Phenylalanine

Standards of amino acids (AA) namely phenylalanine (Phe), tryptophan (Trp), isoleucine (Ile), leucine (Leu), asparagine (Asn), methionine (Met), valine (Val), proline (Pro), tyrosine (Tyr), alanine (Ala), threonine (Thr), glycine (Gly), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), arginine (Arg), glutamine (Gln), lysine (Lys), histidine (His), cysteine (Cys) and cystine (Cys2),were purchased from Sigma-Aldrich (Steinheim, Germany). Acetonitrile (ACN) and water (LC-MS grade) was purchased from Carlo Erba (Milan, Italy). Formic acid (LC-MS additive), TGA (thioglycolic acid; ≥98%), and ammonium formate (NH₄HCO₂) were purchased from Sigma-Aldrich (Steinheim, Germany). Analytical grade hydrochloric acid (HCl) 37% w/w was also supplied from Carlo Erba (Milan, Italy). The chromatographic column HILIC amide BEH, Acquity UPLC 1.7 m, 2 × 150 mm (Waters) were used.

To evaluate conidial germination, conidia of C. gloeosporioides, A. alternata, and L. theobromae were harvested from 10- to 30-day-old PDA plates. Each fungal sample was diluted to 5 × 10⁵ conidia/mL. The fungal conidial suspension (10 μL) was placed on a glass slide with 8 mM phenylalanine (Sigma, St. Louis, MO, USA) solution (10 μL), and incubated at room temperature in a humid chamber for 20 h, with three biological repeats. The percentage of conidial germination was examined under a light microscope (×40; Leica DM500, Heerbrugg, Switzerland). The averages and SEs of conidia germination percentages were calculated from three biological repetitions for both 8 mM phenylalanine and the water control, and each repeat was counted in three different fields.

Adult male Sprague-Dawley rats weighing 230 ± 10 g were obtained from the Animal Breeding Centre of Beijing Vital River Laboratories Company (Beijing, China). The project identification code was 20162005. All animals were housed at 22 ± 2°C with a relative humidity of 50 ± 10% and a 12 h light/12 h dark cycle. The animals had free access to water and fodder (Beijing Keaoxieli Co. Ltd.). All experimental animal procedures were approved by the China Academy of Chinese Medical Sciences' Administrative Panel on Laboratory Animal Care and performed in accordance with institutional guidelines and ethics of the committee as part of the China Academy of Chinese Medical Sciences (February 1^st, 2016).
Ten AA standards including alanine, valine, taurine, leucine, isoleucine, glutamine, glutamate (glutamic acid), phenylalanine, (S)-tyrosine, and tryptophan (purity > 99%, all) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile and methanol were provided from Fisher Scientific (Shanghai, China). Formic acid, ammonium formate, and phenylalanine-d5 (internal standard (IS)) were purchased from Toronto Research Chemicals (YTO, Canada). All chemicals used throughout this study were of HPLC grade unless stated otherwise. BNC (batch number: 140156) was provided from Buchang Pharma Co. Ltd.

HPLC-grade formic acid was obtained from Sigma-Aldrich (USA). HPLC/MS-grade formic acid, ammonium formate, and acetonitrile were acquired from Fisher Scientific (USA). The internal standards Labeled Carnitine Standards (L-carnitine (N-trimethyl-D₉), L-acetylcarnitine (N-methyl-D₃), L-propionylcarnitine (Nimethyl-D₃), L-butyrylcarnitine (N-methyl-D₃), L-isovalerylcarnitine (N-trimethyl-D₉), L-octanoylcarnitine (N-methyl-D₃), L-myristoylcarnitine (N-trimethyl-D₉), L-palmitoylcarnitine (N-methyl-D₃)) were attained from Cambridge Isotope Laboratories. Seventeen amino acid standards (alanine, valine, leucine, isoleucine, phenylalanine, methionine, proline, glycine, serine, threonine, cysteine, tyrosine, histidine, lysine, arginine, aspartic acid, glutamic acid) were purchased from Sigma-Aldrich. Eight amino acid standards (tryptophan, asparaginate, glutamine, ornithine, taurine, citrulline, cysteine, γ-aminobutyric acid) were gained from J&K Scientific. Ultrahigh purity water was prepared by Millipore-Q Water Purification System (Millipore, Germany).

LC-MS grade methanol (LC-MS LiChrosolv^®, Merck (≥99.97%), formic acid (98–100%), water (LC-MS Chromasolv), trans-zeatin (≥97%), solasodine (≥95%), tomatidine hydrochloride (≥85%), α-solanine (≥95%), solanidine (≥95%), phenylalanine (≥98%), tyrosine (≥98%), tryptophan (≥95%), methyl dihydrojasmonate (≥96%), and DMSO (≥99.9%) were purchased from Sigma-Aldrich (St. Louis, MO, United States) and α-chaconine (≥95%) (PhytoLab, Germany).