Vanillin

Capsaicin, vanillin, vanillic acid, acetaminophen, estradiol, estriol, 5-pregnan-3β-OL-2O-one, progesterone, 4-androstene-3,17-dione and insulin (human recombinant) were all obtained from Sigma-Aldrich (St. Louis, MO, USA). estradiol (E2)-6-HRP (NOV-ND-R0951) was acquired from Enzo Biochem (Farmingdale, NY, USA). Estrogen receptor alpha (ER alpha) recombinant protein was obtained from Invitrogen-Thermofisher (Waltham, MA, USA).
Enzyme-linked immunosorbent assay (ELISA) was used to investigate whether CAP acts as a competitive inhibitor or allosteric regulator in the presence of estrogen binding to the ESR1. An estrogen linked to horseradish peroxidase (EST-HRP) served as both the source of estrogen and a means by which to evaluate changes in the binding affinity of estrogen to its receptor in the presence of other ligands, such as CAP and ACET. To begin, the ESR1 was dissolved in a 1:1 solution of water to ethanol at a 1/100 dilution. Then, 100 μL of this solution was pipetted in duplicate into 10 wells of a Costar round-bottomed 96-well ELISA plate and incubated for one hour. Two wells in each row received only the ethanol–water solution and acted as controls. Any excess receptor was triply washed out using a 1% Tween 20 solution (in phosphate buffer) and a plate washer. Then, 200 μL of blocking agent (2% polyvinyl alcohol in phosphate buffer) was added to every well, incubated for an hour and then triply washed. Next, CAP was dissolved at a concentration of 0.1 mg/mL ( $3.27\times {10}^{-4}\mathrm{M}$ ) in a 1:1 solution of water to ethanol which acted as the base solution for EST-HRP dilution. The preparation of the EST-HRP dilutions was also carried out in a 0.01 mg/mL ( $3.27\times {10}^{-5} \mathrm{M}$ ) and 0.001 mg/mL ( $3.27\times {10}^{-6} \mathrm{M}$ ) CAP base solution to investigate the magnitude by which varying levels of CAP can shift the binding curve. A base solution of 1:1 water to ethanol without CAP served as the control. Beginning at a 1/100 dilution, the EST-HRP was then diluted by thirds ten times. An amount of 100 μL of each dilution was added to a well, incubated for an hour and triply washed. Finally, 100 μL of ABTS reagent was added to each well, incubated for an hour and the plate was read at 405 nm in a Spectramax UV-VIS scanning spectrophotometer.
The same competitive-binding ELISA procedure was used to investigate the effect of other ligands on the binding affinity of estrogen to the ESR1. The other ligands investigated were vanilloids with a similar structure to CAP, specifically VAN and VAN ACID, and known competitive inhibitors of the receptor, specifically ACET. These compounds were also tested against the same hormones as CAP using UV spectroscopy to investigate their binding activity.
UV spectroscopy in a crystal plate was used to investigate whether CAP has specificity for estrogen alone or whether it can bind to other naturally occurring hormones. To start, CAP was dissolved in a 1:1 solution of water to ethanol at 1 mg/mL ( $3.27\times {10}^{-3} \mathrm{M}$ ) and then diluted by thirds eight times. Next, the hormones were dissolved in a 1:1 solution of water to ethanol at a 1/100 dilution. An amount of 50 μL of each CAP dilution was added to 3 wells of a crystal plate, with one well serving as the control, with another 50 μL of 1:1 water to ethanol added, and the other two serving as experimental duplicates, with 50 μL of the prepared hormone solution added. Then, two additional wells were prepared to serve as controls: one containing 100 μL of 1:1 water to ethanol and the other containing 50 μL of prepared hormone solution and 50 μL of 1:1 water to ethanol. Finally, the plate was incubated for 1 h and read in a Spectramax UV-VIS scanning spectrophotometer, spanning wavelengths from 190 to 400 nm. Data analysis involved the subtraction of observed absorbance values from expected absorbance values generated from the control wells ([CAP + 1:1 solution] + [hormone + 1:1 solution] − [1:1 solution]) and plotting them as a function of CAP concentration. Because the solvent (1:1 solution of water–ethanol) was used as a control and subtracted from all wells, the resulting data reflected only the absorbances of the solute compounds. Since each compound has a different lambda maximal absorbance, analysis of the resulting data required finding one common wavelength at which the additions and subtractions of the spectra could be carried out and at which the absorbance of each compound was maximized. In practice, the best common wavelengths were found to be between 205 and 210 nm. These wavelengths also suggest that the dominant form of binding involves hydrogen and/or ionic bonding rather than interactions such as charge transfer complexing that would be dominant at higher wavelengths around 270–280 nm.
Data were analyzed and graphed using Microsoft Excel and diagrams were created on Microsoft PowerPoint (Redmond, WA, USA).
Similarity searches involving TRPV1, insulin and the estrogen receptor (SwissProt accession numbers provided in the relevant figures) were carried out using LALIGN (https://www.ebi.ac.uk/jdispatcher/psa/lalign accessed 14 February 2024) with the following parameter settings optimized for finding significant alignments in short sequences of proteins: BLOSUM80, Gap open −12.0, Gap extended −2.0, E value 1.0.

Butylated hydroxyanisole (BHA), 2,2-diphenyl-1-picrylhydrazyl (DPPH), catechin, copper sulfate (CuSO₄), tiobarbituric acid (TBA), rosmarinic acid, chicoric acid, quercetin, caftaric acid, vanillin, caffeic acid, syringic acid, rutin, gallic acid, naringenin, cinnamic acid, sodium carbonate (Na₂CO₃), ascorbic acid, potassium ferricyanide (K₃Fe(CN)₆), ferric chloride (FeCl₃), trichloroacetic acid, aluminum chloride (AlCl₃), and Folin–Ciocalteu reagent were purchased from MERCK (Lyon, France). All chemicals, including solvents, were of analytical grade.

Methanol and water (LC-MS grade) were purchased from HiPerSolv CHROMANORM, VWR Chemicals BDH (Amsterdam, The Netherlands). Formic acid 98–100% was purchased from Merck (Darmstadt, Germany). For the determination of phenolic compounds, apigenin 98%, caffeic acid 98%, catechin 97%, cinnamiccinnamic acid 97%, chrysin 98%, diosmin 97%, epicatechin, epigallocatechin 97%, ferulic acid 98%, epicatechin gallate 98%, hesperidin 98% (internal standard), kaempferol 98%, luteolin 98%, myritecin 97%, myricitrin 97%, naringin 98%, p-coumaric acid 98%, quercetin 98%, quercitrin 99%, rosmarinic acid 98%, protocatechuic acid 97%, rutin 98%, sinapic acid 98%, syringaldehyde 97%, syringic acid 98%, taxifolin 98%, vanillic acid 98%, and vanillin 98% were used and were purchased from Sigma-Aldrich (Stenheim, Germany). Stock standard solutions of all the analytical standard compounds were prepared with LC-MS-grade methanol at 1000 mg/L and were afterward stored in dark brown glass bottles at −20 °C.