α ionone

Antibodies against the following proteins were obtained from Cell Signaling Technology (Danvers, MA, USA): p38 (cat. #9212; 1:1,000 dilution), phospho-p38 (#4511; 1:1,000 dilution), Jun N-terminal kinase (JNK, #9252; 1:1,000 dilution), phospho-JNK (#9251; 1:1,000 dilution), extracellular signal-related kinase (ERK, #4696; 1:1,000 dilution), phospho-ERK (#4370; 1:1,000 dilution), c-Fos (#4384; 1:1,000 dilution), phospho-c-Fos (#5348; 1:1,000 dilution), c-Jun (#9165; 1:1,000 dilution), phospho-c-Jun (#9164; 1:1,000 dilution), SMAD family members 2 and 3 (SMAD2/3, #5678; 1:1,000 dilution), phospho-SMAD2/3 (#8828; 1:1,000 dilution), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, #2118; 1:5,000 dilution), and tubulin (#2148; 1:5,000 dilution). A horseradish peroxidase–conjugated anti-rabbit IgG antibody (1:5,000 dilution) purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) was used as secondary antibody. DMSO (cat. #D2650), an MTT solution (#M2128), and α-ionone (#W259403) were provided by Sigma-Aldrich (St. Louis, MO, USA).

The following standards were used for liquid chromatography analyses: xanthines (caffeine, theobromine and theophylline), monomeric flavan-3-ols ((+)-catechin, catechin gallate, (−)-Epicatechin, Epicatechin-3-gallate, epigallocatechin, epigallocatechin-3-gallate (EGCg), green tea catechin mix), theaflavins (theaflavin, theaflavins mix (tea extract from Camellia sinensis)) and gallic acid. They were purchased from Merck (Barcelona, Spain) and Phytolab (Vestenbergsgreuth, Germany) for the identification and/or quantification of characteristic bioactive components from tea (Camellia sinensis). Trifluoroacetic acid, acetic acid, acetonitrile, dimethyl sulfoxide and water of chromatographic quality were purchased from VWR (Barcelona, Spain).
Regarding gas chromatography, the standards used were as follows: 1-penten-3-ol, n-hexanal, n-hexanol, cisZ-3-hexenal, transE-2-hexenal, cisZ-3-hexenol, transE-2-hexenol and pentanol, and for Group II, linalool, linalool oxides, methyl salicylate, phenyl acetaldehyde, geraniol, benzyl alcohol, 2-phenylethanol, benzaldehyde, α-ionone and β-ionone were purchased from Sigma Aldrich, Steinheim, Germany. Methanol, acetic acid and furfural were obtained from VWR, Darmstadt, Germany.

Octan-2-ol (97%), 1-hexanol (99%), cis-3-hexenol (98%), trans-3-hexenol (97%), vanillin (99%), 2,6-dimethoxyphenol (99%), linalool (97%), terpinen-4-ol (≥95%), α-terpineol (90%), nerol (≥97%), geraniol (98%), linalool oxide (≥97%), β-citronellol (95%), p-cymene (99%), terpinolene (≥85%), γ-terpinene (≥97%), limonene (97%), 1,8-cineole (99%), 1,4-cineole(≥98.5%), β-damascenone (≥98%), isoamyl alcohol (98%), benzyl alcohol (≥99%), 2-phenylethanol (≥99%), ethyl acetate (99%), ethyl butanoate (99%), ethyl 3-methyl butanoate ((≥98%), isoamyl acetate (≥95%), ethyl hexanoate (≥95%), phenylethyl acetate (99%), n-hexyl acetate (≥98%), ethyl lactate (≥98%), ethyl octanoate (≥98%), ethyl decanoate (≥98%), hexanoic acid (≥99%), octanoic acid (≥98%), α-phellandrene (95%), p-menthane-1,8-diol (97%), 3-methylbutanoic acid (99%), α-ionone (90%), 1-pentanol (99%), 1-butanol (≥99%), 2-butanol (≥99%), ethyl guaiacol (≥99%), vinyl guaiacol (≥98%), methyl-vanillate (99%) and ethyl vanillate (99%), were supplied by Sigma Aldrich (Milan, Italy). Dichloromethane (≥99.8%) and methanol (≥99.8%), were provided by Honeywell (Seelze, Germany). Sodium chloride (≥99.5%) was supplied by Sigma Aldrich (Milan, Italy).

α-Ionone (> 96%) (14), β-ionone (96%) (15), dihydro-β-ionone (90%) (16), β-damascone (95%) (17), and β-damascenone (> 98%) (10), were purchased from Sigma-Aldrich. Megastigmatrienone/tabanone (mixtures of isomers) (11) was kindly provided by Lothar Streeck GmbH & Co. KG. Flavors & Fragrances (Germany, Hamburg).

α-ionone and β-ionone were purchased from Sigma (St Louis, MO or NU-chek prep, INC. Elysian, MO), dissolved in DMSO and stored at −80 °C. Specific antibodies against AR, PSGR, Phosphorylated P70 S6 kinase, phosphorylated mTOR, and phosphorylated 4EBP1 were obtained from Abcam (Cambridge, MA). The antibody against PSGR for immunohistochemistry was from Novus Biologicals (Littleton, CO).

All reagents were analytical grade, and all solvents were HPLC grade. Anhydrous sodium sulfate was from Panreac Química (Barcelona, Spain). The water was Milli-Q^® grade (Millipore, Bedford, MA, USA). The identification and quantification of the volatile compounds by gas chromatography were performed using commercial pure standards from Sigma-Aldrich (St. Louis, MO, USA): α-terpineol, eucalyptol, terpinen-4-ol, citronellal, geraniol, methyl eugenol, nerol, α-pinene, citronellol, linalool, limonene, β-pinene, eugenol, theaspirane, α-ionone, β-ionone, 2-phenylethanol, and benzyl alcohol. As internal standards, we utilized 3-octanol and 4-methyl-2-pentanol (Sigma-Aldrich).

Five-year-old tea plants of Camellia sinensis cv. “Shu Cha Zao” grown at the experimental farm of Anhui Agricultural University in Hefei, China, were used in this study. Standards of geranyl diphosphate (GDP), farnesyl diphosphate (FDP), β-myrcene, β-ocimene, linalool, geraniol, α-citral, β-citral, methyl geranate, nerol, D-limonene, trans-linalool oxide (furanoid), neryl acetate, L-terpineol, α-terpinen, geranyl acetone, thymol, (Z)-nerolidol, α-farnesene, β-farnesene, (E)-nerolidol, humulene, α-ionone, β-ionone, (E)-γ-bisabolene, cadinol and caryophyllene were purchased from Sigma-Aldrich (St. Louis, MO, USA).