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Received: 21 August 2019
Received in revised form: 13 April 2020
Accepted: 18 May 2020
Keywords
high-throughput, total phenolic content, honey, Folin-Ciocalteu assay, microdetermination
Abstract
Polyphenols are major antioxidant activity-associated bioactive substances in honey. Measurement of the total phenolic content in honey is of great importance for predicting antioxidant activity and characterising or selecting honey samples for their use as antioxidants or natural food preservatives. Considering that the original Folin-Ciocalteu (F-C) assay for total phenolic content determination is either time-consuming or high-cost, a microplate reader-based study was conducted to modify and validate the original F-C assay for the microdetermination of total phenolic content in honey samples. The linearity (R2 = 0.9982), detection limit (8.61 ± 0.33 mg gallic acid equivalents (GAEpkg-1 honey), quantitation limit (26.08 ± 0.99 mg GAE>kg-1 honey), and recovery rate (93.46 - 109.99%) were acceptable. The results suggested that the proposed F-C assay is fast, sensitive, precise, and repeatable. The method was successfully applied to analyse a total of 33 honey samples from 9 floral origins and 12 geographical regions. The modified F-C assay is low cost and easy to operate, and therefore could be applied to routine analysis.
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Introduction
The Folin-Ciocalteu (F-C) assay is commonly used to evaluate the antioxidant activity of natural products by quantifying the total phenolic content (Magalhâes et al., 2006), which is based on the reaction of phenolic compounds with a colorimetric reagent (phosphotungstic-phosphomolybdic acid) and subject to spectrophotometric measurements. It has been reported that phenolic compounds reduce phosphomolybdate-phosphotungstic acid under alkaline conditions and form blue compounds, which can be spectroscopically determined at approximately 760 nm (Singleton and Rossi, 1965; Singleton et al., 1999). This method was originally developed for the colorimetric determination of tyrosine and then improved to indirectly determine total protein concentration by measuring the content of tyrosine and tryptophan (Folin and Macallum, 1912a; 1912b; Folin and Denis, 1912; Folin and Ciocalteu, 1927). The F-C assay is sensitive and reproducible, and thereafter has been widely employed for total phenolic content quantification. Singleton and Rossi (1965) introduced a F-C assay that was applied to estimate the total phenolic content in plant products, grapes, and wines, and Kähkönen et al. (1999) and Gao et al. (2019) used this method to...