Prominence lc 20a

Polyamines were determined according to the method of Hu et al., with slight modification^{53 (link)}. Leaves (1 g) were extracted in 5% cold perchloric acid (PCA) for 1 h. The extract was centrifuged at 12, 000 × g for 30 min. The supernatants (1 mL) were mixed with 2 mL of 2 M NaOH and 10 μL of benzoyl chloride. The benzoylation action was terminated using 4 mL of saturated NaCl solution after 30 min at 37 °C. The benzoyl PAs were extracted with 3 mL of diethyl ether by centrifuging at 3000 × g for 5 min. A volume of 2 mL of the ether phase was evaporated to dryness and the residue re-dissolved in 500 μL methanol. Polyamines were assayed by high-performance liquid chromatography (LC-20A Prominence, Shimadzu Co. Ltd., Japan). A volume of 10 µL of the benzoyl PAs in methanol were injected into a 20 mL loop, loaded onto a 4.6 × 250 mm, 5 mm particle size, reverse phase Kromasil C18 column (Eka Chemicals, Bohus, Sweden). Column temperature was maintained at 25 °C. Samples were eluted from the column with 64% methanol at a flow rate of 0.7 mL min⁻¹ using a Dionex P680 pump. Polyamine peaks were detected with a UV detector at 254 nm.

The HPLC apparatus consisted of an LC-20A Prominence (Shimadzu^®, Kyoto, JPN) equipped with a quaternary pump and a DAD model SPD-M20A (Shimadzu^®). A 15 µm Phenomenex C18 column 250 × 21.2 mm I.D. (Torrance, CA, USA) connected to a fraction collector FRC-10A (Shimadzu^®, Kyoto, Japan). Elution was performed with a mixture of solvents according to Silva al. [6 (link)] with small modifications: solvent A was 1% formic acid, and solvent B, 80% methanol at a linear gradient (0–25 min, 11–55% of solvent B). The sample injection volume was 400 µL at 3.5 mL/min flow rate, and separations were monitored at 536 nm. Red-violet-colored fractions containing betanin were concentrated by rotary evaporation, and pure and concentrated betanin was suspended in deionized water, freeze-dried, and stored at −80 °C for further analysis (Figure S1).

High-performance liquid chromatography (HPLC) analysis for the simultaneous determination of four reference components in the CRE sample was conducted by using an LC-20A Prominence HPLC system (Shimadzu Co., Kyoto, Japan) equipped with binary pumps, a column oven, an autosampler, and a photodiode array (PDA) detector. In brief, 25 mg of the lyophilized CRE was dissolved in 25 mL of 70% methanol and extracted for 60 min using an ultrasonicator (Branson 8510E-DTH, Danbury, CT, USA), and the extracted solution was then filtered through a 0.2 mm membrane filter (PALL Life Sciences, Ann Arbor, MI, USA). Then, the CRE sample and the four reference components (jatrorrhizine, coptisine, palmatine, and berberine) were subjected to analysis in an HPLC-PDA system. They were separated on a SunFire C18 column (4.6 × 250 mm, 5 mm; Milford, MA, USA) maintained at 30 °C. The mobile phase was eluted with 30 mM ammonium bicarbonate and 0.1% (v/v) aqueous triethylamine (A) and acetonitrile (B) in gradient elution mode. The flow rate was 1.0 mL/min with the following linear gradient: 0 to 15 min, 90–75% A and 10–25% B; 15 to 25 min, 75–70% A and 25–30% B; 25 to 40 min, 70–55% A and 30–45% B; 40 to 45 min, 55% A and 45% B; and 45 to 60 min, 55–90% A and 45–10% B. CRE was detected using a photodiode array at 200–500 nm (Figure 1).

The gel was dissolved in water at a concentration of 25% (w/v) and then centrifuged at 4,500×g for 10 min. The supernatant was filtered throughout a 0.45-µm cellulose membrane filter (MF Millipore^®) and then diluted at 1:8,000 ratio. The ${\text{NO}}_{\text{3}}^{-}$ analysis was performed as described previously by Li et al. (15 ), with some adaptations. ${\text{NO}}_{\text{3}}^{-}$ present in the filtered supernatant was enzymatically converted to nitrite ( ${\text{NO}}_{\text{2}}^{-}$ ) by nitrate reductase (EC 1.6.6.2, from Aspergillus sp.) (Roche Diagnostics, Mannheim, Germany). After converting ${\text{NO}}_{\text{3}}^{-}$ to ${\text{NO}}_{\text{2}}^{-}$ , 100 µL of the filtered sample were incubated at 24°C with 10 µL of 316 mM of 2,3-diaminonaphthalene in 0.62 M HCl for 10 min, followed by the addition of 5 µL of 2.8 M NaOH. Samples were immediately analyzed by reverse-phase HPLC (LC-20A Prominence, Shimadzu^®, Japan). The HPLC device was equipped with a 5-µm C8 Discovery^® column (150×4.6 mm, I.D.) guarded by a 5-µm reversed-phase C18 guard column Ascentis^® (50×4.6 mm, I.D.) and a fluorescence detector model RF-10AXL (Shimadzu^®, Japan) monitoring excitation and emission wavelengths at 375 and 415 nm, respectively. The mobile phase (1.3 mL/min) was 15 mM sodium phosphate buffer (pH7.5) and methanol (50:50, v/v) at gradient elution. Triplicate measures were performed and the nitrate contents were expressed as mmol 100 g⁻¹.

Synthetic peptides were analyzed by mass spectrometry to confirm peptide mass and amino acid sequence. Hs01-04 were individually mixed and co-crystallized with α-cyano-4-hydroxycinnamic acid matrix (Fluka) at 10 mg.mL^-1 in a MALDI target plate. Experiments were carried out in an UltrafleXtreme MALDI-TOF/TOF (Bruker Daltonics). Peptides monoisotopic mass were obtained in reflector mode over a range of 700–3500 m/z with external calibration using Peptide Calibration Standard II (Bruker Daltonics). Peptide primary structures were inferred by means of manual interpretation of fragmentation spectra.
Reverse phase HPLC (RP-HPLC) of the synthetic peptides was performed in a LC-20A Prominence (Shimadzu Co.) using a Jupiter C₁₈ 10 μm column (Phenomenex) at a flow rate of 10 mL.min^-1. Ultrapure Milli-Q water and acetonitrile (J.T. Baker) added with 0.1% (v/v) trifluoroacetic acid (TFA) were used as solvent A and B, respectively. Fractions were manually collected and analyzed by mass spectrometry to confirm the elution time of each synthetic peptide.
Peptides containing Trp or Tyr residues were quantified using calculated molar absorption coefficients [43 (link)]. The remaining peptides were quantified using the UV absorbance of the peptide bond according to the literature [44 (link)].

Extracellular glucose, xylose and xylonic acid were analyzed by HPLC (LC-20A Prominence—Shimadzu) equipped with a Rezex ROA-Organic Acid (300 × 7.8 mm) column maintained at 55 °C and using 5 mM H₂SO₄ as mobile phase at a flow rate of 0.4 mL min⁻¹ [32 (link)]. Glucose and xylose peaks were detected with a refractive index detector (RID-10A), whereas xylonic acid peaks were detected and quantified from an UV/VIS detector (SPD-20A). Once xylonic acid and xylose have the same retention time, when the former was present the latter was indirectly quantified by subtracting the xylonic acid peak (quantified by UV) from the combined xylose and xylonic acid peaks detected by RID [34 (link)].

The DAD chromatography procedure utilized a Shimadzu LC 20 A Prominence^® (Kyoto, Japan) liquid chromatograph equipped with a photodiode array (PDA) Shimadzu^® (Kyoto, Japan) detector, Shimadzu^® column oven, Shimadzu^® automatic injector, Shimadzu Lab Solutions integration system^® (Kyoto, Japan), and a Shimadzu vacuum degasser^® (Kyoto, Japan).