Fluorometric Quantification of Ferulic Acid Concentrations Based on Deconvolution of Intrinsic Fluorescence Spectra

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  • INOKUCHI Reina
    International Photosynthesis Industrialization Research Center, Graduate School and Faculty of Environmental Engineering, The University of Kitakyush
  • TAKAICHI Hiroshi
    International Photosynthesis Industrialization Research Center, Graduate School and Faculty of Environmental Engineering, The University of Kitakyush
  • KAWANO Tomonori
    International Photosynthesis Industrialization Research Center, Graduate School and Faculty of Environmental Engineering, The University of Kitakyush

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Ferulic acid (FA) is one of phenolics found in most higher plants. It is important to quantify the internal FA level in vegetables and fruits, since it was epidemiologically demonstrated and a number of study supported that consumption of fruits and vegetables rich in phenolic acids including FA is associated with the prevention of chronic diseases such as cancer and cardiovascular disease. In order to allow handling of the intact fresh produces, non-invasive methods are desired. Previously, 355 nm ultraviolet (UV) laser-induced fluorescence spectrum revealed that living plants contain fluorophore corresponding to blue-green fluorescence (shown to be FA). However, quantification of FA based on fluorescence in UV-excited leaves can be hardly achieved since FA fluorescence measured at fixed excitation and emission can be applied only to the limited range of FA concentration. Here, we report a model experiment for fluorometric quantification of FA in solution in vitro which may provide a series of useful information required for estimation of FA concentrations in vivo fluid inside the vegetables. Based on deconvolution of intrinsic fluorescence spectra, we observed that FA fluorescence signals can be deciphered to determine the concentration of FA. By viewing that the recorded FA fluorescence (h) is reflecting the primitive function (f) corresponding to FA concentrations and kernel function (g) determining the spike position in the spectra. Thus, f should be obtained as f=h×g−1. In practice, cumulative curves of fluorescence signals at fixed emission wavelength (460 nm) along with the changes in excitation wavelength (200–400 nm) were plotted and the midpoints (along the scale of excitation wavelength) in the resultant curves corresponding to different FA concentration were graphically determined. FA’s concentration-specific changes in fluorescence profiles must be due to the fact that FA possesses multiple fluorophores within the molecule despite its simple structure. Lastly, simplified protocol for determination of FA concentration using dual UV excitation wavelengths was proposed. In this assay, ratio of 460 nm fluorescence intensities induced by two distinct excitation wavelengths (short, 260 nm; long, 330–380 nm) were shown to be highly correlated with FA concentration ranged from μM to mM orders.

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