Ferulic acid

Arabidopsis thaliana (ecotype Col-0) was grown under controlled conditions and pooled after harvest. Methanolic extracts were prepared from ground seed and leaf tissue. o-Anisic acid, biochanin A, p-coumaric acid, ferulic acid, N-(3-indolylacetyl)-L-valine, kinetin, indole-3-acetonitrile, indole-3-carbaldehyde, kaempferol, phloretin, phlorizin and phenylglycine, rutin, and phenylalanine-d5 were used as marker compounds. The chromatographic separations were performed on an Acquity UPLC system (Waters) equipped with a modified C₁₈ column with a 20 min water/acetonitrile gradient. The eluted compounds were detected by a Bruker MicrOTOF-Q in positive ion mode at a scan rate of 3 Hz. Mass calibration was performed against lithium formiate. The detailed experimental setup is available as Additional file 1.
Sample 1 A mixture containing each of the fourteen marker compounds (referred to as MM14) at a concentration of 20 μM was prepared and analysed by UPLC/ESI-QTOF-MS.
Sample set 2 Mixtures containing solvent and seed or leaf extracts were prepared with following volume portions (solvent/seed/leaf, v/v/v): 0/100/0, 25/75/0, 50/50/0, 75/25/0, 0/0/100, 25/0/75, 50/0/50, 75/0/25. The sample set (8 samples) was analysed by UPLC/ESI-QTOF-MS in ten technical replications.
Sample set 3 Mixtures containing solvent, seed, and leaf extracts were prepared with following volume portions (solvent/seed/leaf, v/v/v): 75/0/25, 0/75/25, 0/50/50. The sample set (3 samples) was analysed by UPLC/ESI-QTOF-MS in ten technical replications.
All files were acquired in centroid mode and converted to mzData file format using Bruker CompassXport software. The data sets are available at .

Free and bound phenolics were extracted as described in Verardo et al. (2011) (link). The analysis of BSG free and bound polyphenols was carried out with the use of an ACQUITY Ultra Performance LC system equipped with photodiode array detector with a binary solvent manager (Waters Corporation, Milford, MA, United States) series with a mass detector Q/TOF micro mass spectrometer (Waters) equipped with an electrospray ionization (ESI) source operating in negative mode at the following conditions: capillary voltage, 2300 kV; source temperature, 100°C; cone gas flow, 40 L/Hr; desolvatation temperature, 500°C; desolvatation gas flow, 11,000 L/h; and scan range, m/z 50–1500. Separations of individual polyphenols were carried out using an ACQUITY UPLC BEH Shield RP18 column (1.7 μm, 2.1 mm × 100 mm; Waters Corporation, Milford, MA, United States) at 40°C. The elution gradient was carried out using water containing 1% acetic acid (A) and acetonitrile (B), and applied as follows: 0 min, 1% B; 2.3 min, 1% B; 4.4 min, 7% B; 8.1 min, 14% B; 12.2 min, 24% B; 16 min, 40% B; 18.3 min, 100% B, 21 min, 100% B; 22.4 min, 1% B; 25 min, 1% B. The sample volume injected was 2 μL and the flow rate used was 0.6 mL/min. The compounds were monitored at 280 nm. Integration and data elaboration were performed using MassLynx 4.1 software (Waters Corporation, United States). For the quantification of phenolic compounds, solutions of ferulic acid, chlorogenic acid, catechin, and quercetin in methanol were prepared and used as standard.

250 μl of thawed hydrolysate and 15 μl of sorbitol (0.1000 g/100 ml aqueous) were transferred to a vial and concentrated to dryness under a stream of N₂. The internal standard was added to correct for subsequent differences in derivatization efficiency and changes in sample volume during heating. Dried extracts were dissolved in 500 μl of silylation–grade acetonitrile followed by the addition of 500 μl N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) (Thermo Scientific, Bellefonte, PA), and samples then heated for 1 h at 70°C to generate trimethylsilyl (TMS) derivatives [43 (link)]. After 1 day, 1-μl aliquots were injected into an Agilent Technologies Inc. (Santa Clara, CA) 5975C inert XL gas chromatograph-mass spectrometer, fitted with an Rtx-5MS with Integra-guard (5% diphenyl/95% dimethyl polysiloxane) 30 m × 250 μm × 0.25 μm film thickness capillary column. The standard quadrupole GCMS was operated in the electron ionization (EI) (70 eV) mode, with 6 full-spectrum (50–650 Da) scans per second. Gas (helium) flow was 1.33 ml per minute with the injection port configured in the splitless mode. The injection port, MS Source, and MS Quad temperatures were 250°C, 230°C, and 150°C, respectively. The initial oven temperature was held at 50°C for 2 min and was programmed to increase at 20°C per min to 325°C and held for another 11 min, before cycling back to the initial conditions. A large user-created database (>1600 spectra) of mass spectral EI fragmentation patterns of TMS-derivatized compounds, as well as the Wiley Registry 8th Edition combined with NIST 05 mass spectral database, were used to identify the metabolites of interest to be quantified. Peaks were reintegrated and reanalyzed using a key selected ion, characteristic m/z fragment, rather than the total ion chromatogram, to minimize integrating co-eluting metabolites. The extracted peaks of known metabolites were scaled back up to the total ion current using predetermined scaling factors. Unidentified metabolites used the scaling factor for the internal standard (sorbitol) and were denoted by their RT as well as key m/z fragments. The mass-to-charge ratios used as extracted ions were as follows: iso-sinapyl alcohol (354), iso-sinapic acid (368), iso-syringin (354), 5-hydroxyconiferyl alcohol-4-O-glucoside (412), 5-hydroxyconiferyl alcohol-4-O-glucoside (412), 3,4-dihydroxybenzoic acid (370), xanthine (368), hypoxanthine (265), succinic acid (247), guanosine (324), uracil (241), citraconic acid (259), guanine (352), 5-hydroxyferulic acid (411), uridine (258), maleic acid (245), secoisolariciresinol (560), 5-oxo-proline (156), adenine (264), 1-O-trans-feruloylglycerol (249), vanillin (297, 194), ferulic acid (338), adenosine (236), p-coumaric acid (308), caffeic acid (396), p-hydroxybenzaldehyde (392, 194), coniferyl alcohol (324), 5-hydroxyconiferyl alcohol (412), coniferyl aldehyde (323), guaiacylglycerol (297), sinapyl aldehyde (353), syringylglycerol (327), p-hydroxyphenylpyruvic acid (396), syringaresinol (327), pinoresinol (502), hydroxymethylfurfural (183). Peaks were quantified by area integration and the concentrations were normalized to the quantity of the internal standard recovered, volume of sample extracted, derivatized, and injected.

Thin film hydration method was used to prepare ferulic acid mixed micelles. First, the required amount of FA was accurately weighed, according to Table 1, and dissolved in 10 ml methanol. The drug solution was then placed into a round-bottom flask (250 ml) containing the Pluronics mixture; composed of Pluronic F127 and Pluronic P123, and sonicated until the Pluronics mixture was completely dissolved. The solvent was then subjected to evaporation under vacuum using a rotary vacuum evaporator (Heidolph, Germany) till a dry thin film was formed. Ten ml of distilled water was used for the hydration of the film, and rotation was continued for 1 h under normal pressure (1 atmosphere). Filtration was then done using a Millipore® filter of 0.45µm, to remove the free unentrapped drug, and the ferulic acid polymeric mixed micelles was then stored at 4 ºC^{12 (link)}.

¹H NMR (400 MHz, DMSO-D6) δ 12.14 (s, 1H, COOH), 9.56 (s, 1H, H-11), 7.49 (d, J = 15.9 Hz, 1H, H-7), 7.28 (d, J = 2.0 Hz, 1H, H-2), 7.08 (dd, J = 8.3, 2.0 Hz, 1H, H-5), 6.79 (d, J = 8.1 Hz, 1H, H-6), 6.37 (d, J = 15.9 Hz, 1H, H-8), 3.81 (s, 3H, OCH3-12). ¹³C NMR (100 MHz, DMSO-D6) δ 168.08 (C-9), 149.13 (C-3), 147.96 (C-4), 144.59 (C-7), 125.82 (C-1), 122.91 (C-6), 115.67 (C-5), 115.55 (C-8), 111.14 (C-2), 55.71 (C-12).

The n-butanol fraction was prepared from Liatris spicata (L.) (10 g) as mentioned in our published data²⁰ and it was portioned on a diaion HP-20 AG (250 g, 5 × 120 cm) using water, followed by water/methanol (1:1 v/v) and finally with 100% methanol. The water/methanol fraction was then purified several times over Sephadex LH—20 columns using water/methanol (1:1 v/v) as eluent to give white crystals (120 mg, R_f = 0.72 on TLC in methylene chloride/methanol (80:20 v/v)).
For identification of the compound, it was analyzed using ¹HNMR and ¹³CNMR analysis, where the following data were obtained:
¹H NMR (400 MHz, DMSO): δ = 3.82 (s, 3H, –OCH₃), 6.35 (d, J = 15.8 Hz, 1H, 8-H), 6.79 (d, J = 8.1 Hz, 1H, 5-H), 7.07 (dd, J = 8.1, 1.5 Hz, 1H, 6-H), 7.28 (d, J = 1.5 Hz, 1H, 2-H), 7.48 (d, J = 15.8 Hz, 1H, 7-H).¹³C NMR (125 MHz, DMSO): δ = 56.13 (–OCH3), 126.2 (C-1), 111.6 (C-2), 148.3 (C-3), 149.5 (C-4), 116.0 (C-5), 123.2 (C-6), 144.9 (C-7), 115.9 (C-8), 168.4 (–COOH).