A new bicyclic lactone and other polyphenols from the commercial vegetable Anthriscus cerefolium

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Reagents and materials

The fresh cut chervil was received from Frøvoll Farm, Randaberg, located in southwestern Norway (59°0′56.3″ North, 5°37′25.8″ East). Experimental research and field studies on plants, including collection of plant material, complied with relevant institutional, national and international guidelines and legislation. Ferric chloride hexahydrate (Fluka), potassium hexacyanoferrate, 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), potassium peroxodisulfate, gum arabic, hydrochloric acid, phosphoric acid 85%, gallic acid , Trolox, potassium phosphate, sodium chloride, chlorogenic acid and formic acid were supplied by Merck, Norway. Methanol (Rathburn) and acetonitrile (Rathburn) were supplied by Teknolab AS, Norway.

Sample preparation and analysis

Sample preparations were performed as described by Slimestad et al.14. Fresh garden chervil, including leaves and stems, was freeze-dried for 72 h using a freeze-dryer (CoolSafe 4 ScanVac, ScanLaf AS, Denmark). The dry matter contents were determined on the basis of the weights of the samples before and after lyophilization. Dried plant material was chopped in a bowl chopper and ground (Bosch KM13, Slovenia). For determination of total phenolic content, radical scavenging capacity and UHPLC analysis, 200mg of the sample was extracted with 10ml of methanol in a 20ml test tube at room temperature for 48h in darkness. Samples were filtered through 0.4 μm syringe filters prior to analysis. Two parallel samples were analyzed14.

Splitting and isolation

Fractionation and isolation of phenolic compounds from garden chervil were based on extracts obtained from 500 g of fresh plant material (160 g dry weight). Plant material was minced (about 20 mm) and extraction was performed twice for 24 h in the dark using 2 x 500 mL portions of methanol. The extract was filtered (folded filter grade 315, VWR Norway), concentrated to a volume of 100 ml on a rotavapor (Büchi, Switzerland) and partitioned against equal volumes of dichloromethane, in order to remove chlorophylls and the content lipophilic. The aqueous phase was then concentrated to a total volume of 50 mL, and the concentrated aqueous extract was applied to a 0.5 kg bed of Amberlite XAD-7 HP (Sigma) in a glass column at 5 × 60 cm open top, rinsed with 2 L of distilled water and eluted using 2 L of MeOH as the mobile phase. The purified XAD-7 extract was finally concentrated to a volume of 50 mL.

Further purification was performed by size exclusion chromatography using a 5 × 100 cm open-top column packed with 500 g Sephadex LH20 (GE Healthcare, Norway). Step gradient elution was used with increasing concentrations of methanol in the mobile phase (0, 20, 40, 60, and 80%; 0.1% TFA)14. Pure compounds were isolated by preparative HPLC. The HPLC instrument was equipped with a 250 × 20 mm Ascentis C18 column. Two solvents were used for elution; mobile phase A (water-TFA 99.9: 0.1 v/v) and mobile phase B (methanol-TFA 99.9: 0.1 v/v). Portions of 200 µL were manually injected into the HPLC column and the collected fractions were transferred to HPLC vials for purity check using analytical HPLC14.

Total phenolic content

The total phenolic content was determined according to the method of Price and Butler with stabilization of the Prussian Blue complex as described by Graham12.13 and Slimestad et al.14. 100 μL of sample was diluted with 3 mL of deionized water and mixed with 1 mL of a 0.1 M solution of ferric chloride in a 0.1 M hydrochloric acid solution with 1 mL of a solution of 8 mM potassium ferricyanide. The reaction was allowed to proceed for fifteen minutes at room temperature. 5 ml of an acid gum arabic solution was added (1 g of gum arabic dissolved in 100 ml of hot water. 10 ml of this solution was mixed with 10 ml of 85% phosphoric acid and 30 ml of ‘water). Absorbance at 700 nm was measured using an Agilent 8453 spectrophotometer (Agilent Technologies, Matriks, Norway). Samples were measured against a standard curve of gallic acid, and outputs are given in gallic acid equivalents, mg GAE g−114.

Radical trapping

The TEAC test (Jrolox Eequivalent Aantiradical VScapacity) was performed according to the procedures previously described by Slimestad et al.14 and Re et al.15. 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was dissolved in water to a 7 mM solution with potassium persulfate at a concentration of 2.45 mM. The solution was kept at room temperature for about 16 h. ABTS+ solution was diluted with PBS (phosphate buffered saline: 100 mM KH2Purchase order4-buffer, pH 7.4 and 150 mM NaCl), at an absorbance of 0.70 (±0.02) at 734 nm. The samples were diluted such that after introducing a 10 µL aliquot of each extract into the assay, they produced between 20 and 80% inhibition of blank absorbance. After addition of 1.0 mL of diluted ABTS+ 10 µL solution of trolox extracts or standards (final concentration 0–15 µM) in PBS, absorbance reading was taken at 6 min. Appropriate PBS blanks were run in each test. Final TEAC bleach assay values ​​were calculated against a standard curve of 0–10 mg Trolox in 100 mL methanol, and percent inhibition of absorbance at 734 nm was expressed as mg TEAC 100 g−1 DW14.15.

U(H)PLC-MS

The relative qualitative and quantitative content of individual flavonoids and other phenolic compounds was determined using an Agilent 1290 Infinity II instrument equipped with a 6120 quadrupole mass detector.14. The separation was performed with a Zorbax Eclipse XDB-C8 column (2.1 × 100 mm, 1.8 μm, Agilent Technologies). Water with 0.02% HCOOH (solvent A) and acetonitrile (solvent B) were used for gradient elution with the following time schedule (% B in A): from 0 to 10 ( in 1 min), from 10 to 25 (in 25 min), from 25 to 95 (in 2 min), from 95 to 0 (in 1 min), and finally isocratic reconditioning for 1 min. The flow rate was set at 0.300 mL/min (maximum back pressure of 540 bar) and 5 μL injections were used. UV detection was performed at 280, 320 and 360 nm with a bandwidth of 4 nm. Masses in the range of 250-800 Da were detected using a 500 ms scan time, 70 V fragmenter, and detection was in both positive and negative mode14. The temperature of the gas source was set at 350°C with a flow rate of 10 L min−1nebulizer pressure was 35 psi, while capillary voltage was 4 kV14.

High resolution mass spectrometry (LC-HRMS) was used for the exact determination of the mass of the isolated compounds. An iClass UPLC (Waters) equipped with a C18 BEH column (1.7 µm, 2.1 × 50 mm, Waters) was used to introduce the samples into the mass spectrometer. A gradient of A) 0.2% formic acid and B) acetonitrile was used as follows (% B in A): 1 (isocratic for 0.5 min), from 1 to 90 (in 2 min) . The mass spectrometer (timsTOF, Bruker) was operated in ESI+ mode with ionization at 2 kV and with a full scan from 100 to 2000 Da with a resolution R=50,000 (FWHM) at 1000 Da. Accuracy at RMS 14.

NMR

NMR samples were prepared by dissolving the isolated compounds in deuterated dimethyl sulfoxide (DMSO-D6; 99.96 atom % D, Sigma-Aldrich). 1D 1H and 2D 1H–13C HMBC, the 2D 1H–13C HSQC, the 2D 1H–13C HSQC-TOCSY, the 2D 1H–13C H2BC, 2D 1H–13C 1.1 Adequate, 2D 1H–1H COZY and 2D 1H–1ROESY H NMR experiments were obtained at 850 MHz at 298 K on a Bruker 850 MHz instrument equipped with a 1H, 13VS, 15N triple resonance cryogenic probe14.

Cytotoxicity assays

The cell lines used to study cytotoxicity were: Molm-1316, MV4-11 human monocytic leukemia cells (ATCC, CRL-9591), OCI-AML3 human acute myeloid leukemia cell line (ATCC, ACC-582), NRK rat renal epithelial cells (ATCC, CRL-6509) and cardiomyoblasts H9c2 (ATCC, CRL-1446). See Bjørnstad et al. 2019 for a description of culture conditions and media for the different cell lines17. Cytotoxicity measurements were performed as previously described18. Briefly, the compound was dissolved in DMSO and then diluted in DMSO. Using a robot pipette (Mosquito High Volume, SPT Labtech), 1 L was transferred into 96-well plates to create identical plates to be tested on different cells. The cell suspension was then added and the cells incubated for 72 h before viability assessment using the WST-1 cell proliferation assay (Roce Applied Sciences). Some experiments were also performed with dilution series performed in culture medium to exclude the effect of DMSO on cell viability. After recording the WST-1 signal, cells were fixed, DNA stained using Hoechst 33342, and cell death confirmed by microscopic assessment of nuclear and surface morphology18.

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