FACTORS AFFECTING THE PHENOLIC ACID CONTENT

IV. FACTORS AFFECTING THE PHENOLIC ACID CONTENT

Accumulation of phenolic acids in fruits and vegetables varies strongly in relation to different factors: (1) the genetic background, (2) the stage of development of the plant organ, and (3) the environmental and culture conditions. All these changes involve the regulation of phenolic metabolism (biosynthesis and degradation) and its integration in the program of cell and tissue differentiation, the control of gene expression, and the regulation of enzyme activities and of their compartmentation. The biosynthetic pathway of phenolic compounds is now well known and is not discussed here as it is not specific to fruits or vegetables. The deamination of phenylalanine, previously formed via the shikimate pathway, yields the nonphenolic cinnamic acid that is the direct precursor of the different HCAs and of their coenzyme A (CoA) esters through the general phenylpropanoid metabolism [64]. CoA esters of HCAs are common precursors of various other classes of phenolic compounds (e.g., HBA, anthocyanins, tannins, lignins). The structure and regulation of genes encoding the enzymes of the general phenylpropanoid metabolism from a number of plant species have been studied. Gene expression and enzyme activity are subject to large fluctuations in relation to endogenous and external factors (e.g., temperature, light, various types of stress) [64]. Furthermore, the enzymatic oxidation of phenolic compounds is of vital importance to the quality of fruits, vegetables, and their products, because of the formation of undesirable color and flavor and the loss of nutrients during processing (see Secs. V and VI).

A. Changes in the Phenolic Acid Patterns

According to Species and Cultivars Numerous factors may influence considerable qualitative and quantitative modifications in the phenolic acid patterns of fruits and vegetables from different species and cultivars. Although HBAs are present in most fruits and vegetables, the HBA content of fruits is generally low, except in certain fruits of the Rosaceae family and in particular blackberry, in which protocatechuic and gallic acid content may be very high: respectively, 189 and 67 mg/kg fresh weight in the richest cultivars (Table 1). Great interspecific differences in HBA exist in fruits and vegetables with regard to both quality and quantity, and such differences are also found among the varieties of the same species. In fact, qualitative and quantitative investigation of the native molecules of HBA derivatives is still inconclusive and it is difficult to draw general and final conclusions.

Comparing HCA content in numerous fruits and vegetables reveals enormous variations among species (Tables 2 and 3). Chlorogenic acid itself is especially abundant in coffee beans (6–10% on a dry matter basis), C. olitorius leaves (3.8 g/kg fresh weight [FW]), blueberries (2 g/kg FW), corn salad (approximately 1 g/kg FW), loquat fruit (329–907 mg/kg FW), eggplant (575–632 mg/kg FW), purple carrot (541 mg/kg FW), and artichoke (433 mg/kg FW), whereas it is present only as traces in Cucurbitaceae [1,2,4,24,40,43]. Similar variations are also frequently reported between cultivars of the same species, for example, 26 to 510 mg/kg chlorogenic acid in apples [2,65–68] or 6 to 621 mg/kg caftaric acid in grapes [2].

The relative proportions of each HCA are a characteristic of fruit in the mature stage. Caffeic acid is frequently the most abundant phenolic acid. It commonly exceeds 75% of total HCA in numerous fruits and vegetables (e.g., apple, plum, tomato, grape, red cabbage, endive, artichoke, potatoes) and may even form almost the entire HCA content in extreme cases, such as eggplant or certain blueberries. In some cases (e.g., pineapple, white currants, savoy cabbage, faba bean pod, spinach), p-coumaric acid is predominant, and in rare cases—in a few varieties of raspberry, for example—only traces of caffeic acid are found, whereas the other HCAs are dominant. Whereas ferulic acid usually forms only a small percentage of total HCA in fruits and vegetables, it can reach and even exceed 50% in peppers, some citrus, and some white grape cultivars [27]. Sinapic acid has been reported more rarely in fruits and is generally only observed as traces [31,32], whereas it may be abundant in various Brassica vegetables (Table 3).

The balance of the various HCA conjugates also characterizes fruit and vegetable species and cultivars. Thus, the HCA quinic ester patterns of stone and pome fruit differ considerably: the 3-isomers are major constituents in cherry and plum, whereas the 5-isomers are principally found in apple and pear [[1,2,65,69,70] (Table 2)]. An identical difference is observed between Brassica (rich in 3-isomers) and Asteraceae (rich in 5-isomers) vegetables (Table 3). In most cases (e.g., apple, tomato, artichoke, carrot), glycosylated derivatives are distinctly less abundant than quinic esters, whereas the opposite proportion is more rarely observed (kale, raspberry). The relative proportions of glucose esters and glucosides of both HBAs and HCAs are also variable with the different species of fruits and vegetables (Tables 1, 2, and 3). HCA ester content can be selected, among other parameters, to discriminate between grape species, but the most reliable criterion when comparing cultivars of the same species appears to be the percentage of each HCA, as shown in the case of V. vinifera, where the percentage of p-coumaroyl and caffeoyltartaric esters can be used to discriminate between varieties for taxonomic purposes [27].

B. Changes with Tissular Localization

The highest levels of HCA derivatives are frequently found in the external part of fleshy fruits, as shown for chlorogenic acid in pear and peach and 3-caffeoylquinic acid in cherry [2,70,71]. On the contrary, chlorogenic acid is often more abundant in the core than in the peel of apple, although this distribution depends on the cultivar [67,72]. Tomato is one of the better-known examples of HCA distribution: quantity of quinic esters in ripe fruit was found to be higher in the pulp than in the pericarp, whereas the opposite was found for glucose derivatives [2]. Tissue compartmentation of p-coumaroylglucose, caffeoyl, and 3,4-dimethoxycinnamoyl glycosides makes it possible to discriminate clearly between the placenta and the pericarp of Capsicum frutescens [15]. In grape, although the level was always higher in skin than in pulp, the percentage of caffeoyltartaric acid was higher in the pulp, whereas the opposite was true for p-coumaroyltartaric acid [27]. p-Coumaroylgalactaric and feruloylgalactaric acids are also more abundant in the outer part of flavedo and albedo of citrus [2], and ferulic and sinapic acid concentrations are higher in sour orange peel [73]. Distribution of HCA derivatives is even more complex in certain cases, such as pineapple: in addition to the gradients between the inside and outside, there are very important longitudinal gradients, probably related to different stages of maturity of each of individual fruits that make up the pineapple [2].

The outer part of many plant organs consumed as vegetables also shows the highest concentrations of HBA and HCA conjugates. For example, puree from nonpeeled carrot roots contained 104 mg/kg chlorogenic acid, whereas only 28.3 mg/kg were found after removing approximatively 2 mm of periderm tissue [74]. In potato tuber, about 50% of the phenolic compounds (mostly chlorogenic acid and other mono- or di-caffeoylquinic esters) were located in the peel; the remainder decreased in concentration from the outside to the center of 18 Fleuriet and Macheix tubers [11]. In cereals, bran always presents the highest antioxidant activity, which is due to the localization of bound phenolic acids in the grain: the outer layers (husk, pericarp, testa, and aleurone layer) contain the greatest concentrations of total phenolics, whereas concentrations are considerably lower in the endosperm. About 80% of ferulic acid of both rye and wheat grain was found in the bran [25,75].

C. Changes with Physiological Stage

Concentrations of phenolic acids in a plant organ result from a balance between biosynthesis and further metabolism, including turnover and catabolism. Considerable variations are generally observed in the amount of phenolic acids according to the physiological stage when plant organs are picked up to be consumed or transformed by humans. This may concern each type of organ (leaves, flowers, stalks, tubers, roots, etc.), but the most spectacular cases are those of fruits, as considerable variations in phenolic compounds occur during maturation.

Concentrations of soluble forms of phenolic acid conjugates (expressed per unit of fresh or dry weight) are generally highest in young fruits, with a maximum during the early weeks after blossoming and a rapid decrease during fruit development [2,27]. In different apple cultivars, for example, maximal concentrations of chlorogenic acid, p-coumaroylquinic acid, and p-coumaroylglucose were found in very young fruits, followed by a constant decrease [2,72].

These changes make it possible to divide the life of a fruit into two main periods.

During the first (approximately 2 months in apple), HCA derivatives accumulate in the fruit with a positive balance among in situ biosynthesis, migration, and possible reutilization. However, in the second period, this balance becomes negative and the overall HCA content in the fruit falls.

Loquat fruit represents an exception as the concentration of chlorogenic acid increased during maturation and became more prominent than neochlorogenic acid and all other phenolic compounds identified in this fruit [24]. The late accumulation of chlorogenic acid in this fruit results from the activation of its metabolism and especially from an increase in the enzyme activities of phenylalanine ammonia-lyase, 4-coumarate:CoA ligase, and hydroxycinnamoyl CoA:quinate hydroxycinnamoyltransferase. Consequently, the metabolism of chlorogenic acid may be considered to be a biochemical marker for maturation of loquat fruit. In certain fruits the disappearance of phenolic acids may occur in relation to the biosynthesis of other phenolic compounds, for example, the decrease in hydroxycinnamic conjugates during growth and maturation of Vitis vinifera berries and the rapid increase in anthocyanin levels that occurs at the same time in the red cultivars [2]. In fruits of chili pepper (Capsicum frutescens) the onset of capsaicinoid accumulation and ‘‘ligninlike’’ material parallels the disappearance of the three cinnamoyl glycosides, which may be considered a source of precursors in capsaicinoid biosynthesis [15].

Quantitative changes are sometimes accompanied by qualitative ones. Thus, in tomato (cv. cerasiforme), most HCA conjugates appear during growth, and ripe fruit contains 11 different conjugates, whereas very young green fruit contains only chlorogenic acid [2]. Some compounds are characteristic of a physiological stage: chlorogenic acid forms 76% of total HCA derivatives in the unripe fruit, then falls to 15% in ripe fruit. By contrast, HCA glucosides form 23% and 84% during the same periods, a finding that may suggest certain metabolic relations between these compounds. Thus, growth and matutation of the tomato fruit are characterized by different expressions of the metabolism of HCA derivatives. In growing pulp it is mainly oriented toward the accumulation of quinic derivatives, whereas glucose derivatives (particularly glucosides) accumulate in the pericarp during maturation. A good correlation between variations in activities of enzymes of phenylpropanoid pathway and accumulation of phenolic compounds is observed in tomato. These data led to the notion that phenolic acids and their metabolism may be suitable markers of maturation.

Variations in the phenolic acid levels during growth and development of vegetables and cereals are not so homogeneous as those reported for fleshy fruits. It depends mainly on two parameters: the nature of the plant organ (leaves, tubers, grains, etc.) and the subcellular localization of the phenolic conjugates that accumulate either as soluble forms in the vacuole or linked to the cell wall.

During the development of soft or durum wheat grains, ferulic acid is mainly present as bound forms and accumulates during the milk stage, in correlation with high activities of phenylalanine and tyrosine-ammonia-lyases [76,77]. A decrease in ferulic acid level is then observed during grain ripening, but this may be due in part to the formation of alkali-resistant bonds in crosslinked polymers in cell walls, parallel to the progressive decrease in the grain water content. Peroxidases should play a role in the formation of these covalent linkages [56] as their activity occurs even in the last stages of grain ripening [77]. Changes in the ratios of different dehydrodiferulic acids were also observed in the cell walls during the growth period of sugar beet root along with a decrease of more than 50% [78]. These changes may be related to the major expansion of the storage root during the latter part of the growth period.

The integration of phenolic metabolism in the program of plant development raises the question of the possible role of these substances in physiological regulations. They have sometimes been implicated in the control of growth, maturation, and abscission [2,64], but these aspects are rather speculative and are not discussed here. HCA derivatives and coumarins may act as in situ inhibitors of seed germination in berries or other fleshy fruits, either directly or indirectly through the control of oxygen consumption [79]. It is also well known that various phenolic compounds play a role in the interactions between plants and microorganisms [64], and allelopathic effects of HBA and HCA have been reported in wheat root exudates [80]. Furthermore, they clearly participate in the resistance of plants to biological and environmental stresses [64] and play an important role in the browning capacity of plant organs [2].

D. Changes with Environmental Factors

Secondary metabolism, and in particular phenolic metabolism, largely depends on external factors such as light, temperature, and various stresses [64]. Although this has been mainly studied in relation to flavonoid production, we report here only information related to phenolic acids. Caffeoyl quinic ester concentrations of potato tubers steadily increased after light exposure, whereas they decline during prolonged storage in the dark [81,82]. Rates of accumulation were influenced by cultivar, storage period, and light source. Furthermore, ratios of 5-:4-:3-caffeoylquinic esters were modified, but this had no effect on blackening of tubers. In Vitis vinifera berries, some phenolic components of aroma (e.g., methyl vanillate, methyl syringate) were significantly less abundant after bunch shading, whereas this treatment did not modify some other compounds (e.g., methyl salicylate, 4-vinylphenol) [83]. g-Irradiation treatment degradates phenolic acids in a first step, but it later stimulates phenylalanine ammonia-lyase and the biosynthesis of p-coumaric acid, flavanones, and flavones in clementines [84]. p-Coumaric content was then particularly high in irradiated fruits after 49 days of storage at 3 jC and could be related to better resistance to pathogens. Similar data have been obtained in potato: irradiation of tubers to inhibit sprouting caused an initial reduction in phenolics, but an increased formation of chlorogenic acid and its isomers was later observed during storage [11].

Two major types of observations reveal relations between phenolic acids and temperature. First, various data link the overall effects of climate and local environmental conditions with the accumulation of phenols. Another group of results is taken from the frequent use of low temperatures in postharvest storage of fruits and vegetables. In this case, physiological disturbances may occur even when temperatures are maintained above 0 jC. Such effects are generally referred to as chilling injury, and they frequently take the form of discoloration, for which phenolic compounds may be directly responsible. Several examples show great variations in phenolic compounds or in the enzymes of their metabolism during cold, but the changes vary greatly, depending on the species and cultivar [2]. Cold storage of apples did not induce important variations in their chlorogenic acid content for up to 9 months, and the health benefits of phenolics should be maintained during long-term storage, although the response may slightly differ with cultivars [66,67]. On the contrary, numerous other examples indicate an increase in phenolic acids during cold storage: chlorogenic acid in Anjou pear [2], ellagitannins and p-coumaroylglucose in strawberries [19], a sucrose ester of ferulic acid in the peel of beetroot [53], chlorogenic acid in different cultivars of potato tubers stored at 0 jC [11,85], whereas a decrease was also observed at 5 jC in other cultivars [82]. p-Coumaric and caffeic derivatives also accumulate in pineapple during storage at 8 jC, and the content in phenolic acids was multiplied 10-fold 15 days after the fruits were returned at 20 jC [2]. In tomato, a fairly specific action of low-temperature storage was observed on chlorogenic acid metabolism. Among the enzymes tested, levels of phenylalanine ammonia-lyase and hydroxycinnamoyl-quinate transferase, two enzymes that allow synthesis of chlorogenic acid, increased considerably, in relation to chlorogenic acid accumulation [2].

Storage of bean seeds at high temperature (35 jC) and humidity causes textural defects along with an increase in free phenolic acids (caffeic, pcoumaric, ferulic and sinapic acids), a decrease in soluble esters, and a strong increase in ferulic acid bound to soluble pectins [59]. These modifications result in poor soaking imbibition of seeds and in prolonged cooking time.

Phenolic acids are directly implied in the response of plant organs to different kinds of stresses [64]: mechanical (wounding), chemical (various types of treatment), or microbiological (pathogen infection). Phenolic acids are involved in resistance in two ways: (1) by contributing to the healing of wounds by lignification of cell walls around wounded zones [86] and (2) through the antimicrobial properties demonstrated for many of them [87]. The compounds involved can be classified in three groups: (1) some are already present in the plant, and their level generally increases after stress; (2) others are formed only after injury but are derived from existing substances by hydrolysis or oxidation; (3) still others are biosynthesized de novo and can be classified as phytoalexins.

The effect of wounding has been particularly well studied in fruits [2] and in minimally processed fruits and vegetables [88]. The most immediate response to wounding is the oxidation of preexisting phenolic compounds and hence their degradation. Thus, the chlorogenic acid content of tomato fruit pericarp falls for 6 h after wounding, and activation of phenolic metabolism occurs later, with an increase in phenylalanine ammonia-lyase activity and consequent accumulation of chlorogenic acid, feruloyl-, p-coumaroyl, and sinapoyl-glucose [2]. Such an accumulation of caffeoylquinic acids or caffeoyl-and dicaffeoyl tartaric acids is also observed in shredded carrots and in minimally processed lettuce leaves [89–91], although its intensity may depend on storage conditions, either in air or in controlled atmospheres. Along with polyphenoloxidase and peroxidase activities, this increase in phenolic substrates is responsible of the browning of wounded tissues that shortens storage life of the product. Finally, the third aspect of response to wounding is the formation of healing tissues (‘‘wound lignin’’ or suberin) that protect plant organs from water loss and also form a physiological The increase in phenolic content with pathogen infection has been well documented in cell suspension cultures, especially at a molecular level [64]. It was postulated that as a defense mechanism, ferulic and p-coumaric acids are esterified to wall polysaccharides, possibly rendering the wall resistant to fungal enzymes either by masking the substrate or by altering the solubility properties of these wall polysaccharides [56,64]. Phenolic acids also possess antimicrobial properties, and chlorogenic acid and related compounds may function to arrest Molinia fructicola, in quiescent infections associated with immature and ripening peach fruits [92]. Chlorogenic and p-coumaroylquinic acids in apple are inhibitors of both Botrytis sp. spore germination and mycelial growth [2]. When their effects on growth of certain fungi are compared, p-coumaroylquinic is more inhibitory than chlorogenic at the same concentrations for B. cinerea and Alternaria sp., whereas P. expansum is less sensitive. Both quinic derivatives are stimulatory at low concentrations for Botrytis and Penicillium sp.

The acquisition of antimicrobial properties by phenolic compounds may derive from oxidation or hydrolysis. First, o-quinones are generally more active than o-diphenols and browning intensity is often greatest in highly resistant plants, suggesting that black and brown pigments contribute to resistance. Hydrolysis may be carried out by fungal pectic enzymes, as suggested by the appearance of free p-coumaric, caffeic, and ferulic acids in apple infected with P. expansum [2]. In this case, the damage does not cause much browning around the infection site because of the inhibition of the phenolase system by acids released after hydrolysis of chlorogenic and p-coumaroylquinic acids in the fruit. Again in apple, antifungal compounds, such as 4-hydroxybenzoic acid produced after infection with Sclerotinia fructigena, are thought to be derived from the transformation of chlorogenic acid by the fungus. Free phenolic acids are the best inhibitors of growth of the fungi appearing during the postharvest storage, and their structure/activity relationships have been studied in vitro [87].

An additional methoxy group caused increased activity of HBA and HCA derivatives. Thus, ferulic and 2,5-methoxybenzoic acids showed a strong inhibition against all fungi tested. Certain phenolic compounds can be biosynthesized de novo after infection.

These compounds, which do not exist before infection and which have antimicrobial properties, are called phytoalexins. They are produced by plants as defense mechanisms in response to microbial infection [64,93], but the accumulated compounds are often flavonoids or coumarins, although benzoic acid itself has been shown in apple after infection.

Both constitutive phenolic acids and phytoalexins may be involved in the resistance of potatoes to Erwinia sp. [94]. This resistance could result from the increase of caffeic, chlorogenic, and ferulic acids and the formation of suberized barriers after wound injury or pathogen attacks [11,63,94]. Both constitutive phenolic acids and phytoalexins may be involved in the resistance of potatoes to Erwinia sp. [94]. This resistance could result from the increase of caffeic, chlorogenic, and ferulic acids and the formation of suberized barriers after wound injury or pathogen attacks [11,63,94]. Both polyesterforming HCAs and ligninlike monolignols of potato suberin constitute a dense covalent network capable of repelling water and protecting the cell wall from pathogenic attack [63]. The involvement of phenolic acids in potato resistance is nevertheless selective as they protect against some but not all pathogens: although levels of chlorogenic acid increased after infection of potato tubers with the fungus Phytophtora infestans, no differences in the levels were observed in resistant or nonresistant cultivars [95].