Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112879

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

journal homepage: www.elsevier.com/locate/jpba

Optimization of the extraction procedure for the determination of

phenolic acids and flavonoids in the leaves of globe artichoke (Cynara
cardunculus var. scolymus L.)
Beate Stumpf a,∗ , Margitta Künne a,1 , Lan Ma a,1 , Menglu Xu a,1 , Feng Yan a ,
Hans-Peter Piepho b , Bernd Honermeier a
a
Institute of Agronomy and Plant Breeding I, Justus Liebig University, Schubertstrasse 81, 35392, Giessen, Germany
b
Biostatistics Unit, Institute of Crop Science, University of Hohenheim, Fruwirthstrasse 23, 70599, Stuttgart, Germany

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

Article history: Globe artichoke (Cynara cardunculus var. scolymus L.) is not only used as a vegetable and ornamental plant,
Received 3 June 2019 but also as important medicinal plant for the treatment of dyspeptic disorders. The European Pharma-
Received in revised form 8 September 2019 copoeia describes a method for the quality assessment of dry artichoke leaves, which is time-consuming
Accepted 9 September 2019
and requires huge amounts of organic solvents. In this study, an ultrasound-assisted extraction method
Available online 10 September 2019
was studied, which proved to be more efficient than the standard protocol of the European Pharma-
copoeia, since it led to comparable results, was faster and easier to handle, and was more sustainable due
Keywords:
to a reduced need for organic solvents.
Artichoke leaf extracts
European Pharmacopoeia © 2019 Published by Elsevier B.V.
Hot water extraction
Sonication

1. Introduction
Globe artichoke (Cynara cardunculus var. scolymusL.) is an
important vegetable and medicinal plant belonging to the Aster-
aceae family. Besides the use of green flower heads in vegetable
production, the leaves and leaf extracts of artichoke plants are
traditionally used for the treatment of dyspeptic disorders [1]. In
addition, artichoke leaf extracts are supposed to reduce the amount
of LDL (low density lipoprotein) cholesterol in the blood [2,3], and
they display antimicrobial [4], antioxidant and anti-carcinogenic
properties [5].
The health beneficial effects of artichoke leaves are mainly
ascribed to phenolic secondary metabolites, such as phenolic acids
and flavonoids, but also to sesquiterpene lactones and inulin [6].
Among phenolic acids, chlorogenic acid (CA) is recognized as lead
∗ Corresponding author at: Institute of Agronomy and Plant Breeding I, Biomed-
ical Research Center, Justus Liebig University, Schubertstrasse 81, 35392, Giessen,
Germany.
E-mail addresses: beate.stumpf@ernaehrung.uni-giessen.de
(B. Stumpf), margitta.kuenne@ernaehrung.uni-giessen.de (M. Künne),
lan.ma@ernaehrung.uni-giessen.de (L. Ma), menglu.xu@ernaehrung.uni-giessen.de
(M. Xu), feng.yan@agrar.uni-giessen.de (F. Yan), piepho@uni-hohenheim.de
(H.-P. Piepho), bernd.honermeier@agrar.uni-giessen.de (B. Honermeier).
1
These authors contributed equally to this work.
substance and used as marker substance for the quality control in
the trade of artichoke herbs. According to the European Pharma-
copoeia (Ph. Eur.), artichoke leaves as herbal drugs require at least
0.7% CA [7]. Although other parts of artichoke plants also contain
CA, their CA concentrations are significantly lower, e. g. in artichoke
heads concentrations ranged from no CA to 0.46% CA [8,9]. Also in
globe artichoke for vegetable production, lower concentrations of
CA up to 0.21% have been found in the leaves [10]. Therefore, they
are not used in pharmaceutical industry.
For the determination of chlorogenic acid, the Ph. Eur. describes
a heat reflux based method [7]. The recommended procedure
includes two times heating the sample in methanol up to 70 ◦ C for
1 h. Therefore, this procedure is time-consuming, and it requires
huge amounts of organic solvents, which can be harmful for
the environment. Furthermore, it has been reported that individ-
ual phenolic compounds react differently to heat treatments. For
example, the concentration of chlorogenic acid in Citrus paradisi
peel extracts decreased due to heat treatment, whereas the concen-
trations of other phenolic compounds increased [11]. In addition,
heat treatment of strawberry (Fragaria x ananassa) juice resulted
in enhanced concentrations of p-hydroxybenzoic acid and reduced
concentrations of ellagic acid [12]. Bilia et al. [13] stated that ele-
vated storage temperatures up to 60 ◦ C led to a decomposition of
compounds in artichoke tinctures. However, it is not clear if pheno-

https://doi.org/10.1016/j.jpba.2019.112879
0731-7085/© 2019 Published by Elsevier B.V.
2 B. Stumpf, M. Künne, L. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112879
lic compounds are partially degraded by the extraction procedure
described in the Ph. Eur.
It is time to rethink the official Ph. Eur. protocol and to evaluate if
it can be replaced by another one which is easier to handle, requires
less time and chemicals, and is more preservative to the extracted
compounds. Many different methods are commonly applied for the
extraction of phenolic compounds from plant material. Compar-
ing maceration, boiling, Soxhlet extraction and ultrasound-assisted
extraction (UAE) for the extraction of chlorogenic acid from arti-
choke leaves, Saleh et al. [14] described UAE as the most efficient
one of these methods. They explain the principle of the UAE as fol-
lows: Compounds diffuse from the particles of the samples into
the solvent. A stagnant solvent layer forms around each particle,
preventing further diffusion. Cavitation bubbles burst near the par-
ticles in an asymmetric manner, creating a high speed jet. These
jets can disrupt the stagnant layer, break the particle surface, and
enlarge cell pores of the particle, so the solvent can easier enter
into the particle and further diffusion takes place [14]. Thus, UAE
seems to be a good alternative to the traditional extraction meth-
ods. Therefore, we compared the official method of the Ph. Eur. to
another promising protocol, using an ultrasound-assisted extrac-
tion procedure. In addition, many companies produce artichoke leaf
extracts by hot water extraction, omitting the utilization of organic
solvents. Therefore, it would be interesting to know how close the
results of a hot water extraction and a methanol extraction are
related. Thus, an additional hot water extraction was implemented
in the present study.
Not only the concentration of distinct compounds, but also their
antioxidant properties are interesting for the quality determina-
tion of artichoke leaf extracts. For the evaluation of antioxidant
properties, it can be distinguished between electron transfer
(ET) based methods, which measure the reducing capacity of an
antioxidant, and H-atom transfer (HAT) based methods, which
describe the ability of an antioxidant to scavenge peroxyl radi-
cals [15]. Huang et al. [16] recommended to measure both total
phenolic concentration (TPC) as an ET based method, as well
as oxygen radical absorbance capacity (ORAC) assay as a HAT
based analysis. Therefore, both analyses were carried out in this
study.
Although the only requirement of the Ph. Eur. for arti-
choke leaves is their concentration of chlorogenic acid, other
caffeoylquinic acids (CQA) and flavonoids, such as luteolin-7-
O-glucoside (cynaroside), might also be of interest, since they
contribute to the antioxidant properties of the extracts. Therefore,
in addition to CA, the concentrations of total CQA, cynaroside, and
total flavonoids were also determined by high performance liquid
chromatography (HPLC).
The main aim of this study was to clarify whether an ultrasound-
assisted extraction would be suited to replace the common Ph.
Eur. procedure for the extraction of phenolic compounds from
artichoke leaves, regarding the concentrations of distinct phenolic
compounds as well as their antioxidant properties. Two addi-
tional aspects were considered in this study: the milling procedure
and the solvent. For the milling of samples, a coffee grinder is a
common tool in most laboratories. However, due to their hairy
surface, it is hardly possible to obtain a homogeneous product by
grinding artichoke leaves with a coffee grinder. Therefore, samples
obtained from a coffee grinder and a ball mill, respectively, were
compared within this study. Although organic solvents are usu-
ally employed for laboratory analysis of phenolic compounds in
plants, hot water is commonly used in industry to extract pheno-
lic compounds from herbal drugs. Therefore, it was tested whether
the yield of phenolic compounds using methanol extraction was
comparable to the yield of phenolic compounds from hot water
extraction.
2. Material and methods
All chemicals were of HPLC grade. In total, 26 dry artichoke
leaf samples were provided by different companies. The artichoke
plants were cultivated in Germany under conventional conditions
and harvested several times in 2017. The used leaves were ground
for one minute with a coffee grinder (Type 4142, Braun, Germany)
or for 30 s with a frequency of 30 Hz in a ball mill (MM400, Retsch,
Germany), respectively, to elucidate the influence of the grind-
ing procedure on the extraction of phenolic compounds. Phenolic
compounds were extracted from ground samples according to
three different protocols: the standard protocol of the Ph. Eur.,
an ultrasound-assisted extraction (UAE) method, and a hot water
(HW) extraction. Each extraction was performed in triplicate. Dis-
tinct phenolic compounds were analyzed by HPLC. In addition,
antioxidant properties of the artichoke leaf extracts were exam-
ined using a single electron transfer based method (TPC), and an H
atom transfer based method (ORAC). All results are presented on a
dry matter (DM) basis.
2.1. Extraction procedure according to Ph. Eur
500 mg of the ground sample were mixed with 50 ml methanol
and heated to 70 ◦ C in a reflux condenser for 1 h. The extract was
centrifuged for 2 min at 1218 g and 10 ◦ C. The supernatant was fil-
tered through a pleated filter (615 ¼, Macherey Nagel, Germany),
and the residue was extracted once more with 50 ml methanol
at the same conditions. The second extract was filtered through
a pleated filter and pooled with the first extract. The volume of the
combined extracts was made up to 250 ml with water. The resulting
concentration of methanol was 40%.
2.2. Ultrasound-assisted extraction
100 mg of the ground leaves were mixed with 25 ml 80%
methanol, and sonicated for 30 min. The ultrasonic bath (35 kHz;
Bandelin Sonorex Digitec, Germany) was cooled with ice, and the
temperature was kept below 40 ◦ C. The extract was diluted with
25 ml water, resulting in a final methanol concentration of 40% in
the extract solution. The extract was filtered through a pleated filter
for further analysis.
2.3. Hot water extraction
50 mg ground artichoke leaves were extracted with 15 ml pre-
heated water in a water bath for 1 h at 85 ◦ C. The extracts were
filtered through a pleated filter.
All extracts were directly used for the determination of total
phenolic concentration and antioxidant capacity. For HPLC mea-
surements, all extracts were further filtered through a syringe filter
(PVDF, pore size 0.45 ␮m), and stored at −20 ◦ C until analysis.
2.4. HPLC analysis
The HPLC system (manager 5050, pump 1050, diode array UV
detector 2600, and autosampler Azura 3950, Knauer, Germany;
thermostat K7, Techlab, Germany) was equipped with a ReproSil-
Pur C18-AQ column (Techlab, Germany), and separation was
carried out at 30 ◦ C. The mobile phase consisted of A) 0.5%
H3 PO4 and B) acetonitrile. The gradient is shown in Table 1.
Hot water extracts were diluted 1:2 with 80% methanol prior to
HPLC measurements to obtain the same methanol concentration
(40%) as the other extracts and the HPLC standards. Chlorogenic
acid (EDQM, France) [17] and cynaroside (Extrasynthese, France)
served as reference standards for CQA and flavonoids, respectively.
Chromatograms were recorded at 330 nm. Chlorogenic acid and
 B. Stumpf, M. Künne, L. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112879 3

Table 1 formed to compare extraction method × milling methods when the

HPLC gradient for the determination of phenolic acids and flavonoids in the leaves
interaction was significant. Comparisons were performed among
of artichoke. A: 0.5% H3 PO4 ; B: acetonitrile.
extraction methods for each milling method and vice versa. These
Time (min) A (%) B (%) Flow (ml/min) analyses were implemented using the GLIMMIX procedure of SAS
0.00 94 6 1 [19]. Correlations among the parameters CA, CQA, cynaroside,
0.64 94 6 1 flavonoids, TPC, and ORAC were calculated separately for each of
2.95 88 12 1 the six combinations of extraction and milling method using the
3.86 82 18 1
program R [20]. Correlation coefficients are given as Pearson’s r.
8.39 82 18 1
8.85 78 22 1
13.34 78 22 1
14.06 0 100 1
3. Results
15.88 0 100 1
15.90 94 6 1 In most cases, the concentrations of phenolic compounds and
29.00 94 6 1 the values for TPC and ORAC were significantly higher in extracts
produced from material which was prepared by ball mill com-
pared to a coffee grinder, irrespective of the extraction procedure
cynaroside were identified by their respective retention times and
(Table 2). In addition, using a ball mill instead of a coffee grinder
absorbance spectra. The concentrations of chlorogenic acid, cynaro-
notably improved the reproducibility of all extraction procedures.
side, total CQA, and total flavonoids were calculated via their peak
Therefore, it will only be referred to ball mill extracts in the follow-
area. The limit of quantitation, calculated according to Eurachem
ing.
[18], was about 0.40 ␮g ml−1 and 0.81 ␮g ml−1 for chlorogenic acid
The chosen raw material provided a wide range of concentra-
and cynaroside, respectively. Calibration was done in the range
tions for phenolic compounds. The concentration of chlorogenic
from 3.2 to 64.3 ␮g ml−1 and 1.2 to 23.6 ␮g ml−1 for chlorogenic
acid ranged between 0.80 and 3.38% DM in the Ph. Eur. extracts.
acid and cynaroside, respectively.
Thus, the maximum was approximately four times as high as the
minimum value.
2.5. Total phenolic concentration (TPC)
The concentrations of CA and total CQA, as well as the antiox-
idant capacity were comparable for Ph. Eur. and UAE extracts
TPC is often referred to as “total phenolic content”. In fact, TPC
(Table 2). TPC and the concentrations of cynaroside and total
is no content but a concentration. Therefore, it will be termed as
flavonoids were significantly lower for UAE compared to Ph. Eur.
“total phenolic concentration” throughout this study. For TPC anal-
extracts (cynaroside: -16%; total flavonoids: -11%; TPC: -2.5%)
ysis, 200 ␮l of the extracts (for HW extracts 100 ␮l) were mixed
(Table 2). On average, CQA concentrations were 4.5 and 5.1 times
with 3 ml H2 O (for HW extracts 3.1 ml) and 200 ␮l Folin-Ciocalteu’s
as high as flavonoid concentrations for Ph. Eur. and UAE extracts,
phenol reagent (Merck, Germany), and incubated for 5 min at ambi-
respectively (Table 2).
ent temperature. After the addition of 600 ␮l saturated Na2 CO3
All investigated parameters were significantly lower in the HW
solution, the reaction mixtures were kept at 40 ◦ C for 30 min. Mea-
extracts compared to the Ph. Eur. extracts (CA: -9.45%; total CQA:
surements were carried out with a spectrophotometer (Specord
-25.0%; cynaroside: -33.6%; total flavonoids: -22.2%; TPC: -9.7%;
205, Analytik Jena, Germany) at 765 nm. Gallic acid served as stan-
ORAC: -15.6%) (Table 2). Two additional peaks of CQA derivatives
dard. Therefore, results are presented as gallic acid equivalents
appeared in the chromatograms of the HW extracts compared to
(GAE).
the Ph. Eur. extracts (Fig. 1).
All measured parameters were closely correlated to each other,
2.6. Antioxidant capacity
irrespective of the extraction method. This is exemplarily shown
in Fig. 2 for the Ph. Eur. extraction. The strongest correlation was
Antioxidant capacity was determined using the oxygen radical
found for CQA and TPC with a Pearson’s r of 0.993. The weakest cor-
absorbance capacity (ORAC) assay. 25 ␮l extract were mixed with
relation (r = 0.735) was observed between total CQA and cynaroside
150 ␮l 0.02 ␮M fluorescein (2-(6-Hydroxy-3-oxo-(3 H)-xanthen-9-
concentration (Fig. 2).
yl)benzoic acid) in potassium phosphate buffer (75 mM, pH 7.4),
and incubated at 37 ◦ C for 30 min. 150 ␮l AAPH (2,2 -Azobis (2-
methylpropionamidine) dihydrochloride) was added as a radical 4. Discussion
generator, and the decrease in fluorescence was measured every
90 s for 90 min in a 96 well microplate reader (Fluoroskan Ascent Ph. Eur. extracts revealed CA concentrations in artichoke leaves
FL, Thermo Scientific, Finland) with an excitation wavelength of between 0.80% and 3.38%. According to the European Pharma-
485 nm and an emission wavelength of 538 nm. The vitamin E copoeia, artichoke leaves should contain at least 0.7% CA [7].
analogue Trolox ((+/-)-6-Hydroxy-2,5,7,8-tetramethylchromane- Therefore, all samples fulfilled the requirements of the Ph. Eur.,
2-carboxylic acid) served as standard, and results are presented and they covered a wide range which makes them suitable for the
as Trolox equivalents (TE). comparison of different extraction methods. For other plant organs
of artichoke, lower concentrations of CA have been reported in
2.7. Statistical analysis literature [8–10].
Milling with a coffee grinder resulted in an inhomogeneous con-
As extraction and milling methods were applied to the same sistency of the material, with a fine powder and a “wooly” part. In
samples, a multivariate analysis of variance was performed using a contrast, samples ground with a ball mill produced a fine homo-
linear mixed model implementation based on residual maximum geneous powder. Therefore, the reproducibility was much better
likelihood estimation of the variance parameters. The linear pre- for the latter samples. In addition, using a ball mill required less
dictor comprised main effects for extraction method and milling time, and the device did not get hot, in contrast to the coffee grinder
method as well as their interaction. The variance-covariance matrix which needed to cool down between samples. The ball mill samples
for the six combinations of extraction and milling method per also displayed a larger surface, which can explain a higher effi-
sample was unstructured. Denominator degrees of freedom were ciency in extracting phenolic compounds. The observed differences
determined using the Kenward-Roger method. Tukey test was per- between coffee grinder and ball mill extracts emphasize the impor-
4 B. Stumpf, M. Künne, L. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112879

Table 2
Means and standard errors for the concentrations of phenolic compounds in artichoke leaves and their antioxidant properties, comparing three extraction procedures (Ph.
Eur.: extraction according to the European Pharmacopoeia; UAE: ultrasound assisted extraction; HW: hot water extraction) and two grinding methods (ball mill and coffee
grinder). Means in the same column followed by no common uppercase letter are significantly different (alpha = 5%) by the Tukey test. Means in the same row followed by no
common lowercase letter are significantly different (alpha = 5%) by the Tukey test. CQA: caffeoylquinic acids; TPC: total phenolic concentration; GAE: gallic acid equivalents;
ORAC: oxygen radical absorbance capacity; TE: Trolox equivalents.

Parameter Ph. Eur. UAE HW

Ball mill 1.51 ± 0.13 bB 1.51 ± 0.13 bB 1.37 ± 0.13 aB

Chlorogenic acid (% DM)
Coffee grinder 1.45 ± 0.13 bA 1.45 ± 0.13 bA 0.95 ± 0.08 aA
Ball mill 3.09 ± 0.24 bB 3.09 ± 0.24 bB 2.32 ± 0.23 aB
Total CQA (% DM)
Coffee grinder 2.92 ± 0.24 bA 2.88 ± 0.24 bA 1.47 ± 0.14 aA
Ball mill 0.46 ± 0.03 cB 0.38 ± 0.02 bA 0.31 ± 0.02 aB
Cynaroside (% DM)
Coffee grinder 0.43 ± 0.02 cA 0.39 ± 0.02 bA 0.25 ± 0.01 aA
Ball mill 0.68 ± 0.04 cB 0.60 ± 0.03 bA 0.53 ± 0.03 aB
Total flavonoids (% DM)
Coffee grinder 0.62 ± 0.03 bA 0.62 ± 0.03 bA 0.43 ± 0.02 aA
Ball mill 32.7 ± 1.8 cB 31.9 ± 1.7 bB 29.5 ± 1.8 aB
TPC (mg GAE g−1 DM)
Coffee grinder 27.7 ± 1.4 bA 28.6 ± 1.6 cA 25.0 ± 1.4 aA
Ball mill 524 ± 30 bB 518 ± 30 bB 442 ± 27 aB
ORAC (␮mol TE g−1 DM)
Coffee grinder 460 ± 26 bA 464 ± 25 bA 367 ± 22 aA

Fig. 1. Overlay of the chromatograms of a Ph. Eur. extract (upper chromatogram) and a hot water extract (lower chromatogram) from the same artichoke leaf sample. 1, 2,
6 and 7: caffeoylquinic acid derivatives; 3: chlorogenic acid; 4: cynaroside; 5: unknown flavonoid.

tance of standardization of the grinding method or of the particle
size to determine the quality of herbal drug raw materials.
In the present study, no significant differences could be observed
in the concentrations of chlorogenic acid or total CQA, respectively,
for the Ph. Eur. and UAE extracts (Table 2), although Ph. Eur. extrac-
tion was carried out at higher temperatures. Heat susceptibility
of phenolic compounds depends on the number and type of sub-
stituents [21,22]. In a microwave-assisted extraction experiment,
p-coumaric acid, which had the lowest number of substituents, was
the most stable compound among the investigated hydroxycin-
namic acids, whereas sinapic acid, possessing the highest number of
substituents, was completely degraded at 175 ◦ C [22]. In addition,
ferulic acid, showing a hydroxyl and a methoxyl group, was less
heat susceptible than caffeic acid with two hydroxyl groups [22]. In
contrast to the present study, Xu et al. [11] reported decreasing con-
centrations of chlorogenic acid in heat treated Citrus paradisi peel,
whereas the concentrations of other free phenolic acids increased.
However, heat treatments were carried out between 90 ◦ C and
150 ◦ C, which was higher than the applied temperature for the
Ph. Eur. extraction in the present study. Increasing the tempera-
ture from 40 to 60 ◦ C for the preparation of ethanol extracts from
grape (Vitis vinifera) marc enhanced the yield of total phenolics
[23]. In addition, increasing the extraction temperature for red dis-
tilled grape byproducts from 25 to 50 ◦ C in methanol, ethanol and
water extracts, respectively, resulted in higher concentrations of
total phenolic compounds irrespective of the solvent [24]. There-
fore, 70 ◦ C can be recognized as a moderate heat treatment which
does not impair the extraction of CA in methanol extracts.
Concentrations of cynaroside and total flavonoids were lower
in the UAE extracts than in the Ph. Eur. extracts (Table 2). This
is in accordance with literature, since flavonoid concentration has
been reported to be decreased in UAE extracts compared to heat
reflux extracts [21]. The extent of degradation is depended on
the number and positions of OH groups. Higher numbers of OH
groups and OH groups at position 3 of the flavonoids tended to
facilitate degradation, whereas sugar moieties prevented this pro-
cess [21]. Comparing UAE of Sophora japonica buds with pure
methanol and water, respectively, water extracts showed lower
rutin concentration. This was attributed to the formation of reactive
hydroxyl radicals in sonicated aqueous solutions, which degrade
rutin molecules [25]. A similar effect might be a possible explana-
tion for lower flavonoid concentrations of the UAE extracts in the
present study, since UAE was not carried out in pure methanol, but
in a mixture of methanol/water (80:20 v/v). In addition, it cannot
 B. Stumpf, M. Künne, L. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112879 5

Fig. 2. Correlations between the concentrations of chlorogenic acid (CA), total caffeoylquinic acids (CQA), cynaroside (Cyn), total flavonoids (F), total phenolic concentration
(TPC), and oxygen radical absorbance capacity (ORAC) for samples which were extracted by the standard protocol of the European Pharmacopoeia. The lower left part of the
matrix displays scatter plots; the upper right part shows the corresponding Pearson’s r. All correlations were highly significant (p < 0.001).

be excluded that the extraction efficiency for flavonoids was lower
for UAE than for Ph. Eur. extraction.
However, the only criterion of the Ph. Eur. is a CA concentra-
tion of at least 0.7% in the dry artichoke leaves [7]. Since there
are strong correlations between CA concentration and TPC (Pear-
son’s r = 0.952) and CA concentration and ORAC (r = 0.957), the
concentration of CA is a good indicator for general antioxidant
properties of the extracts. The correlations between flavonoids
and TPC or flavonoids and ORAC were weaker (Fig. 2). This is in
accordance with additional measurements of pure chlorogenic acid
and cynaroside standards, respectively (data not shown), which
revealed a close correlation between the concentrations of these
phenolic compounds and their corresponding TPC and ORAC val-
ues. The closest correlation was found for CA concentration and
TPC with a Pearson’s r of 0.99996. The correlation between cynaro-
side concentration and TPC was slightly weaker but still very high
(r = 0.992). Therefore, it is most important to find an extraction
procedure which results in CA concentrations similar to those of
the Ph. Eur. extracts. Consequently, the tested UAE seems to be an
appropriate alternative to the Ph. Eur. extraction.
TPC and ORAC were closely correlated with r = 0.976, 0.965, and
0.974 for Ph. Eur., UAE and HW extracts, respectively. Other studies
also reported close correlations for TPC and ORAC in many differ-
ent plant extracts [15,26,27]. However, there does not necessarily
need to be a close correlation between TPC and ORAC, since sim-
ple phenols, which are no good radical scavengers, are also able
to react with Folin-Ciocalteu’s phenol reagent [16]. For example,
in an UAE of Origanum vulgare leaves, the correlation between TPC
and ORAC (r = 0.60) was weaker than the correlation between these
parameters in the present study [28]. A possible explanation for
this difference is the composition of phenolic substances in the
extracts. In the Origanum experiment, the correlation between the
lead substance rosmarinic acid and TPC was 0.39 [28]. In contrast,
correlation between CA, the lead substance of the artichoke leaf
extracts, and TPC was 0.952, 0.951, and 0.942 for Ph. Eur., UAE,
and HW extracts, respectively. A very close correlation for TPC
and ORAC has also been found in the pure standards (CA stan-
dards r = 0.99946; cynaroside standards r = 0.975). Therefore, TPC
of artichoke leaf extracts gives a good estimation for their radical
scavenging activity.
In the present study with artichoke leaves, all parameters were
significantly lower in the HW extracts than in the Ph. Eur. extracts
(Table 2). HW extraction was carried out at higher temperatures
than Ph. Eur. extraction. It has been shown that flavonoids are
susceptible to heat and their degradation products can display
lower, equal, or higher antioxidant activity compared to the original
molecules [29]. Higher temperatures for HW extraction compared
to Ph. Eur. extraction could therefore be one of the reasons for lower
concentrations of phenolic compounds. In contrast, in an experi-
ment with fresh artichoke heads, cooked artichokes reached higher
TPC values than raw samples [8]. The authors related this phe-
nomenon to inactivation of polyphenol oxidases due to heat, and
to tissue break-down resulting in enhanced availability of phenolic
compounds [8]. However, these two experiments are hardly com-
parable, since they differed in organs (heads vs. leaves) and form of
the samples (fresh and intact vs. dry and ground).
Another reason for lower values in the HW extracts could be
the solubility of phenolic acids and flavonoids in different solvents.
According to Wang et al. [27], polar organic solvents are better
extractants for phenolic compounds than pure water. Increasing
methanol concentrations increased the concentration of flavonoids
in the extracts of maize kernels, with an optimum methanol con-
centration of 70% for luteolin [21]. Similar results were obtained
from an experiment with grape (Vitis vinifera) marc extracts, where
6 B. Stumpf, M. Künne, L. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112879
the concentration of phenolic compounds in ethanol/water extracts
increased from 10% to 30% water, and decreased at water concen-
trations higher than 50% [23]. In addition, boiling water proved to
be less efficient than a reflux system with methanol for the extrac-
tion of rutin from Sophora japonica flower buds [25]. Using water
instead of 80% methanol as solvent for the extraction of freeze dried
Moringa oleifera leaves led to decreased total phenolic and flavonoid
contents, as well as to a lower reducing power [30]. Methanol seems
to be a more potent solvent than water for the extraction of phe-
nolic compounds. Therefore, methanol extraction bears the risk of
overestimating the concentration of phenolic compounds which
can be extracted by HW.
Not only was the concentration of CA decreased in the HW
extracts, but also additional peaks of CQA derivatives appeared
in the HW chromatograms (Fig. 1). This finding is in agreement
with the report of Dawidowicz and Typek [31], who observed
hydroxylation reactions as well as the conversion of trans-5-O-
caffeoylquinic acid to cis-5-O-caffeoylquinic acid, formation of
trans-3-O-caffeoylquinic acid and trans-4-O-caffeoylquinic acid,
and hydrolysis of chlorogenic acid to caffeic acid and quinic acid
by heating an aqueous CA solution. These derivatives displayed dif-
ferent retention times compared to CA [31]. In addition, in coffee
beans roasted at 200 ◦ C, dehydration, epimerization, acyl migra-
tion and transesterification products of CA have been reported [32].
Therefore, it is likely that similar reactions took place during HW
extraction in the present study, like trans to cis conversion of the
caffeoyl moiety in CQA derivates or transfer of caffeoyl to another
position of the quinic acid part, resulting in the additional CQA
peaks which were observed in the chromatograms.
The error-proneness is higher for the Ph. Eur. method compared
to UAE, since more steps are required, like the transfer of the extract
into suitable tubes for centrifugation, and the transfer of residue
after centrifugation back to the flask for repeated extraction under
reflux. Furthermore, in contrast to the UAE method, the Ph. Eur.
extracts are diluted after filtration, which increases the risk of los-
ing a part of the sample in the filter paper. Another important point,
which should not be forgotten, is the cost efficiency. It takes more
time to extract each sample by the Ph. Eur. method. In addition,
the reflux system used for Ph. Eur. extraction requires much more
space than the ultrasound bath, and a smaller number of sam-
ples can be extracted simultaneously, which makes it even more
time-consuming. The Ph. Eur. extraction procedure does not only
require more time than the UAE, but it also needs five times as much
methanol per sample. Thus, the costs per sample as well as envi-
ronmental pollution can be reduced by performing UAE instead of
Ph. Eur. extraction.
5. Conclusion
Despite higher acquisition costs, the use of a ball mill can be
recommended for the analysis of herbal drugs, since the grind-
ing procedure is faster, requires smaller amounts of samples, and
results in a more homogenous product compared to a coffee
grinder.
The HW extraction method which was applied in this experi-
ment was less efficient than methanol extraction. Therefore, there
might be a risk of overestimating the concentration of phenolic
compounds which can be obtained from HW extraction. However,
there are many different ways to perform HW extraction, so the
results obtained from the method used herein can hardly be gen-
eralized.
Both Ph. Eur. extraction and UAE method showed similar results.
However, in comparison to Ph. Eur. extraction the UAE method
proved to be easier to handle, less time-consuming, and needed less
pollutive organic solvents. Therefore, UAE can be recommended to
replace the standard protocol of the European Pharmacopoeia in
long term.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Declaration of Competing Interest
None.
Acknowledgement
We would like to thank the anonymous reviewers for their valu-
able suggestions which helped to improve this manuscript.
References
[1] V. Lattanzio, P.A. Kroon, V. Linsalata, A. Cardinali, Globe artichoke: a
functional food and source of nutraceutical ingredients, J. Funct. Foods 1
(2009) 131–144, http://dx.doi.org/10.1016/j.jff.2009.01.002.
[2] B. Wider, M.H. Pittler, J. Thompson-Coon, E. Ernst, Artichoke leaf extract for
treating hypercholesterolaemia, Cochrane Database Syst. Rev. (3) (2013),
http://dx.doi.org/10.1002/14651858.CD003335.pub3.
[3] R. Mocelin, M. Marcon, G.D. Santo, L. Zanatta, A. Sachett, A.P. Schönell, F.
Bevilaqua, M. Giachini, R. Chitolina, S.M. Wildner, M.M.M.F. Duarte, G.M.M.
Conterato, A.L. Piato, D.B. Gomes, W.A. Roman Junior, Hypolipidemic and
antiatherogenic effects of Cynara scolymus in cholesterol-fed rats, Rev. Bras.
Farmacogn. 26 (2016) 233–239, http://dx.doi.org/10.1016/j.bjp.2015.11.004.
[4] X. Zhu, H. Zhang, R. Lo, Phenolic compounds from the leaf extract of artichoke
(Cynara scolymus L.) and their antimicrobial activities, J. Agric. Food Chem. 52
(2004) 7272–7278, http://dx.doi.org/10.1021/jf0490192.
[5] Z. Velez, M.A. Campinho, Â.R. Guerra, L. García, P. Ramos, O. Guerreiro, L.
Felício, F. Schmitt, M. Duarte, Biological characterization of Cynara
cardunculus L. methanolic extracts: antioxidant, anti-proliferative,
anti-migratory and anti-angiogenic activities, Agriculture 2 (2012) 472–492,
http://dx.doi.org/10.3390/agriculture2040472.
[6] B. de Falco, G. Incerti, M. Amato, V. Lanzotti, Artichoke: botanical,
agronomical, phytochemical, and pharmacological overview, Phytochem. Rev.
14 (2015) 993–1018, http://dx.doi.org/10.1007/s11101-015-9428-y.
[7] Ph. Eur. 9, Monographie 9.0/1866 Artischockenblätter (Cynarae folium), in:
Eur. Arzneib, 9th ed., Deutscher Apotheker Verlag, Stuttgart, 2017, pp.
1930–1932.
[8] M. Lutz, C. Henríquez, M. Escobar, Chemical composition and antioxidant
properties of mature and baby artichokes (Cynara scolymus L.), raw and
cooked, J. Food Anal. 24 (2011) 49–54, http://dx.doi.org/10.1016/j.jfca.2010.
06.001.
[9] G. Pandino, F.L. Courts, S. Lombardo, G. Mauromicale, G. Williamson,
Caffeoylquinic acids and flavonoids in the immature inflorescence of globe
artichoke, wild cardoon, and cultivated cardoon, J. Agric. Food Chem. 58
(2010) 1026–1031, http://dx.doi.org/10.1021/jf903311j.
[10] G. Pandino, S. Lombardo, G. Mauromicale, Globe artichoke leaves and floral
stems as a source of bioactive compounds, Ind. Crops Prod. 44 (2013) 44–49,
http://dx.doi.org/10.1016/j.indcrop.2012.10.022.
[11] G. Xu, X. Ye, J. Chen, D. Liu, Effect of heat treatment on the phenolic
compounds and antioxidant capacity of citrus peel extract, J. Agric. Food
Chem. 55 (2007) 330–335, http://dx.doi.org/10.1021/jf062517l.
[12] I. Odriozola-Serrano, R. Soliva-Fortuny, O. Martín-Belloso, Phenolic acids,
flavonoids, vitamin C and antioxidant capacity of strawberry juices processed
by high-intensity pulsed electric fields or heat treatments, Eur. Food Res.
Technol. 228 (2008) 239, http://dx.doi.org/10.1007/s00217-008-0928-5.
[13] A.R. Bilia, M.C. Bergonzi, G. Mazzi, F.F. Vincieri, Analysis and stability of the
constituents of artichoke and St. John’s Wort tinctures by HPLC–DAD and
HPLC–MS, Drug Dev. Ind. Pharm. 28 (2002) 609–619, http://dx.doi.org/10.
1081/DDC-120003457.
[14] I.A. Saleh, M. Vinatoru, T.J. Mason, N.S. Abdel-Azim, E.A. Aboutabl, F.M.
Hammouda, A possible general mechanism for ultrasound-assisted extraction
(UAE) suggested from the results of UAE of chlorogenic acid from Cynara
scolymus L. (artichoke) leaves, Ultrason. Sonochem. 31 (2016) 330–336,
http://dx.doi.org/10.1016/j.ultsonch.2016.01.002.
[15] S. Dudonné, X. Vitrac, P. Coutière, M. Woillez, J.-M. Mérillon, Comparative
study of antioxidant properties and total phenolic content of 30 plant extracts
of industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC assays, J. Agric.
Food Chem. 57 (2009) 1768–1774, http://dx.doi.org/10.1021/jf803011r.
[16] D. Huang, B. Ou, R.L. Prior, The chemistry behind antioxidant capacity assays, J.
Agric. Food Chem. 53 (2005) 1841–1856, http://dx.doi.org/10.1021/jf030723c.
[17] EDQM, Ph. Eur. reference standard for chlorogenic acid, catalogue code
Y0000569, in: European Directorate for the Quality of Medicines &
Healthcare, 2017, 7, Allée Kastner CS 30026, F-67081 Strasbourg (France).
 B. Stumpf, M. Künne, L. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 177 (2020) 112879
[18] B. Magnusson, U. Ornemark, Eurachem Guide: The Fitness for Purpose of
Analytical Methods – A Laboratory Guide to Method Validation and Related
Topics, 2nd ed. https://www.eurachem.org/images/stories/Guides/pdf/MV
guide 2nd ed EN.pdf, 2014 (Accessed 29. 07. 2019).
[19] SAS 9.4, SAS Institute Inc., Cary, NC, USA, 2016.
[20] R Core Team, R: A Language and Environment for Statistical Computing, R
Foundation for Statistical Computing, Vienna, Austria, 2015 https://www.R-
project.org/.
[21] M. Biesaga, Influence of extraction methods on stability of flavonoids, J.
Chromatogr. A 1218 (2011) 2505–2512, http://dx.doi.org/10.1016/j.chroma.
2011.02.059.
[22] A. Liazid, M. Palma, J. Brigui, C.G. Barroso, Investigation on phenolic
compounds stability during microwave-assisted extraction, J. Chromatogr. A
1140 (2007) 29–34, http://dx.doi.org/10.1016/j.chroma.2006.11.040.
[23] G. Spigno, L. Tramelli, D.M. De Faveri, Effects of extraction time, temperature
and solvent on concentration and antioxidant activity of grape marc
phenolics, J. Food Eng. 81 (2007) 200–208, http://dx.doi.org/10.1016/j.
jfoodeng.2006.10.021.
[24] M. Pinelo, M. Rubilar, M. Jerez, J. Sineiro, M.J. Núñez, Effect of solvent,
temperature, and solvent-to-solid ratio on the total phenolic content and
antiradical activity of extracts from different components of grape pomace, J.
Agric. Food Chem. 53 (2005) 2111–2117, http://dx.doi.org/10.1021/jf0488110.
[25] L. Paniwnyk, E. Beaufoy, J.P. Lorimer, T.J. Mason, The extraction of rutin from
flower buds of Sophora japonica, Ultrason. Sonochem. 8 (2001) 299–301,
http://dx.doi.org/10.1016/S1350-4177(00)00075-4.
[26] C.A. Ballus, A.D. Meinhart, F.A. de Souza Campos, H.T. Godoy, Total phenolics
of virgin olive oils highly correlate with the hydrogen atom transfer
7
mechanism of antioxidant capacity, J. Am. Oil Chem. Soc. 92 (2015) 843–851,
http://dx.doi.org/10.1007/s11746-015-2629-0.
[27] T. Wang, R. Jónsdóttir, G. Ólafsdóttir, Total phenolic compounds, radical
scavenging and metal chelation of extracts from Icelandic seaweeds, Food
Chem. 116 (2009) 240–248, http://dx.doi.org/10.1016/j.foodchem.2009.02.
041.
[28] F. Yan, A. Azizi, S. Janke, M. Schwarz, S. Zeller, B. Honermeier, Antioxidant
capacity variation in the oregano (Origanum vulgare L.) collection of the
German National Genebank, Ind. Crops Prod. 92 (2016) 19–25, http://dx.doi.
org/10.1016/j.indcrop.2016.07.038.
[29] H. Chaaban, I. Ioannou, L. Chebil, M. Slimane, C. Gérardin, C. Paris, C.
Charbonnel, L. Chekir, M. Ghoul, Effect of heat processing on thermal stability
and antioxidant activity of six flavonoids, J. Food Process. Preserv. 41 (2017),
e13203, http://dx.doi.org/10.1111/jfpp.13203.
[30] P. Siddhuraju, K. Becker, Antioxidant properties of various solvent extracts of
total phenolic constituents from three different agroclimatic origins of
drumstick tree (Moringa oleifera Lam.) leaves, J. Agric. Food Chem. 51 (2003)
2144–2155, http://dx.doi.org/10.1021/jf020444+.
[31] A.L. Dawidowicz, R. Typek, Thermal stability of 5-o-caffeoylquinic acid in
aqueous solutions at different heating conditions, J. Agric. Food Chem. 58
(2010) 12578–12584, http://dx.doi.org/10.1021/jf103373t.
[32] R. Jaiswal, M.F. Matei, A. Golon, M. Witt, N. Kuhnert, Understanding the fate of
chlorogenic acids in coffee roasting using mass spectrometry based targeted
and non-targeted analytical strategies, Food Funct. 3 (2012) 976–984, http://
dx.doi.org/10.1039/c2fo10260a.