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].
