Naringenin

Liquid chromatography was carried out using a UFLC with an autosampler (Shimadzu Corporation, Kyoto, Japan). A Waters Atlantis T3 (Waters Corporation) column (2.1 × 150 cm, 3 μm) was used at ambient temperature. The injected volume of sample was 10 μL. The elution gradient was carried out with binary solvent system consisting of 0.02% acetic acid in H₂O (solvent A) and 0.02% acetic acid in MeCN (solvent B) at a constant flow rate of 250 μL/min. A linear gradient profile with the following proportions (v/v) of solvent B was applied: gradient profile 0 to 5 min and 0% of B, 5 to 8 min and 0% to 16% of B, 8 to 20 min and 16% to 100% of B, 20 to 25 min and 100% of B, 25 to 28 min and 100% to 0% of B, and 28 to 32 min with 4 min for re-equilibration and 0% of B.
To diagnose the hormone precursor-to-product ion transitions, mixtures of 150 ng/mL of the standard compounds dissolved in 50% MeCN were directly infused into a hybrid triple quadrupole/linear ion trap mass spectrometer (ABI 4000 Q-Trap, Applied Biosystems, Foster City, CA, USA) outfitted with an electrospray ion source. The analysis parameters were optimized for the production of characteristic precursor-to-product ion transitions in negative or positive ionization modes. ABA, IAA, IAA-Asp, JA, JA-Ile, SA, sakuranetin, naringenin, and their internal standards were scanned in the negative mode, whereas momilactone A and MeJA were analyzed in the positive mode. The mixtures of standard compounds were separated by reversed-phase UFLC and analyzed by ESI-MS/MS in the MRM mode with 50 ms dwell time, 5 ms of pause time between mass ranges, and 700 ms of settle time for switching polarities. The identities of phytohormones and metabolites in the crude plant extracts were confirmed by analysis of product ion fragments obtained by the hybrid triple quadrupole/linear ion trap mass spectrometer, operating in the IDA mode, with a source voltage of 4.5 kV and source temperature of 550. In the ''Enhanced Product Ion" scan mode, precursor ions were fragmented with collision energy +25 kV or -25 kV and products in the range of 50 to 500 m/z were detected.
UFLC-ESI-MS/MS assays were repeated twice biologically, with each repetition having three replicates. Similar results were obtained in repeated experiments; only the result in one repetition was presented.

The standard IAA, IAA-Asp, JA, MeJA, and SA were purchased from Sigma-Aldrich (St. Louis, MO, USA), and ABA and JA-Ile were from OlChemIm (OlChemIm, Olomouc, Czech Republic). The internal standards were ²H₆ABA (Olchemin) for ABA, 10-dihydro-JA (DHJA; Olchemin) for JA, JA-Ile, and MeJA, D₂-IAA (Sigma-Aldrich) for IAA and IAA-Asp, and naphthalene acetic acid (NAA; internal standards) for SA. The standard momilactone A was kindly provided by Dr. Morifumi Hasegawa of College of Agriculture, Ibaraki University. The standard naringenin and sakuranetin were purchased from Extrasynthèse (Genay, France).

Lyophilized lettuce leaves from 18-week old cv. Firecracker, kfoA, kfoB and nco plants were ground using a mortar and pestle. Fifty mg leaf powder was placed in a plastic tube and subjected to acid hydrolysis, based on the method of Hertog et al.^{31 (link)}. In short, 4 ml solvent (methanol/water; 62.5:37.5 v/v, 2 g/l tert-butylhydroquinone, Sigma Aldrich) was added, then the mix was acidified with 1.0 ml 6 M HCl, vortexed for a few seconds, and placed in a 90 °C water bath for 2 h. Afterwards, 100% methanol was used to make up the volume of the extract to 10 ml. The extract was then sonicated for 5 min, centrifuged at 2500 rpm for 8 min, and filtered through 0.45 μm PTFE filters (Fisher Scientific) for UPLC-MS/MS analysis. Total polyphenol content of the extracts was measured by a modified Folin-Ciocalteu assay^{17 (link)} based on^{79 (link)} and^{80 (link)}.
Extracts were separated and analyzed by a UPLC-MS/MS consisting of the Dionex® UltiMate 3000 RSLC ultra-high-pressure liquid chromatography system, consisting of a workstation with ThermoFisher Scientific’s Xcalibur v. 4.0 software package combined with Dionex^®’s SII LC control software, solvent rack/degasser SRD-3400, pulseless chromatography pump HPG-3400RS, autosampler WPS-3000RS, column compartment TCC-3000RS, and photodiode array detector DAD-3000RS. After passing through the photodiode array detector, the eluent flow was guided to a Q Exactive Plus Orbitrap high-resolution high-mass-accuracy mass spectrometer (MS). Mass detection was full MS scan with low energy collision induced dissociation (CID) from 100 to 1000 m/z in either positive, or negative ionization mode with electrospray (ESI) interface. Sheath gas flow rate was 30 arbitrary units, auxiliary gas flow rate was 7, and sweep gas flow rate was 1. The spray voltage was 3500 volts (−3500 for negative ESI) with a capillary temperature of 275 °C. The mass resolution was 140,000. Column and run conditions were identical to^{78 (link)} apart from that the average pump pressure was 3900 psi for the initial conditions.
Putative formulas of flavonoids and other compounds were determined by isotope abundance analysis on the high-resolution mass spectral data with Xcalibur v.4.0 software and reporting the best fitting empirical formula. Database searches were performed using reaxys.com (Elsevier RELX Intellectual Properties SA) and SciFinder (American Chemical Society).
Quantification was based on external standards of commercially available compounds. Naringenin and naringenin chalcone were purchased from Cerilliant, quercetin from Tocris, kaempferol and cyanidin chloride from Sigma Aldrich. Standards were dissolved in anhydrous methanol (naringenin, naringenin chalcone) or ethanol (cyanidin chloride, quercetin, kaempferol). Additionally, pelargonidin was quantified in cyanidin equivalents.

The combinational antibacterial activity of naringenin and naringenin nanoconjugates was evaluated using the well diffusion assay against four bacterial pathogens: Staphylococcus aureus (NCIM 2492), Bacillus subtilis, Escherichia coli (NCIM2139), and Pseudomonas aeruginosa (NCIM 2200). In this assay, the bacterial inoculum of ~10⁵ CFU/mL was spread evenly on the agar plates. Wells were made in the agar, and into these wells, 50 µL of naringenin-nano Ag and Au conjugates and standard naringenin (10 µg/mL) were added. The plates were then incubated at 37 °C for 24 h. After the incubation period, the diameter of the inhibition zones around each well was measured. And MIC and MBC of conjugates were also determined as per standard method [30 (link),31 ]. The experiments were performed in triplicate, and the data were presented as mean ± SD. To assess the synergistic or enhanced effect of naringenin-nano Ag and Au conjugates, the activity enhancement was calculated using the formula shown below [32 (link),33 (link)].
$$Increases fold activity Ag=\frac{[{\left(Zone of inhibition of naringenin-nano Ag conjugate\right)}^2-{(Zone of inhibition of standard naringenin)}^2}{{\left(Zone of inhibition of standard naringenin\right)}^2}$$

An amount of 0.1 g of sample powder was dissolved in 2 mL of methanol and extracted for 2 h through heating reflux at 70 °C. The solution was dried in a water bath and then filtered through a 0.45 µm microporous filter membrane. The residue was similarly filtered with 0.5 mL of methanol. Subsequently, all the filtrates were preserved in a refrigerator (4 °C) until use. Finally, the naringenin content in the filtrate was determined by using an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA). The component separation was achieved using a Waters Xbridge-C18 column (4.6 mm × 250 mm, 5 μm) (Waters Corporation, Milford, MA, USA). The main methods were as follows: The mobile phase consisted of 0.2% phosphoric acid (A) and methanol (B). The gradient elution modes were 25% B at 0–5 min and 25–30% B at 5–10 min. The flow rate was set to 1.0 mL min⁻¹, and the column temperature was 25 °C. Additionally, the injection volume was set to 20 μL, and the ultraviolet spectrum of the sample was set at 290 nm. Each sample was measured three times, and the average was used for further analysis.

Naringenin titers were detected at 290 nm using an Agilent 1260 HPLC system (Waldbronn, Germany) equipped with a diode array detector (DAD) 1260 model VL + (G7115A) and a C18 column (3 × 100 mm 2.7 µm). The column was eluted with gradient elution at 30°C and 0.3 mL min⁻¹ flow rate: 10% to 40% acetonitrile/water (vol/vol) for 5 min, 40% acetonitrile (vol/vol) for 7 min, 40% to 95% acetonitrile (vol/vol) for 3 min, and 95% to 10% acetonitrile (vol/vol) for 3 min. It was noted that 0.3% acetic acid (vol/vol) was added to above mobile phases, including the acetonitrile or water, which contributed to naringenin separation.