Safranal

The saffron (Crocus sativus L.) stigmas were extracted in the factory of Pharmactive Biotech Products S. L. in Madrid (Spain) to produce the commercial saffron extract, branded as affron®, and dosed in tablets as preferred galenic form for the in vitro digestion and the clinical trial. Each tablet contained 14 mg of dried affron® standardised to ≥3.5% lepticrosalides®, defined as the content of bioactive compounds including Safranal and crocin by HPLC [12 (link), 13 (link)], together with the following excipients: microcrystalline cellulose, calcium hydrogen phosphate, povidone, croscarmellose sodium, colloidal anhydrous silica, and magnesium stearate as excipients and coated with hypromellose (HPMC), macrogol (PEG) 8000, and carnauba wax.
Safranal, crocetin, p-nitroaniline, and digestive enzymes were purchased from Merck (Darmstadt, Germany). Kaempferol was purchased from Extrasynthese (Genay, France), and trans-crocin-4 was purchased from Phytolab (Vestenbergsgreuth, Germany). All the solvents used for the chromatographic analyses were of HPLC grade and purchased from Merck and VWR (Pennsylvania, United States).

Analytical standards of crocin 1 (#80391; crocetin bis (gentiobiosyl) ester, purity ≥ 95%), crocin 2 (#80392; crocetin gentiobiosylglucosyl ester, purity ≥ 95%), and picrocrocin (#89256; purity ≥ 95%) were purchased from PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany), while safranal (2,3-dihydro-2,2,6-trimethylbenzaldehyde, purity ≥ 90%) was purchased from Merck KGaA (Darmstadt, Germany). Solvents, such as HPLC grade acetonitrile, methanol, and formic acid were supplied by Merck KGaA.

Safranal was purchased from Sigma Aldrich (MO, USA), with a purity of ≥90.0%. DMEM, FBS, penicillin, and streptomycin were purchased from Gibco (Grand Island, NY/Carlsbad, CA, USA). Escherichia coli LPS was obtained from Sigma Chemical Co. (MO, USA). Milli-Q water was supplied from a water purification system (Millipore, MA, USA). The HRP-conjugated goat anti-rabbit IgG was purchased from Invitrogen (Carlsbad, CA, USA). DSS (molecular weight of 36,000–50,000 Da) was obtained from MP Biomedicals (Santa Ana, CA, USA).

Samples. Saffron from the Protected Designation of Origin (PDO) “Azafrán de La Mancha” was directly purchased from the producer Agrícola Técnica de Manipulación y Comercialización (Minaya, Albacete, Spain). Category I saffron was ground and characterized according to ISO 3632:2011 [25 ] ( $A_{1\mathrm{cm}}^{1\%}$ 440 nm: 235 ± 2, $A_{1\mathrm{cm}}^{1\%}$ 257 nm: 97 ± 3, and $A_{1\mathrm{cm}}^{1\%}$ 330 nm: 22 ± 1). Cured ham was obtained from the company Benibaldo, S.A.U. (Albacete, Spain), and rice flour obtained from Panificadora Conquense Agrícola, S.A. (Cuenca, Spain), a usual product in the ham industry, was used in this study as a solid support to retain the patterns and for solid dilution of saffron.
Patterns. A total of four patterns of the major aromatic components of saffron were used. Safranal (purity: 90%), linalool (purity: 97%), isophorone (purity: 97%), and 2,6,6-trimethyl-2-cyclohexene-1,4-dione (purity: 98%) were obtained from Sigma-Aldrich (Madrid, Spain).
Solvent. Cyclohexane of analytical grade was obtained from Panreac (Barcelona, Spain).

Standard 2‐hexanone, hexanal, 1H‐1‐ethyl‐pyrrole, 2‐hexenal, cis‐3‐hexen‐1‐ol, heptanal, 6‐methyl‐5‐hepten‐2‐one, 2‐pentyl‐furan, 2‐ethyl‐1‐hexanol, benzeneacetaldehyde, cis‐linalool oxide, trans‐linalool oxide, linalool, 3‐octen‐2‐ol, phenylethyl alcohol, menthol, α‐terpineol, safranal, decanal, camphene, geraniol and indole were purchased from Sigma Co. Ltd. Benzyl alcohol, trans‐β‐damascenone, trans‐α‐ionone, cis‐geranylacetone, trans‐β‐ionone, 2,4‐ditert‐butylphenol, cedrol, and caryophyllene oxide were purchased from Alfa Aesar Co. Ltd. Standard chemical series of C₈–C₂₀ alkanes that were used to determine the liner retention index (RI) and the internal standard cyclohexanone were obtained from Sigma Co. Ltd. Cis‐pyranoid‐linalool oxide and trans‐pyranoid‐linalool oxide were purchased from Sinopharm Chemical Reagent Co. Ltd.