Hippuric acid

Metabolomic profiling was performed using three separate mass spectrometry platforms run in parallel essentially as described previously (Evans et al 2009 (link)). Starting with 100 μl of plasma, small molecules were extracted in an 80 % methanol solution containing four standards (tridecanoic acid, 4-Cl-phenylalanine, 2-flurophenylglycine, and d6-cholesterol) used to monitor extraction efficiency. Clarified supernatant was split into three aliquots and dried under N₂. Additional internal standards (Standards for negative ion mode analyses included d7-glucose, d3-methionine, d3-leucine, d8-phenylalanine, d5-tryptophan, Cl-phenylalanine, Br-phenylalanine, d15-octanoic acid, d19-decanoic acid, d27-tetradecanoic acid, and d35-octadecanoic acid. Standards for positive ion mode analyses included d7-glucose, fluorophenylglycine, d3-methionine, d4-tyrosine, d3-leucine, d8-phenylalanine, d5-tryptophan, d5-hippuric acid, Cl-phenylalanine, Br-phenylalanine, d5-indole acetate, d9-progesterone, and d4-dioctylpthalate.) were added to each of three aliquots to control the quality of the chromatographic and mass spectrometric analyses. Each of the three aliquots were analyzed via a unique mass spectrometry assay: (1) gas chromatography coupled mass spectrometry (GC-MS) (2) liquid chromatography coupled mass spectrometry in positive ion mode (LC-MS pos), and (3) LC-MS in negative ion mode (LC-MS neg). For GC-MS analysis, analytes were derivatized using bistrimethyl-silyl-trifluoroacetamide and analyzed on a Trace DSQ fast-scanning single-quadruple mass spectrometer (Thermo-Finnigan). For LC-MS analyses one specimen was resuspended in 50 μl of 6.5 mM ammonium bicarbonate, pH 8, for liquid chromatography mass spectrometry (LC/MS) analysis in negative ion mode the other was resuspended in 50 μl of 0.1 % formic acid in 10 % methanol for LC/MS analysis in positive ion mode. Both resuspension buffers contained instrument internal isotopic standards used to monitor performance and serve as retention index markers. Standards for negative ion mode analyses included d7-glucose, d3-methionine, d3-leucine, d8-phenylalanine, d5-tryptophan, Cl-phenylalanine, Br-phenylalanine, d15-octanoic acid, d19-decanoic acid, d27-tetradecanoic acid, and d35-octadecanoic acid. Standards for positive ion mode analyses included d7-glucose, fluorophenylglycine, d3-methionine, d4-tyrosine, d3-leucine, d8-phenylalanine, d5-tryptophan, d5-hippuric acid, Cl-phenylalanine, Br-phenylalanine, d5-indole acetate, d9-progesterone, and d4-dioctylpthalate. Internal standards were chosen based on their broad chemical structures, biological variety and their elution spectrum on each of the arms of the platform. Chromatographic separation was completed using an ACQUITY UPLC (Waters) equipped with a Waters BEH C18 column followed by analysis with an Orbitrap Elite high resolution mass spectrometer (Thermo-Finnigan) (Evans et al 2009 (link)). For all analytic methods, metabolites were identified by matching the ion chromatographic retention index, accurate mass, and mass spectral fragmentation signatures with reference library entries created from authentic standard metabolites under the identical analytical procedure as the experimental samples (Dehaven et al 2010 (link)).

Antioxidant capacity was measured by the ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) assay previously optimised for cereal samples [9 ] with results expressed as ascorbic acid (AA) equivalents/L. Anti-hypertensive capacity was evaluated by the angiotensin converting enzyme (ACE) inhibitory test [10 (link)] with minor modifications. ACE enzymatic activity was assayed by monitoring the amount of hippuric acid derived from the hydrolysis of the substrate hippuryl-histidyl-leucine in the presence of the sample. Pyridine and benzene sulfonyl chloride (40% and 20%, respectively, on the final assay volume) were added and the absorbance of the developed yellow coloured solution was measured at 410 nm. The percent inhibition curves were plotted using a minimum of five increasing concentrations for each sample and the IC50 value was calculated. Anti-tyrosinase activity was assessed by an optimised tyrosinase inhibition assay [11 (link)]. The kinetic of brown colour formation was evaluated (490 nm absorbance measurement) in a reaction containing 10 U of tyrosinase and 2 mM L-DOPA in the presence of the sample. The results were expressed as kojic acid (KA, a well-known tyrosinase inhibitor) equivalents/L by means of a dose-response calibration curve (between 1 and 10 μg of KA). A bioluminescent cell-based assay for anti-inflammatory activity was performed using human embryonic kidney HEK293 cells (ATCC, American Type Culture Collection, Manassas, VA, USA) routinely grown in Dulbecco Modified Essential Medium (DMEM high glucose 4.5 g/L, GE Healthcare, Milan, Italy), supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 50 U/μL penicillin, and 50 μg/mL streptomycin. The day before transfection, HEK293T cells were plated in a 24-well plate at a density of 8 x 10⁴ per well. Cells were co-transfected with plasmid pGL4.32[luc2P/NF-κB-RE/Hygro] containing five copies of the NF-κB response element (NF-κB-RE) driving transcription of the luc2P reporter gene (Promega, Madison, WI, USA), and plasmid pmcherryPRET9 expressing the fluorescent protein mcherry-C1 (Clontech, Mountain View, CA, USA) and a red thermostable P. pyralis luciferase mutant [12 (link)], obtained by standard molecular biology procedures. Co-transfections were performed by using FuGENE®HD according to the manufacturer's instructions and incubated at 37°C with 5% CO₂ for 24 h. Forty-eight hours post-transfection, cells were co-incubated for 20 hours with 500 μL of fresh medium containing sample (1:20 dilution) and 20 ng/mL TNFα. After incubation at 37°C, cells were detached with trypsin-EDTA 1X in PBS, resuspended in 100 μL PBS 0.1 M pH 7.5 and then transferred to black 96-well microplates. Fluorescence (FL) and bioluminescence (BL) measurements were performed with a Varioskan™ Flash Multimode Reader. The FL signal was obtained exciting samples at 570 nm and acquiring the signal at 610 nm, while the BL signal was acquired with band pass green and red filters after injection of the substrate BrightGlo [13 ]. Statistical analysis was performed by using one-way Anova with p < 0.05 accepted as significant. Basal activation of NF-κB or activation with 20 ng/mL TNFα were used to calculate fold of induction of treated cells vs control cells. Cytotoxicity and irritation tests were performed on Sterlab Reconstructed Human Epidermis (RHE). For cytotoxicity evaluation, human tissues were placed in a 24-well plate with medium and exposed topically to pure samples for 24 hours at 37°C. After washing with PBS, a cell viability test was performed. For irritation evaluation, human tissues were placed in a 24-well plate with medium and each tested substance was topically applied for 42 min at room temperature. Exposure to the substance was followed by rinsing with PBS and mechanical drying. RHEs were transferred to fresh medium and incubated at 37°C for 42 additional hours. Then a cell viability test was performed. For each test the cell viability was assessed by incubating the tissues for 3 hours with 0.3 mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (0.5 mg/mL). Formazan crystals were extracted for 2 hours at room temperature using 1.5 mL isopropanol and quantified by spectrometry at 550 nm absorbance. SDS 0.1% (w/v) and PBS were used as positive and negative controls, respectively. For each treated tissue, the cell viability was expressed as percentage of the mean negative control tissues. A cell viability above 50% indicated the not toxicity or the not irritancy potential of the tested substance.

Hippuric acid (Analytical grade), benzene sulfonyl chloride (BSC), and pyridine were obtained from Sigma-Aldrich. Methanol (HPLC grade), hydrochloric acid (HCl), and sodium hydroxide were supplied from Merck Company. Doubled distilled water was used from a milli-Q system (Bedford, USA). Standard solutions of HA (1000 mg L⁻¹) were obtained using double-distilled water. The daily standards were prepared from the stock solution. The prepared stock solution was stored in the refrigerator for 10 days. The stability of the stock solution was checked by preparing a solution with a specific concentration of the analyte and comparing the results of absorbance during the time.

We implemented the method used by Cushman & Cheung (1971) with some modifications [30 (link),31 (link)]. We added 40 mL of each sample to 100 mL of HHL substrate (hippuryl-L-histidyl-L-leucine) in a 0.1 M sodium borate buffer solution with 0.3 sodium chloride at a pH of 8.3. Subsequently, we added 2 mU of the ACE (ACE 3.4.15.1, 5.1 U/mg) (Sigma; Germany), which was dissolved in 50 % glycerol. The reaction was run at 37 °C for 30 min. We lowered the pH by adding 150 mL of 1 N HCl to inactivate the enzyme and the resulting hippuric acid was extracted with 1000 mL of ethyl acetate. 750 mL of the organic phase was collected after stirring and subsequent centrifugation at 4000×g for 10 min at room temperature. This volume was evaporated by heating it at 95 °C for 15 min. The hippuric acid residue was dissolved in 800 mL of distilled water. After stirring, absorbance was measured at 228 nm.
ACE inhibitory activity is calculated using Equation (4), while the EC₅₀ value is calculated as the amount of soluble protein required to inhibit 50 % of the enzyme. The activity of each sample was determined in triplicate. We used Equation (4) to calculate the inhibitory activity of each sample. $Inhibitoryactivity(\%)=\frac{{Abs}_{Control}-{Abs}_{sample}}{{Abs}_{Control}-{Abs}_{blank}}*100$ ${Abs}_{Control}$ represents the absorbance of hippuric acid after ACE action without inhibitors. ${Abs}_{blank}$ corresponds to the absorbance of unreacted hippuryl-L-histidyl-L-leucine (HHL) extracted with ethyl acetate. ${Abs}_{sample}$ is the absorbance of the hippuric acid formed after ACE action in the presence of inhibitory substances.