by Merck Group (1 mentions)
Avance 500 spectrometer, by Bruker (1 mentions)
Waters alliance 2695 hplc system, by Waters Corporation (1 mentions)
Xselect c18 column, by Waters Corporation (1 mentions)
3 protocols using waters 2998 pda
1
Cyanobacterial Extract Fractionation
2
Isolation and Characterization of Oleuropein
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The sub-fraction of interest (30 mg) was obtained using CHCl3:CH3OH:H2O in a ratio of 3:4:3, then injected into a preparative high performance liquid chromatograph (Waters 600 E multisolvent delivery system, Waters 600 E pump and Waters 2998 PDA) which was employed using Lichrospher 100 RP-18 (250 mm × 10 mm i.d.; 10 µm) (Merck KGaA, Darmstadt, Germany). The mobile phase used was composed of 0.2% H3PO4 (v/v), methanol and acetonitrile in a ratio of 96:2:2. NMR spectra were obtained using a Bruker Avance 500 spectrometer (Bremen, Germany) 5 mm-Zgrad probe, operating at 500.13 MHz for 1H and 125.77 MHz for 13C. The purity of oleuropein was confirmed using analytical HPLC (Agilent Technologies, Waldbronn, Germany), equipped with a PDA detector G 1314 C (SL). Chromatographic separation was carried out on a Superspher 100 RP-18 (75 mm × 4 mm i.d.; 4 μm) column (Merck, Darmstadt, Germany) using mobile phases: (A) 2% acetic acid (pH 2.6) and (B) 80% methanol. A gradient starting from 5% B to 50% B was employed for the elution process with 100 μL/min flow rate at 30°C and compared vs standard material (Sigma Aldrich) using HPLC. Confirmation of oleuropein identity was carried out by comparing its spectral data to the obtained literature[36 (link)].
3
HPLC Analysis of Phytochemical Standards
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Preparation of Standard Solutions. A mixed standard stock solution containing uridine, inosine, guanosine, safflomin A, and brazilin was prepared in MeOH/H2O (50 : 50, v/v). The standard solutions were filtered through a 0.22 μm membrane prior to injection. All solutions were stored in a refrigerator at 4°C before analysis.
HPLC Analysis of Sample. Analysis was performed on Waters 2695 Alliance HPLC system (Waters Corp., Milford, MA), consisting of a quaternary pump solvent management system, an online degasser, and an autosampler. The raw data were detected by Waters 2998 PDA, acquired, and processed with Empower Software. A Waters X-select C18 column (5 μm, 4.6 mm × 250 mm) preceded by a RP C18 guard column (5 μm, 3.9 mm × 20 mm) was applied for all chromatographic separation. Detection wavelength was set at 260 nm for all compounds. The mobile phase was composed of A (methanol) and B (0.1% aqueous ethylic acid) with a linear gradient elution: 0–20 min, 5% A; 20–75 min, 5–50% A, then keeping 50% A for 10 min to clean the column. MeOH/H2O (10 : 90, v/v) were used as solutions for cleaning the injection needle. The flow rate was set at 0.80 mL/min and the injection volume was 10 μL. The column temperature was maintained at 30°C.
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Preparation of Standard Solutions. A mixed standard stock solution containing uridine, inosine, guanosine, safflomin A, and brazilin was prepared in MeOH/H2O (50 : 50, v/v). The standard solutions were filtered through a 0.22 μm membrane prior to injection. All solutions were stored in a refrigerator at 4°C before analysis.
HPLC Analysis of Sample. Analysis was performed on Waters 2695 Alliance HPLC system (Waters Corp., Milford, MA), consisting of a quaternary pump solvent management system, an online degasser, and an autosampler. The raw data were detected by Waters 2998 PDA, acquired, and processed with Empower Software. A Waters X-select C18 column (5 μm, 4.6 mm × 250 mm) preceded by a RP C18 guard column (5 μm, 3.9 mm × 20 mm) was applied for all chromatographic separation. Detection wavelength was set at 260 nm for all compounds. The mobile phase was composed of A (methanol) and B (0.1% aqueous ethylic acid) with a linear gradient elution: 0–20 min, 5% A; 20–75 min, 5–50% A, then keeping 50% A for 10 min to clean the column. MeOH/H2O (10 : 90, v/v) were used as solutions for cleaning the injection needle. The flow rate was set at 0.80 mL/min and the injection volume was 10 μL. The column temperature was maintained at 30°C.
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