Diode array detector

The HPLC and denaturing HPLC (dHPLC) separation was carried out using the XBridge OST with a RP C18 column (2.5 µm in particle size, 4.6 × 50 mm) (Waters, Milford, MA, USA) and phase A: 50 mM TEAA (triethylamine acetate) and 1% Acetonitrile (ACN) (v/v) in water, B: 80% ACN (v/v) in water. For purification (before radiation experiment) and to check materials, a gradient of 0–20% Phase B at 25 °C was used at a 1 mL/min flow rate. Analysis of material after radiation was performed at the same condition but at an elevated temperature of 80 °C (dHPLC) to ensure the DNA melted into single strands. Separation was performed on a DionexUltiMate 3000 System (Thermo Fisher, Waltham, MA, USA) with Diode Array Detector (Thermo Fisher, Waltham, MA, USA) monitoring at 260 nm wavelength.

Sweet Portuguese oranges (Newhall variety) were purchased from the local supermarket in December 2016. The peels were obtained after processing the fruits into juices and then the raw material was crushed in a knife mill followed by dehydration in a freeze drier. OPE was obtained using supercritical CO₂ and ethanol as co-solvent 20% (w/w) at 25 MPa and 45 °C, after a pre-treatment with CO₂ during 20 min at 45 °C, under atmospheric pressure, as previously described [30 (link)]. After 30 min of extraction, the collected fraction was concentrated by rotary evaporation and a stock solution of 150 mg/mL was prepared in ethanol and stored at −20 °C until further use.
The PMF content of OPE was determined by high-performance liquid chromatography with diode array detection (HPLC-UV/DAD), as previously described [30 (link)], using a Surveyor apparatus with a diode array detector (Thermo Fisher Scientific, San Jose, CA, USA). PMF content of the extracts was determined by analyzing the peak area at 320 nm—through the data acquisition system, Chromquest 4.0 (Thermo Fisher Scientific, San Jose, CA, USA)—and comparing with the calibration curve of each compound (0.1–100 mg/L). Final results were expressed as milligrams of nobiletin, tangeretin, sinensetin, or scutellarein tetramethylether per gram of dry extract.

HPLC analysis and fractionation were performed with the Vanquish Flex UHPLC System using the Diode Array Detector (Thermo Fisher Scientific, Waltham, MA, USA), equipped with Luna^® 5 µm C18(2) 100 Å, 250 × 4.6 mm column (Phenomenex, Torrance, CA, USA).
Mass spectra were collected using maXis II 4G ETD mass spectrometer (Bruker Daltonics, Bremen, Germany) and UltiMate 3000 chromatograph (Thermo Fisher Scientific, Waltham, MA, USA), equipped with Acclaim RSLC 120 C18 2.2 µm 2.1 × 100 mm column (Thermo Fisher Scientific, Waltham, MA, USA). Spectrum registration mode: ESI ionization mode, full scan from 100–1500 m/z, MS/MS with selection of the three most intense ions, dissociation type: CID 10–40 eV, nitrogen collision gas. Mass spectra were processed using OpenChrom Lablicate Edition (1.4.0.202201211106), TOPPView v. 2.6.0 [58 (link)]. The chemical structures were identified using the GNPS [59 (link)], NPAtlas [60 (link),61 (link)] and Dictionary of Natural Products 31.1 (https://dnp.chemnetbase.com (accessed on 10 March 2022)) [38 (link)] databases.

LC–QTOF–MS analysis was performed according to our previous study [21 (link)]. Briefly, an Ultimate 3000 UHPLC system equipped with Ultimate 3000 degasser, pump, RS autosampler, and RS column compartment, coupled with diode-array detector (Thermo Fisher, Osterode am Harz, Germany). Waters Acquity BEH C₁₈ column (2.1 × 150 mm i.d., 1.7 μm, Waters, Milford, MA, USA) was used for sample separation. The mobile phase was consisted of 0.2% acetic acid in water (A) and acetonitrile (B) with gradient elution: 0–8 min, 3% B-22% B; 8–25 min, 22% B-25% B; 25–40 min, 25% B-35% B; 40–65 min, 35% B-100% B, 65–70 min, 100% B. The flow rate was 0.3 mL/min, injection volume was 1 μL, column temperature was 25 ℃ and detection wavelength was set at 257 nm.
A high-resolution impact HD QTOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) equipped with an electrospray ionization (ESI) source was operated in the negative ion mode. The mass range was set at m/z 100–1500 in the full scan mode. The capillary voltage was set at 3500 V. The fragmentation mode was CID. The source temperature was set at 250 ℃. Nitrogen was used as the drying gas. The gas flow rate was set at 8 L/min. MS² data analysis of the three highest intensive ion fragments was intelligently performed in real time.

Crude venoms (2 mg) were reconstituted in ultrapure water, centrifuged to remove debris, and separated by reverse phase HPLC using Symmetry C18 column (250 × 4.6 mm, 5 μm particle size, 300 Å pore size) (Waters, Milford, MA, USA) and Thermo Scientific UltiMate 3000 high-performance gradient system equipped with a Diode-Array Detector (Thermo Fisher Scientific, Waltham, MA, USA). Elution was carried out at a flow rate of 1 mL/min using Solution A containing 0.1% (v/v) trifluoroacetic acid (TFA) and Solution B containing 0.1% TFA in 80% acetonitrile. The gradient used for the separation of venom proteins was 20–55% Solution B for 100 min and 55–80% for 20 min. Protein detection was performed spectrophotometrically at 215 nm and fractions were collected for further analysis. As a comparator, the venom sample of N. naja (Irula Co-op, Vadanemmeli, Tamil Nadu, India) was also subjected to reverse-phase HPLC using the same chromatographic conditions.

Optical rotations were measured using a Jasco P-2000 polarimeter (Jasco Inc., Easton, MD, USA). UV spectra were measured using a Beckman Coulter DU-800 spectrophotometer (Beckman Coulter Inc., Brea, CA, USA). NMR spectra were collected using a Bruker 800 MHz NMR instrument (Bruker, Rheinstetten, Germany) equipped with a cryoprobe with CDCl₃ and DMSO-d₆ as the internal standard (δ_C 77.0, δ_H 7.26; δ_C 39.5, δ_H 2.50). A Varian 500 MHz NMR spectrometer equipped with a 5 mm, room temperature OneNMR probe was utilized for certain experiments (Varian Inc., Palo Alto, CA, USA). HRESIMS analysis was performed using a AB SCIEX TripleTOF 4600 mass spectrometer (SCIEX, Framingham, MA, USA) with Analyst TF software. Semi-preparative HPLC was carried out using a Dionex Ultimate 3000 HPLC system equipped with a micro vacuum degasser, an autosampler and a diode–array detector (Thermo Scientific, Waltham, MA, USA).

Organic acids were analyzed by high performance liquid chromatography (HPLC) equipped with a low-pressure quadruplex pump, autosampler, column temperature chamber, and diode array detector (Thermo Fisher, Bremen, Germany). The column used was a XSelect HSS T3 column (100 Å, 5-μm particle size, 4.6 mm × 250 mm, Waters, Milford, MA, USA) with detection at 210 nm [11 (link)]. After filtration through a 0.22 μm organic Nylon 66 membrane filter (Jinlong, Tianjin, China), 20 μL of the samples was injected into the HPLC apparatus, and elution was performed isocratically with a mixture of 0.04 mol/L potassium dihydrogen phosphate (pH adjusted to 2.7 by phosphoric acid) and acetonitrile (96.5:3.5 v/v) as the mobile phase at a flow rate of 0.4 mL/min. Peaks were identified through a comparison of their retention times with standard organic acids analyzed under the same conditions. Quantification was carried out using the external standard method with standard curves constructed from organic acids.