Preparation of Terminalia chebula extract
Dried ripen fruits of T. chebula were collected from the local market and authenticated by a professor from the Botany Department, Cooch Behar Panchanan Barma University (CBPBU). Appropriate T. chebula fruits (those which are well ripened, not moldy or rotten and free from any kind of insect attack) were selected, then seeds were removed, grounded to coarse powder using a domestic grinder. The powder thus obtained was stored in vacuum desiccators for subsequent use.
At first 1.5 gm of T. chebula powder was weighed and, taken in a conical flask containing 100 mL of double distilled water. The mix was kept in the conical flask for 24 h at room temperature and filtered. The filtrate thus obtained was preserved in the refrigerator for further use.
Characterization
The T. chebula raw extract was initially column chromatographed using RP-HPLC (reverse phase high performance liquid chromatography) [Shimadzu SPD-20A UV detector and Shimadzu LC-20AR series pumping system (Shimadzu Corporation, Kyoto, Japan) with Eclipse XDB-C18 (reverse phase) and C18 monochromatic column, respectively], then the active ingredient was identified by treatment of Fe3+ and Fe2+ solutions to different collected fractions from HPLC. The active ingredient of the raw extract for iron sensing was further purified by preparative RP-HPLC with MeOH as mobile phase (isocratic system) on a C18 column (25 × 250 mm), at UV detector: 292 nm, flow rate: 1.0 ml/min with a runtime of 20 min. Subsequently, the purity of the isolated product was checked using the analytical column SB C18-column (ZORBAX SB-C18, 4.6 × 250 mm, 5 μm, UV detector: 292 nm, flow rate: 1.1 ml/min, tR = 2.5 min). Then the chemical structure of the isolated fraction was characterized and confirmed by FAB-MS spectroscopy (Fast Atom Bombardment Mass Spectrometry) (the Korea Basic Science Institute at Daegu, S. Korea) and was found to be m/z 637.9 [M + H]+, and calculated m/z 636.1 for C27H24O18.
UV–Vis titration measurements of Terminalia chebula extract with Fe2+ and Fe3+
For the UV–Vis titration, the prepared extract was diluted with double distilled water in a ratio of 1:250. The desired concentrations of FeSO4 and FeCl3 solutions were prepared for the colorimetric estimation. 2.4 mL of the prepared T. chebula extract was taken and 100–1000 µL of the metal ion solution (final concentration 1.44 × 10–5–1.44 × 10–4 M) was added to it. After mixing and keeping solutions for 5 min in the dark, UV–Vis spectra were measured at room temperature (You et al. 2015; Kang and Kim 2018).
Competitive experiments
For this experiment, the extract was diluted in the ratio of 5:250 and metal ion solutions of NiCl2, CoCl2, HgCl2, CdCl2, Al2(SO4)3, MnSO4, Cr2O3, LaCl3, MgCl2, CaCl2, NaCl, KCl were prepared in distilled water (0.004 M). 1 mL of each metal solution was taken in a test tube containing 1 mL 0.004 M of Fe2+ or Fe3+ solution, followed by addition of 1 mL of the extract. After mixing and keeping for 5 min in dark, the UV–visible spectra of each mixture were taken at room temperature (Gao et al. 2017).
Determination of Fe2+ and Fe3+ in water samples
UV–Vis spectra of water samples containing Fe2+ and Fe3+ were carried out by adding 1 mL of diluted (5:250) stock solution of T. chebula extract and 2 mL of sample solutions. Mixed homogenously, the solutions were allowed to stand for 5 min at room temperature before recording UV–visible spectra (You et al. 2015).
Colorimetric determination of iron concentration by naked eye
A colorimetric technique was utilized to determine the content of Fe2+ and Fe3+ in aqueous solution by the naked eye. Different concentrations of iron solutions (10, 50, and 100 ppm) were prepared, then 200 μL of T. chebula raw extract was added to each solution, to a final volume of 2.5 mL. Color changes were observed after 5 min of waiting. The same technique was performed using a paper strip, at first 200 μL T. chebula raw extract was added onto the strip and dried in the oven. Following that, Fe2+ and Fe3+ solution (50 μL) separately of various concentrations (10, 50, and 100 ppm) were added to the strips, dried and the colors for different concentrations of Fe2+ and Fe3+ were monitored.