Polyamide 6

NMR spectra were recorded in either CDCl₃ or MeOD, on a Bruker AVANCE III HD 400 (Bruker Corp., Billerica, MA, USA), at resonance frequency of 400.17 MHz for ¹H. Optical rotation was determined in MeOH on a PolAAr31 polarimeter (Optical Activity Ltd., England). RP-HPLC separations were performed using an Agilent 1200 Series HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a diode array detector. Analytical chromatographic separations were carried out on either a Kinetex XB-C18 column (4.6 × 250 mm, 5 μm total particle size; Phenomenex Inc., Torrance, CA, USA; nonpolar compounds) or a Zorbax Eclipse XDB-C18 column (4.6 × 150 mm; Agilent Technologies, Santa Clara, CA, USA; phenolic compounds). Semipreparative RP-HPLC was conducted on a Synergi 4μ Fusion-RP, 80 A, 250 × 10 mm column (Phenomenex Inc., Torrance, CA, USA), with an isocratic elution, using MeOH-H₂O mixtures of different polarities. Conventional column chromatography (CC) was carried out on Silica gel 60 (0.063–0.2 mm, Merck, Germany), Polyamide 6 (Sigma-Aldrich Co., Saint Louis, MO, USA) and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden). TLC separations were performed using precoated plates (Silica gel 60 without fluorescence indicator, Art. No 5553; Merck, Darmstadt, Germany).

Mandarin peel extract (25 g) was applied to polyamide 6 (Sigma-Aldrich, Munich, Germany) column chromatography (250 g), eluted by water to yield 12.5 g of sticky material, mainly sugars, and then eluted with 30% ethanol in order of decreasing polarity to yield 4.2 g of yellow amorphous powder, then 60% ethanol to yield 3.3 g, and finally 100% ethanol 2.1g after evaporation of the eluent under vacuum using a Heidolph rotatory evaporator (Schwabach, Germany).
The presence of hesperidin was checked using comparative paper chromatography (Whatman filter paper sheets No.1, United Kingdom) using 15% acetic acid as aqueous eluent and butanol/acetic acid/water (BAW) in portions (4:1:5, respectively) as organic eluent. The 30% ethanol fraction (4 g) was applied to Sephadex LH-20 column chromatography (Pharmacia Company, Uppsala, Sweden) and eluted with 50% ethanol and monitored by UV lamp (365 nm). Then, the collected fractions were checked by comparative paper chromatography using hesperidin as a standard sample (friendly, 2 mg obtained from the Department of Phytochemistry and Plant Systematics, National Research Center, Dokki, Cairo, Egypt), whereby 750 mg of hesperidin-containing sub-fractions was collected. The hesperidin sub-fraction was applied to repeated Sephadex LH-20 column chromatography using butanol saturated with water as eluent to finally yield 400 mg of hesperidin.

Silica gel 60 (particle size 0.063–0.2 mm, 70–230 mesh) (Fluka, St. Louis, MO, USA), Polyamide-6 and Sephadex LH-20 (Sigma-Aldrich, Taufkirchen, Germany) were utilized for column chromatography. TLC plates (Si 60 F₂₅₄, Merck, Darmstadt, Germany) and analytical-grade solvents were used in the study. Visualization of the spots was carried out using p-anisaldehyde spray reagent [37 ].
The UV investigation was carried out in methanol utilizing a Shimadzu UV1, 601PC UV–visible scanning spectrophotometer (Shimadzu Corp., Tokyo, Japan). Optical rotation was measured using a 341 Perkin Elmer polarimeter (Darmstadt, Germany). HRESIMS was executed on Agilent LC/Q-TOF, 6530 (Santa Clara, CA, USA). All 1D and 2D NMR spectra were recorded on a Bruker Avance III 400 MHz (Bruker AG, Fällanden, Switzerland) and analysed using Topspin 3.1 software (Bruker AG, Fällanden, Switzerland). Deuterated methanol and chloroform (Cambridge Isotopes, Andover, MA, USA) were used.
Aluminium trichloride (AlCl₃.6H₂O), anhydrous sodium carbonate (Na₂CO₃), ascorbic acid, DPPH, ABTS (Sigma, PN: A3219, St. Louis, MO, USA), Folin–Ciocalteu, gallic acid, rutin, sodium hydroxide (NaOH), and sodium nitrite (NaNO₂) were purchased from Merck (Rahway, NJ, USA) and Sigma-Aldrich (St. Louis, MO, USA).

Thiophene (≥99%, Sigma-Aldrich, Saint Louis, MI, USA), anhydrous ferric chloride (≥99%, Sigma-Aldrich, Saint Louis, MI, USA), polyamide 6 (Sigma-Aldrich, Saint Louis, MI, USA), formic acid (97%, Sigma-Aldrich, Saint Louis, MI, USA) and methanol (≥99%, Sigma-Aldrich, Saint Louis, MI, USA) were used as received. Chloroform was dried over phosphorus pentoxide under reflux and used immediately after distillation.

Polyamide-6 (MW: 150,000, density: 1.184 g/cm³) was purchased from Merck (made in Germany). Acetic acid was also purchased from Merck (made in Germany, Darmstadt, Germany); formic acid was purchased from Sigma Aldrich (St. Louis, MO, USA).