Food Chemistry 173 (2015) 501–513

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Review

Bioactivity of phenolic acids: Metabolites versus parent

compounds: A review
Sandrina A. Heleno a,b, Anabela Martins a, Maria João R.P. Queiroz b, Isabel C.F.R. Ferreira a,⇑
a
Centro de Investigação de Montanha, Escola Superior Agrária, Campus de Santa Apolónia, apartado 1172, 5301-854 Bragança, Portugal
b
Centro de Química, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: Phenolic acids are present in our diet in different foods, for example mushrooms. Due to their bioactive
Received 8 August 2014 properties, phenolic acids are extensively studied and there is evidence of their role in disease prevention.
Received in revised form 17 September Nevertheless, in vivo, these compounds are metabolized and circulate in the organism as glucuronated, sul-
2014
phated and methylated metabolites, displaying higher or lower bioactivities. To clarify the importance of
Accepted 13 October 2014
Available online 19 October 2014
the metabolism of phenolic acids, knowledge about the bioactivity of metabolites is extremely important.
In this review, chemical features, biosynthesis and bioavailability of phenolic acids are discussed, as well
as the chemical and enzymatic synthesis of their metabolites. Finally, metabolite bioactive properties are
Keywords:
Mushrooms
compared with that of the corresponding parental compounds.
Phenolic acids Ó 2014 Elsevier Ltd. All rights reserved.
Biosynthesis/bioavailability
Metabolites
Chemical/enzymatic synthesis
Bioactivity

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501
2. Chemical features and biosynthesis of phenolic acids usually found in mushrooms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 502
3. In vivo human metabolism of phenolic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 502
3.1. Bioavailability of phenolic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 502
3.2. Conjugation reactions for metabolite formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
4. Bioactive properties of phenolic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506
5. Controversy on in vivo bioactivity of polyphenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
6. Chemical and enzymatic synthesis of phenolic acid metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510
7. Bioactivity of phenolic acids versus their metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510
8. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511

1. Introduction Almeida, Ferreira, Martins, & Vasconcelos, 2012), antimicrobial

(Alves et al., 2013), and antioxidant (Piazzon et al., 2012).
Mushrooms are rich sources of bioactive compounds with an However, very little is known about the phenolic acid bioactive
enormous variety of chemical structures (Ferreira, Barros, & forms in vivo and the mechanisms by which they may contribute
Abreu, 2009). In particular, different bioactive properties have been towards disease prevention. Moreover, several studies dealing with
attributed to phenolic acids from mushrooms, namely antitumor the biological effects of phenolic acids have ignored the question of
(Heleno, Ferreira, Calhelha, Esteves, & Queiroz, 2014a; Vaz, their achievable concentrations in the circulation after ingestion as
well as the possibility of metabolism (Rechner et al., 2002). There is
accumulating evidence suggesting that phenolic acids are rapidly
⇑ Corresponding author. Tel.: +351 273 303219; fax: +351 273 325405. metabolized in the human body (Nardini et al., 2009; Rechner
E-mail address: [email protected] (I.C.F.R. Ferreira).
et al., 2002; Scalbert & Williamson, 2000).

http://dx.doi.org/10.1016/j.foodchem.2014.10.057
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
502 S.A. Heleno et al. / Food Chemistry 173 (2015) 501–513

After absorption from the gastrointestinal tract, these molecules ferulic acids. It is believed that phenolic compounds were funda-
suffer conjugation reactions causing several changes in their initial mental for plants to conquer their terrestrial environment, such
structure, and circulate in human plasma in their conjugated as lignin which stimulates the development of the vascular system,
forms, such as glucuronide, methylated and sulphated derivatives. giving stiffness to the vessels (Gross, 1985).
These changes in their structures may increase or decrease the bio- Phenolic acids are synthesized from the shikimate pathway
activity of the initial phenolic acids (Piazzon et al., 2012; Rechner from L-phenylalanine or L-tyrosine (Rice-Evans, Miller, & Paganga,
et al., 2002). 1996) (Fig. 2 and Table 1). Phenylalanine and tyrosine are very
Therefore, detailed knowledge concerning the conjugative and important amino acids in this pathway since these amino acids
metabolic events and resulting plasma levels following the inges- are the common precursors for the majority of the natural phenolic
tion of a polyphenol-rich diet is crucial for understanding their products (Fig. 2 and Table 1).
bioactivity (Rechner et al., 2002). Despite the large amount of data Firstly, a deamination of the phenylalanine and/or the tyrosine
concerning the bioactivity of phenolic acids, only a few studies deal occurs giving cinnamic and/or p-coumaric acids, respectively. Cin-
with the bioactive properties of their metabolites, especially as namic and p-coumaric acid aromatic rings are then hydroxylated
most of those molecules are not commercially available (Piazzon and methylated to form its derivatives e.g., ferulic and caffeic acids.
et al., 2012). Deamination, hydroxylation and methylation are the main three
In this review, several features of the phenolic acids found in reactions involved in the formation of phenolic acids (Fig. 2 and
mushrooms will be discussed, namely their chemical characterisa- Table 1) (Gross, 1985). Benzoic acid formation can result from
tion, biosynthetic pathways, bioavailability and metabolism, as degradation of the side chain of cinnamic acid. As mentioned for
well as the chemical and enzymatic synthesis of glucuronated, sul- cinnamic and p-coumaric acids, the same hydroxylation and meth-
phated and methylated metabolites of different phenolic acids. The ylation reactions can occur in the aromatic ring of benzoic acid
antioxidant, antimicrobial and antitumor properties of the metab- giving the correspondent derivatives e.g., protocatechuic and
olites will be discussed and compared with the bioactivities of the p-hydroxybenzoic acids (Gross, 1985).
corresponding parental phenolic acid.

3. In vivo human metabolism of phenolic acids

2. Chemical features and biosynthesis of phenolic acids usually
found in mushrooms 3.1. Bioavailability of phenolic acids

Mushrooms have been extensively studied during the last few Despite the extensive literature describing the biological effects
decades due to their bioactive potential (Ferreira et al., 2009), attrib- of phenolic acids, little is known about how they are absorbed from
uted to different molecules including phenolic acids. These com- diet.
pounds (Fig. 1) have been identified in different mushroom species Phenolic acids are present in almost all plant-derived foods,
around the world (Kim et al., 2008; Puttaraju, Venkateshaiah, representing a significant portion of the human diet. The average
Dharmesh, Urs, & Somasundaram, 2006; Ribeiro, Valentão, phenolic acid intake in humans has been reported to be in the
Baptista, Seabra, & Andrade, 2007; Valentão et al., 2005). order of 200 mg/day depending on diet habits and preferences
Phenolic acids can be divided into two major groups, hydroxy- (Clifford & Scalbert, 2000). The most frequently encountered and
benzoic acids and hydroxycinnamic acids, which are derived from studied phenolic acids are caffeic and ferulic acids. Caffeic acid is
non-phenolic molecules of benzoic and cinnamic acid, respectively. also found in the form of esters, chlorogenic acid being the most
Chemically, these compounds have at least one aromatic ring in frequently encountered (Clifford & Scalbert, 2000). Coffee is
which at least one hydrogen is substituted by a hydroxyl group normally studied for the absorption of these molecules since it is
(Fig. 1). a good source of bound phenolic acids, such as caffeic, ferulic and
Phenolic compounds, including phenolic acids, are secondary p-coumaric acids (Nardini, Cirillo, Natella, & Scaccini, 2002). In
metabolites from plants and fungi. These compounds are produced patients that ingested a specific quantity of coffee, (Marmet,
for protection against UV light, insects, viruses and bacteria. There Actis-Goretta, Renouf, & Giuffrida, 2014) several methylated,
are even certain plant species that develop phenolic compounds to glucuronated and sulphated metabolites of phenolic acids circulat-
inhibit the growth of other plant competitors (allelopathy). Exam- ing in plasma, were identified. In another study, Fumeaux et al.
ples of phenolic acids with this allelopathic action are caffeic and (2010) described that after a specific dose of coffee, several

R1 O R1 O
2
R R2
OH OH

R3 R3
4
R R4
Substitution Cinamic acid derivatives Benzoic acid derivatives
R1=OH o-Coumaric acid -
R3=OH p- Coumaric acid p- Hydroxybenzoic acid
R3=R4=OH Caffeic acid Protocatechuic acid
R2=OCH3,R3=OH Ferulic acid Vanillic acid
R2=R3=OCH3 - Veratric acid
R2=R3=R4=OH - Gallic acid
R1=R4=OH - Gentisic acid
R2=R4=OCH3, R3=OH Sinapic acid Syringic acid
R1=OH, R4=HSO3 - 5- Sulphosalicylic acid
R2=R3=OH 3,4 or 5-O-caffeoylquinic acid * -
* The carboxylic group is esterified with quinic acid.

Fig. 1. Chemical structures of benzoic and cinnamic acid derivatives usually found in mushrooms.
 S.A. Heleno et al. / Food Chemistry 173 (2015) 501–513 503

Shikimic acid

Phenylacetic acids L- Phenylalanine

1 NH3 L- Tyrosine

C2H4 Cinnamic acid OH

10 NH3
2
9
Benzoic acid
OH 3 p-Coumaric acid
Other
derivatives p-Hydroxybenzoic acid
OH 11
Quinic acid
Other
OH 4 Caffeic acid derivatives
14 12
CH3
6
Protocatechuic or/ H
CH3 and Gentisic acid 3,4 or 5-O-
Ferulic acid
OH 5 caffeoylquinic
H OCH3 13
Vanillic acid Gallic acid
Sinapic acid
7
CH3 H

OCH3 8
Veratric acid

Syringic acid

Fig. 2. General representation of the phenolic acids biosynthesis derived from the shikimic acid pathway. The numbers find the correspondence in the entries of Table 1.

hydroxycinnamic acid sulphate and glucuronide conjugates were The bioavailability of polyphenols is crucial to their biological
present in biological fluids. Nardini et al. (2009) studied the properties. To study the bioavailability of these molecules, differ-
absorption of phenolic acids from white wine and reported that ent authors measured their concentrations in the plasma and urine
after the consumption of a single glass of wine, hydroxycinnamic after ingestion of a known content of pure compounds or incorpo-
acids are absorbed from the gastrointestinal tract and circulate in rated in food (Pérez-Jiménez et al., 2010).
the blood after being largely metabolized to glucuronide and sul- When ingested in the free form phenolic acids are rapidly
phate conjugates. absorbed by the small intestine and later conjugated (Scalbert &
In the past few decades, mushrooms have received special Williamson, 2000). Nevertheless, the chemical structures of the
attention because they are described as being rich sources of phe- compounds can also influence the conjugation reactions as well
nolic acids that are amongst the major contributors to their medic- as the amount of metabolites formed by the gut microflora in the
inal properties (Ferreira et al., 2009). Extensive knowledge of colon (Scalbert & Williamson, 2000). For example, in chlorogenic
phenolic acid bioavailability is essential to understand the conju- acid (a caffeic acid ester linked to quinic acid), the bound ester
gations and bioactivities in the organism. Phenolic acids are a con- can change its biological properties. In human tissues there are
siderable group inside the polyphenol family, and there is evidence no esterases able to release caffeic acid from chlorogenic acid
that when they are absorbed in the free form (as are mostly found (Plumb et al., 1999). Thus, the only local reaction for chlorogenic
in mushrooms), their absorption and conjugation, (specially glu- acid metabolism is in the colon by bacteria.
curonation), follows the same pathways as that of flavonoids and In agreement with this, ferulic or other hydroxycinnamic acids
other polyphenols (Cremin, Kasim-Karakas, & Waterhouse, 2001). bound to cell walls are also not released by human endogenous
Thus, during absorption, polyphenols are conjugated in the enzymes and require release by enzymes, such as xylanases and
small intestine, and later in the liver, where methylation, sulfation esterases of the colonic microflora (Kroon, Faulds, Ryden,
and glucuronidation, as the main conjugation reactions, take place Robertson, & Williamson, 1997). When these molecules are hydro-
(Manach, Scalbert, Morand, Rémésy, & Jiménez, 2004). This is a lysed by the colonic flora, the efficiency of their absorption is
very important process in not only detoxification, to avoid any reduced because flora can degrade the aglycones, releasing simple
potential toxic effects, but also because increasing their hydrophi- aromatic acids (Scalbert & Williamson, 2000). Therefore, the effi-
licity they can easily be eliminated by the biliary or the urinary ciency of absorption of phenolic acids is markedly reduced when
route. These conjugation mechanisms are very efficient, and agly- they are present in the esterified form rather than in the free forms
cones are present in the blood in very low concentrations after (Azuma et al., 2000; Olthof, Hollman, & Katan, 2001). Olthof et al.
the consumption of polyphenols. (2001) reported that in patients with colonic ablation, caffeic acid
Circulating polyphenols are conjugated derivatives that are was better absorbed than chlorogenic acid. In another report,
extensively bound to albumin. Generally polyphenols are secreted where chlorogenic acid was given to rats, no intact compound
by the biliary pathway into the duodenum, in the distal segments was detected in the plasma in the following 6 h, and the maximum
of the intestine where they are exposed to the action of bacterial concentrations of metabolites obtained after the administration of
enzymes, particularly b-glucuronidase. After this step, they can caffeic acid under the same conditions, were much higher (Azuma
be reabsorbed which may lead to a longer presence of polyphenols et al., 2000). Glucuronated metabolites, for example acyl glucuron-
within the body (Manach et al., 2004). ides can react with sulfhydryl and hydroxyl groups and can be
504 S.A. Heleno et al. / Food Chemistry 173 (2015) 501–513

Table 1
Main enzymes involved in the biosynthesis of phenolic acids through shikimate pathway from L-phenylalanine or L- tyrosine.

Entry Starting molecule Enzyme Final compound

1 O Phenylalanine ammonia lyase (PAL) O

OH OH

NH2

Phenylalanine Cinnamic acid

2 O Oxidase (presumed b-oxidation) O

OH OH

Cinnamic acid Benzoic acid

3 O Benzoic acid 4-hydroxylase O

OH OH

HO

Benzoic acid p-Hydroxybenzoic acid

4 O p-Hydroxybenzoic acid 3-hydroxylase O O

O HO
OH OH OH

HO O OH

p-Hydroxybenzoic acid Gentisic acid

5 O Protocatechuic acid 5-hydroxylase O

HO HO
OH OH

HO HO

Protocatechuic acid OH

Gallic acid
6 O Protocatechuic acid 3-O-methyltransferase O

HO H3CO
OH OH

HO HO

Protocatechuic acid Vanillic acid

7 O Vanillic acid 4-O-methyltransferase O

H3CO H3CO
OH OH

HO H3CO

Vanillic acid Veratric acid

8 O Vanillic acid 5 hydroxylase and vanillic acid O
5-O-methyltransferase
H3CO H3CO
OH OH

HO
HO
Vanillic acid
OCH3

Syringic acid
 S.A. Heleno et al. / Food Chemistry 173 (2015) 501–513 505

Table 1 (continued)

Entry Starting molecule Enzyme Final compound

9 O Cinnamic acid 4-hydroxylase O

OH OH

HO
Cinnamic acid p-Coumaric acid
10 O Tyrosine ammonia lyase (TAL) O

OH
OH
NH2
HO
HO
L- Tyrosine
p-Coumaric acid
11 O p-Coumaric acid 3-hydroxylase O

HO
OH OH

HO HO

p-Coumaric acid Caffeic acid

12 O Caffeic acid 3-O-methyltransferase O

HO H3CO
OH OH

HO
HO

Caffeic acid Ferulic acid

13 O Ferulic acid 5-hydroxylase and caffeic/5- O
hydroxyferulic acid O-methyltransferase
H3CO (COMT) H3CO
OH OH

HO HO

Ferulic acid OCH3

Sinapic acid
14 O 4-Caffeate CoA ligase and quinate O- O
O hydroxycinnamoyltransferase
HO OH
HO OH
HO +
OH O
HO OH
HO OH
O OH

OH
Caffeic + quinic acid HO

OH

3,4 or 5-O-caffeoylquinic acid

hydrolysed back to the aglycone (Spahn-Langguth & Benet, 1992). acyl groups occurs in plasma, bile and urine (Faed, 1984). Addition-
These molecules can go through covalent binding to plasma ally, if b-glucuronidase hydrolysis is used to liberate the aglycones
proteins (Zhou et al., 2001), and react with the glutathione and in a sample containing the rearranged isomers, the concentrations
transacylate cellular macromolecules (Faed, 1984). Moreover, acyl of glucuronic acid conjugates of carboxylic acid might be underes-
glucuronides go through rearrangements due to intramolecular timated (Faed, 1984).
acyl migration from –OH to the adjacent OH, giving different
positional isomers. 3.2. Conjugation reactions for metabolite formation
The rearrangement of glucuronides through the biosynthetic
C-1 isomers to other positional isomers is very important After ingestion and absorption, phenolic acids are conjugated by
because only 1-O-substituted acyl glucuronides are substrates of methylation, sulfation and glucuronidation reactions that are
b-glucuronidase, an enzyme commonly used to identify these con- controlled by specific enzymes that catalyse these steps (Fig. 3).
jugates (Sinclair & Cardwell, 1982) when measuring the concentra- Catechol-O-methyltransferase catalyses the transfer of a methyl
tions of phenolic acids present in biological fluids. The migration of group from S-adenosyl-L-methionine to polyphenols that have an
506 S.A. Heleno et al. / Food Chemistry 173 (2015) 501–513

Dietary phenolic acids

Small Intestine
Glucoronidation→ UDP- glucoronosyltransferases
Methylation → Catechol-O-methyl transferases

Bile

Liver Colon
Hydrolysis → Esterases
Glucoronidation → UDP- glucoronosyltransferases
Methylation → Catechol-O-methyl transferases
Sulfation → Sulfotransferases

Feces

Kidneys
Methylation → Catechol-O-methyl Urine
Tissues
transferases

Fig. 3. Possible pathways for phenolic acids metabolism.

o-diphenolic moiety (Wu, Cao, & Prior, 2002). Methylation gener- 4. Bioactive properties of phenolic acids
ally occurs in the 30 position of the polyphenol with a minor propor-
tion of 40 -O-methylated product also formed. These enzymes Phenolic acids are often included in the human diet and have
showed the highest activity in the liver and kidneys (Piskuta & been largely studied due to their bioactivities, such as antioxidant
Terrao, 1998). (Ferreira et al., 2009; Rice-Evans et al., 1996), antitumor (Carocho &
Sulfotransferases catalyse the transfer of a sulphate moiety Ferreira, 2013; Heleno et al., 2014a) and antimicrobial (Alves et al.,
from 30 -phosphoadenosine-50 -phosphosulfate to a hydroxyl group 2013) properties, amongst others. In particular, mushrooms are a
on the polyphenols. This conjugation reaction occurs in the liver source of these molecules in diet (Table 2).
(Piskuta & Terrao, 1998). Gallic acid, besides having astringent and styptic uses, also has
The membrane-bound enzymes UDP-glucuronosyltransferases several reported bioactivities, such as antineoplastic, bacterio-
that are located in the endoplasmic reticulum in many tissues cata- static, antimelanogenic and antioxidant properties (Kim, 2007).
lyse the transfer of a glucuronic acid from UDP-glucuronic acid to This molecule showed anticancer properties in prostate carcinoma
polyphenols. The presence of glucuronidated metabolites in the cells (Kaur, Velmurugan, Rajamanickam, Agarwal, & Agarwal,
mesenteric or portal blood after perfusion of polyphenols in the 2009). Grupta, Grupta, and Mahmood (2007), reported gallic acid
small intestine of rats shows that glucuronidation of polyphenols as a potent inhibitor of brush border sucrose and other disaccha-
first occurs in the enterocytes before conjugation in the liver rides in the mammalian intestine, while Kratz et al. (2008)
(Crespy et al., 2001). described its promising activity as an anti-HSV-2 (Herpes simplex
Conjugation reactions appear to vary according to the nature of virus) agent. Furthermore, Lu et al. (2010) proposed gallic acid as a
the polyphenol and the dose ingested. Sulfation generally has a candidate for the treatment of brain tumours, due to its ability to
higher-affinity and lower-capacity pathway than glucuronidation, suppress cell viability, proliferation, invasion and angiogenesis in
so that when the ingested dose is increased, a rise from sulfation human glioma cells. In another study, this phenolic acid proved
toward glucuronidation occurs (Koster, Halsema, Scholtens, to induce HeLa cervical cancer cell death by apoptosis and necrosis
Knippers, & Mulder, 1981). (You, Moon, Han, & Park, 2010).
Identification of circulating metabolites has been undertaken p-Hydroxybenzoic acid has been reported to have antioxidant
for only a few polyphenols. Further investigation is needed to activities against free radicals (Rice-Evans et al., 1996), antimicro-
know not only the nature and number of the conjugating groups, bial activities against pathogenic bacteria and fungi (Heleno et al.,
but also the positions of these groups on the polyphenol molecule, 2013a), amongst other bioactivities, such as estrogenic and anti-
as these positions can affect the biological properties of the conju- mutagenic properties (Pugazhendhi, Pope, & Darbre, 2005).
gates (Day, Bao, Morgan, & Williamson, 2000). There are only a few Protocatechuic acid possesses several bioactivities, such as anti-
studies describing the proportions of the various conjugates and oxidant (Ferreira et al., 2009), antimicrobial (Alves et al., 2013), cyto-
the percentages of the free forms in plasma, but the main circulat- toxic (Yip, Chan, Pang, Tam, & Wong, 2006), chemopreventive,
ing compounds are generally glucuronides (Zhang, Hendrich, & apoptotic (Yin, Lin, Wu, Tsao, & Hsu, 2009), neuroprotective (An
Murphy, 2003). et al., 2006) properties, and also a LDL oxidation inhibitor (Hur
To be eliminated these metabolites can follow two pathways: et al., 2003).
the biliary or the urinary pathway. Large extensively conjugated Another phenolic acid with beneficial properties is vanillic acid
metabolites are more likely to be eliminated in the bile, whereas that showed antisicking and anthelmintic activities, and is also able
small conjugates, such as monosulphates, are preferentially to suppress hepatic fibrosis in chronic liver injury (Itoh et al., 2010).
excreted in the urine (Crespy et al., 2003). Furthermore, this compound proved to be able to inhibit snake
 S.A. Heleno et al. / Food Chemistry 173 (2015) 501–513 507

Table 2
Phenolic acids identified in mushroom species. Adapted from Ferreira et al. (2009).

Phenolic acid Mushroom species Reference

Gallic acid Termitomyces heimii, Termitomyces mummiformis, Lactarius deliciosos, Pleurotus Puttaraju et al. (2006) and Kim et al. (2008)
sajor-caju, Hydnum repandum, Lentinus squarrulosus, Sparassis crispa, Morchella
conica, Russula brevipes, Geastrum arinarius, Cantharellus cibarius, Lactarius
sanguifluus, Macrolepiota procera, Cantharellus clavatus, Auricularia polytricha,
Pleurotus djamor, Lentinus sajor-caju, Termitomyces tylerance, Morchella
angusticeps, Termitomyces microcarpus, Helvella crispa, Termitomyces shimperi,
Pleurotus ostreatus, Agaricus bisporus, Flammulina velutipes, Pleurotus eryngii,
Lentinus edodes, Agaricus blazei, Phellinus linteus, Ganoderma lucidum, Inonotus
obliquus
p-Hydroxybenzoic acid Agaricus bisporus (white), Agaricus bisporus (brown), Lentinus edodes, Russula Mattila et al. (2001), Ribeiro et al. (2006, 2007) and Heleno
cyanoxantha, Tricholoma equestre, Amanita rubescens, Suillus granulatus, et al. (2012a, 2012b, 2013a, 2013b)
Agaricus arvensis, Agaricus silvicola, Agaricus romagnesii, Lactarius deliciosus,
Lepista nuda, Lycoperdon molle, Sarcodon imbricatus, Ramaria botrytis,
Tricholoma acerbum, Sparassis crispa, Phellinus linteus, Inonotus obliquus,
Ganoderma lucidum, Coprinopsis atramentária, Lactarius bertillonii, Lactarius
vellereus, Rhodotus palmatus, Xerocomus chrysenteron, Morchella esculenta
Protocatechuic acid Agaricus bisporus (white), Agaricus bisporus (brown), Lentinus edodes, Mattila et al. (2001), Puttaraju et al. (2006), Kim et al. (2008)
Termitomyces mummiformis, Boletus edulis, Lactarius deliciosos, Pleurotus sajor- and Heleno et al. (2012b, 2013a, 2013b)
caju, Lentinus squarrulosus, Hydnum repandum, Sparassis crispa, Morchella
conica, Russula brevipis, Lentinus sajor-caju, Lactarius sanguifluus, Macrolepiota
procera, Cantharellus clavatus, Auricularia polytricha, Pleurotus djamor,
Termitomyces tylerance, Morchella angusticeps, Termitomyces microcarpus,
Helvella crispa, Termitomyces shimperi, Termitomyces heimii, Lepista nuda,
Ramaria botrytis, Pleurotus ostreatus, Flammulina velutipes, Pleurotus eryngii,
Agaricus blazei, Inonotus obliquus, Phellinus linteus, Ganoderma lucidum,
Lactarius bertillonii, Lactarius vellereus, Rhodotus palmatus, Xerocomus
chrysenteron, Morchella esculenta
Vanillic acid Pleurotus sajor-caju, Hydnum repandum, Lentinus squarrulosus, Morchella Puttaraju et al. (2006) and Barros, Dueñas, Ferreira, Baptista,
conica, Russula brevipis, Lactarius sanguifluus, Macrolepiota procera, and Santos-Buelga (2009)
Cantharellus clavatus, Auricularia polytricha, Pleurotus djamor, Helvella crispa,
Termitomyces microcarpus, Termitomyces shimperi, Lentinus sajor-caju,
Termitomyces heimii, Lycoperdon molle, Tricholoma acerbum
Syringic acid Termitomyces mummiformis, Hydnum repandum, Morchella conica, Russula Puttaraju et al. (2006) and Kim et al. (2008)
brevipes, Lactarius sanguifluus, Macrolepiota procera, Cantharellus clavatus,
Pleurotus djamor, Lentinus sajor-caju, Termitomyces tylerance, Morchella
angusticeps, Termitomyces microcarpus, Agaricus blazei, Sparassis crispa
Veratric acid Sparassis crispa Kim et al. (2008)
Gentisic acid Termitomyces heimii, Termitomyces mummiformis, Lactarius deliciosus, Pleurotus Puttaraju et al. (2006) and Kim et al. (2008)
sajor-caju, Hydnum repandum, Lentinus squarrulosus, Sparassis crispa, Morchella
conica, Russula brevipes, Lactarius sanguifluus, Macrolepiota procera, Cantharellus
clavatus, Auricularia polytricha, Pleurotus djamor, Lactarius sanguifluus,
Termitomyces tylerance, Morchella angusticeps, Termitomyces microcarpus,
Helvella crispa, Termitomyces shimperi, Agaricus blazei
Cinnamic acid Agaricus bisporus (white), Agaricus bisporus (brown), Termitomyces heimii, Puttaraju et al. (2006), Kim et al. (2008) and Heleno et al.
Termitomyces mummiformis, Termitomyces shimperi, Pleurotus sajor-caju, (2012a, 2012b)
Hydnum repandum, Lentinus squarrulosus, Sparassis crispa, Lactarius
sanguifluus, Cantharellus clavatus, Pleurotus djamor, Agaricus arvensis, Agaricus
silvicola, Agaricus romagnesii, Cantharellus cibarius, Lycoperdon perlatum,
Macrolepiota procera, Agaricus blazei, Ganoderma lucidum, Coprinopsis
atramentaria, Lactarius bertillonii, Lactarius vellereus, Rhodotus palmatus,
Xerocomus chrysenteron
p-Coumaric acid Cantharellus cibarius, Termitomyces heimii, Boletus edulis, Sparassis crispa, Valentão et al. (2005), Puttaraju et al. (2006), Ribeiro et al.
Geastrum arinarius, Lactarius sanguifluus, Macrolepiota procera, Pleurotus (2007), Kim et al. (2008) and Heleno et al. (2012a, 2012b,
djamor, Lentinus sajor-caju, Fistulina hepatica, Agaricus arvensis, Agaricus 2013a, 2013b)
silvícola, Lepista nuda, Sparassis crispa, Ganoderma lucidum, Coprinopsis
atramentaria, Lactarius bertillonii, Lactarius vellereus, Xerocomus chrysenteron,
Morchella esculenta
o-Coumaric acid Inonotus obliquus Kim et al. (2008)
Ferulic acid Termitomyces heimii, Termitomyces microcarpus, Termitomyces shimperi, Puttaraju et al. (2006) and Kim et al. (2008)
Lactarius deliciosus, Pleurotus sajor-caju, Lentinus squarrulosus, Sparassis crispa,
Morchella conica, Cantharellus cibarius, Lactarius sanguifluus, Macrolepiota
procera, Cantharellus clavatus, Pleurotus djamor, Flammulina velutipes, Inonotus
obliquus
Caffeic acid Termitomyces heimii, Termitomyces tylerance, Termitomyces microcarpus, Puttaraju et al. (2006), Ribeiro et al. (2007) and Kim et al.
Termitomyces shimperi, Boletus edulis, Lentinus squarrulosus, Morchella conica, (2008)
Russula brevipes, Cantharellus cibarius, Lactarius sanguifluus, Macrolepiota
procera, Cantharellus clavatus, Pleurotus djamor, Lentinus sajor-caju, Morchella
angusticeps, Fistulina hepatica, Flammulina velutipes, Sparassis crispa, Phellinus
linteus

(continued on next page)

508 S.A. Heleno et al. / Food Chemistry 173 (2015) 501–513

Table 2 (continued)

Phenolic acid Mushroom species Reference

5-Sulfosalicylic acid Flammulina velutipes, Sparassis crispa, Phellinus linteus, Ganoderma lucidum Kim et al. (2008)
3,4 or 5-O- Cantharellus cibarius, Pleurotus ostreatus, Flammulina velutipes, Phellinus linteus Valentão et al. (2005) and Kim et al. (2008)
caffeoylquinic acid

venom 50 -nucleotidase (Dhananjaya, Nataraju, Gowda, Sharath, & mushrooms are a rich source of these molecules, we can conclude
D’Souza, 2009). that mushrooms are a good option to include in our daily diet.
Syringic acid shows antioxidant, antibacterial and hepatopro-
tective activities (Itoh et al., 2010; Kong, Zhao, Shan, Xiao, & Guo, 5. Controversy on in vivo bioactivity of polyphenols
2008).
Gentisic acid has been described as having anti-inflammatory, As mentioned above, phenolic acids represent a significant por-
antirheumatic, analgesic activities, and is also a cytostatic agent tion of polyphenols in our diet. Their bioactivity, specially antioxi-
and capable to inhibit low density lipoprotein oxidation in human dant properties, are related with the phenolic hydroxyl groups
plasma (Ashidate et al., 2005). attached to ring structures. These molecules can act as reducing
Lin and Nakatsui (1998) described that salicylic acid has analge- agents, hydrogen donators, singlet oxygen quenchers, superoxide
sic, antipyretic, anti-inflammatory, antiseptic and antifungal radical scavengers and metal chelators over hydroxyl and peroxyl
properties. radicals, superoxide anions and peroxynitrites (Terpinc et al., 2011).
Besides being an antioxidant (Ferreira et al., 2009; Rice-Evans Nevertheless, there has been some controversy about the bioac-
et al., 1996), cinnamic acid has also been reported to be an antitu- tivity of polyphenols after metabolism. Once ingested, these
mor agent by Vaz et al. (2012), and verified its capacity to inhibit molecules are metabolized and transformed into methylated, glu-
cell growth in a non-small lung cancer cell line (NCI-H460). curonated and sulphated metabolites (Manach et al., 2004).
Heleno et al. (2014a), in agreement with the aforementioned Glucuronidation and sulfation conjugation reactions are
authors, referred the cytotoxicity of cinnamic acid against the same described to have a significant impact on the bioactivity of poly-
cell line, also revealing activities against colon (HCT15) and cervi- phenols. These conjugation reactions significantly reduce the poly-
cal (HeLa) carcinoma cell lines. Ekmekcioglu, Feyertag, and Marktl phenols antioxidant activity, since both sulfation and
(1998), reported that this compound was able to inhibit prolifera- glucuronidation occur at the reducing hydroxyl groups in the phe-
tion and modulate brush border membrane enzyme activities in nolic structure. These groups are mainly responsible for the antiox-
human colon adenocarcinoma cells (CaCo-2). idant properties of polyphenols (Piazzon et al., 2012).
Different authors (Alves et al., 2013; Heleno et al., 2013a) There are only a few studies about the biological properties of
described cinnamic acid as an antimicrobial agent, showing the conjugated derivatives of polyphenols, where phenolic acids
activities against Gram positive and Gram negative bacteria (either are included, present in plasma or tissues due to the lack of precise
clinical isolates or collection microorganisms), and fungi. identification and commercial standards.
p-Coumaric acid also revealed antioxidant activities against Polyphenols are expected to act at water–lipid interfaces and
free radicals (Rice-Evans et al., 1996), and antitumor activities may also be involved in oxidation regeneration pathways with
against breast (MCF7), NCI-H460 and HCT15 carcinoma cell lines vitamins C and E, since the hydrophobicity of these compounds
(Heleno et al., 2014a). In a study about antimicrobial activity is in between that of vitamin C (highly hydrophilic) and that of
performed by Lou et al. (2012), the authors reported that p-cou- vitamin E (highly hydrophobic) (Manach et al., 2004).
maric acid had dual mechanisms of bactericidal activity: it was Glucuronidation and sulfation render polyphenols more hydro-
able to disrupt bacterial cell membranes, and bind to bacterial philic, which can affect their site of action and interactions with
genomic DNA to inhibit cellular functions, leading to cell death. other antioxidants (Manach et al., 2004).
Heleno et al. (2014b) also described a promising antimicrobial For phenolic acids, bearing a carboxyl function in addition to
activity of the hydroxyl groups, glucuronidation can occur at the reducing
p-coumaric acid against several pathogenic bacteria and fungi. hydroxyl group (phenyl-O-glucuronides) and at the carboxylic
Caffeic and ferulic acids have also been accounted for their group (acyl glucuronides). Therefore, acyl glucuronides retain all
bioactive properties. These phenolic acids showed antioxidant activ- the free reducing hydroxyl functions of the parent compound,
ities measured by common assays, such as the DPPH (2,2-diphenyl- while in the case of phenyl-O-glucuronides, at least one hydroxyl
1-picrylhydrazyl radical), ABTS (2,20 -azino-bis(3-thylbenzothiazo- group of the phenolic acid is bound to the glucuronate moiety
line-6-sulphonic acid)) and ORAC (oxygen radical absorbance (Piazzon et al., 2012).
capacity) assays (Piazzon et al., 2012; Vilãno, Fernández-Pachón, Cren-Olive, Teissier, Duriez, and Rolando (2003), described that
Troncoso, & García-Parrilla, 2005). These compounds have been methylated polyphenols present a decreased ability to protect low
also reported as antimicrobial agents against pathogenic bacteria density lipoproteins (LDL) from in vitro oxidation than the respec-
and fungi (Alves et al., 2013). tive parental compound. On the other hand, several authors
Yagasaki, Miura, Okauchi, and Furuse (2000) studied the activ- reported an increase in antioxidant capacity of plasma after the
ity of chlorogenic acid and its related compounds, such as caffeic consumption of various polyphenol-rich foods, meaning that some
acid, and proved that both chlorogenic acid as well as the released of the polyphenol metabolites retain antioxidant activity (Serafini,
compound caffeic acid were able to significantly reduce the inva- Laranjinha, Almeida, & Maiani, 2000).
sion of a rat ascites hepatoma cell line (AH109A), without altering Nevertheless, conjugation reactions might enhance certain spe-
the cell proliferation. cific bioactivities. For example, Koga and Meydani (2001),
The diverse biological functions of these phenolic acids suggest described that the plasma metabolites of catechin have an inhibi-
potential pharmacological activities (Khadem & Marles, 2010). tory effect on monocyte adhesion to interleukin 1 in b-stimulated
Thus, looking at all the promising bioactivities and knowing that human aortic endothelial cells, while catechin had no effect. In
 S.A. Heleno et al. / Food Chemistry 173 (2015) 501–513 509

another study, quercetin 3-O-glucuronide presented vascular protective. The bioactivity of phenolic acids and metabolites will
smooth muscle cell hypertrophy by angiotensin II (Yoshizumi be compared and discussed in Section 7.
et al., 2002). In contrast, Spencer et al. (2001), reported that two Further investigation on the bioactivities of these molecules
flavonoid metabolites (5-and 7-O-glucuronides of epicatechin) is needed to better understand the effects on the type and posi-
were unable to protect fibroblasts and neuronal cells from oxida- tion of conjugation on the various potential activities of
tive stress in vitro, while epicatechin and methylepicatechin were polyphenols.

A B H
C C C
O
MeOOC OMe H OH
OH O CA
AcO HO MeOOC
AcO O
OMe AcO
AcOBr COOMe AcO
i) ii)
O O OAc
AcOBr
i) OAc 1equiv.
O O AcO
OH FAGP
FA
COOMe O
ii) O H
OMe O C C C
OAc
OAc OMe
HO H
O AcO
COOH CAM
O O OH
OH CAGP
O HO
i) K2CO3 1.5 equivs, DMSO, Ar, RT, 24h;
FAG
ii) MeOH; H 2SO4 , 5 days, R.T.
i) ACN, H2O, NaOH
ii) KOH, H2O,HCl

C D
O
HO C
O OH
H
HO C C C
H OH HA
CoA
MeOOC OMe
O MeO
MeOOC OMe S
MeO AcO
AcO O
O S
AcO AcOBr i) O
AcO O HO ii) iii) 1equiv.
HO AcOBr i) O
ii) iii) 1equiv. 1.5equiv.
1.5equiv.
O
H O MeO C
MeO C C C O O COOMe OMe
O
COOMe O H OMe O C
O O H HO HAM2
HO C C C AcO OAc OMe
OAc CoAM2 OAc
OAc H OMe HAPG
O AcO HAM1 iv)
CoAM1
CoAGP iv)
i) K2CO3 1.5 equivs, DMSO, Ar, RT, 24h;
i) K2CO3 1.5 equivs, DMSO, Ar, RT, 24h; ii) MeOH; H2SO4 , 5 days, R.T. O
ii) MeOH; H2SO4 , 5 days, R.T. iii) K2CO3, 2 equivs, acetone, 50ºC, 24h; MeO C
H O OH
iii) K 2CO3, 2 equivs, acetone, 50ºC, 24h; MeO C C C iv) NaOH, 3 equivs, EtOH, 65ºC, 3h
iv) NaOH, 3 equivs, EtOH, 65ºC, 3h H OH HAM3
CoAM3

E OH
MeO OSO3H
OMe

O i)
OH O
OH
FA Ferulic acid-4'-O-Sulf ate

OH
HO3SO OH HO OSO3H
OH
+
i)
O
OH O O
OH OH
CA caf f eic acid-3'-O-Sulf ate caf f eic acid-4'-O-Sulf ate

i) ClSO3H, pyridine, 20ºC

Fig. 4. Glucuronidation, methylation and sulfation of ferulic, cinnamic, p-coumaric and p-hydroxybenzoic acids. (A) (i) Glucuronidation of ferulic acid (FA); FAGP, 2,3,4-tri-O-
acetyl-1-feruloyl-D-glucuronic acid methyl ester; (ii) deprotection of FAGP; FAG – ferulic acid 1-O-acyl-glucuronide (Piazzon et al., 2012). (B) (i) Glucuronidation of cinnamic
acid (CA). CAGP – cinnamic acid glucuronide protected form, 2,3,4-tri-O-acetyl-1-cinnamoyl-D-glucuronic acid methyl ester (Heleno et al., 2013a); (ii) methylation of CA. CAM
– methyl-3-phenylacrylate (Heleno et al., 2014a). (C) (i) Glucuronidation of p-coumaric acid (CoA). CoAGP – p-coumaric acid glucuronide protected form, 2,3,4-tri-O-acetyl-1-
p-coumaroyl-D-glucuronic acid methyl ester (Heleno et al., 2014a); (ii–iv) Methylations of CoA. CoAM1 – 3-(4-hydroxyphenyl)acrylate, CoAM2 – methyl-(4-methoxyphenyl)
acrylate, CoAM3 – 3-(4-methoxyphenyl) acrylic acid (Heleno et al., 2014a). (D) (i) Glucuronidation of p-hydroxybenzoic acid (HA). HAGP – p-hydroxybenzoic acid protected
form, 2,3,4-tri-O-acetyl-1-p-hydroxybenzoyl-D-glucuronic acid methyl ester (Heleno et al., 2013a); (ii–iv) Methylations of HA. HAM1 – methyl-4-hydroxybenzoate, HAM2 –
methyl-p-anisate, HAM3 – 4-methoxybenzoic acid (Heleno et al., 2014a). (E) (i) Sulfation of FA and CA (Piazzon et al., 2012).
510 S.A. Heleno et al. / Food Chemistry 173 (2015) 501–513

6. Chemical and enzymatic synthesis of phenolic acid acids namely, ferulic acid-40 -O-glucuronide (FFG), caffeic acid-30 -
metabolites O-glucuronide and caffeic acid-40 -O-glucuronide.
The antioxidant activity of the glucuronated and sulphated
There are only a few reports in the literature describing the syn- compounds was measured by both the ferric reducing antioxidant
thesis of metabolites from phenolic acids to evaluate their bioac- power (FRAP, ferric reducing ability) and the ABTS radical scaveng-
tivities in comparison with the corresponding parent compounds. ing assays.
Piazzon et al. (2012), as well as our research group (Heleno Concerning the glucuronide derivatives, the results showed that
et al., 2013a, 2014a), described the chemical or enzymatic synthe- FAG, the acyl glucuronide of ferulic acid was able to retain antiox-
sis of glucuronated, sulphated and methylated derivatives of sev- idant activity, similar to the activity of the parental compound in
eral phenolic acids, obtaining structures similar to those formed the FRAP assay, while in the ABTS assay its activity decreased by
in vivo after phenolic acid metabolism (Fig. 4A–E). almost 50% in comparison with ferulic acid antioxidant activity
For the glucuronidation reactions, the authors reacted the phe- for this assay.
nolic acids with acetobromo-a-D-glucuronic acid methyl ester, However, the phenyl-O-glucuronide of ferulic acid, the ferulic
potassium carbonate and dimethyl sulfoxide (Heleno et al., acid 40 -O-glucuronide, showed a much lower antioxidant activity
2013a) or with sodium hydroxide and acetonitrile (ACN) (Piazzon than ferulic acid in both antioxidant activity assays. Caffeic acid
et al., 2012) at room temperature, to obtain the glucuronide pro- 30 -O-glucuronide also retained a strong antioxidant activity in
tected forms of the parental acids (Fig. 4A–D). Piazzon et al. comparison with the parental phenolic acid activity for the FRAP
(2012) continued the reaction to obtain the final deprotected glu- assay, while for the ABTS assay its activity was also half the activity
curonide using potassium hydroxide, water and chloridric acid as of caffeic acid.
reaction conditions to obtain the final deprotected glucuronide Caffeic acid 40 -O-glucuronide displayed a good antioxidant
(Fig. 4A). activity with ABTS assay, which is about a third with respect to that
The same authors also synthesized glucuronides from FA using of caffeic acid, while with the FRAP assay, it was about 20-fold
mouse liver microsomes. After extraction procedures and HPLC lower than caffeic acid.
analysis with electrochemical or UV detection, FA, FAG and another Regarding the sulphate derivatives of ferulic and caffeic acids,
compound, the ferulic acid 40 -O-glucuronide (FFG) were identified Piazzon et al. (2012) verified that their activity is lower when com-
and quantified, being the ratio between FFG and FAG of 2.4:1, pared with the antioxidant activities of the parental phenolic acids
residual FA was also quantified (Piazzon et al., 2012). for both assays. These results showed the importance of the reduc-
For the methylation reactions, the authors performed several ing hydroxyl groups in the antioxidant activities of phenolic acids
different steps in order to obtain a complete series of methylated and their metabolites.
compounds derived from each phenolic acid. CAM, CoAM1 and Considering all the metabolites tested the acyl glucuronide of
HAM1 were obtained by reacting the parental compound with sul- ferulic acid and the phenyl 30 -O-glucuronide of caffeic acid retained
phuric acid in methanol at room temperature, affording the desired a strong antioxidant capacity. These compounds contained the
derivatives that have the carboxylic group from the parental 40 -hydroxyl groups in the aromatic ring that is determinant for
compound replaced by a methoxy group, HAM1 and CoAM1 have the antioxidant activity (Hodnick, Milosevljevic, Nelson, &
a free hydroxyl group in the p-positions (Fig. 4B–D) (Heleno Pardini, 1988; Pulido, Bravo, & Saura-Calixto, 2000).
et al., 2014a). Heleno et al. (2013a) and Heleno et al. (2014b) evaluated the
CoAM2 and HAM2 were achieved by reacting the parental antimicrobial activity of p-hydroxybenzoic, cinnamic and p-cou-
compounds with dimethyl sulphate and potassium carbonate in maric glucuronated protected forms and methylated metabolites,
acetone at 45–50 °C, affording the desired compounds that have and compared their antimicrobial activities with those of their
both carboxylic and hydroxyl groups replaced by methoxyl groups parental compounds. The authors measured the antimicrobial
(Fig. 4C and D) (Heleno et al., 2014a). activity against Gram-positive bacteria (Staphylococcus aureus,
To afford CoAM3 and HAM3, the compounds HAM2 and HAM3 Bacillus cereus, Listeria monocytogenes, and Micrococcus flavus),
were reacted with NaOH and ethanol at 65 °C affording the desired Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli,
compounds in which the carboxylic group remains but the hydro- Salmonella typhimurium, Enterobacter cloacae), and fungi (Aspergil-
xyl group from the p-position is replaced with a methoxyl group, lus fumigatus, Aspergillus versicolor, Aspergillus ochraceus, Aspergillus
completing the series (Fig. 4C and D) (Heleno et al., 2014a). niger, Trichoderma viride, Penicillium funiculosum, Penicillium
Piazzon et al. (2012) synthesized ferulic acid sulphate ester and ochrochloron, Penicillium verrucosum var. cyclopium), and also the
caffeic acid monosulphate esters according to procedures previ- demelanizing activity against the mentioned fungi.
ously described by Todd, Zimmerman, Crews, and Alberte (1993). Regarding the antibacterial activity, the glucuronide protected
Briefly, the sulfurochloridic acid (ClOS3H) was added dropwise to forms of p-hydroxybenzoic, cinnamic and p-coumaric acids (HAGP,
ferulic and caffeic acids in pyridine and the reaction left stirring CAGP and CoAGP) revealed lower activity than the parental com-
at 20 °C. The authors obtained ferulic acid 40 -O-sulphate, caffeic pounds, with the exception of CoAGP that maintained the antibac-
acid 40 -O-sulphate and caffeic acid 30 -O-sulphate (Fig. 4E). terial activity of p-coumaric acid.
Methylated derivatives of p-hydroxybenzoic acid (HAM1,
HAM2 and HAM3) presented lower antibacterial activities than
7. Bioactivity of phenolic acids versus their metabolites the parental compound HA. Cinnamic acid presented an excellent
antibacterial activity against all the tested bacteria, while its meth-
All the glucuronated, methylated and sulphated compounds ylated derivative revealed a much lower activity. Methylated
mentioned above, and the respective parental phenolic acids, were derivatives of p-coumaric acid (CoAM1, CoAM2 and CoAM3)
studied for their bioactivities namely, antioxidant (Piazzon et al., revealed higher activity than the parental compound.
2012), antimicrobial (Heleno et al., 2013a, 2014a) and antitumor Concerning the antifungal activity, glucuronated protected
(Heleno et al., 2014a) properties, in order to compare the biological forms, HAGP, CAGP and CoAGP, revealed higher activities than
activities of the parental compounds before and after metabolism the respective parental compounds against almost all the fungi
in vivo. tested.
Piazzon et al. (2012) also evaluated the antioxidant activity of Regarding the methylated derivatives, all the p-hydroxybenzoic
some commercial glucuronides derived from ferulic and caffeic acid derivatives showed lower activities than the parental com-
 S.A. Heleno et al. / Food Chemistry 173 (2015) 501–513 511

pound as well as the methylated derivative of cinnamic acid, CAM. their bioactivity after metabolism. Furthermore, future studies are
Nevertheless, all the p-coumaric acid derivatives, CoAM1, CoAM2 needed in order to clarify specific mechanistic pathways of these
and CoAM3 showed higher activities than the parental compound. molecules.
The compounds HAM1 and HAM3 showed demelanizing effects
on A. fumigatus, lowering the amount of conidia and giving nude Acknowledgements
vesicles without conidia, as well as the compounds CoAM1,
CoAM2, HAM3 and CAM on P. verrucosum. The authors are grateful to Fundação para a Ciência e a Tecno-
Heleno et al. (2014a), reported the cytotoxicity of p-hydroxy- logia (FCT, Portugal) and FEDER-COMPETE/QREN/EU for the finan-
benzoic, cinnamic and p-coumaric acid glucuronated protected cial support through the research centres (PEst-C/QUI/UI0686/
forms (HAGP, CAGP and CoAGP) and methylated derivatives 2011 and PEst-OE/AGR/UI0690/2011). S.A. Heleno (BD/70304/
(HAM1, HAM2, HAM3, CAM, CoAM1, CoAM2 and CoAM3), against 2010) also thanks FCT, POPH-QREN and FSE for her grant.
five human tumour cell lines namely, MCF-7 (breast adenocarci-
noma), NCI-H460 (non-small cell lung carcinoma), HCT15 (colon References
carcinoma), HeLa (cervical carcinoma) and HepG2 (hepatocellular
carcinoma). Alves, M. J., Ferreira, I. C. F. R., Froufe, H. J. C., Abreu, R. M. V., Martins, A., & Pintado,
The authors reported that the glucuronated protected forms M. (2013). Antimicrobial activity of phenolic compounds identified in wild
mushrooms, SAR analysis and docking studies. Journal of Applied Microbiology,
presented a higher cytotoxicity than the parental molecules 115, 346–357.
against all the tested cell lines. Regarding the methylated deriva- An, L. J., Guan, S., Shi, G. F., Bao, Y. M., Duan, Y. L., & Jiang, B. (2006). Protocatechuic
tives of p-hydroxybenzoic acid, HAM1 and HAM2, showed a higher acid from Alpinia oxyphylla against MPP+-induced neurotoxicity in PC12 cells.
Food and Chemical Toxicology, 44, 436–443.
cytotoxicity than the parental phenolic acids, as well as p-coumaric Ashidate, K., Kawamura, M., Mimura, D., Tohda, H., Miyazaki, S., Teramoto, T., et al.
acid methylated derivatives. The presence of an ester group (2005). Gentisic acid, an aspirin metabolite, inhibits oxidation of low-density
increased the growth inhibitory activity of the compounds; lipoprotein and the formation of cholesterol ester hydroperoxides in human
plasma. European Journal of Pharmacology, 513, 173–179.
HAM1 and CoAM1 have the carboxylic group replaced by an ester Azuma, K., Ippoushi, K., Nakayama, M., Ito, H., Higashio, H., & Terrao, J. (2000).
group and an hydroxyl group in the para position, while HAM2 and Absorption of chlorogenic acid and caffeic acid in rats after oral administration.
CoAM2 also have a carboxylic group replaced by an ester group and Journal of Agricultural and Food Chemistry, 48, 5496–5500.
Barros, L., Dueñas, M., Ferreira, I. C. F. R., Baptista, P., & Santos-Buelga, C. (2009).
a methoxy group in the para position. Phenolic acids determination by HPLC-DAD-ESI/MS in sixteen different
However, HAM3 and CoAM3 retains the carboxylic group of CoA Portuguese wild mushrooms species. Food and Chemical Toxicology, 47,
but with a methoxyl group in the para positions, and showed no, or 1076–1079.
Carocho, M., & Ferreira, I. C. F. R. (2013). The role of phenolic compounds in the fight
very weak, activity. Cinnamic acid methylated derivative, CAM,
against cancer – A review. Anti-Cancer Agents in Medicinal Chemistry, 13,
that has also the carboxylic group replaced by an ester and no 1236–1258.
groups in para position presented lower activity than cinnamic Clifford, M. N., & Scalbert, A. (2000). Ellagitannins-occurrence in food,
acid. bioavailability and cancer prevention. Journal of the Science of Food and
Agriculture, 80, 1118–1125.
All the tested compounds revealed no toxicity on non-tumour Cremin, P., Kasim-Karakas, S., & Waterhouse, A. L. (2001). LC/ES–MS detection of
porcine liver cells. hydroxycinnamates in human plasma and urine. Journal of Agricultural and Food
Chemistry, 49, 1747–1750.
Cren-Olive, C. C., Teissier, E., Duriez, P., & Rolando, C. (2003). Effect of catechin O-
methylated metabolites and analogues on human LDL oxidation. Free Radical
8. Concluding remarks Biology and Medicine, 34, 850–855.
Crespy, V., Morand, C., Besson, C., Cotelle, N., Vézin, H., Demigné, C., et al. (2001).
The splanchnic metabolism of flavonoids highly differed according to the nature
Overall, the results from the antioxidant activities revealed that, of the compound. American Journal of Physiology, 284, G980–G988.
although ferulic and caffeic acids are extensively metabolized after Crespy, V., Morand, C., Besson, C., Manach, C., Demigne, C., & Remesy, C. (2003).
absorption, their glucuronated metabolites can retain a strong Comparison of the intestinal absorption of quercetin, phloretin and their
glucosides in rats. Journal of Nutrition, 131, 2109–2114.
antioxidant activity and might still exert a significant antioxidant Day, A. J., Bao, Y. P., Morgan, M. R. A., & Williamson, G. (2000). Conjugation position
action in vivo. These two phenolic acids are the most representa- of quercetin glucuronides and effect on biological activity. Free Radical Biology
tive in the human diet and, after absorption, they are metabolized and Medicine, 29, 1234–1243.
Dhananjaya, B. L., Nataraju, A., Gowda, C. D. R., Sharath, B. K., & D’Souza, C. J. M.
and circulate in human plasma in conjugated forms. Thus, the
(2009). Vanillic acid as a novel specific inhibitor of snake venom 50 -
strong antioxidant activity exhibited by some of these metabolites nucleotidase: A pharmacological tool in evaluating the role of the enzyme in
might contribute to the increase of plasma antioxidant activities snake envenomation. Biochemistry (Mosc.), 74, 1315–1319.
measured after the intake of phenolic acids rich-foods (e.g., mush- Ekmekcioglu, C., Feyertag, J., & Marktl, W. (1998). Cinnamic acid inhibits
proliferation and modulates brush border membrane enzyme activities in
rooms) as described before. Caco-2 cells. Cancer Letters, 128, 137–144.
Regarding the antimicrobial activity, the methylation reactions Faed, E. M. (1984). Properties of acyl glucuronides: Implications for studies of the
in the parental molecules have considerably increased the activity pharmacokinetics and metabolism of acidic drugs. Drug Metabolism Reviews, 15,
1213–1249.
of CoA. However, the inclusion of acetyl groups increased the anti- Ferreira, I. C. F. R., Barros, L., & Abreu, R. M. V. (2009). Antioxidants in wild
fungal activity but maintained the antibacterial effects. In the case mushrooms. Current Medicinal Chemistry, 16, 1543–1560.
of HA and CA, despite the inclusion of methyl groups did not Fumeaux, R., Menozzi-Smarrito, C., Stalmach, A., Munari, C., Kraehenbuehl, K.,
Steiling, H., et al. (2010). First synthesis, characterization, and evidence for the
increase the antimicrobial activity, however demelanizing activity presence of hydroxycinnamic acid sulfate and glucuronide conjugates in human
of the parental compounds increased. biological fluids as a result of coffee consumption. Organic and Molecular
Concerning the antitumor potential, in most of the cases the Chemistry, 8, 5199–5211.
Gross, G. G. (1985). Biosynthesis and metabolism of phenolic acids and
substitution of the carboxylic group (in parental organic acids) monolignols. In Takayoshi Higuchi (Ed.), Biosynthesis and biodegradation of
for an ester (in methylated derivatives) increased the cytotoxicity wood components (pp. 229–271). Elsevier.
of the parental compounds. Glucuronated protected derivatives Grupta, N., Grupta, S., & Mahmood, A. (2007). Gallic acid inhibits brush border
disaccharides in mammalian intestine. Nutrition Research, 27, 230–235.
showed an increase in cytotoxicity of the respective parental com-
Heleno, S. A., Barros, L., Martins, A., Queiroz, M. J. R. P., Santos-Buelga, C., & Ferreira,
pounds due to the inclusion of acetyl molecules in the parental I. C. F. R. (2012a). Fruiting body, spores and in vitro produced mycelium of
compound. Ganoderma lucidum from Northeast Portugal: A comparative study of the
These reports allow a comparison between parental molecules antioxidant potential of phenolic and polysaccharidic extracts. Food Research
International, 46, 135–140.
and derived metabolites. It is extremely important to understand Heleno, S. A., Barros, L., Martins, A., Queiroz, M. J. R. P., Santos-Buelga, C., & Ferreira,
the behaviour of organic acids (including phenolic acids) regarding I. C. F. R. (2012b). Phenolic, polysaccharidic, and lipidic fractions of mushrooms
512 S.A. Heleno et al. / Food Chemistry 173 (2015) 501–513

from Northeastern Portugal: Chemical compounds with antioxidant properties. Pérez-Jiménez, J., Hubert, J., Hooper, L., Cassidy, A., Manach, C., Williamson, G., et al.
Journal of Agricultural and Food Chemistry, 60, 4634–4640. (2010). Urinary metabolites as biomarkers of polyphenols intake in humans: A
Heleno, S. A., Ferreira, I. C. F. R., Calhelha, R. C., Esteves, A. P., & Queiroz, M. J. R. P. systematic review. American Journal of Clinical Nutrition, 92, 801–809.
(2014a). Cytotoxicity of Coprinopsis atramentaria extract, organic acids and their Piazzon, A., Vrhovsek, U., Masuero, D., Mattivi, F., Mandoj, F., & Nardini, M. (2012).
synthesized methylated and glucuronate derivatives. Food Research Antioxidant activity of phenolic acids and their metabolites: Synthesis and
International, 55, 170–175. antioxidant properties of the sulfate derivatives of ferulic and caffeic acids and
Heleno, S. A., Ferreira, I. C. F. R., Ćirić, A., Glamočlija, J., Martins, A., Queiroz, M.-J. R. of the acyl glucuronide of ferulic acid. Journal of Agricultural and Food Chemistry,
P., et al. (2014b). Coprinopsis atramentaria extract, organic acids, synthesized 60, 12312–12323.
glucuronated and methylated derivatives as antibacterial and antifungal agents. Piskuta, M. K., & Terrao, J. (1998). Accumulation of ()-epicatechin metabolites in
Food & Function, 5, 2521–2528. rat plasma after oral administration and distribution of conjugation enzymes in
Heleno, S. A., Ferreira, I. C. F. R., Esteves, A. P., Ćirić, A., Glamočlija, J., Martins, A., rat tissues. Journal of Nutrition, 128, 1172–1178.
et al. (2013a). Antimicrobial and demelanizing activity of Ganoderma lucidum Plumb, G. W., Garcia Conesa, M. T., Kroon, P. A., Rhodes, M., Ridley, S., & Williamson,
extract, p-hydroxybenzoic and cinnamic acids and their synthetic acetylated G. (1999). Metabolism of chlorogenic acid by human plasma, liver, intestine and
glucuronide methyl esters. Food and Chemical Toxicology, 58, 95–100. gut microflora. Journal of the Science of Food and Agriculture, 79, 390–392.
Heleno, S. A., Stojković, D., Barros, L., Glamočlija, J., Soković, M., Martins, A., et al. Pugazhendhi, D., Pope, G. S., & Darbre, P. D. (2005). Oestrogenic activity of p-
(2013b). A comparative study of chemical composition, antioxidant and hydroxybenzoic acid (common metabolite of paraben esters) and methylparaben
Antimicrobial properties of Morchella esculenta (L.) Pers. from Portugal and in human breast cancer cell lines. Journal of Applied Toxicology, 25, 301–309.
Serbia. Food Research International, 51, 236–243. Pulido, R., Bravo, L., & Saura-Calixto, F. (2000). Antioxidant activity of dietary
Hodnick, W. F., Milosevljevic, E. B., Nelson, J. H., & Pardini, R. S. (1988). polyphenols as determined by a modified ferric reducing/antioxidant power
Electrochemistry of flavonoids. Biochemical Pharmacology, 37, 2606–2611. assay. Journal of Agricultural and Food Chemistry, 48, 3396–3402.
Hur, J. M., Park, J. G., Yang, K. H., Park, J. C., Park, J. R., Chun, S. S., et al. (2003). Puttaraju, N. G., Venkateshaiah, S. U., Dharmesh, S. M., Urs, S. M., & Somasundaram,
Effect of methanol extract of Zanthoxylum piperitum leaves and of its R. (2006). Antioxidant activity of indigenous edible mushrooms. Journal of
compound, protocatechuic acid, on hepatic drug metabolizing enzymes and Agricultural and Food Chemistry, 54, 9764–9772.
lipid peroxidation in rats. Bioscience, Biotechnology, and Biochemistry, 67, Rechner, A. R., Kuhnle, G., Bremner, P., Hubbard, G. P., Moore, K. P., & Rice-Evans, C.
945–950. A. (2002). The metabolic fate of dietary polyphenols in humans. Free Radical
Itoh, A., Isoda, K., Kondoh, M., Kawase, M., Watari, A., Kobayashi, M., et al. (2010). Biology and Medicine, 33, 220–235.
Hepatoprotective effect of syringic acid and vanillic acid on CCl4-induced liver Ribeiro, B., Rangel, J., Valentão, P., Baptista, P., Seabra, R. M., & Andrade, P. B. (2006).
injury. Biological and Pharmaceutical Bulletin, 33, 983–987. Contents of carboxylic acids and two phenolics and antioxidant activity of dried
Kaur, M., Velmurugan, B., Rajamanickam, S., Agarwal, R., & Agarwal, C. (2009). Gallic Portuguese wild edible mushrooms. Journal of Agricultural and Food Chemistry,
acid, an active constituent of grape seed extracts, exhibits anti-proliferative, 54, 8530–8537.
pro-apoptotic and antitumorigenic effects against prostate carcinoma xenograft Ribeiro, B., Valentão, P., Baptista, P., Seabra, R. M., & Andrade, P. B. (2007). Phenolic
growth in nude mice. Pharmaceutical Research, 26, 2133–2140. compounds, organic acids profiles and antioxidative properties of beefsteak
Khadem, S., & Marles, R. J. (2010). Monocyclic phenolic acids, hydroxy- and fungus (Fistulina Hepatica). Food and Chemical Toxicology, 45, 1805–1813.
polyhydroxybenzoic acids: Occurrence and recent bioactivity studies. Molecules, Rice-Evans, C. A., Miller, N. J., & Paganga, G. (1996). Structure – Antioxidant activity
15, 7985–8005. relationships of flavonoids and phenolic acids. Free Radical Biology and Medicine,
Kim, Y. J. (2007). Antimelanogenic and antioxidant properties of gallic acid. 20, 933–956.
Biological and Pharmaceutical Bulletin, 30, 1052–1055. Scalbert, A., & Williamson, G. (2000). Dietary intake and bioavailability of
Kim, M. Y., Seguin, P., Ahn, J. K., Kim, J. J., Chun, S. C., Kim, E. H., et al. (2008). Phenolic polyphenols. Journal of Nutrition, 130, 2073S–2085S.
compound concentration and antioxidant activity of edible and medicinal Serafini, M., Laranjinha, J. A. N., Almeida, L. M., & Maiani, G. (2000). Inhibition of
mushrooms from Korea. Journal of Agricultural and Food Chemistry, 56, human LDL lipid peroxidation by phenol-rich beverages and their impact on
7265–7270. plasma total antioxidant capacity in humans. Journal of Nutritional Biochemistry,
Koga, T., & Meydani, M. (2001). Effect of plasma metabolites of (+)-catechin and 11, 585–590.
quercetin on monocyte adhesion to human aortic endothelial cells. American Sinclair, K. A., & Cardwell, J. (1982). The formation of b-glucuronidase resistant
Journal of Clinical Nutrition, 73, 941–948. glucuronides by the intramolecular rearrangement of glucuronide acid
Kong, W. K., Zhao, Y. L., Shan, L. M., Xiao, X. H., & Guo, W. Y. (2008). Thermochemical conjugates at mild alkaline pH. Biochemical Pharmacology, 31, 953–957.
studies on the quantity-antibacterial effect relationship of four organic acids Spahn-Langguth, H., & Benet, L. Z. (1992). Acyl glucuronides revisited: is the
from Radix isatidis on Escherichia coli growth. Biological and Pharmaceutical glucuronidation process a toxification as well as detoxification mechanism?
Bulletin, 31, 1301–1305. Drug Metabolism Reviews, 24, 5–48.
Koster, H., Halsema, I., Scholtens, E., Knippers, M., & Mulder, G. J. (1981). Dose- Spencer, J. P., Schroeter, H., Crossthwaithe, A. J., Kuhnle, G., Williams, R. J., & Rice-
dependent shifts in the sulfation and glucuronidation of phenolic compounds in Evans, C. (2001). Contrasting influences of glucuronidation and O-methylation
the rat in vivo and in isolated hepatocytes. The role of saturation of of epicatechin on hydrogen peroxide-induced cell death in neurons and
phenolsulfotransferase. Biochemical Pharmacology, 30, 2569–2575. fibroblasts. Free Radical Biology and Medicine, 31, 1139–1146.
Kratz, J. M., Andrighetti-Frohner, C. R., Leal, P. C., Nunes, R. J., Yunes, R. A., Trybala, E., Terpinc, P., Polak, T., Šegatin, N., Hanzlowsky, A., Ulrih, N. P., & Abramovič, H. (2011).
et al. (2008). Evaluation of anti-HSV-2 activity of gallic acid and pentyl gallate. Antioxidant properties of 4-vinyl derivatives of hydroxycinnamic acids. Food
Biological and Pharmaceutical Bulletin, 31, 903–907. Chemistry, 128, 62–68.
Kroon, P. A., Faulds, C. B., Ryden, P., Robertson, J. A., & Williamson, G. (1997). Release Todd, J. S., Zimmerman, R. C., Crews, P., & Alberte, R. S. (1993). The antifouling
of covalently bound ferulic acid from fiber in the human colon. Journal of activity of natural and synthetic phenolic acid sulphate esters. Phytochemistry,
Agricultural and Food Chemistry, 45, 661–667. 34, 401–404.
Lin, A. N., & Nakatsui, T. (1998). Salicylic acid revisited. International Journal of Valentão, P., Andrade, P. B., Rangel, J., Ribeiro, B., Silva, B. M., Baptista, P., et al.
Dermatology, 37, 335–342. (2005). Effect of the conservation procedure on the contents of phenolic
Lou, Z., Wang, H., Rao, S., Sun, J., Ma, C., & Li, J. (2012). P-coumaric acid kills bacteria compounds and organic acids in Chanterelle (Cantharellus cibarius) mushroom.
through dual damage mechanisms. Food Control, 25, 550–554. Journal of Agricultural and Food Chemistry, 53, 4925–4931.
Lu, Y., Jiang, F., Jiang, H., Wu, K., Zheng, X., Cai, Y., et al. (2010). Gallic acid suppresses Vaz, J. A., Almeida, G. M., Ferreira, I. C. F. R., Martins, A., & Vasconcelos, M. H. (2012).
cell viability, proliferation, invasion and angiogenesis in human glioma cells. Clitocybe alexandri extract induces cell cycle arrest and apoptosis in a lung
European Journal of Pharmacology, 641, 102–107. cancer cell line: Identification of phenolic acids with cytotoxic potential. Food
Manach, C., Scalbert, A., Morand, C., Rémésy, C., & Jiménez, L. (2004). Polyphenols: Chemistry, 132, 482–486.
Food sources and bioavailability. American Journal of Clinical Nutrition, 79, Vilãno, D., Fernández-Pachón, M. S., Troncoso, A. M., & García-Parrilla, M. C. (2005).
727–747. Comparison of antioxidant activity of wine phenolic compounds and
Marmet, C., Actis-Goretta, L., Renouf, M., & Giuffrida, F. (2014). Quantification of metabolites in vitro. Analytica Chimica Acta, 538, 391–398.
phenolic acids and their methylates, glucuronides, sulfates and lactones Wu, X., Cao, C., & Prior, R. L. (2002). Absorption and metabolism of anthocyanins in
metabolites in human plasma by LC–MS/MS after oral ingestion of soluble elderly women after consumption of elderberry of blueberry. Journal of
coffee. Journal of Pharmaceutical and Biomedical Analysis, 88, 617–625. Nutrition, 132, 1865–1871.
Mattila, P., Konko, K., Eurola, M., Pihlava, J. M., Astola, J., Vahteristo, L., et al. (2001). Yagasaki, K., Miura, Y., Okauchi, R., & Furuse, T. (2000). Inhibitory effects of
Contents of vitamins, mineral elements, and some phenolic compounds in chlorogenic acid and its related compounds on the invasion of hepatoma cells in
cultivated mushrooms. Journal of Agricultural and Food Chemistry, 49, culture. Cytotechnology, 33, 229–235.
2343–2348. Yin, M. C., Lin, C. C., Wu, H. C., Tsao, S. M., & Hsu, C. K. (2009). Apoptotic effects of
Nardini, M., Cirillo, E., Natella, F., & Scaccini, C. (2002). Absorption of phenolic acids protocatechuic acid in human breast, lung, liver, cervix, and prostate cancer
in humans after coffee consumption. Journal of Agricultural and Food Chemistry, cells: Potential mechanisms of action. Journal of Agricultural and Food Chemistry,
50, 5735–5741. 57, 6468–6473.
Nardini, M., Forte, M., Vrhovsek, U., Mattivi, F., Viola, R., & Scaccini, C. (2009). White Yip, E. C. H., Chan, A. S. L., Pang, H., Tam, Y. K., & Wong, Y. H. (2006). Protocatechuic acid
wine phenolics are absorbed and extensively metabolized in humans. Journal of induces cell death in HepG2 hepatocellular carcinoma cells through a c-Jun N-
Agricultural and Food Chemistry, 57, 2711–2718. terminal kinase-dependent mechanism. Cell Biology and Toxicology, 22, 293–302.
Olthof, M. R., Hollman, P. C. H., & Katan, M. B. (2001). Chlorogenic acid and caffeic Yoshizumi, M., Tsuchiya, K., Suzaki, Y., Kirima, K., Kyaw, M., Moon, J. H., et al. (2002).
acid are absorbed in humans. Journal of Nutrition, 131, 66–71. Quercetin glucuronide prevents VSMC hypertrophy by angiotensin II via the
 S.A. Heleno et al. / Food Chemistry 173 (2015) 501–513 513

inhibition of JNK and AP-1 signaling pathway. Biochemical and Biophysical Zhou, S. F., Paxton, J. W., Tingle, M. D., Kestell, P., Jameson, M. B., Thompson, P. I.,
Research Communications, 293, 1458–1465. et al. (2001). Identification and reactivity of the major metabolite (beta-1-
You, B. R., Moon, H. J., Han, Y. H., & Park, W. H. (2010). Gallic acid inhibits the growth glucuronide) of the antitumour agent 5,6-dimethylxanthenone-4-acetic acid
of HeLa cervical cancer cells via apoptosis and/or necrosis. Food and Chemical (DMXAA) in humans. Xenobiotica, 31, 277–293.
Toxicology, 48, 1334–1340.
Zhang, Y., Hendrich, S., & Murphy, P. A. (2003). Glucuronides are the main isoflavone
metabolites in women. Journal of Nutrition, 133, 399–404.