Serine

The inhibition of acetylcholinesterase was evaluated spectrophotometrically following the method of Ellman et al. [13 (link)] with slight modifications. Acetylcholinesterase of Electrophorus electricus (electric ceel Type-VI-S, Sigma Chemical Co, St. Louis, MO, USA) was used. The acetylcholinesterase activity was quantified using 15mM 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB, Sigma Chemical Co, St. Louis, MO, USA). The hydrolysis of acetylcholine iodide was monitored by the formation the anion 5-tio-2-methyl nitrobenzoate (yellow colour), measured at 412 nm. The percentage of inhibition was determined by comparing the rates of reaction of samples (final concentrations of 1000, 750, 500, 250, 50, 10, 1.5 μg/mL) with those of the blank (ethanol in phosphate buffer (Merck and Riedel-de Haën, 0.2 M, pH = 8) using the following formula, where E is the activity of enzyme without test sample and S is the activity of enzyme with the sample under analysis: $\frac{E-S}{E}\times 100$
The extract concentration providing 50% inhibition (IC₅₀) was calculated by interpolation from the graph of I% against extract concentration, following linear regression analysis with 95% of confidence level using the standard curve built with serine (Sigma-Aldrich (Madrid, Spain)) as reference.

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).

The following chemicals were purchased from Sigma-Aldrich (St. Louis, MO): methanol and acetonitrile (MS grade), ammonium acetate, ascorbic acid, β-mercaptoethanol, formic acid and standards, 5-adenosyl-methionine, 5-adenosyl-homocysteine, ATP, betaine, choline, cyanocobalamin, cystathionine, cysteine, dihydrofolate, dimethylglycine, dUMP, folic acid, folinic acid, glycine, homocysteine, methionine, methylcobalamine, methyl-tetrahydrofolate, NADPH, pyridoxal 5-phosphate, riboflavin, serine, taurine, tetrahydrofolate, thymidine 5-phosphate and Dulbecco's modified Eagle's medium mixed 1:1 with Ham's F-12 (DMEM/F12). Ultrapure type 1 water was obtained from a Milli Q water system (Merck Millipore, Darmstadt, Germany). Matrigel (growth factor-reduced) was purchased from BD Biosciences (San Jose, CA). Recombinant basic fibroblast growth factor (bFGF) was purchased from Mylteni (San Diego, USA).

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone Laboratories, Inc. (Logan, UT, USA). 1% penicillin/streptomycin was obtained from GE Healthcare (Chicago, IL, USA). Dimethyl sulfoxide (DMSO), ethanol, lipopolysaccharides (LPS), nitrite assay kit, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), spermidine, histidine, serine, asparagine, glucose, citrulline, glutamate, glucose-6-phosphate, methionine, itaconate, tryptophan and FMOC-glycine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lysine, arginine, aspartate, threonine, glutamine, proline, creatine and tyrosine were purchased from Alfa Aesar (Ward Hill, MA, USA). Apigenin, apigetrin, apiin, bergapten and xanthotoxin were purchased from Toronto Research Chemicals (North York, ON, Canada).