Analysis of Nonextractable Phenolic Compounds in Foods: The

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Analysis of Nonextractable Phenolic Compounds in Foods: The Current State of the Art Jara Perez-Jimenez* and Josep Lluís Torres Institute for Advanced Chemistry of Catalonia (IQAC), CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain ABSTRACT: More than 500 phenolic compounds have been reported as present in foodstuffs, and their intake has been related to the prevention of several chronic diseases. Most of the literature on phenolic compounds focuses on those present in the supernatant of aqueousorganic extractions: extractable phenolics. Nevertheless, significant amounts of phenolic compounds remain in the solid residues after such extractions. These nonextractable phenolics are mostly proanthocyanidins, phenolic acids, and hydrolyzable tannins that are closely associated with the food matrix. Studies of this fraction of dietary phenolic compounds are scarce, and the few there are usually refer to particular types of phenolics rather than to the fraction as a whole. The present review reports the stateof-the-art methods that currently exist for analyzing nonextractable phenolic compounds in foods. KEYWORDS: nonextractable phenolics, nonextractable proanthocyanidins, bound phenolics, insoluble phenolics

1. INTRODUCTION Phenolic compounds are a large group of secondary plant metabolites. More than 10,000 structures have been described as phenolics that occur in nature,1 and more than 500 are reported to occur in foodstuffs.2 Besides the protection that phenolic compounds provide to plants, they may also have beneficial preventive effects on animals. Phenolics have attracted huge interest over recent decades due to the possible association between their intake and the prevention of certain major chronic diseases, such as cardiovascular disease or certain kinds of cancer, as suggested by clinical trials and epidemiological studies.36 The group of phenolic compounds includes a wide range of structures from small molecules, such as phenolic acids, to large complex polymers, such as high-molecular-weight proanthocyanidins (PA) and thearubigins. To analyze such a diverse family of compounds in food, several methods have been proposed: from those that provide a global estimation of a range of compounds (e.g., pH differential assays for anthocyanins or Folin assays for total antioxidants) to specific methods targeted at individual phenolics that are usually based on HPLC or GC techniques.2 All of the methods are most commonly performed on food extracts, which may be obtained by using different combinations of water and organic solvents, and certain optimized techniques have been developed to extract specific classes of phenolics.7,8 The phenolic compounds detected in such food extracts constitute the “extractable phenolics” (EP) and include phenolic compounds that belong to different classes, as summarized in Figure 1. The solid residues from such extractions are generally not considered a source of bioactive compounds and are ignored. However, significant amounts of phenolic compounds may remain in the residues, associated with the food matrix; these constitute the “nonextractable phenolics” (NEP). Studies of NEP are scarce in comparison to the work published on EP; indeed, NEP were recently described as the “hidden face” of food phenolics.1 NEP include several classes of phenolic compounds, mostly PA, hydrolyzable tannins, and phenolic acids (Figure 1). r 2011 American Chemical Society

NEP form an interesting group of compounds from a nutritional point of view for two reasons: (a) some of them may be hydrolyzed by intestinal enzymes in the small intestine and thereby become bioaccessible and potentially bioavailable;9,10 (b) the fraction that reaches the colon intact may be released from the food matrix by the action of colonic microbiota and transformed into small phenolics and metabolites that are subsequently absorbed.11,12 Since microbial metabolites are increasingly recognized as relevant to the reported health effects of phenolic compounds,13,14 the importance of the contribution of the largely neglected NEP is becoming increasingly clear. Finally, the overall bioavailability of NEP has been demonstrated both directly by determining individual phenolics in plasma or urine15,16 or indirectly by evaluating plasma antioxidant capacity.17 NEP and dietary fiber are intimately associated and exert a mutual influence: first, NEP modify the structure of the dietary fiber, affecting properties such as its reticulation, molecular weight, and water solubility;18 second, the dietary fiber acts as a carrier of NEP.19 In fact, a significant fraction of NEP are constituents of the dietary fiber, and together, the two should be considered as a particular type of dietary fiber with a specific influence on health. The existence of NEP, in particular, of nonextractable proanthocyanidins or NEPA, was first suggested nearly 40 years ago, as a result of studies of several species of herbaceous Leguminoseae.20 A few papers published during the 1990s also addressed this topic and reported the presence of NEP in barks and in some foodstuffs.21,22 However, it was not until more recent years that an increasing number of groups turned their attention to this fraction of food phenolics, which has been given different names: nonextractable,23 unextractable,24 insoluble,25 or bound26 phenolics. It is now becoming evident that NEP are more abundant than EP in many foodstuffs; as indicated by comparing the antioxidant Received: August 22, 2011 Accepted: November 9, 2011 Revised: November 8, 2011 Published: November 09, 2011 12713

dx.doi.org/10.1021/jf203372w | J. Agric. Food Chem. 2011, 59, 12713–12724

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Figure 1. Extractable and nonextractable phenolics in foods.

capacity of EP and that of either extraction residues2729 or whole samples.30 As NEP have been largely ignored despite being a significant portion of dietary phenolic compounds, it is necessary to study the individual constituents in order to advance our knowledge. Precisely because of their nonextractable nature, the task is particularly complicated, and new analytical methods need to be developed. To the best of our knowledge, to date there has been no review of current knowledge concerning NEP analysis. The different classes of putative NEP constituents (i.e., PA, hydrolyzable tannins, and phenolic acids) have been considered separately but not as constitutive parts of the wider category of NEP. The present work aims to provide such an overview of the current state of the art of the analysis of NEP in foods.

2. NONEXTRACTABLE PROANTHOCYANIDINS (NEPA) PA are polymers of flavan-3-ols that are widely distributed in plant-based foodstuffs, and they can present considerable structural differences according to the degree of hydroxylation of the aromatic rings, the stereochemistry of the C in the central ring, the presence of esterified galloyl substituents, and the kind of interflavan linkages involved.31 They can be divided into extractable PA (EPA), commonly extracted by acetone/water/acetic acid (70:29.5:0.5, v/v),32 and NEPA (Figure 1). This division is not primarily related to the molecular weight of the PA since high-molecular-weight PA with a mean degree of polymerization

(DP) higher than 25 may be extractable1 but to their associations with other components of the food matrix. NEPA include both PA bound to the cell wall and PA associated with proteins that cannot be easily disrupted.1 Moreover, a fraction of EPA may be strongly linked to NEPA associated with the cell wall.21 Interactions with the cell wall will mostly be via hydrogen bonds or hydrophobic interactions; in the latter case including possible encapsulation into hydrophobic pockets,33 while the existence of covalent links to the cell wall has yet to be proven. The adsorption of PA onto cell walls is affected by different PA compositional characteristics, such as their DP (which increases the number of hydroxyl groups that are available to form hydrogen bonds and also increases in the number of aryl groups capable of establishing hydrophobic interactions), the degree of galloylation, or the proportion of (+)-catechin in relation to that of ()-epicatechin.33,34 Similarly, different types of dietary fiber interact differently with NEPA; higher proportions of pectin impart higher flexibility to the structure, thus allowing for a higher contact surface area, while higher proportions of lignin and cellulose, with greater structural rigidity, allow for fewer surface-mediated interactions.35 Other factors related to the food itself, such as the stage of ripeness, also influence the degree to which such interactions occur.36,37 Finally, they may also be affected by food processing; drying, for instance, reduces the number of associations due to a reduction of available pores, although the affinity of each association may be increased.33 12714

dx.doi.org/10.1021/jf203372w |J. Agric. Food Chem. 2011, 59, 12713–12724

Journal of Agricultural and Food Chemistry Several methods for analyzing NEPA have been developed and are described in the remainder of this section. They are all carried out on the residues of aqueousorganic extractions, e.g., treatment with butanol-HCl, followed by spectrophotometric measurement (the Porter method);38,39 enzymatic or alkaline hydrolysis, followed by NP-HPLC;26,39 and acid-catalyzed depolymerization, followed by RP-HPLC.24 Briefly, the Porter method is the most drastic and therefore allows the most efficient release of PA by acid depolymerization into monomeric anthocyanins which are nonspecifically quantified by spectrophotometry. Hydrolysis, either alkaline or enzymatic, yields PA which may be separated by NP-HPLC according to their DP. Acidcatalyzed depolymerization in the presence of thiols (e.g., thiolysis with benzylthioether) yields the constituent monomeric units, and subsequent RP-HPLC analysis provides the mean DP. Some examples of the results obtained by these three methods are shown in Table 1. 2.1. Treatment with Butanol-HCl, Followed by Spectrophotometric Measurement. A drastic acid hydrolysis in butanol (butanol/HCl, 97.5:2.5, in the presence of FeCl3, 100 °C, 1 h) is performed on residues from aqueousorganic extractions to yield soluble red anthocyanins, which are determined spectrophotometrically, and a phlobaphene powder, a polymeric material derived from side reactions that has not yet been well characterized.38,39,49 The Porter method is commonly used to estimate EPA content in several foodstuffs.50,51 Its application to the residue of aqueousorganic extraction from leaves had already been suggested,52 but it was not applied to the residues of aqueous organic extractions from foods until more recently.39,53 Some results of this method are shown in Table 1. Although a direct comparison between data for NEPA content obtained by this spectrophotometric method and those obtained by HPLC for EPA content cannot be made, when NEPA in a selection of 13 foodstuffs and food byproducts were determined by the butanol acid assay, the values obtained were significantly higher in half of the products than those reported in the USDA database for EPA.39 This shows just how important this fraction of dietary PA may be. This acidolytic method revealed that NEPA are constituents of the water-insoluble fraction of cell walls from postharvest banana,41 that the exotic fruit ac-aí contains as much as 1400 mg NEPA/100 g dw,42 and that the mixture of fruits consumed in the Spanish diet includes an unusually high 514 mg NEPA/100 g dw.40 After subjecting different typical European diets to a process emulating digestion and to in vitro fermentation, significant amounts of NEPA remained in the residues of fermentation.43 This method is fast and releases NEPA very efficiently due to the drastic conditions employed. However, the treatment converts the original PA into anthocyanidins so that the information about the original polymer size distribution is lost. Also, such drastic conditions may cause partial degradation of the anthocyanins released, which would contribute to an underestimation of NEPA content. Moreover, the butanolic solution obtained after applying the Porter method cannot be used for further activity evaluation; only the antioxidant capacity assay ABTS could be performed on the resulting colored supernatant.42 Another problem with the acidolytic method is the lack of a universal standard because PA with different structural units present different reaction yields,49 and PA linkages (A-type) are more resistant than others (B-type).54 The use of anthocyanidins as standards leads to overestimation of PA content,39,49 and therefore, the use of PA concentrates of plant origin has been

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suggested as a better standard.39,55 However, these PA concentrates are not commonly commercially available, and low purity may cause an overestimation of PA content. Overall, it has been suggested that, although this method may not be suitable for providing absolute content values for different foodstuffs containing different kinds of PA, it may be appropriate for obtaining information about the relative amounts of PA in different food samples.39,56 2.2. Enzymatic Treatments, Followed by Normal-Phase HPLC. Enzymatic treatments of the residues of aqueousorganic extractions partly release intact PA that can be separated according to their DP by NP-HPLC, but the yields are low. Again, although results obtained by HPLC and those by the spectrophotometric assay cannot be directly compared, when a mixture of pectinase, protease, cellulase, and esterase was used on apple, the resultant NEPA content was much lower than that obtained for the same sample by the butanol-HCl method (Table 1).39 Moreover NP-HPLC analyses of PA present the additional problem of the strong absorption of the polymers in the stationary phase.1 This problem is shared with other methods that yield intact unmodified NEPA. 2.3. Alkaline Cleavage, Followed by Normal-Phase HPLC. A study of the best conditions for alkaline hydrolysis to release NEPA by White et al.26 concluded that 2 M NaOH at 60 °C for 15 min was the best choice. A higher temperature or longer reaction time resulted in lower yields of NEPA due to partial degradation. The resulting NEPA were separated according to their DP by NP-HPLC. The mechanism of NEPA release by NaOH is a combination of enhanced extraction of bound PA and partial depolymerization. Moreover, solubilization of the cell walls within samples may contribute to the release of NEPA, as it is known that dilute NaOH extracts hemicelluloses from cell wall material.57 This method was used to determine the NEPA content of cranberry pomace,26 and a value of 1685 mg/100 g dw was obtained (Table 1). Degradation of PA may also take place under these conditions, e.g., A-ring cleavage products such as catechinic acid may be generated and cause an underestimation of NEPA.26 However, since the conditions are less drastic than those of the Porter method and the reactions are mostly oxidations, degradation may be partially prevented if the tests are performed in a nitrogen atmosphere.26 Moreover, although this method can be considered a means of obtaining accurate estimations of total NEPA, the DP distribution is not representative of the original composition of the sample since the treatment itself causes depolymerization of PA.26 Similarly, dimers with A-type linkages are resistant to these hydrolysis conditions, while dimers with B-type linkages may be hydrolyzed to monomers. 2.4. Acid-Catalyzed Depolymerization in the Presence of Nucleophiles, Followed by Reversed-Phase HPLC. The acid treatment of PA in the presence of a nucleophilic reagent, e.g., benzylmercaptan (or toluene α-thiol; thiolysis) or phloroglucinol (phloroglucinolysis) yields flavan-3-ol extension units and terminal units flavan-3-ols. The mean DP of the PA in the sample is calculated as the ratio of total units (i.e., extension and terminal units) to terminal units. This method, commonly used for the analysis of EPA, was used to analyze both EPA and NEPA in pine bark21 and in grape skin and seed during grape ripening.36,37 Similarly, Guyot et al.34,45 used this method to analyze PA content in whole apple, including, therefore, NEPA. By using thiolysis, Hellstr€om and Mattila24 presented the most systematic determination of NEPA in foods at the time. NEPA were analyzed in more than 50 foods, and the results show that NEPA are a substantial 12715



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Table 1. NEPA Content in Several Foods Reported in the Literature According to Different Methodologiesa content sample

methodology

(mg/100 g FW)

standard

ref

Treatment of the Residue with Butanol-HCl, Followed by Spectrophotometry banana

residue of aqueousorganic extraction subjected to butanol/HCl

980 ( 45b

carob pod PA concentrate

39

514 ( 11b


40

15 ( 0.1

banana PA concentrate

41

1,240 ( 140b


42

4856


43

1823


44


39

cococa PA concentrate

26

3743

C, EC, EC benzylthioether

45

6 ( 0.8

E, EC, ECG, EGC, EC

24

(97.5:2.5, v/v) treatment at 100 °C during 60 min, followed by spectrophotometric determination at 555 nm fruits consumed in the Spanish diet

residue of aqueousorganic extraction subjected to butanol/HCl (97.5:2.5, v/v) treatment at 100 °C during 60 min, followed by spectrophotometric determination at 555 nm

water insoluble fraction of postharvest banana cell wall

residue of organic extraction, subjected to hydrolysis with TFA 2 M (80 °C, 2 h), followed by treatment with butanol/HCl (97.5:2.5, v/v) treatment at 95 °C during 120 min, followed by spectrophotometric determination at 555 nm

ac-aí fruit


foods of vegetal origin consumed in European diets

residues of aqueousorganic extraction after in vitro digestion and fermentation subjected to butanol/HCl (97.5:2.5, v/v) at 100 °C during 180 min, followed by spectrophotometric determination at 555 nm

apple pomace


Enzymatic Hydrolysis, Followed by NP-HPLC apple

residue of aqueousorganic extraction subjected to enzymatic

5 ( 1b

treatment with a mixture of pectinase, proteasa, cellulase and esterase, followed by NP-HPLC analysis Alkaline Hydrolysis of the Residue, Followed by NP- HPLC cranberry pomace

residue of aqueousorganic extraction subjected to treatment with

1685b

2 N NaOH (60 °C, 15 min), followed by defatting, extraction with ethyl acetate and NP-HPLC analysis Acid-Catalyzed Depolimerization of the Residue, Followed by RP-HPLC apple-flesh

whole flesh apple subjected to thioacidolysis (5% solution of benzylmercaptan in methanol containing 1% HCl at 40 °C during 30 min),

apple

followed by RP- HPLC analysis residue of aqueousorganic extraction subjected to thioacidolysis (5% solution of benzylmercaptan in methanol containing

benzylthioether

1% HCl at 40 °C during 30 min), followed by NP-HPLC analysis cocoa powder

residue of aqueousorganic extraction subjected to thioacidolysis

602 ( 13

(5% solution of benzylmercaptan in methanol containing

E, EC, ECG, EGC, EC

46

benzylthioether

1% HCl at 40 °C during 30 min), followed by NP-HPLC analysis strawberry

whole sample subjected to acidolysis (0.5% solution of phloroglucinol

54163

in methanol containing 0.1 N HCl at 50 °C during 10 min), followed by RP-HPLC analysis quince

residue of organic extraction, subjected to acidolysis (5% solution of benzylmercaptan in metanol containing 0.2 HCl at 50 °C during 1 h),

EC, EC phloroglucinol

47

adduct 190

E, EC, ECG, EGC, EC

48

benzylthioether

followed by RP-HPLC analysis a

C, (+)-catechin; EC, ()-epicatechin; EGC, ()-epigallocatechin; ECG, ()-epicatechin gallate; PA, proanthocyanidins. b Results expressed as dry weight.

portion of total PA; as high as 100% in banana, 71% in redcurrant, 63% in red grape, and 40% in cocoa powder46 (Table 1). Recently,47 a direct analysis of total PA in whole strawberries was carried out using this method, and the total content obtained was

53.9163.2 mg/100 g fw (Table 1) with a mean DP of 3.75.8; most probably, if NEPA had been specifically analyzed, a higher mean DP would have been obtained. Recently, NEPA content was analyzed using this method after extracting with 80% ethanol 12716



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Table 2. Nonextractable Hydrolyzable Tannin Content in Several Foods Reported in the Literature According to Different Methodologies content sample

methodology

(mg/100 g FW)

compd

ref

Acid Hydrolysis of the Residue, Followed by Spectrophotometric Determination fruits consumed in the Spanish diet

residue of aqueousorganic extraction subjected to methanol/H2SO4 (90:10, v/v) treatment at 85 °C during 20 h, followed by Folin assay

697 ( 68a

gallic acid

53

onion

residue of aqueousorganic extraction subjected to methanol/H2SO4

410 ( 20a

gallic acid

62

542.8

ellagic acid

63

108191

ellagic acid and

64

(90:10, v/v) treatment at 85 °C during 20 h, followed by Folin assay Acid Hydrolysis of the Residue, Followed by HPLC-MS Analysis walnut

whole sample subjected to HCl 2 M treatment at 85 °C during 20 h, followed by HPLC-MS analysis

walnut

residue of aqueousorganic extraction subjected to 2 N HCl treatment at 95 °C during 8 h, followed by HPLC-MS analysis

apple

valoneic acid


3(1

dilactone gallic acid

23

11 ( 1

gallic acid

40

210

gallic acid and

65

(90:10, v/v) treatment at 85 °C during 20 h, followed by HPLC-MS analysis fruits consumed in the Spanish diet brazil nut

residue of aqueousorganic extraction subjected to methanol/H2SO4 (90:10, v/v) treatment at 85 °C during 20 h, followed by HPLC-MS analysis residue of aqueousorganic extraction subjected to 4 M NaOH treatment at room temperature during 4 h, followed by HPLC-MS analysis

a

ellagic acid

Results expressed in dry weight.

(the common procedure to obtain soluble dietary fiber in the supernatnant, leaving insoluble dietary fiber in the corresponding residue), and mean DP for fruits such as blueberry, quince, or pear of 33, 93, and 147 were obtained, respectively.48 The method has some limitations. The relatively mild acid treatment in the presence of nucleophiles compared to butanolHCl or NaOH may not release the same amount of NEPA from the food matrix. Indeed, it has been reported that some of the polymers resist degradation with thiols, as specifically described in plants whose PA originate from aged tissues.58 The relevance of these thiol-resistant PA in foods has not been established. It has also been reported that galloylation may hamper the cleavage of the interflavonoid linkage by this procedure.49 The nucleophilic reagent employed may also affect the results; for instance, phloroglucinolysis produces lower yields than benzylmercaptan.54 Finally, the products present different stabilities, products of phloroglucinolysis being less stable than those of thiolysis.58 2.5. Qualitative Analysis. Several spectrometric techniques provide qualitative information on PA structure, such as the kind of interflavan linkage and the presence of galloyl groups, which cannot be obtained by other methods. MALDI-TOF, usually performed on aqueousorganic extracts, can also be performed directly on the solid sample. To our knowledge, this latter technique has only been applied once to a bark sample from Acacia auriculiformis, and PA dimers to decamers were detected.59 Since the laser ionization was carried out directly on the whole sample, it may be hypothesized that NEPA were also released and, therefore, included in this analysis; however, more MALDI-TOF studies with direct sample analysis performed on food products are needed to determine whether this technique actually allows the analysis of NEPA.

3. NONEXTRACTABLE HYDROLYZABLE TANNINS Hydrolyzable tannins are polyesters of a sugar moiety and an organic acid which have highly variable degrees of esterification

and cross-linking. Gallotannins contain only gallic acid, whereas ellagitannins also include more complex structures such as crosslinked gallic acid (hexahydroxydiphenyl moiety) and ellagic acid (Figure 1). Although they are commonly determined in aqueous organic extracts from foods, only a few studies have determined them in the corresponding residues, but these show that there is also a fraction of nonextractable hydrolyzable tannins, although the way by which they are associated with the food matrix has not yet been established. Current methods for their determination include acid hydrolysis of the residue, followed either by spectrophotometric measurements or HPLC-MS analysis. As the hydrolysis causes depolymerization, quantitative results are expressed as gallic acid or ellagic acid equivalents, and no information about individual tannins is obtained. 3.1. Acid Hydrolysis Followed by Spectrophotometric Analysis. Hartzfeld et al.60 suggested a modification of the Bate-Smith method for determining hydrolyzable tannin content. The modified method includes an initial methanolysis step, in which hydrolyzable tannins are transformed into methyl gallates, followed by reaction with KIO3, and spectrophotometric absorbance measurement. The method accurately measures gallotannins but underestimates the content of ellagitannins since cross-linked and condensed gallic acid structures are missed. Originally, the method was applied directly to the complete sample, so no distinction was made between extractable and nonextractable hydrolyzable tannins. A modification of the method was suggested by Saura-Calixto et al.,53 whereby methanolysis is applied to the residues left after aqueousorganic extractions, to determine the nonextractable hydrolyzable tannins by the spectrophotometric Folin assay, which is commonly used to estimate total phenolic compounds in foods.61 With this method, significant amounts of nonextractable hydrolyzable tannins were found in onion (410 mg gallic acid equivalent/100 g dw) and in fruits (697 mg gallic acid equivalent/100 g dw) (Table 2). These results may be easily compared with those from Folin assays 12717


Journal of Agricultural and Food Chemistry performed on direct aqueousorganic extracts from plant-based foodstuffs to estimate total reducing substances in the samples. The major drawback of this procedure is the lack of selectivity of the Folin assay, as it is sensitive to all reducing substances in the sample, not just to phenolics, and therefore, it can overestimate the phenolic content if samples are not purified prior to the procedure.66 3.2. Acid Hydrolysis Followed by HPLC-MS Analysis. Other authors have combined the methanolysis described above or other hydrolysis procedures with analysis by HPLC-MS of either ellagic or gallic acid.6365 This method has been used to determine the nonextractable hydrolyzable tannin content of several types of fruits and nuts (Table 2). Li et al.64 quantified ellagic acid from walnuts in three different fractions: soluble, soluble-bound (released after acid hydrolysis), and insoluble-bound, a protocol commonly used for the analysis of phenolic acids (see Nonextractable Phenolic Acids) but not for hydrolyzable tannins.25,67 They concluded that the ellagic acid and a valoneic acid derivative (an ellagic acid equivalent) found in the insoluble fraction constituted 3260% of the total ellagic acid in walnut.64 As HPLC-MS is a more selective analytical method, the results obtained are significantly lower than those obtained when applying the Folin assay after acid hydrolysis of the residue.53,68 A recent study used alkaline hydrolysis of the extraction residue to estimate hydrolyzable tannin content in Brazil nut.65

4. NONEXTRACTABLE PHENOLIC ACIDS (OR INSOLUBLE PHENOLIC ACIDS) Phenolic acids are particularly abundant in cereals and have commonly been divided into three fractions, depending on their potential interactions with other components in the food matrix: soluble phenolic acids, which may be directly measured in the supernatants of aqueousorganic extractions; esterified soluble phenolic acids, which are measured in the same supernatants after performing alkaline hydrolysis; and insoluble phenolic acids, which remain in the residues from the extraction and hydrolysis. Insoluble phenolic acids therefore constitute another kind of nonextractable phenolics, which we will refer to as nonextractable phenolic acids. They are mainly found in cereals and mostly consist of ferulic acid derivatives, such as feruloylated oligosaccharides (e.g., feruloyl-arabinofuranosyl-xylopyranosylxylose) or sugar esters of ferulic acid (e.g., 5-O-feruloyl-L-arabinofuranose and feruloyl-arabinoxylan). In all of these conjugates, the ferulic acid is linked by ester bonds to polymers in the plant cell wall, mostly to hemicelluloses, as has been known for over a decade.69 Similarly, oligomers (dimers, trimers, and tetramers) of ferulic acid generated by peroxidase-catalyzed oxidations are cross-linking agents that mediate the aggregation of polysaccharides or of polysaccharides with other polymers (e.g., xylanes and lignins).70 This cross-linking is also stabilized by the formation of ether bonds between polysaccharides and the hydroxyl groups of ferulic acid.71 Ferulic acid is also linked to lignin alone through ether and ester bonds,72 and it mediates polysaccharideprotein cross-links within the cell wall. Other phenolic acids are associated with cell-wall polysaccharides.73 In fact, during the lignification process in cereals, peaks may be found in the deposition of several phenolic acids, such as p-hydroxyphenyl, syringyl, feruloyl, or p-coumaroyl moieties,72 and it has been reported that phenolic acids other than ferulic acid constitute 34% of the nonextractable phenolic acids in wheat bran.74 The ripening process tends to reduce the

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nonextractable phenolic acid content.75 Phenolic acids are also present in other plant-based foodstuffs: ferulic and p-coumaric acids in spinach and sugar beet; sinapic, ferulic, and p-coumaric acid in orange flavedo; and p-coumaric acid in lentils.76 Table 3 is an overview of data reported in the literature for nonextractable phenolic acids in several plant-based foodstuffs. The analysis of nonextractable phenolic acids requires, as explained above, hydrolysis treatment of the extraction residue to release the phenolic acids that are bound to the cell wall (either to polysaccharides, lignins, or proteins) or trapped in cores, followed by HPLC-UV or HPLC-MS analysis. The hydrolysis procedure may be acid, alkaline, or enzymatic; the alkaline treatment will tend to cleave ester and ether bonds, while acid hydrolysis targets glycosidic bonds.96 Although most of the studies address the three fractions of phenolic acids previously described separately (soluble, esterified, and insoluble), some of them hydrolyze the whole sample directly, thereby measuring total phenolic acids without distinction.82,89 It should be noted that some papers have determined nonextractable phenolic acids in dietary fiber isolated from cereal flour or fruit.78,81,93,97 The detection of nonextractable phenolic acids in such samples is of particular relevance since it agrees with some recent studies which emphasize the need to consider this fraction of dietary phenolic compounds, part of NEP, and therefore constituent of dietary fiber.18,19 It has been reported, for instance, that the higher the proportion or arabinoxylans in dietary fiber, the greater the amount of nonextractable ferulic acid in it.85 Moreover, the presence of these phenolic acids associated with insoluble dietary fiber would alter its physicochemical characteristics, by modifying its solubility.78 4.1. Alkaline Hydrolysis Followed by HPLC-UV, HPLC-MS, or GC-MS. Alkaline hydrolysis is the most common procedure used to release nonextractable phenolic acids from the cell wall.73,79,82The conversion of vanillic acid to vanillin during alkaline hydrolysis has been reported,87 and the addition of EDTA during the treatment has been suggested in order to reduce any degradation that may occasionally occur.79 Alkaline hydrolysis of extraction residues combined with HPLC-UV or HPLC-MS analysis reveals significant nonextractable phenolic acid content, e.g., 4-hydroxyphenylacetic acid, caffeic acid, p-coumaric acid, ferulic acid, o-, m-, and p-hydroxybenzoic acid, sinapic acid, salicylic acid, syringic acid, and vanillic acid in cereals (Table 3). Additionally, several dimers of ferulic acid, as well as triferulic acid, have been detected in cereal flours, and dehydrodiferulic acid and sinapic acid dehydrodimer were also identified as nonextractable phenolic acids by GC-MS (Table 3). The development of new analytical methods may allow the elucidation of the structures of new nonextractable phenolic acids. In fact, Hosseininan et al.87 report that half of the nonextractable phenolic acid content in triticale bran corresponds to compounds that were not identified by HPLC due to the lack of standards but which exhibited UV spectra similar to those of known phenolic acids. Interestingly, some of these phenolic acids are mainly found in cereals in a nonextractable form; for instance, the proportion of free to soluble conjugate esters to insoluble ferulic acid in different cereal samples was estimated to be 0.1:1:100.25 Nonextractable phenolic acids, as determined after alkaline hydrolysis, have also been identified in red ginseng,80 in beverages such as beer and coffee,79,82 in different types of fruits such as medlar75 or young mandarin,90 and in vegetables such as asparagus.89 Moreover, they were also found in the insoluble 12718



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Table 3. Nonextractable Phenolic Acids Content in Several Foods Reported in the Literature According to Different Methodologies content (mg/100 g sample

methodology

compda,b

FW or 100 mL)

ref

Alkaline Hydrolysis of the Residue, Followed by HPLC, HPLC-MS, or GC-MS Analysis corn refined flour

residue of aqueousorganic extraction subjected to NaOH treatment at room temperature during 4 h, followed by HPLC analysis

209

Fer

67

whole-grain

residue of aqueousorganic extraction subjected to NaOH treatment

32

Fer

77

1260

DehydroDiFer

78

59

DehydroDiFer

78

174 ( 2

Fer

25

14

Fer

79

2 ( 0.1

Fer, Sal, p-HBA,Van

80

4+1

Sin dehydrodimer

81

13

4-OHPhAc, Van, Caf,

82

wheat flour maize insoluble dietary fiber

at room temperature during 2 h, followed by HPLC analysis insoluble dietary fiber (obtained according to AOAC method) subjected to 2 M Na OH treatment during 18 h at room temperature, followed by extraction with diethyl ether and GC-MS analysis

maize soluble dietary fiber corn whole-wheat

soluble dietary fiber (obtained according to AOAC method) subjected to 2 M Na OH treatment during 18 h at room temperature, followed by extraction with diethyl ether and GC-MS analysis residue of aqueousorganic extraction subjected to 2 M NaOH

flour

treatment during 1 h, followed by extraction with ethyl

coffee brew

whole sample subjected to 2 M NaOH treatment containing

acetate and HPLC analysis 10 mM EDTA at 30 °C during 30 min followed by HPLC analysis red ginseng

residue of aqueousorganic extraction subjected to 4 M Na OH treatment at room temperature during 4 h, followed by extraction with diethyl etherether acetate and GC-MS analysis

rice insoluble dietary fiber

residue of organic extraction subjected to 2 M NaOH treatment at room temperature during 18 h, followed by extraction with ethyl acetate and GC-MS analysis

beer

whole sample subjected to 2 M NaOH, 10 mM EDTA treatment at 30 °C during 30 min followed by HPLC analysis

wheat bran

Syr, p-Coum, Fer, Sin

residue of aqueousorganic extraction subjected to 1 M NaOH

26

treatment during 16 h, followed by extraction with ethyl acetate and HPLC analysis rice bran


85-DiFer, 55-DiFer,

83

8-O-4-DiFer, 85-Benz DiFer 243c

Fer, p-Coum

84

60135

p-Coum, Fer, 550 -DiFer,

85

treatment during 4 h, followed by HPLC analysis whole-grain barley flour


8-O-40 -DiFer, 850 -DiFer,

treatment at room temperature during 1 h, followed by extraction with ethyl acetate and HPLC analysis

malt (malted barley) triticale bran

TriFer

whole sample subjected to 2 M NaOH, 10 mM EDTA treatment

7299

Fer, p-Coum

86

137 ( 1

Fer, p-Coum, m-Coum,

87

at room temperature during 24 h, followed by extraction with ethyl acetate and HPLC analysis residue of aqueousorganic extraction subjected to 2 M NaOH, 0.1% EDTA treatment at room temperature during 4 h,

Van, Gal, Chlor

followed by extraction with ethyl acetate and HPLC analysis wheat bran


189239

Fer, p-Coum, Sal, Sin, Van

74

32168

Fer

88

54

880 -DiFer, 850 -DiFer,

89

treatment at room temperature during 4 h, followed by extraction with diethyl ether and ethyl acetate and millet

HPLC analysis residue of aqueousorganic extraction subjected to 2 M NaOH treatment at room temperature during 4 h, followed by defatting, extraction with ethyl acetate and HPLC-MS analysis

asparagus

whole sample subjected to 2 M NaOH treatment at room temperature

550 -DiFer, 8-O-40 -DiFer

during 18 h, followed by extraction with ethyl acetate and HPLC analysis medlar

residue of aqueousorganic extraction subjected to 2 M NaOH treatment at room temperature during 4.5 h, followed by extraction with diethylether and HPLC-MS analysis 12719

0.51.0

Prot, p-HBA, Caf,

75

p-Coum, o-HBA, m-HBA



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Table 3. Continued content (mg/100 g sample young mandarin

methodology

compda,b

FW or 100 mL)

residue of aqueousorganic extraction subjected to 4 M NaOH,

39107

Caf, p-Coum, Fer, Sin,

ref 90

Prot, p-HBA, Van

10 mM EDTA treatment at room temperature during 4 h, followed by HPLC analysis Acid Hydrolysis of the Residue, Followed by HPLC or HPLC-MS Analysis black olive

residue of aqueousorganic extraction subjected to 6 M HCltreatment

1440

Sin

91

during 3 h, followed by extraction with ethyl acetate and HPLC analysis black currant pomace

residue of aqueousorganic extraction subjected to 2 M HCl treatment in methanol 60% at 90 °C during 90 min, followed by HPLC analysis

41 ( 1

Caf equivalents

92

vegetables consumed


267 ( 17

Prot, p-HBA

53

78 ( 6

Feru, p-HBA, Prot, Van

23

74237

Fer, Sal, Sin, Van, Syr, Caf

74

1491 ( 30

Prot, Caf derivative,

68

in the Spanish diet apple

(90:10, v/v) treatment at 85 °C during 20 h, followed by HPLC analysis residue of aqueousorganic extraction subjected to methanol/H2SO4 (90:10, v/v) treatment at 85 °C during 20 h, followed by HPLC-MS analysis

wheat bran

residue of aqueousorganic extraction subjected to 6 M HCl treatment at room temperature during 4 h, followed by extraction

wheat bran


with diethyl ether and ethyl acetate and HPLC analysis (90:10, V/V) treatment at 85 °C during 20 h, followed

Cin, Fer

by HPLC-MS analysis Enzymatic Hydrolysis of the Residue, Followed by HPLC Analysis tomato peel dietary fiber

total dietary fiber (obtained according to AOAC method) subjected

69 ( 15

Chlor, p-Coum,

93

p-Coum der

to treatment with Biocellulase, followed by HPLC analysis Combination of Hydrolysis of the Residues, Followed by HPLC Analysis

barley

whole sample subjected to H2SO4 at 100 °C during 60 min, followed

48

Fer

94

134161c

p-HBA, Fer, Chlor,

95

by α-amylase treatment and HPLC analysis barley

whole sample is subjected to 0.2 N H2SO4 at 100 °C during 2 h, followed by treatment with α-amilase and cellulase and HPLC analysis

wheat bran

residue of aqueousorganic extraction subjected to 6 M HCl treatment at room temperature during 4 h and 2 M NaOH at

Van, p-Coum 212430

Fer, p-Coum, Sal,

74

Van, Syr, Sin

room temperature during 4 h, followed by extraction with diethyl ether and ethyl acetate and HPLC analysis

In decreasing order of content. b 4-OHPhAc, 4-hydroxyphenylacetic acid; 50 -DiFer, 50 -diferulic acid; 550 -DiFer, 550 -diferulic acid; 850 -Benz DiFer, 850 -benzofuran diferulic acid; 850 -DiFer, 850 -diferulic acid; 880 -DiFer, diferulic acid; 8-O-40 -DiFer, 8-O-40 -diferulic acid; Caf, caffeic acid; Cin, cinnamic acid; Chlor, chlorogenic acid; p-Coum, p-coumaric acid; p-Coum der, p-coumaric acid derivatives; DehydroDiFer, dehydrodiferulic acids; Fer, ferulic acid; Gal, gallic acid; m-HBA, m-hydroxybenzoic acid; o-HBA, o-hydroxybenzoic acid; p-HBA, p-hydroxybenzoic acid; Prot, protocatechuic acid; Sal, salicylic acid; Sin, sinapic acid; Syr, syringic acid; TriFer, triferulic acid; Van, vanillic acid. c Results expressed in dry weight. a

cell-wall fraction of postharvest banana.41 In these foodstuffs, nonextractable phenolic acids constitute a smaller fraction of total phenolic acids than in cereals. 4.2. Acid Hydrolysis Followed by HPLC-UV or HPLC-MS. Acid hydrolysis, with either HCl or H2SO4 and usually with heat too, has also been applied to the residues left after extraction prior to the determination of nonextractable phenolic acids by HPLC. Using these methods, caffeic acid, chlorogenic acid, pcoumaric acid, ferulic acid, p-hydroxybenzoic acid, protocatechuic acid, salicylic acid, sinapic acid, syringic acid, and vanillic acid have been identified, mainly in cereal samples but also in apple, fruit or vegetable mixtures, and black olive and blackcurrant pomace (Table 3). Hydrolysis conditions (methanol/ H2SO4 10:1 v/v at 85 °C for 20 h) similar to those employed to hydrolyze polysaccharides in plant-based foodstuffs for chemical

analysis were used by Arranz et al.40 to determine nonextractable phenolic acids in several cereal samples, and the results were compared with those generated by the most common alkaline hydrolysis procedure (2 M NaOH at room temperature for 4 h). Acid hydrolysis released nonextractable phenolic acids more efficiently than alkaline hydrolysis, yet the latter yielded more ferulic acid, the phenolic acid for which the method was originally developed. Similarly, Verma et al.74 reported losses of caffeic acid during alkaline hydrolysis, while the same conditions yielded more p-coumaric acid than acid hydrolysis did. These results show that, to determine all nonextractable phenolic acids present in a food sample, a combination of acid and alkaline hydrolysis procedures may be necessary. 4.3. Enzymatic Hydrolysis Followed by HPLC-UV or HPLC-MS. Enzymatic hydrolysis has also been used to release nonextractable 12720


Journal of Agricultural and Food Chemistry phenolic acids. Bartolome et al.98 compared the efficiency of different enzymes to release ferulic acid and p-coumaric acid from spent barley grain (a byproduct of the brewing process). Ultraflo L (a mixture of β-glucuronidase and xylanase) was the most efficient enzyme mixture, but the release efficiencies for ferulic and p-coumaric acids were only 70% and 8%, respectively, of those obtained using alkaline hydrolysis. In another report, enzymatic treatment of blackcurrant pomace with a cellulase (C013L) was followed by acid hydrolysis of the residue, in which nonextractable phenolic acids, including both hydroxybenzoic and hydroxycinnamic acids, were detected.99 Recently,93 enzymatic extraction, a maceration process, and ultrasound-assisted extraction of phenolic acids from tomato peel dietary fiber were compared; the conclusion was that enzymatic extraction was the least efficient of the three.

5. OTHER NONEXTRACTABLE PHENOLICS Flavonoids other than flavan-3-ols may be found in the nonextractable fraction, although this aspect has not been thoroughly studied. Kapasakalidis et al.99 performed acid hydrolysis on residues of blackcurrant pomace subjected to enzymatic hydrolysis, and they found a flavonol content of 1530 mg/ 100 g fw. They suggested that this may correspond to flavonol glycosides present in cell vacuoles. The presence of flavonols associated with dietary fiber in tomato peel, wine, beer, and roselle tea (made from the flower of Hibiscus sabdariffa) has also been reported,93,100102 although the exact nature of these associations remains to be elucidated; in the case of tomato peel dietary fiber, flavanones were also detected. Finally, nonextractable catechins (monomeric flavanols) have also been detected after performing acid hydrolysis on extraction residues from mixtures of fruits, vegetables, cereals, pulses, or nuts.40 The exact nature of the interactions among monomeric flavanols, flavonols, and other subclasses of flavonoids with the solid matrix and the relevance of these associations deserve further attention. The nonextractable catechins detected might come from terminal units of PA, but this does not seem likely as cleavage of PA under acid conditions similar to those used by Arranz. et al.68 has not been observed. Anthocyanins constitute a separate case in the study of NEP. It was recently reported that data included in the USDA database for the extractable anthocyanin content in banana, as determined after acid hydrolysis, may correspond to the hydrolysis of PA.103 Similarly, some of the nonextractable anthocyanin content reported after acid hydrolysis of extraction residues40,41,92,99 may actually correspond to hydrolyzed PA. In agreement with this, nonextractable anthocyanins were only found after acid hydrolysis and not after alkaline hydrolysis of cereal products.68 Whether there actually is such a fraction of nonextractable anthocyanins remains to be properly established. Moreover, when analyzing this fraction, it should also be considered that the use of glycosidases to release anthocyanins from the food matrix may cause anthocyanins (anthocyanidin glycosides) to transform into their corresponding less stable aglycones or anthocyanidins;99 hydrolysis conditions should therefore be optimized to reduce such degradation. 6. PERSPECTIVES Over recent decades, hundreds of papers have reported the determination of EP in foodstuffs. In contrast, only a few dozen papers have focused on the NEP content, and most of these

REVIEW

studies have focused on particular types of phenolics but not on this fraction of dietary phenolic compounds as a whole. With regard to analytical procedures, only the analysis of insoluble phenolic acids has been satisfactorily standardized because of their significant content in cereals. There is not yet a general analytical method for the analysis of the other classes of NEP (e.g., NEPA), and the diversity of methods makes it difficult to compare results from different groups. Therefore, there is a need, first, to develop and validate techniques for the analysis of NEP and, second, for a systematic analysis of NEP content (including all the different subclasses) of a wide range of samples corresponding to common diets. This would allow the integration of this information into databases for NEP content in foods, as has already occurred for EP.2,104 This in turn would allow a more accurate calculation of the total polyphenol intake of different populations; which is currently clearly underestimated due to the lack of reliable data regarding the nonextractable fraction. The first attempt to consider the whole intake of NEP (phenolic acids, NEPA, and hydrolyzable tannins) in a diet was recently made. However, the authors pointed out that the determinations were carried out either by HPLC-MS or spectrophotometry depending on the type of phenolic and that a single method that is valid for the three groups is still lacking.40 As the role of the nonextractable fraction of food phenolic compounds that are constituents of dietary fiber is increasingly acknowledged,18,19 thorough evaluations of the intake of this fraction as a whole, as well as of its individual constituents, should help to fully characterize the possible associations between the intake of polyphenol-rich dietary fiber and the prevention of disease. Finally, the few studies dealing specifically with the bioavailability of phenolic compounds associated with the food matrix11,15,16,39 suggest that consumption of NEP may lead to the gradual release of phenolic species during transit along the gastrointestinal tract. More studies of the specific bioavailability of NEP are needed to complete the picture of this fraction of dietary phenolic compounds that is emerging as an important component of fruits and vegetables.

’ AUTHOR INFORMATION Corresponding Author

*Phone: (+34) 93 400 61 00. Fax: (+34) 93 204 59 04. E-mail: [email protected]. Funding Sources

This work was supported by the Spanish Ministry of Science and Innovation (AGL2009-12374-C03-03/ALI). J.P.-J. thanks this Ministry and ISCIII for granting her a Sara Borrell postdoctoral contract (CD09/00068).

’ ACKNOWLEDGMENT Language revision by Christopher Evans is appreciated. María Luisa Mateos-Martín is acknowledged for her help in the preparation of Figure 1. ’ ABBREVIATIONS USED 4-OHPhAc, 4-hydroxyphenylacetic acid; 50 -DiFer, 50 -diferulic acid; 550 -DiFer, 550 -diferulic acid; 850 -Benz DiFer, 8 50 -benzofuran diferulic acid; 850 -DiFer, 850 -diferulic acid; 8 80 -DiFer, diferulic acid; 8-O-40 -DiFer, 8-O-40 -diferulic acid; Caf, 12721


Journal of Agricultural and Food Chemistry caffeic acid; Cin, cinnamic acid; Chlor, chlorogenic acid; p-Coum, p-coumaric acid; DehydroDiFer, dehydrodiferulic acids; DP, degree of polymerization; EPA, extractable proanthocyanidins; EP, extractable phenolics; Fer, ferulic acid; Gal, gallic acid; m-HBA, m-hydroxybenzoic acid; o-HBA, o-hydroxybenzoic acid; p-HBA, p-hydroxybenzoic acid; NEPA, nonextractable proanthocyanidins; NEP, nonextractable phenolics; PA, proanthocyanidins; Prot, protocatechuic acidSal, salicylic acid; Sin, sinapic acid; Syr, syringic acid; TriFer, triferulic acid; Van, vanillic acid

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