Methodology optimization for the analysis of phenolic compounds in chestnut (Castanea sativa Mill.)

Phenols are bioactive substances of great interest because of their involvement in plant physiology, their use in many industrial processes, and their impact on human health. This work aims to summarize the varied approaches to the phenolic analysis of chestnut (bark and wood of trunk and branches, leaves, catkins, burs, and fruit) and to collate the optimal conditions into an easy to follow and execute protocol. Phenolic compounds were extracted by solid-liquid extraction and separated by liquid-liquid extraction. Total phenols content was determined by Folin-Ciocalteu assay, condensed tannins by vanillin assay, and hydrolyzable tannins (gallotannins and ellagitannins) by high-performance liquid chromatography quantification of methyl gallate and ellagic acid following acid methanolysis. The lowest temperature for conservation (-80 ℃), lyophilization, and milling (liquid N₂) were the most effective pretreatments for samples. For quantification of tannins, the use of water clearly reduced the sensitivity of the analysis of condensed tannins, whilst the more efficient degradation capacity of sulfuric acid improved the methanolysis of hydrolyzable tannins. These findings were validated using a range of chestnut tissues, and thus confirm the utility and effectiveness of this easy to implement, cost-effective, and efficient protocol.