Antioxidant HPTLC-DPPH Fingerprinting of Honeys and Tracking of Antioxidant Constituents upon Thermal Exposure

Abstract

The use of High-Performance Thin-Layer Chromatography (HPTLC) coupled with the use of DPPH* (2,2-diphenyl-1-picrylhydrazyl) as a derivatisation reagent is a novel approach to the analysis of antioxidant activity of honeys. The method facilitates the visualisation of individual constituents that contribute to the overall antioxidant activity of the honey, even if they are not yet chemically identified, and allows for the quantification of their antioxidant activity as gallic acid equivalents. The method supports a more in-depth study of the antioxidant activity of honey as it allows for a comparative analysis of the antioxidant fingerprints of honeys of different floral origin and is able to capture differences in their individual bioactive constituents. Further, it supports the tracking of changes in antioxidant activity of individual honey constituents over time upon exposure to different temperature conditions, which demonstrates the potential value of the method for in-process quality control.

Australian Honeypot Ant (Camponotus inflatus) Honey—A Comprehensive Analysis of the Physiochemical Characteristics, Bioactivity, and HPTLC Profile of a Traditional Indigenous Australian Food

“…The total antioxidant band activity of these eight ant honey constituents was calculated as 0.24 mg equivalent gallic acid per 100 g ant honey. With this, the total band activity of antioxidant constituents in the ant honey’s organic extract is similar to that found for a honeybee-derived Western Australian multifloral honey and about a quarter of the total band activity recorded for an Australian Manuka honey [ 28 ].…”

“…The total antioxidant activity as measured by the FRAP and DPPH assays does not identify individual active constituents. This makes it difficult to compare the antioxidant activity of different types of honey at a constituent level, as types of honey that yield the same total antioxidant activity may differ in the chemical composition of their active compounds [ 28 ]. Using HPTLC–DPPH fingerprint analysis (Islam et al, 2021), however, the antioxidant constituents of the ant honey’s organic extract are separated from each other during development.…”

The Development and Application of a HPTLC-Derived Database for the Identification of Phenolics in Honey

“…The colours of bands in HPTLC analysis have been used in the qualitative and quantitative analyses of honey; however, they were only described using basic colour descriptions [ 20 , 50 , 51 ]. This study was able to capture more nuanced colour differences in the bands by converting their RGB values into a single hue (H°) value.…”

“…Since these imaging conditions without derivatisation might, nonetheless, not capture all compounds, those that are not detectable at 254 or 366 nm can be visualised after a reaction with a suitable reagent; in this study, either VSA or NP-PEG [ 52 ]. In HPTLC-based honey research, to date, 1% methanolic AlCl 3 , ceric phosphomolybdate, 2-aminoethyl diphenylborate, vanillin sulfuric acid and 2,2-diphenyl-1-picrylhydrazyl (DPPH) appear to be the most popular spraying reagents [ 20 , 37 , 50 , 51 , 52 , 53 ]. In this study, 2-aminoethyl diphenylborate (natural product reagent or Neu’s reagent) and vanillin sulfuric acid were used to derivatise the honey constituents for visualisation.…”

High-Performance Thin-Layer Chromatography (HPTLC) Method for Identification of Meloxicam and Piroxicam

“…Compared with other methods used in chromatography, such as gas chromatography (GC) and high-performance liquid chromatography (HPLC), TLC is the simplest and least expensive to perform [4,5]. At the same time, both TLC and HPTLC have a wide variety of application fields, such as biotechnology, chemistry, phytochemistry, the pharmaceutical industry, the food industry, cosmetics and many others [6][7][8][9][10][11][12][13].…”