3.2. Identification of phlorotannin
The methanolic extract was combined with water and
fractionated with DCM, ethyl acetate, n-butanol and water.
The DCM fraction which showed higher antioxidant property
was further purified by using silica gel column chromatography
to yield 45 fractions. Finally, 7 fractions were obtained
through TLC profile. The UV–Visible spectra for active
column-purified fractions of ethanol were recorded. The
spectral properties are given in Figs. 1–3.
Tentative identification of antioxidant compounds from C.
trinodis was based on TLC and UV–Vis spectra. The four
fractions isolated from the methanolic crude extract by solvent
extraction were separated by using TLC. Blue and orange
spots were observed in the DCM fraction after spraying with
the spray reagent (Table 2). This indicated the presence of
phenolic compounds. Table 2 shows the Rf values and colors
of spots from the TLC chromatograms of the seven subfractions
after application of spray solution.
According to the UV–Visible spectral data (Table 3), the
seven subfractions isolated from the DCM extract contained
different components despite some fractions containing similar
absorption maxima.
The use of modifying reagent NaOH confirmed that the
peak corresponded to a phenolic compound as shifting of
the wavelengths of absorption maxima to longer ones was
observed. This effect is known as a bathochromatic shift
caused by the ionization of the phenolate ion (Waterman