Methods Used To Create Phenol-Explorer

Phenol-Explorer is the most comprehensive electronic database on polyphenol contents in foods and beverages. Content values were retrieved from scientific papers published in peer-review journals. Information on original food description, commercial origin of the sample and number of analysed samples to produce each original content value was registered together with the analytical method used. Data were evaluated and those considered acceptable were used to produce mean content values for the various polyphenols in the main foods and beverages.

The lack of a value for a particular polyphenol in a food does not necessarily imply the absence of this polyphenol, but only that data were not available. Zero value, when reported in original publications, have been included. These zero-values are not shown by default but only upon request using the option "Show zero-value entries".

Food entries were defined according to the availability of data on their polyphenol content. Some individual foods, e.g. the different varieties of a fruit, were not separated under different entries due to the lack of sufficient data to provide content values which would truly reflects differences in polyphenol contents between each of these foods. Similarly no entry could be made for various cooked foods due to the lack of sufficient reliable data. The user of Phenol-Explorer will find information on the factors influencing polyphenol content (varieties, processing, storage conditions, cooking, etc.) together with appropriate literature references in the reports on the different food categories.

Collection of composition data from literature sources

The literature search for published analytical values of polyphenols in foods and beverages was performed using the ISI Web of Knowledge^SM platform (Thomson Reuters). Different queries were built per food group from a template using the most representative food names in the food group of interest, associated to keywords related to the compounds (names, classes and sub-classes) and to quantitative information. The search was limited to peer-reviewed journal articles published in English. Additional searches were made in books and reviews (1). The comprehensiveness was checked in citations of the collected publications.

Critical evaluation of data

Articles containing quantitative data on polyphenols in foods or beverages were critically analysed and evaluated, before inclusion of the data in the database. Data were evaluated considering sampling, polyphenol extraction and analytical method, disclosure of experimental details. Only those values filling specified minimal requirements were selected for aggregation (production of mean values).

Sample

Lack of information on the nature of the samples analysed.
Analysed samples correspond to non-edible parts of the plant.
For non-commercial products, samples obtained experimentally using procedures clearly different from regular industry or domestic practice.

Analytical method

Inaccurate identification of the compounds.
Inappropriate method of polyphenol extraction or lack on information on the method used.
Inappropriate method of analysis (see below).
Lack of information on phenolic standards used for quantification.

Expression of results

Results expressed as dry weight without providing moisture content.
Content values reported in graph.
Mean content values without a description of the number of samples analysed.
Content values corresponding to sums of the content of different individual phenolic compounds.

Food definition and classification

A specific food ontology was built, based on the LanguaL system (2). LanguaL is a food description thesaurus that provides a standardized language using facetted classification. Each food is described by a set of controlled terms, such as the botanical origin, food processing including cooking and conservation. The exact identity of the foods described in the original sources was checked and botanical names added using botanical databases and other resources (3, 4, 5). It was sometimes necessary to separate some specific groups of cultivars because of their clearly different polyphenol profiles (e.g. blond orange and blood orange). However, for other foods, cultivars were not differentiated due to the lack of sufficient analytical data.

Phenolic compounds and their classification

The classification for phenolic compounds was principally derived from reference books, on-line chemical databases (6, 7, 8). Classes, sub-classes and families are named according to structural features of the compounds: presence of functional groups such as acid (COOH), alcohol (OH), aldehyde (CHO) or ketone (CO), methoxylation, alkylations, and polymerization. The compound name is based on the most common name used in the literature. A list of synonyms is also provided.

Cis and trans isomers of phenolic compounds (cinnamic acids, stilbenes) are not given separately but as a total. Indeed these isomers can be converted from one form to the other following exposure to UV light or heat treatment (9, 10), factors not necessarily controlled during the preparation of the samples.

Some phenolic compounds can also be present in different enantiomeric forms (flavanols, lignans). For example, (-)-epicatechin is partially epimerized to (-)-catechin during cocoa processing (11). However these enantiomers have very rarely been analysed separately. For this reason, no distinction is made in the database for the different enantiomers.

Phenolic acids can be present in foods in different forms: some are soluble (free phenolic acids, esters or glycosides) and are usually directly analysed by chromatography. Others are insoluble and esterified to polysaccharides in the cell wall as in cereals. Cell-wall bound phenolic acid esters are generally estimated by hydrolysis of the extractive-free cell-wall material. In Phenol-Explorer, phenolic acid content estimated by chromatography after hydrolysis is the calculated sum of the contents of all esters (soluble and insoluble) and free phenolic acids.

Proanthocyanidins are commonly analysed by two different methods: reverse phase HPLC allows to estimate dimers and trimers. Proanthocyanidins of high polymerization degree can only be separated by normal phase HPLC (12). Contents measured by this last method are given per individual degree of polymerization. In Phenol-Explorer, five categories of proanthocyanidins were made: dimers, trimers, 4-6mers, 7-10mers and polymers as used in the USDA database (13).

Method classification

Analytical methods used to determine polyphenol content in foods were grouped in five categories and data aggregated to produce mean content values per category.

Chromatography. HPLC, the most common technique, followed by GC and CE, are used to estimate phenolic compounds as present in the food. Polyphenol glycosides, phenolic acid esters together with aglycones and free phenolic acids are simultaneously quantified.
Chromatography after hydrolysis. Acid or alkaline hydrolysis is used to cleave glycosylated or esterified polyphenols. It is used with two different purposes: a) to release certain compounds such as insoluble phenolic acids esterified to the cell wall in cereals that cannot be solubilized without hydrolysis; and b) to simplify the analysis of certain flavonoids that are present in the food matrix as several glycosides and that, in this way, can be determined as aglycone equivalents. No distinction was made between acid and alkaline hydrolysis. Lignans are commonly analysed after liberation of the aglycone by a combination of chemical and enzymatic hydrolyses.
Folin assay. It is a spectrophotometric assay commonly used to determine simultaneously all phenolic compounds. It is based on the chemical reduction of the Folin reagent into a coloured product by all phenolic groups. It is not a specific assay for phenolic compounds as many other food constituents such as ascorbic acid may also reduce the Folin reagent. However, it is largely used as a global assay to give a crude estimate of the total concentration of antioxidants. It can be particularly useful in samples containing complex polyphenols such as proanthocyanidins or oxidized polyphenols.
Normal phase HPLC (proanthocyanidins) (14). This method has been used in a few laboratories to estimate proanthocyanidin oligomers according to their degree of polymerization (dimers to decamers), which cannot be easily separated by reverse phase HPLC. Only proanthocyanidin dimers and trimers are individually analysed by reverse phase HPLC (content values reported under “Chromatography” category). A separate category is made here with normal phase HPLC to avoid the user of Phenol-Explorer to sum total proanthocyanidin dimers and trimers as analysed by this method together with the contents of the various individual dimers and trimers as analysed by reverse phase HPLC.
pH Differential method (anthocyanins) (15, 16). It is a spectrophotometric method commonly used to determine total anthocyanin content.

Content values obtained by other analytical methods are not included in the database. Some of these methods are potentially useful but they have so far been used by a too limited number of authors, e.g. thiolysis for proanthocyanidin estimation (17), or total polyphenols estimated after removal of interfering compounds with solid phase extraction (18). Other methods were not considered reliable due to poor stoechiometry (UV spectrophotometry or Prussian blue method for total polyphenols, vanillin-HCl, DMACA-HCl or butanol-HCl assays for proanthocyanidins) (19).

Production of mean content values and content units

Phenol-Explorer gives weighted mean content values and standard deviations which take into account the different number of samples used to generate each original data collected from the publications. The data displayed are expressed in standard units: mg/100 g fresh weight for solid foods and oils and mg/100 mL for beverages and other liquid foods such as vinegar or soy sauce. Various options are propose to either retrieve content values in molar units or to calculate contents in aglycone equivalents when glycosides and esters are analysed by chromatography.

Content values for a given polyphenol are sometimes expressed in the original publication in equivalents of a related compound used as a standard for quantification. They were recalculated on the basis of the molecular weight of the considered polyphenol.

For some cereal products of low moisture content, samples are eventually dried before analysis and the content values expressed per dry wt units without indicating the initial moisture content. Content values have then been converted per fresh weight units using standard moisture contents (20).

Tabulated data were clearly separated where differences in study design meant they could not be directly compared. Data fell into two main categories: those which were obtained after the treatment of biofluids with β-glucuronidase and sulfatase enzymes, and those obtained from untreated biofluids. Enzymatic treatments are used to convert all conjugated polyphenol metabolites to the parent aglycone, which is then measured as a single compound. Because of the common lack of chemical standards for conjugated polyphenols and to the diversity of conjugated compounds that can be formed in tissues, enzymatic deconjugation is often used to make analyses easier. However the precise identities of polyphenol conjugates are therefore lost. Studies in which biofluids were not enzymatically treated allowed the identification of individual conjugated metabolites and were therefore a richer source of qualitative information.

Data are then separated by species from which they were acquired. Also, the nature of the dose should be considered where interpreting or comparing data. Either a single dose was administered at t=0, or repeated doses were administered at regular intervals throughout the intervention.

Polyphenol retention factors were calculated using the following formula: Retention factor (RF) = (Concentration in processed food / Concentration in raw food) * Yield factor

Retention factors describe the loss or gain of a compound during food processing: a value of 0.40 means that 40% of the compound has been retained during processing.

Yield factors describe weight changes of the food mainly due to water movement during processing: a yield factor of 0.95 means the food has lost 5% of its weight during processing.

Publications providing polyphenol concentrations on both raw foods and the corresponding processed food were systematically collected and corresponding data entered into Phenol-Explorer.

Yield factor values, when available in the same publications, were also collected and used to calculate retention factors. If not available, data were extracted from different yield factors tables. Where values for specific foods and processes were missing from these tables, extrapolations were made using yield factors for similar foods or processes. Yield factors and extrapolations used for the calculations of retention factors are available in Phenol-Explorer.

Finally, an average retention factor for each combination of food, compound and process was calculated and categorized according to the analytical method used for polyphenol analysis (with or without hydrolysis).

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