As discussed above, even though polyphenols share the common phenolic feature, due to the structural diversity, these phytochemicals vary significantly in their physicochemical properties. Owing to the chemical complexity and the frequent occurrence of polyphenols in plants, extraction, separation, identification and analysis of polyphenols remain as challenging as ever, despite the recent advances in new instrumentation. The challenge is multiplied when the complex glycosylation and polymerization patterns and the various food matrices are considered. While it is nearly impossible to develop a protocol for all polyphenols, there are some general approaches to these important aspects of polyphenol research. Many good reviews and books are available on this subject, so only a brief summary will be covered here [32-35].
Before polyphenols are extracted, samples containing these compounds must be collected, reserved and prepared properly. It is generally understood that samples (e.g., plants, foods, biological fluids) collected must represent the actual pool. Care must be taken to minimize the loss of compounds of interest during transportation and preservation of the samples. To avoid degradation of native polyphenols, samples are often dried, frozen or lyophilized before extraction because high moisture or water content aids enzyme activities [33] (Figure 10). Heating and exposure to light and oxygen may affect the polyphenolic composition in many cases; therefore high-temperature drying should be avoided as much as possible. Antioxidants such as butylated hydroxytoluene (BHT) and ascorbic acid are often added to samples to avoid oxidation of the polyphenols. Sample pre-treatment may be done by filtration and centrifugation as well [33] (Figure 10).
Many different extraction methods are available for different types of samples [32-34]. For the majority of plant originated food samples, solvent extractions such as liquid/liquid partitioning and solid/liquid extraction are most frequently employed in the laboratory. The phenolic nature of polyphenols makes them relatively hydrophilic, thus free polyphenols, including aglycones, glycosides, and oligomers, are extracted using water, polar organic solvents such as methanol, ethanol, acetonitrile and acetone, or their mixture of water. The liquid extracts are sometimes partitioned with solvents such as ethyl acetate, depending on the solubility of the target polyphenols. Also important is the pH of the extraction solvent. For polyphenols, most extractions are carried out under acidic conditions because they are generally more stable in low pH, and the acidic condition helps polyphenols to stay neutral, thus readily extracted into organic solvents. This is done using weak acid or low concentrations of a strong acid. High acid concentration can cause hydrolysis of glycosides or acylglycosides and thus may give different pictures of native polyphenol profiles. On the other hand, not all polyphenols exist in the free form. Phenolic acids such as ferulic acid and lignans in grains are often bound to structural materials. Hydrolysis using acid or alkaline releases these phenolics which
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are partitioned into ethyl acetate or n-butanol [8-10]. Sometimes, intentional hydrolysis is carried out
to obtain aglycones with enzymes such as -glucosidase, or with strong acid such as 2–4 M HCl at
elevated temperature such as refluxing. This is necessary, particularly when the glycosylation patterns
are extremely complex, and when standard reference materials of polyphenol glycosides are
unavailable. The hydrolysis can simplify the chromatographic profile during separation, and aid
quantification and structural identification of the polyphenols.