Journal of Food Measurement and Characterization

https://doi.org/10.1007/s11694-020-00417-0

ORIGINAL PAPER

Identification and quantification of phenolic acid compounds

of twenty‑six mushrooms by HPLC–DAD
Fatih Çayan1 · Ebru Deveci2 · Gülsen Tel‑Çayan1 · Mehmet Emin Duru3

Received: 26 November 2019 / Accepted: 10 February 2020

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract
Phenolic acids are found in different foods in the human diet, for example mushrooms. Determination of phenolic acids is
important because of their relationship to their role in disease prevention due to their bioactive properties. In this study,
the phenolic acid profile of 26 mushroom species was analyzed by using high-performance liquid chromatography method
coupled with photodiode array detector (HPLC–DAD) and 16 phenolic acid compounds were identified. The chromato-
graphic separation was performed using Intertsil ODS-3 reverse phase ­C18 column (5 µm, 250 mm × 4.6 mm i.d), gradient
solvent system with 1.5 mL/min flow rate and detected at 280 nm. The coefficient of determination (­ R2) was in the range
of 0.9965–0.9999. Limit of detection and quantification ranged from 0.001–0.970 to 0.001–2.940 µg/L, respectively. The
phenolic compounds were characterized according to their retention times and UV data were compared with commercial
standards. S. granulatus (71.79 µg/g) and L. nuda (68.38 µg/g) revealed the highest concentration of total phenolic compounds
among the studied mushrooms. Gallic acid was found as the major phenolic compound in R. aurora (2.96 ± 0.56 µg/g) while
6,7-dihydroxy coumarin was identified as major phenolic compounds in A. tabescens (2.07 ± 0.25 µg/g) and L. leucothites
(9.02 ± 0.87 µg/g). Fumaric acid was found as the most abundant compounds in 16 out of 26 mushrooms. Catechin hydrate
was identified as major phenolic compounds in the rest of mushrooms. This method provided a beneficial standardization
procedure of phenolic acid compounds in mushroom samples.

Keywords HPLC–DAD · Phenolic compounds · Mushroom species · Fumaric acid · Catechin hydrate

Introduction antiatherogenic and hematological properties [3–8]. The

medicinal properties of mushrooms are caused by the bio-
Mushrooms have been used for centuries both as food active compounds such as phenolic compounds, terpenoids,
and medicine all over the world. Mushrooms are valuable lectins, polysaccharides they contain [9, 10]. Among these
healthy foods since they are poor in calories, fat and essen- biologically active substances present in mushrooms, phe-
tial fatty acids, and rich in proteins, vitamin, and minerals nolics have attracted much attention due to their antioxidant,
[1, 2]. In previous studies, it was reported that mushrooms anti-inflammatory, and antitumor effects [11].
have anti-inflammatory, antioxidant, antitumor, antiviral Phenolic compounds are aromatic hydroxylated com-
and antimicrobial effects also, they have hypoglycemic, pounds, possessing one or more aromatic rings with one or
more hydroxyl groups. They can be divided into two classes:
simple phenols and phenolic acids such as gallic acid, ben-
* Gülsen Tel‑Çayan zoic acid, syringic acid, chlorogenic acid, and other associ-
[email protected]
ates and; polyphenols, which are classified into many groups
1
Department of Chemistry and Chemical Processing such as flavonoids, tannins, stilbenes, and so on. Natural
Technologies, Muğla Vocational School, Muğla Sıtkı phenolic compounds are formed as end product in shikimate
Koçman University, 48000 Muğla, Turkey and acetate pathways and can range from relatively simple
2
Chemistry and Chemical Processing Technology molecules (phenolic acids, phenylpropanoids, flavonoids) to
Department, Technical Sciences Vocational School, Konya highly polymerized compounds (lignins, melanins, tannins)
Technical University, 42100 Konya, Turkey
[12, 13].
3
Department of Chemistry, Faculty of Sciences, Muğla Sıtkı
Koçman University, 48121 Muğla, Turkey

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 F. Çayan et al.

Phenolic acids are found as the main phenolic compounds Materials and methods
in mushrooms. Phenolic acids can be divided into two major
groups, hydroxybenzoic acids and hydroxycinnamic acids Chemicals and reagents
which are consist of benzoic and cinnamic acid, respectively.
Hydroxybenzoic acid derivatives generally are present in HPLC grade solvents were obtained from E. Merck (Darm-
the attached form and their structure is similar to lignins stadt, Germany). Gallic acid (≥ 99%), fumaric acid (≥ 99%),
and hydrolyzable tannins. Hydroxycinnamic acid derivatives protocatechuic acid (97%), catechin hydrate (≥ 98%),
mostly occur in the bound form, linked to cell wall structural p-hydroxy benzoic acid (99%), 6,7-dihydroxy coumarin
components, such as cellulose, lignin, and proteins [13, 14]. (98%), caffeic acid (≥ 98%), vanillin (99%), 2,4-dihydroxy
Some findings suggest that biological properties of phe- benzoic acid (98%), p-coumaric acid (≥ 98%), ferulic acid
nolic compounds are associated with to their antioxidant (99%), coumarin (≥ 99%), trans-2-hydroxy cinnamic acid
activity [15]. Antioxidant properties of phenolic compounds (99%), ellagic acid (≥ 98%), rosmarinic acid (≥ 98%) and
have a vital role in the stability of food products, as well as trans-cinnamic acid (99%) were obtained from Sigma
in the antioxidative defence mechanisms of biological sys- Chemical Co. (Sigma-Aldrich GmbH, Sternheim, Germany).
tems [16]. The antioxidative effect of phenolic compounds
in functional foods is caused from a direct free radical scav- Instrument
enging activity, reducing activity and chelating of prooxidant
metal ions [17–19]. Phenolic hydrogen is responsible for The phenolic acid analysis was carried out using a Shi-
free radical scavenging activity of phenolic compounds and madzu 20 AT series high performance liquid chromatograph
the presence of hydroxyl group at ortho and para positions (HPLC–DAD, Shimadzu Cooperation, Japan).
increases antioxidant activity [13]. For chelating metal ions,
the presence of ortho-dihydroxylation on the phenyl ring
in phenolic acids and flavonoids or the presence of a 3- or Mushroom samples
5-hydroxyl group in flavonoids is required [20]. Phenolic
compounds have been reported to prevent of various degen- The species names, collection localities and dates, family
eration of human diseases, such as Alzheimer’s diseases [21, and edibility of 26 mushroom species are listed in Table 1.
22]. Flavonoids are considered to protect against cancer and Voucher specimens were deposited at the fungarium of Nat-
heart diseases [23]. ural Products Laboratory of Muğla Sıtkı Koçman University.
In recent years, the increase in mushroom consumption
in relation to the beneficial effects of bioactive compounds Extraction
such as phenolic compounds on human health has further
increased the studies on mushroom species. Therefore, The phenolic acids were determined according to the method
investigations about the quantification of bioactive com- of Barros et al. [25] with slight modification [26]. The
pounds present in the mushrooms gained importance. One mushroom sample (3 g) was extracted with acetone: water
of the main analytical techniques used to obtain the chemical (80:20 v/v; 30 mL) at − 18 °C for 24 h. After ultrasonic
profile of mushroom is high performance liquid chroma- bath for 15 min, the mushroom extract was centrifuged at
tography (HPLC) [24]. In this context, the objective of the 4000 rpm for 10 min and filtered through Whatman no. 4
present study was to determine of phenolic compounds of paper. The residue was then re-extracted by two additional
26 mushroom species; namely, Agaricus bisporus, Amanita 30 mL of the acetone:water. The combined extracts were
vaginata, Armillaria tabescens, Clitocybe odora, Collybia evaporated at 40 °C under reduced pressure to remove ace-
confluens, Collybia dryophila, Coprinus atramentarius, tone. The obtained extract solved in water:methanol (80:20)
Chroogomphus rutilus, Lactarius deliciosus, Lactarius sal- and filtered through a 0.20 µm disposable LC filter disk for
monicolor, Laetiporus sulphureus, Lepista nuda, Lepista HPLC–DAD.
personata, Leucoagaricus leucothites, Leucopaxillus tri-
color, Marasmius oreades, Morchella elata, Morchella escu- HPLC–DAD conditions
lenta, Pleurotus ostreatus, Ramaria flava, Russula aurora,
Russula azurea, Russula delica, Russula vinosa, Suillus Separation was achieved on an Intertsil ODS-3 reverse phase
granulatus and Tapinella panuoides were collected from ­C18 column (5 µm, 250 mm × 4.6 mm i.d) thermostatted at
Anatolia by using high performance liquid chromatography 40 °C. The solvent flow rate was 1.5 mL/min. The sam-
coupled to photodiode array detector (HPLC–DAD). This ple volume injection was 20 µL. The mobile phases used
described method benefits the analysis of phenolic acids were: (A) 0.5% acetic acid in water, (B) 0.5% acetic acid
in mushroom samples with sensitivity, trusty, rapidity, and in methanol. The elution gradient was as follows: 0–20%
selectivity.

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Identification and quantification of phenolic acid compounds of twenty‑six mushrooms by HPLC–…

Table 1  Collection localities and dates, family and edibility of the studied mushroom species
Code Mushroom name Collection localities and dates Family Edibilty

AB Agaricus bisporus (J.E. Lange) Imbach Uşak-Banaz, December 2013 Agaricaceae Edible
AV Amanita vaginata (Bull.) Lam Uşak-Eşme, June 2014 Pluteaceae Poisonous
AT Armillaria tabescens (Scop.) Emel Uşak-Sivaslı, November 2013 Physalacriaceae Inedible
CO Clitocybe odora (Bull.) P. Kumm Denizli-Buldan, November 2013 Tricholomataceae Poisonous
CC Collybia confluens (Pers.) P. Kumm Uşak-Eşme, November 2013 Tricholomataceae Inedible
CD Collybia dryophila (Bull.) P. Kumm Uşak-Eşme, November 2013 Tricholomataceae Poisonous
CA Coprinus atramentarius (Bull.) Fr Denizli- Honaz, November 2013 Agaricaceae Poisonous
CR Chroogomphus rutilus (Schaeff.) O.K. Mill Uşak-Banaz, May 2014 Gomphidiaceae Edible
LD Lactarius deliciosus (L.) Gray Fethiye-Babadağ, October 2013 Russulaceae Edible
LS1 Lactarius salmonicolor R. Heim & Leclair Bolu, April 2013 Russulaceae Edible
LS2 Laetiporus sulphureus (Bull.) Murrill Uşak-Subaşı, December 2014 Fomitopsidaceae Edible
LN Lepista nuda (Bull.) Cooke Denizli-Babadağ, December 2014 Tricholomataceae Edible
LP Lepista personata (Fr.) Cooke Uşak, December 2013 Tricholomataceae Edible
LL Leucoagaricus leucothites (Vittad.) Wasser Uşak-Banaz, September 2013 Agaricaceae Edible
LT Leucopaxillus tricolor (Peck) Kühner Denizli-İncirpınar, December, 2014 Tricholomataceae Inedible
MO Marasmius oreades (Bolton) Fr Uşak-Eşme, November 2014 Marasmiaceae Edible
ME1 Morchella elata Fr Denizli-Babadag, April 2013 Morchellaceae Edible
ME2 Morchella esculenta (L.) Pers Muğla-Fethiye, April 2013 Morchellaceae Edible
PO Pleurotus ostreatus (Jacq.) P. Kumm Uşak-Eşme, December 2014 Pleurotaceae Edible
RF Ramaria flava (Schaeff.) Quél Muğla, December 2014 Gomphaceae Edible
RA1 Russula aurora Krombh Denizli-Honaz, November 2013 Russulaceae Edible
RA2 Russula azurea Bres Denizli-Honaz, November 2013 Russulaceae Edible
RD Russula delica Fr Muğla, December 2014 Russulaceae Edible
RV Russula vinosa Lindblad Denizli-Honaz, November 2014 Russulaceae Edible
SG Suillus granulatus (L.) Roussel Uşak-Banaz, November 2014 Suillaceae Edible
TP Tapinella panuoides (Fr.) E.-J. Gilbert Uşak-Eşme, November 2013 Tapinellaceae Inedible

B (0–0.01 min); 20–60% B (0.01–2 min); 60–80% B concentration range was studied using mixed standard solu-
(2–15 min); 100% B (15–30 min); 100–10% B (30–35 min); tions ranging from 0.01 to 1 mg/L. The linearity was exam-
10–0% B (35–40 min). Detection was carried out photo- ined using coefficient of determination ­(R2) values. Deter-
diode array detector (PDA) using 280 nm as the preferred mination of signal-to-noise ratio was calculated under the
wavelength. proposed chromatographic condition. LOD was considered
as 3:1 and LOQ as 10:1. The analytical parameters and num-
Method validation bers of phenolic compounds are described in Table 2.

The proposed chromatographic method was validated in Statistical analysis

terms of linearity, LOD, LOQ, and repeatability. The phe-
nolic compounds were characterized according to their All the experiments were carried out at least in triplicate
retention times, and UV data were compared with commer- with constant results. Data were recorded as mean ± S.E.M.
cial standards. Three parallel analyses were performed. For Significant differences between means were determined by
the quantitative analysis of phenolic compounds, calibration Student’s test, p values < 0.05 were regarded as significant.
curves were obtained via the injection of known concentra-
tions (0.0, 0.00782, 0.01563, 0.03125, 0.0625, 0.125, 0.25,
0.5 and 1.0 ppm) of different standards compounds i.e. gallic Results
acid, fumaric acid, protocatechuic acid, catechin hydrate,
p-hydroxy benzoic acid, 6,7-dihydroxy coumarin, caffeic Studies on mushrooms for the preparation of alternative
acid, vanillin, 2,4-dihydroxy benzoic acid, p-coumaric pharmaceutical components in the management of various
acid, ferulic acid, coumarin, trans-2-hydroxy cinnamic acid, diseases are increasing. Among the bioactive compounds
ellagic acid, rosmarinic acid, trans-cinnamic acid. Linear present in mushrooms, phenolics are one of the most

13
 F. Çayan et al.

Table 2  Analytical parameters No Compounds RT R2 Linearity LOD (µg/L) LOQ (µg/L)

for HPLC–DAD analysis range (mg/L)

1 Gallic acid 4.37 0.9986 0.06–1 0.114 0.345

2 Fumaric acid 5.59 0.9999 0.06–3 0.970 2.940
3 Protocatechuic acid 6.87 0.9997 0.01–1 0.021 0.063
4 Catechin hydrate 8.46 0.9965 0.06–1 0.094 0.285
5 p-Hydroxybenzoic acid 10.64 0.9971 0.01–1 0.112 0.342
6 6,7-Dihydroxy coumarin 11.62 0.9998 0.01–1 0.019 0.060
7 Caffeic acid 13.13 0.9999 0.02–1 0.010 0.031
8 Vanillin 14.89 0.9995 0.01–0.5 0.020 0.061
9 2,4-Dihydroxy benzoic acid 15.54 0.9999 0.01–0.5 0.006 0.021
10 p-Coumaric acid 18.74 0.9994 0.01–0.5 0.022 0.067
11 Ferulic acid 19.76 0.9997 0.01–1 0.157 0.475
12 Coumarin 20.96 0.9999 0.01–0.5 0.001 0.001
13 trans-2-Hydroxy cinnamic acid 21.98 0.9998 0.03–0.5 0.018 0.055
14 Ellagic acid 22.54 0.9982 0.02–1 0.079 0.239
15 Rosmarinic acid 23.61 0.9999 0.03–2 0.060 0.181
16 trans-Cinnamic acid 24.52 0.9975 0.01–1 0.199 0.603

RT retention time (min), R2 coefficient of determination, LOD limit of detection, LOQ limit of quantifica-
tion

important and probably the main candidates responsible Fumaric acid ((2E)-but-2-enedioic acid) is one of the
for the most health beneficial properties of the mushrooms. important organic acids due to its antioxidant, antimicro-
The analytical parameters and numbers of phenolic bial and acidifying properties [28]. The highest fumaric acid
compounds are described in Table 2. Phenolic and organic content was found in L. nuda (53.70 ± 3.66 µg/g), whereas
acid compositions of the mushroom species were given in the lowest fumaric acid content was found in L. leucothites
the Table 3. The results were expressed as µg per g of dry (4.42 ± 0.64 µg/g) (Table 3).
weight (dw). Totally 16 phenolic and organic acid com- Protocatechuic acid (3,4-dihydroxy benzoic acid) is
pounds, namely; gallic acid, fumaric acid, protocatechuic known to have a range of bioactivities including anti-
acid, catechin hydrate, p-hydroxy benzoic acid, 6,7-dihy- inflammatory, antioxidant, antimicrobial, free radical-scav-
droxy coumarin, caffeic acid, vanillin, 2,4-dihydroxyben- enging activities, peroxidation inhibition and estrogenic/
zoic acid, p-coumaric acid, ferulic acid, coumarin, trans- antiestrogenic activity [29]. The protocatechuic acid con-
2-hydroxy cinnamic acid, ellagic acid, rosmarinic acid, tent of the mushroom species ranged from 0.75 ± 0.06 to
and trans-cinnamic acid were identified in the mushroms. 4.89 ± 0.32 µg/g (Table 3).
Figure 1 shows the HPLC–DAD chromatogram of stand- Catechin ((2R,3S)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-
ard phenolic compounds. 2H-chromene-3,5,7-triol) is a natural secondary metabolite.
According to obtained results, the highest level of total In previous studies, catechin was reported to show antioxi-
phenolic compounds was determined in S. granulatus dant, antimicrobial, anti-allergy and anticancer effects. Also,
(71.79 µg/g), L. nuda (68.38 µg/g), R. vinosa (58.41 µg/g), consuming catechin rich teas lowers the blood glucose levels
R. azurea (45.05 µg/g), L. personata (43.44 µg/g), C. and prevents from type-2 diabetes [30]. The lowest and high-
rutilus (40.33 µg/g), and A. vaginata (39.60 µg/g), est catechin amounts in mushroom species were found to be
respectively. in range of 1.33 ± 0.31–20.50 ± 1.26 µg/g. These values were
Gallic acid (3,4,5-trihydroxy benzoic acid) is a trihydroxy determined in L. personata and A. vaginata, respectively
benzoic acid, a type of phenolic acid. Earlier studies have (Table 3).
shown that gallic acid indicated various bioactivities includ- p-Hydroxy benzoic acid is an organic compound can
ing anticancer, anti-HIV, anti-inflammatory, antimicrobial, be obtained naturally or synthesized chemically. Vari-
and antifungal. Furthermore, gallic acid is used in skin and ous pharmacological activities of p-hydroxy benzoic acid
leather industry as a chelating agent and preservative in food include antimicrobial, antialgal, antimutagenic, anties-
and beverages [27]. When R. aurora (2.96 ± 0.56 µg/g) has trogenic, hypoglycemic, anti-inflammatory, anti-platelet
the higher level of gallic acid, R. delica (0.07 ± 0.01 µg/g) aggregating, nematicidal, antiviral and antioxidant [31].
has the lower level of the gallic acid (Table 3). p-Hydroxy benzoic acid contents of the mushrooms ranged

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 Table 3  Composition (µg/g) of phenolic and organic acids
Compounds

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total

AB 0.83 ± 0.02 10.10 ± 0.98 nd 2.28 ± 0.57 nd 9.28 ± 0.76 nd nd 0.40 ± 0.01 nd nd 0.04 ± 0.01 nd nd 0.05 ± 0.01 0.45 ± 0.02 23.43
AV 0.82 ± 0.12 17.67 ± 1.17 nd 20.50 ± 1.26 0.08 ± 0.02 0.38 ± 0.04 0.08 ± 0.01 nd nd nd nd nd nd nd nd 0.07 ± 0.01 39.60
AT 2.26 ± 0.43 nd 1.37 ± 0.18 nd nd 2.07 ± 0.25 nd nd nd nd nd 0.06 ± 0.02 nd nd nd 0.09 ± 0.01 5.85
CO 0.13 ± 0.01 nd 0.76 ± 0.09 2.14 ± 0.21 0.56 ± 0.12 nd nd nd nd nd nd nd nd 0.79 ± 0.48 nd nd 4.38
CC 0.13 ± 0.03 5.59 ± 0.47 0.75 ± 0.06 2.14 ± 0.21 0.55 ± 0.12 0.37 ± 0.14 nd 0.09 ± 0.01 nd 0.06 ± 0.01 nd nd nd nd 0.16 ± 0.04 0.02 ± 0.01 9.86
CD 1.86 ± 0.14 29.95 ± 2.15 nd 3.72 ± 0.18 0.52 ± 0.07 nd nd 0.08 ± 0.02 0.09 ± 0.01 0.08 ± 0.01 0.02 ± 0.01 0.02 ± 0.01 nd nd nd 0.01 ± 0.01 36.35
CA 1.44 ± 0.32 8.93 ± 0.87 2.01 ± 0.39 7.04 ± 0.85 2.02 ± 0.16 3.44 ± 0.23 0.25 ± 0.12 0.23 ± 0.10 0.14 ± 0.07 0.03 ± 0.01 0.03 ± 0.01 nd 0.18 ± 0.04 nd nd 0.08 ± 0.02 25.82
CR 1.20 ± 0.10 27.82 ± 2.08 1.18 ± 0.22 7.81 ± 0.78 0.27 ± 0.06 nd nd nd 1.33 ± 0.65 0.05 ± 0.01 nd 0.36 ± 0.08 nd nd 0.31 ± 0.05 nd 40.33
LD 0.69 ± 0.11 6.95 ± 0.89 0.85 ± 0.11 15.82 ± 1.25 1.14 ± 0.12 0.73 ± 0.17 nd 0.10 ± 0.03 0.61 ± 0.09 0.02 ± 0.01 nd 0.09 ± 0.01 nd nd 0.09 ± 0.01 0.16 ± 0.04 27.25
LS1 0.43 ± 0.04 11.01 ± 0.85 0.87 ± 0.08 1.44 ± 0.21 0.44 ± 0.07 nd nd nd 0.28 ± 0.06 nd nd 0.03 ± 0.01 nd nd 0.18 ± 0.04 0.04 ± 0.01 14.72
LS2 0.24 ± 0.02 5.72 ± 0.58 nd 4.01 ± 0.35 0.14 ± 0.02 0.28 ± 0.03 0.16 ± 0.01 nd nd nd nd 0.01 ± 0.01 nd 0.20 ± 0.02 nd nd 10.76
LN 1.90 ± 0.35 53.70 ± 3.66 1.90 ± 0.35 2.76 ± 0.41 5.44 ± 0.67 1.11 ± 0.12 nd nd 0.99 ± 0.10 0.05 ± 0.01 nd nd 0.30 ± 0.13 nd 0.07 ± 0.01 0.16 ± 0.02 68.38
LP 1.71 ± 0.16 34.27 ± 2.54 4.03 ± 0.68 1.33 ± 0.31 0.30 ± 0.08 0.29 ± 0.07 nd 0.22 ± 0.06 nd 0.04 ± 0.01 0.32 ± 0.06 nd nd nd 0.85 ± 0.15 0.08 ± 0.01 43.44
LL 0.21 ± 0.02 4.42 ± 0.64 0.91 ± 0.14 nd 0.47 ± 0.15 9.02 ± 0.87 nd nd 0.13 ± 0.02 nd nd nd nd 0.34 ± 0.08 nd 0.38 ± 0.09 15.88
LT 1.18 ± 0.12 nd 1.95 ± 0.19 2.11 ± 0.27 0.41 ± 0.09 nd nd nd 0.29 ± 0.06 nd nd nd nd 0.25 ± 0.05 nd nd 6.19
MO nd 25.85 ± 1.14 2.83 ± 0.32 nd nd nd nd nd nd nd 0.05 ± 0.01 0.01 ± 0.01 0.10 ± 0.02 nd 0.20 ± 0.02 0.01 ± 0.01 29.05
ME1 1.17 ± 0.15 nd 3.85 ± 0.26 5.04 ± 0.41 0.17 ± 0.02 nd 0.18 ± 0.02 nd nd 0.08 ± 0.02 0.01 ± 0.01 nd nd nd nd 0.02 ± 0.01 10.52
ME2 1.32 ± 0.32 nd 1.98 ± 0.22 10.24 ± 0.78 nd nd nd nd nd 0.11 ± 0.02 nd nd nd 0.39 ± 0.09 0.04 ± 0.01 nd 14.08
PO 1.38 ± 0.06 4.86 ± 0.37 2.07 ± 0.24 1.79 ± 0.20 nd nd nd nd nd nd nd nd nd 0.27 ± 0.02 nd 0.04 ± 0.01 10.41
Identification and quantification of phenolic acid compounds of twenty‑six mushrooms by HPLC–…

RF 0.29 ± 0.02 4.72 ± 0.31 0.89 ± 0.10 5.77 ± 0.41 nd nd nd nd nd 0.01 ± 0.01 nd 0.09 ± 0.02 nd nd nd 0.05 ± 0.01 11.82
RA1 2.96 ± 0.56 nd nd nd nd nd nd nd nd nd nd nd nd 0.45 ± 0.14 0.59 ± 0.16 0.39 ± 0.12 4.39
RA2 1.45 ± 0.06 41.76 ± 3.02 nd nd nd 0.49 ± 0.04 nd nd nd 0.07 ± 0.01 0.11 ± 0.02 nd nd 0.73 ± 0.41 0.09 ± 0.02 0.35 ± 0.08 45.05
RD 0.07 ± 0.01 15.59 ± 1.21 4.89 ± 0.32 2.27 ± 0.20 nd nd nd nd nd nd 0.35 ± 0.07 nd nd nd nd 0.05 ± 0.01 23.22
RV 2.50 ± 0.41 52.08 ± 3.21 nd 3.65 ± 0.21 nd nd nd nd nd 0.01 ± 0.01 nd nd nd nd nd 0.17 ± 0.02 58.41
SG nd 48.38 ± 1.28 2.11 ± 0.18 16.59 ± 0.71 2.55 ± 0.23 nd nd nd 0.91 ± 0.18 nd nd nd nd 0.84 ± 0.10 0.29 ± 0.07 0.12 ± 0.02 71.79
TP 0.18 ± 0.01 nd 0.39 ± 0.05 3.32 ± 0.49 nd nd nd nd nd nd nd nd 0.27 ± 0.14 nd 2.76 ± 0.27 0.15 ± 0.04 7.07

Values represent the means ± S.E.M. of three parallel measurements (p < 0.05)
n.d. not detected

13
 F. Çayan et al.

Fig. 1  HPLC–DAD chroma-

togram of standard phenolic
compounds

from 0.08 ± 0.02 to 5.44 ± 0.67 µg/g. The lowest and highest Coumarins are seconder metabolites of plants and fruits.
p-hydroxy benzoic acid levels observed in A. vaginata and It has been reported that coumarins show cyclooxygenase
L. nuda, respectively. The minimum and maximum contents inhibition, antimutagenic, scavenging of reactive oxygen
of 2,4-dihydroxy benzoic acid were 0.09 ± 0.01 µg/g in C. species, anti-inflammatory, anticoagulant, lipoxygenase,
dryophila and 1.33 ± 0.65 µg/g in C. rutilus, respectively CNS stimulants, antithrombotic, vasodilatory and anticancer
(Table 3). activity [36]. The highest and lowest concentration of cou-
Caffeic acid (3,4-dihydroxy cinnamic) is mostly found in marin were detected in C. rutilus (0.36 ± 0.08 µg/g) and L.
plants. Caffeic acid is also found in many food sources such sulphureus and M. oreades (0.01 ± 0.01 µg/g), respectively.
as coffee, blueberries, apples and cider. It is known that caf- A. bisporus indicated the highest amount of 6,7-dihydroxy
feic acid has antioxidant and antimicrobial activities in vivo, coumarin with the value of 9.28 ± 0.76 µg/g, whereas L.
protects against atherosclerosis and cardiovascular diseases. sulphureus indicated the lowest amount of 6,7-dihydroxy
Moreover, caffeic acid is used as photo-protective agents coumarin with the value of 0.28 ± 0.03 µg/g (Table 3).
[32]. Caffeic acid was only found in A. vaginata, C. atra- Ellagic acid (2,3,7,8-tetrahydroxy-chromeno[5,4,3-cde]
mentarius, L. sulphureus, and M. elata mushrooms and caf- chromene-5,10-dione) is a lactone derivative found in vari-
feic acid contents were 0.08 ± 0.01, 0.25 ± 0.12, 0.16 ± 0.01 ous plants and fruits. Ellagic acid is famous for its biologi-
and 0.18 ± 0.02 µg/g, respectively (Table 3). cal and pharmacological activities such as antimutagenic,
Vanillin (4-hydroxy-3-methoxy benzaldehyde), com- anticarcinogenic, antioxidant and anti-inflammatory [37].
monly used as a flavouring agent in food industry since The minimum and maximum contents of ellagic acid were
ancient times and it has antimicrobial, anticancer and anti- 0.20 ± 0.02 µg/g in L. sulphureus, and 0.79 ± 0.48 µg/g in C.
oxidant activities [33]. The minimum and maximum con- odora, respectively (Table 3).
tents of vanillin were 0.08 ± 0.02 µg/g in C. dryophila and Rosmarinic acid ((R)-( +)-3-(3,4-dihydroxy phenyl)
0.23 ± 0.10 µg/g in C. atramentarius, respectively (Table 3). lactic) is a phenolic ester derivate, found in many plants.
p-Coumaric acid is mostly found in plants and mush- Well documented biological activities of rosmarinic acid
rooms in free or bound form. p-coumaric acid is known to are antioxidant, anti-inflammatory, antimutagenic, anti-
exhibit a range of bioactivities including antioxidant, anti- tumor, antigenotoxic, cytotoxic, antimetastatic, antian-
inflammatory, antimutagenic, anti-ulcer, antiplatelet and giogenic, neuroprotective, antimicrobial, immunomodula-
anti-cancer [34]. The highest p-coumaric acid content was tory, melanogenic and antivenom effects [38]. Rosmarinic
found in M. esculenta (0.11 ± 0.02 µg/g), whereas the lowest acid contents of mushroom samples were found between
p-coumaric acid content was found in R. flava and R. vinosa 0.04 ± 0.01–2.76 ± 0.27 µg/g. The lowest and highest ros-
(0.01 ± 0.01 µg/g) (Table 3). marinic acid values were observed in M. esculenta and T.
Ferulic acid (4-hydroxy-3-methoxy cinnamic acid) is panuoides, respectively (Table 3).
naturally occurring phenolic compound, abundant in cit- Cinnamic acid ((2E)-3-Phenylprop-2-enoic acid) is
rus fruits, banana, coffee, orange juice, eggplant, bamboo widely distributed in plants. The literature survey reveals
shoots, beetroot, cabbage, spinach and broccoli. Ferulic acid cinnamic acid derivatives have various biological properties
is also used as a food preservative in Japan. Recent studies such as antibacterial, antifungal, antioxidant, anti-inflamma-
have indicated that ferulic acid possesses anti-atherosclerotic tory and antitumor [39]. The highest amount of cinnamic
and antioxidant activities. Besides its biological properties acid was found in A. bisporus (0.45 ± 0.02 µg/g) while the
ferulic acid has protective properties against Alzheimer’s, lowest amount of cinnamic acid was found in C. dryophila,
inflammatory disease, diabetes, and hypertension [35]. and M. oreades (0.01 ± 0.01 µg/g). Trans-2-hydroxy cin-
Value of ferulic acid content was ranged from 0.01 ± 0.01 namic acid was only observed in C. atramentarius, L. nuda,
to 0.35 ± 0.07 µg/g for mushroom species. The highest and M. oreades and T. panuoides mushrooms and trans-2-hy-
lowest of ferulic acid concentrations were obtained in R. droxy cinnamic acid values were 0.18 ± 0.04, 0.30 ± 0.13,
delica and M. elata, respectively (Table 3). 0.10 ± 0.02 and 0.27 ± 0.14 µg/g, respectively (Table 3).

13
Identification and quantification of phenolic acid compounds of twenty‑six mushrooms by HPLC–…

Discussion benzoic acid, homogentisic acid, protocatechuic acid,

catechin, and pyrogallol. In a study on C. atramentarius,
There are many studies about phenolic compounds of p-hydroxy benzoic acid, p-coumaric acid, and cinnamic
mushroom species. A summary of the literature studies on acid contents have been reported [43]. The phenolic acid
phenolic compounds of the studied mushroom species is compositions such as gallic acid, gentisic acid, 2,3,4-tri-
shown in the Table 4. The phenolic acid compositions of A. hydroxy benzoic acid as of C. rutilus have been studied by
bisporus collected from different countries i.e. Northeast Islam et al. [44]. Another studies, by Palacios et al. [40],
Portugal, Spain, Korea, and Australia have been reported Taofiq et al. [42] and Barros et al. [25] reported phenolic
by Reis et al. [2], Palacios et al. [40], Kim et al. [41], acid compositions such as caffeic acid, chlorogenic acid,
Taofiq et al. [42], Barros et al. [25]. They studied phenolic ferulic acid, gallic acid, gentisic acid, p-hydroxy benzoic
compounds, namely; gallic, p-coumaric acid, cinnamic acid, homogentisic acid, protocatechuic acid, and pyro-
acid, caffeic acid, chlorogenic acid, ferulic acid, p-hydroxy gallol in L. deliciosus species. p-Hydroxy benzoic acid
and cinnamic acid compositions of L. salmonicolor have

Table 4  Phenolic compositions of studied mushroom species

Mushroom species Phenolic compounds Collected area References

A. bisporus Gallic acid, p-coumaric acid, cinnamic acid Northeast Portugal Reis et al. [2]
A. bisporus Caffeic acid, catechin, chlorogenic acid, p-coumaric acid, ferulic acid, Spain Palacios et al. [39]
gallic acid, p-hydroxy benzoic acid, homogentisic acid, protocatechuic
acid, pyrogallol
A. bisporus Gallic acid, pyrogallol, protocatechuic acid Korea Kim et al. [40]
A. bisporus Cinnamic acid Australia Taofiq et al. [41]
A. bisporus p-Hydroxy benzoic acid Northeast Portugal Barros et al. [24]
C. atramentarius p-Hydroxy benzoic acid, p-coumaric acid, cinnamic acid Northeast Portugal Heleno et al. [42]
C. rutilus Gallic acid, gentisic acid, 2,3,4-trihydroxy benzoic acid China Islam et al. [43]
L. deliciosus Caffeic acid, chlorogenic acid, ferulic acid, gallic acid, gentisic acid, Spain Palacios et al. [39]
p-hydroxy benzoic acid, homogentisic acid, protocatechuic acid, pyro-
gallol
L. deliciosus p-Hydroxy benzoic acid, cinnamic acid Spain Taofiq et al. [41]
L. deliciosus p-Hydroxy benzoic acid Northeast Portugal Barros et al. [24]
L. salmonicolor p-Hydroxy benzoic acid, cinnamic acid Northeast Portugal Vaz et al. [44]
L. sulphureus Caffeic acid, gallic acid, p-hydroxy benzoic acid Ethiopia Woldegiorgis et al. [45]
L. sulphureus p-Hydroxy benzoic acid, p-coumaric acid, salicylic acid Poland Nowacka et al. [46]
L. sulphureus Protocatechuic acid Poland Sulkowska-Ziaja et al. [47]
L. nuda Protocatechuic acid, p-hydroxy benzoic acid, p-coumaric acid Northeast Portugal Barros et al. [24]
L. leucothites Catechin, p-coumaric acid Dumlupinar-Turkey Aslim and Ozturk [48]
M. oreades p-Hydroxybenzoic acid, p-coumaric acid, vanillic acid, salicylic acid Poland Nowacka et al. [46]
M. esculenta Gallic acid, protocatechuic acid, p-hydroxybenzoic acid, p-coumaric acid, Turkey Yıldız et al. [49]
chlorogenic acid, ferulic acid, epicatechin
M. esculenta Gallic acid, protocatechuic acid, p-hydroxy benzoic acid, vanillic acid, China Islam et al. [43]
caffeic acid
M. esculenta cinnamic acid Northeast Portugal Taofiq et al. [41]
P. ostreatus Protocatechuic acid, p-hydroxy benzoic acid, p-coumaric acid, cinnamic Northeast Portugal Reis et al. [2]
acid
P. ostreatus p-Coumaric acid, ferulic acid, gallic acid, gentisic acid, homogentisic Spain Palacios et al. [39]
acid, p-hydroxy benzoic acid, protocatechuic acid
P. ostreatus Gallic acid, p-hydroxy benzoic acid, caffeic acid Ethiopia Woldegiorgis et al. [45]
P. ostreatus Protocatechuic acid, catechin, vanillic acid, p-coumaric acid, cinnamic Trabzon-Turkey Kolayli et al. [50]
acid
P. ostreatus Gallic acid, homogentisic acid, protocatechuic acid, chlorogenic acid Seoul-Korea Kim et al. [40]
P. ostreatus p-Hydroxy benzoic acid, p-coumaric acid, cinnamic acid Australia Taofiq et al. [41]
R. delica p-Hydroxy benzoic acid, syringic acid, vanillic acid, caffeic acid, cin- Greece Kalogeropoulos et al. [51]
namic acid, chlorogenic acid, ferulic acid, p-coumaric acid

13
 F. Çayan et al.

been studied [45]. In other studies, on L. sulphureus col- from Romania were reported as 77.01, 36.79, 292.44, 55.33,
lected different area, caffeic acid, gallic acid, p-hydroxy 71.11, 45.57 and 109.09 mg/100 g FW, respectively [56].
benzoic acid, protocatechuic acid compositions have been The phenolic profiles of the methanol extracts of twelve
reported [46–48]. The phenolic acid contents i.e. protocat- cruciferous vegetables, pakchoi (Brassica. rapa var. chin-
echuic acid, p-hydroxy benzoic acid, p-coumaric acid of ensis) (2472.00 ± 433.94 µg/g), choysum (B. rapa var.
L. nuda have been studied by Barros et al. [25]. Catechin parachinensis) (4377.62 ± 400.24 µg/g), Chinese cabbage
and p-coumaric acid contents of L. leucothites have been (B. rapa var. pekinensis) (9608.09 ± 3614.89 µg/g), kailan
reported [49]. In another recent study, p-hydroxy benzoic (B. oleracea var. alboglagra) (22,591.87 ± 1755.35 µg/g),
acid, p-coumaric acid, vanilic acid, salicylic acid compo- Br ussels sprout (B. oleracea var. gemmifera)
sitions of M. oreades using LC–ESI–MS/MS have been (38,487.84 ± 2681.78 µg/g), cabbage (B. oleracea var. capi-
studied by Nowacka et al. [47]. The phenolic acid compo- tata) (46,021.61 ± 16,141.09 µg/g), cauliflower (B. olera-
sitions such as gallic acid, protocatechuic acid, p-hydroxy cea var. botrytis) (7090.92 ± 1980.38 µg/g), broccoli (B.
benzoic acid, p-coumaric acid, chlorogenic acid, ferulic oleracea var. italica) (34,947.07 ± 7592.93 µg/g), rocket
acid, epicatechin, vanillic acid, caffeic acid, cinnamic acid salad (Eruca sativa) (9253.61 ± 3175.72 µg/g), red cherry
of M. esculenta have been reported by Yildiz et al. [50], radish (Raphanus sativus) (1.68 ± 0.24 µg/g), daikon rad-
Islam et al. [44], and Taofiq et al. [42]. There are several ish (Raphanus sativus) (1.02 ± 0.32 µg/g), and watercress
publications of phenolic acid compositions of P. ostreatus. (Nasturtium officcinale) (6777.73 ± 3252.94 µg/g) purchased
Protocatechuic acid, p-hydroxy benzoic acid, p-coumaric from various supermarkets in Singapore were reported by Li
acid, cinnamic acid by Reis et al. [2], p-coumaric acid, et al. [57]. The total amount of phenolic compounds of medi-
ferulic acid, gallic acid, gentisic acid, homogentisic acid, cally important plants Sideritis albiflora and Sideritis lepto-
p-hydroxy benzoic acid, protocatechuic acid by Palacios clada collected from Turkey were determined as 22.257 and
et al. [40], gallic acid, p-hydroxy benzoic acid, caffeic acid 39.245 µg/g in our previous research [58]. In a study of our
by Woldegiorgis et al. [46], protocatechuic acid, catechin, group on the phenolic compounds of mushrooms carried out
vanillic acid, p-coumaric acid, cinnamic acid by Kolaylı by Çayan et al. [59], the total amount of phenolic compounds
et al. [51], gallic acid, homogentisic acid, protocatechuic of Agrocybe cylindracea, Coprinus comatus, Clathrus rub-
acid, chlorogenic acid by Kim et al. [41], p-hydroxy ben- ber, Hypholoma fasciculare, Lentinus tigrinus, Mitrophora
zoic acid, p-coumaric acid, cinnamic acid by Taofiq et al. semilibera were calculated as 16.18, 84.63, 115.90, 11.68,
[42] have been reported. Another study, Kalogeropoulos 146.55 and 40.84 µg/g, respectively. It is supported by this
et al. [52] reported phenolic acids such as p-hydroxy ben- study that mushrooms have a significantly high amount of
zoic acid, syringic acid, vanilic acid, caffeic acid, cinnamic phenolic compounds when compared to different food, fruit,
acid, chlorogenic acid, ferulic acid, p-coumaric acid in R. plant and mushroom species.
delica. The results obtained with this study show similari-
ties and differences with the literature. Cultivation tech-
niques, growing conditions, ripening process, processing
and storage conditions, extraction methods, different spe- Conclusions
cies, as well as stress conditions such as UV radiation,
infection by pathogens and parasites, wounding air pollu- Phenolic compounds of 26 mushrooms collected from Ana-
tion and exposure to extreme temperatures are important tolia were detected by using HPLC–DAD. To the best our
factors that can explain these similarities and differences knowledge, phenolic acids of A. vaginata, A. tabescens, C.
between the obtained results and the literature [53, 54]. odora, C. confluens, C. dryophila, L. personata, L. leuco-
Phenolic acids are found in the human diet in different thites, L. tricolor, M. elata, R. flava, R. aurora, R. azurea, S.
foods such as mushrooms, plants, vegetables, fruits, spices granulatus and T. panuoides have been studied for the first
and cereals. Because of their bioactive importance, phenolic time. The total amount of phenolic compounds of the studied
acids are extensively studied and there is evidence of their mushroom species was determined to be comparable to other
role in disease prevention [55]. As it seen Table 3, S. granula- foods. It can be suggested that the differences in the qualita-
tus (71.79 µg/g) and L. nuda (68.38 µg/g) revealed the high- tive and quantitative composition of phenolic compounds
est concentration of total phenolic compounds among the can be caused by genetic differences between species, habi-
studied mushrooms. Total amount of phenolic compounds tats, growth conditions, and maturation of mushrooms. Our
of the methanol extracts of sea-buckthorn (Hippophaë findings suggest that these mushroom species can be used as
rhamnoides), hawthorn (Crataegus monogyn), wild grown an alternative source of natural phenolic compounds for the
European blackberry (Rubus fruticosus), Cornelian cherry food, cosmetic and pharmaceutical industries.
(Cornus mas), blackthorn (Prunus spinosa), dog rose (Rosa
canina), hackberry (Prunus padus) wild fruits collected

13
Identification and quantification of phenolic acid compounds of twenty‑six mushrooms by HPLC–…

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