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Journal of Chromatography A, xxx (2017) xxx–xxx

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Antibacterial potential of the Cistus incanus L. phenolics as studied

with use of thin-layer chromatography combined with direct
bioautography and in situ hydrolysis
Ágnes M. Móricz a,∗ , Dariusz Szeremeta b , Magdalena Knaś b , Ewa Długosz c , Péter G. Ott a ,
Teresa Kowalska b , Mieczysław Sajewicz b
a
Department of Pathophysiology, Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Herman O. Street 15, 1022
Budapest, Hungary
b
Institute of Chemistry, University of Silesia, 9 Szkolna Street, 40-006 Katowice, Poland
c
Department of Retail Pharmacy, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia in Katowice, 3
Kasztanowa Street, 41-200 Sosnowiec, Poland

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

Article history: The main aim of this study was to detect and identify antibacterial components of fraction I derived
Received 7 December 2017 from eleven commercial C. incanus herbal teas. Fraction I obtained by a well-established phytochemical
Received in revised form protocol of a multi-step extraction was expected to contain flavonoid aglycons alone. Antibacterial profile
20 December 2017
of fraction I was demonstrated by means of thin-layer chromatography – direct bioautography (TLC-DB)
Accepted 21 December 2017
using a Gram positive B. subtilis and a Gram negative A. fischeri strain. Six chromatographic zones of
Available online xxx
fraction I exhibited a well pronounced antibacterial potential. In qualitative terms, a good agreement
was observed among chromatographic fingerprints and the corresponding bioautograms of the eleven
Keywords:
Hairy rockrose (Cistus incanus L.)
samples. The compounds isolated from the six zones were analyzed by HPLC- diode array detector (DAD)-
Thin-layer chromatography–direct electrospray ionization (ESI)-MS. High numerical m/z values valid for certain constituents of these isolates
bioautography suggested that some selected antibacterial components are, unexpectedly, flavonoid glycosides. In order
Multi-development and two-dimensional to confirm this suggestion, three independent HPTLC methods (multi-development on amino phase and
HPTLC two two-dimensional developments on silica gel phase) were devised to in situ hydrolyze flavonoid
In situ acidic hydrolysis glycosides and then separate and visualize the liberated glucose and some other building blocks of the
Flavonoid aglycons zones’ components. Additionally, the sensitivity of glucose detection with p-aminobenzoic acid reagent
Acylated flavonoid glycosides
was enhanced by paraffin. In that way, the presence of the kaempferol glycosides (and not only the
aglycones alone) in fraction I was confirmed. Beside kaempferol, p-coumaric acid as a building block unit
was shown by HPLC-DAD-MS analysis of the hydrolyzed isolates. Results proved apigenin, kaempferide
and acylated kaempferol glycosides (cis- and trans-tiliroside and their conjugates with p-coumaric acid)
to be antibacterial components of fraction I. Because isomers of the coumaric acid conjugated tiliroside
were detected only in fraction I and not in the crude C. incanus extract, they are regarded as artifacts
produced through fractionation.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction
For the millennia, many thousand medicinal plants have been
used within the framework of traditional medicines across differ-
ent cultures around the globe. Due to the very high number of
medicinal plants, so far not all herbal materials have been systemat-
ically investigated for their beneficial effects and healing potential.
∗ Corresponding author.
E-mail address: moricz.agnes@agrar.mta.hu (Á.M. Móricz).
In order to make up for this evident delay, a strategy has been
developed which includes a fast and efficient screening of biological
activity of herbal extracts as an important preliminary procedure.
It is based on thin-layer chromatography (TLC) combined with
various bioactivity assays, constituting a high-throughput method-
ology that yields a bioprofile either of a crude plant extract or
the different fractions thereof. The method to localize antimicro-
bial activity directly on the thin-layer chromatographic adsorbent
(direct bioautography, DB) has been introduced as early as in the
seventies of the past century [1–3]. With time, TLC-DB has become a
well-established bioassay and it has found its well-deserved posi-

https://doi.org/10.1016/j.chroma.2017.12.056
0021-9673/© 2017 Elsevier B.V. All rights reserved.

Please cite this article in press as: Á.M. Móricz, et al., Antibacterial potential of the Cistus incanus L. phenolics as studied
with use of thin-layer chromatography combined with direct bioautography and in situ hydrolysis, J. Chromatogr. A (2017),
https://doi.org/10.1016/j.chroma.2017.12.056
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Table 1
Consecutive steps of the HPTLC method on the amino-modified silica gel 60 F254 s
combined with the in situ acidic hydrolysis to prove the presence of glucose in active
compounds.
1. Application of the isolated compounds
2. Incubation in cc. HCl vapor for 10 min
3. Heating for 8 min at 100 ◦ C (covered with a glass sheet)
4. Heating for 2 min at 100 ◦ C (uncovered)
5. Pre-development with acetonitrile up to 75 mm
6. Drying for 5 min by cold air stream
7. Development with acetonitrile – water 7:3 (v/v) up to 75 mm
8. Drying for 5 min by cold air stream
9. Heating for 20 min at 170 ◦ C
10. Dipping into paraffin – n-hexane 1:2 (v/v) and drying
tion in chemistry of natural products as a relatively inexpensive
and an easy to use tool for non-targeted screening of antimicro-
bial components of a complex matrix (e.g., [4–8]), as extensively
discussed in the monograph [9]. Furthermore, TLC as a flexible,
open-bed system enables the in situ pre- or post-chromatographic
derivatizations [10,11], two-dimensional (2D) and multiple devel-
opments (MD) [12], as well as the elution or desorption of the
compounds present in an active zone [7,13–15], which makes their
highly targeted characterization and identification possible.
An important representative of the Cistus genus is the hairy rock-
rose (Cistus incanus L.; syn.: Cistus creticus L. [16]), which belongs
to the Cistaceae family and is well recognized for its therapeutic
activity. This medicinal plant is very popular in its natural habitats:
eastern parts of the Mediterranean basin (including Greek Islands)
and the Middle East [17], and it has been traditionally used as an
anti-inflammatory, anti-allergic, antiulcerogenic, wound-healing,
antimicrobial and cytotoxic agent [18]. Antimicrobial potential of
the non-polar organic [19–21], methanolic [22] and the aque-
ous methanolic [23,24] extracts as well as essential oil [19,25,26]
derived from C. incanus leaves and flowers had been investigated
in a number of studies carried out against Gram positive and
Gram negative bacterial strains. It has been established that the
main activity of the essential oil can be attributed to monoter-
penes and diterpenes. Direct antibacterial potential of the aqueous
methanol extracts was demonstrated against Streptococcus mutans
[23], Staphylococcus aureus and Staphylococcus epidermidis [24].
Additional in situ experiments showed that rinses with the C.
incanus infusion reduced an initial bacterial colonization of tooth
enamel samples. It was also established that antibacterial poten-
tial of the alcoholic extracts was higher against the Gram positive
bacteria than the Gram negative ones [22,24]. Moreover, infusions
acted as growth inhibitors of yeast (e.g., Candida albicans and C.
glabrata, [25]) and of fungi such as the Aspergillus molds [27,28].
Table 2
Consecutive steps of the two-dimensional HPTLC method on silica gel 60 F254 combined with the in situ acidic hydrolysis to prove the presence of the glucose (a) and
kaempferol (b) conjugates in active zones.
a b
1. Development with chloroform – methanol – ethyl acetate
75:15:10 (v/v/v) on silica gel layer up to 75 mm (1st
dimension)
2. Incubation in cc. HCl vapor for 10 min
3. Heating for 8 min at 100 ◦ C (covered with a glass sheet)
4. Heating for 2 min at 100 ◦ C (uncovered)
5. Pre-development with acetonitrile in orthogonal direction
up to 85 mm (2nd dimension)
6. Drying for 5 min by cold air stream
7. Development with acetonitrile – water 4:1 or 35:10 (v/v)
in orthogonal direction up to 70 mm (2nd dimension)
8. Drying for 5 min by cold air stream
9. PABA(140 ◦ C, 5 min), PABA(140 ◦ C, 5 min)-paraffin or DPA
(110 ◦ C, 5 min) reagents
Please cite this article in press as: Á.M. Móricz, et al., Antibacterial potential of the Cistus incanus L. phenolics as studied
with use of thin-layer chromatography combined with direct bioautography and in situ hydrolysis, J. Chromatogr. A (2017),
https://doi.org/10.1016/j.chroma.2017.12.056
As the aqueous methanol provided high extraction yields with the
phenolic compounds including ellagitannins, flavanols, and glyco-
sylated flavonols, a strong correlation was demonstrated between
the antimicrobial activity of the C. incanus extracts and phenolic
contents of this plant [23,24,27,28].
As a non-selective total extraction of a whole plant (with use of
such potent extractants as methanol, or aqueous methanol) results
in a very complex mixture of herbal constituents, application of
TLC-DB to these samples in most cases allows a rough fractionation
only and the message derived from the respective bioautograms
usually is not informative enough. In order to overcome this short-
coming, a selective multi-step extraction of the C. incanus herbal
material is recommended for the investigation of its phenolics with
use of TLC-DB. For this purpose, a popular and elaborate protocol
[29–32] was utilized that offers separation of the plant phenolics
into six different fractions, which include flavonoid aglycons (I),
free phenolic acids (II), non-polar flavonoid glycosides (III), polar
flavonoid glycosides (IV), and phenolic acids obtained from acidic
(V) and basic hydrolysis (VI). Among them, fraction I displayed the
most diverse antibacterial potential in TLC-B. subtilis and TLC-A.
fischeri bioassays [22].
In the first part of this study, we focused on the TLC-DB screening
of fraction I obtained from eleven C. incanus samples (originating
from a number of local discount stores) for antibacterial com-
pounds and on identification of the constituents of this fraction.
However, the HPLC-MS analysis of the compounds eluted from the
active TLC zones unexpectedly resulted in m/z signals too high for
flavonoid aglycons. Therefore in the further characterization strat-
egy, the presence of the flavonoid condensates (dimers and trimers)
or glucose conjugates was anticipated. In the second part, an easy
and fast method was elaborated to discover and characterize com-
plex antibacterial components of a plant matrix, such as fraction I
of C. incanus, by means of TLC-DB, MD-HPTLC, and 2D-HPTLC com-
bined with an in situ acidic hydrolysis. The obtained results were
confirmed by parallel HPLC-MS analyses.
2. Experimental
2.1. Materials
The aluminium foil-backed silica gel 60F254 plates with normal
(TLC) and fine particle size (HPTLC), and the glass-backed amino-
modified HPTLC silica gel 60 F254 s plates were acquired from Merck
(Darmstadt, Germany). All solvents used for the TLC separations,
formic acid, 38% hydrochloric acid, aluminium chloride, ferric chlo-
ride, glucose, o-phosphoric acid and glacial acetic acid were of
analytical grade (Reanal, Budapest, Hungary). Sodium bicarbon-
Development with chloroform – methanol – ethyl acetate
75:15:10 (v/v/v) on silica gel layer up to 75mm (1st
dimension)
Incubation in cc. HCl vapor for 120 min
Drying for 5 min by cold air stream
Development with toluene – i-propyl acetate – formic acid
3:2:0.5 (v/v/v) in orthogonal direction up to 75 mm (2nd
dimension)
Drying for 5 min by cold air stream
Aluminium chloride reagent
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ate, sodium carbonate, sodium sulfate and diethyl ether were of
analytical purity (PPH POCH, Gliwice, Poland). Water used for the
preparation of herbal extracts was double distilled and deionized
under laboratory conditions using an Elix Advantage model Mil-
lipore system (Molsheim, France). p-Aminobenzoic acid (PABA),
aniline, diphenylamine (DPA) and the test substances apigenin
(≥97%), kaempferol (≥97%), and p-coumaric acid (≥98%) were
from Sigma–Aldrich (Budapest, Hungary). The dye reagent MTT (3-
[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), the
natural product (NP) reagent (diphenylboryloxyethylamine, or
diphenylboric acid ␤-ethylamino ester) and polyethylene gly-
col 400 (PEG 400) were purchased from Carl Roth (Karlsruhe,
Germany). Paraffin was purchased from a drugstore. For the HPLC
analysis, the gradient grade acetonitrile was purchased from Fisher
Scientific (Pittsburg, PA, USA) and pure water was produced by a
Millipore Direct-Q 3 UV system (Merck). The Gram negative, nat-
urally luminescent marine bacterium Aliivibrio fischeri (DSM-7151,
German Collection of Microorganisms and Cell Cultures, Berlin,
Germany) and the Gram positive soil bacterium Bacillus subtilis
F1276 (gift from the late József Farkas, Central Food Research Insti-
tute, Budapest, Hungary) were used for the bioassays.
2.2. Sample preparation
Dried herbal teas of the C. incanus L. species consisting of the
coarse-grained leaf, stem and flower particles were obtained from
the local market supplied by different manufacturers. According to
the distributors’ information, the plant material originated from
Turkey (samples T1–T5), Albania (samples A1–A4), and Greece
(sample G1), and one sample came from a not specified geograph-
ical location (sample ND1).
Isolation of the phenolics from the defatted, commercial C.
incanus L. samples (10 g) was carried out by liquid-solid extraction
in a Soxhlet apparatus using methanol (10 mL) followed by liquid-
liquid extraction. A detailed protocol of the selective multi-step
extraction of phenolic acids and flavonoids was elaborated based
on the literature [29–32]. Briefly, the production of fraction I started
with washing of the dry residue of the methanol extract (10 mL) by
hot water (4 × 25 mL). Then the merged water solution was cooled
for 24 h, filtered and extracted with diethyl ether (3 × 20 mL). The
combined ether fraction was extracted with 5% sodium bicarbon-
ate (3 × 20 mL) and then with 5% sodium carbonate (3 × 20 mL). The
merged aqueous carbonate layer was acidified with 18% hydrochlo-
ric acid (pH = 2) and then extracted with diethyl ether (3 × 20 mL).
The anhydrous combined organic layer was filtered, dried and re-
dissolved in methanol (10 mL). In that way, fraction I was obtained,
expectedly including flavonoid aglycons.
2.3. Thin-layer chromatography
Samples were applied with a Linomat IV sample applicator
(CAMAG, Muttenz, Switzerland) in the range of 5–10 ␮L at 8-mm
distance from the lower plate edge in 7-mm bands, with 5-mm
track distance. One-dimensional TLC or HPTLC separations were
achieved in a 20 cm × 10 cm twin trough chamber (CAMAG) sat-
urated for 10 min with chloroform – methanol – ethyl acetate,
75:15:10 (v/v/v) as a mobile phase. The chromatographic plates
developed to the distance of 75 mm were dried for 5 min by a
cold air stream from a hair-dryer and visualized using an UV lamp
(␭ = 254, or 365 nm) (CAMAG). The chromatographic zones were
documented through bioassays, or after derivatization with alu-
minium chloride (1% methanolic solution), ferric chloride (0.5 g
FeCl3 in 2.5 mL water and 47.5 mL ethanol), NP-PEG (0.5% methano-
lic NP solution, and after drying, 5% ethanolic PEG solution), PABA
(0.5 g PABA in 18 mL glacial acetic acid diluted with 20 mL water,
plus 1 mL o-phosphoric acid and 60 mL acetone), or DPA reagents
Please cite this article in press as: Á.M. Móricz, et al., Antibacterial potential of the Cistus incanus L. phenolics as studied
with use of thin-layer chromatography combined with direct bioautography and in situ hydrolysis, J. Chromatogr. A (2017),
https://doi.org/10.1016/j.chroma.2017.12.056
3
(1 g aniline and 1 g diphenylamine in 100 mL acetone and 10 mL
o-phosphoric acid, heated for 5 min at 110 ◦ C), by dipping the
chromatoplates in these solutions. Derivatization with the PABA
reagent was followed by an immersion of the chromatoplates in n-
hexane – paraffin, 1:2 (v/v). The enhancement effect of paraffin was
evaluated by videodensitometry, using the ImageJ program (NIH,
Bethesda, MA, U.S.A.).
For the preparative isolation of the zones of interest, 130-␮L
aliquots of the flavonoid fractions of A1 or T3 were applied as 80-
mm bands and after the development, the appropriate zones (c1 to
c4 from sample A1/I and c5 and c6 from T3/I) and the background
sample (control, #) were scraped off and loaded into a syringe
attached to a Nylon filter (0.22 ␮m, Phenomenex, Torrance, CA,
USA). Active compounds were eluted by adding 300 ␮L ethanol that
was forced through the membrane filter. The eluate was directly
analyzed by the TLC-based methods and the HPLC-DAD-MS system.
The presence of glucose conjugates in the antibacterial zones
was confirmed by applying the isolated compounds and glucose
onto an amino-modified HPTLC plate, in situ hydrolyzing the iso-
lated compounds on the adsorbent layer, upon which the plate was
double developed with acetonitrile and acetonitrile – water mix-
ture. The Maillard reaction between the reducing sugar and the
NH2 groups of the NH2 -type HPTLC plate produces a local fluores-
cent signal. The fluorescence can be enhanced with paraffin [33].
Detailed steps of this procedure are given in Table 1.
Additionally, to prove that kaempferol and glucose were con-
stituents of the compounds present in certain antibacterial zones,
a 2D-HPTLC method was developed that needed no prior isola-
tion of these compounds. The one-dimensional separation of the
flavonoid fraction I was performed on a 10 cm × 10 cm HPTLC silica
layer, followed by an in situ acidic hydrolysis and then an orthog-
onal development using a toluene – i-propyl acetate – formic acid
mixture, or acetonitrile and a subsequent acetonitrile – water mix-
ture. Kaempferol or glucose were applied at a height of 80-mm, just
above the track of the fraction, for their development in the sec-
ond (orthogonal) development direction. After the development,
an appropriate derivatization was applied. Detailed steps of these
procedures are given in Table 2.
2.4. TLC-direct bioautography
Bioassays were performed with B. subtilis and A. fischeri test bac-
teria, utilizing the TLC-DB methods described in papers [34] and
[35], respectively. Briefly, the developed and dried chromatoplates
were immersed in one of the bacterial cell suspensions. Then the A.
fischeri bioautograms were placed in a humid glass cage and doc-
umented instantly by a cooled low-light camera IS-4000 (Alpha
Innotech, San Leandro, CA, USA) at an exposure time of 2 min. The B.
subtilis bioautograms were visualized after 2 h incubation (at 28 ◦ C,
100% humidity), by dipping them in an aqueous solution of the MTT
vital dye (1 mg mL−1 ), followed by 0.5 h incubation and documenta-
tion by a Cybershot DSC-HX60 digital camera (Sony, Neu-Isenburg,
Germany). The living active cells are the only ones to emit light
or to reduce the yellow MTT to the bluish MTT-formazan, so that
the antibacterial compounds appear as dark spots against a bright
background (in the A. fischeri assay), or as bright spots against a
bluish background (in the B. subtilis assay).
2.5. HPLC-DAD-ESI-MS system
Compounds present in bioactive zones were analyzed by a
single-quadrupole HPLC–MS system (the LC–MS-2020 model, Shi-
madzu, Kyoto, Japan) equipped with a binary gradient solvent
pump, a vacuum degasser, a thermostatted autosampler, a col-
umn oven, a diode array detector (DAD) and a mass analyser with
electrospray ionization (ESI-MS). Data was acquired and processed
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Fig. 1. The components of flavonoid fraction I of the C. incanus samples separated by means of TLC with chloroform – ethyl acetate – methanol, 75:10:15 (v/v/v), documented
at UV 365 nm (a), after derivatization with aluminium chloride at UV 365 nm (b), at UV 254 nm (c) and using antibacterial assays against B. subtilis (d) and A. fischeri recorded
instantly (e) and after 20 min (f). (For interpretation of the references to colour in the text, the reader is referred to the web version of this article.)

using the LabSolutions 5.72v software (Shimadzu, Kyoto, Japan).
The separation was carried out at 35 ◦ C on a Kinetex C18 column
(100 mm × 3 mm, 2.6 ␮m particle size) protected by a C18 guard
column (4 mm × 3 mm) purchased from Phenomenex. Eluent A
was 5% aqueous acetonitrile with 0.05% formic acid and eluent
B was acetonitrile with 0.05% formic acid. The flow rate was
0.8 mL min−1 and the gradient was as follows: 0–2 min, 10–30%
B; 2–10 min, 30–35% B; 10–11 min, 35–100% B; 11–14 min, 100%
B and 14–17 min, 10% B. The sample injection volume was from 1
to 5 ␮L. The working ESI conditions were as follows: temperature,
350 ◦ C; the applied voltage, 4.5 kV; the desolvation line tempera-
ture, 250 ◦ C; the heat block temperature, 400 ◦ C; the nebulizer gas
(N2 ) flow rate, 1.5 L min-1 ; the drying gas (N2 ) flow rate, 15 L min-1 .
The full mass scan spectra were recorded in the positive and nega-
tive ionization modes over the range of m/z 100–2000.
Hydrolysis of the isolated compounds was carried out in Eppen-
dorf tubes by mixing 80 ␮L eluate, 20 ␮L water and 20 ␮L 38%
hydrochloric acid and heating the tubes on a water bath (Büchi
B-480, Flawil, Switzerland) for 30 min at 80 ◦ C.
3. Results and discussion
3.1. TLC-DB screening for antibacterial compounds
A total of eleven C. incanus herbal tea samples were extracted
and fractionated in order to obtain their supposedly flavonoid
aglycons-rich fraction I which had shown a noteworthy antibacte-
rial profile, as demonstrated in our previous study that comprised
the TLC-DB test of the six phenolic fractions (I–VI) of sample A3
originating from Albania [22]. TLC fingerprints of fraction I were
obtained with the use of chloroform – methanol – ethyl acetate,
75:15:10 (v/v/v), as a mobile phase. Most fractions I (with an excep-
tion of those derived from T2, T4 and G1) showed similar TLC
fingerprints, seen under UV light before and after derivatization
(Fig. 1a–c). This observation confirmed the statement emphasized
by Wittpahl et al. [23] that the contents of the C. incanus extracts
originating from different commercial sources are qualitatively
very similar. Using aluminium chloride, six characteristic yellow
zones were visualized (most probably corresponding to flavonoids)
and denoted as c1–c6 at hRF 28, 36, 40, 46, 67 and 71, respectively
(Fig. 1b). With each sample, the chemical profile of fraction I was in
close correlation with its antibacterial profile obtained by the Gram
positive B. subtilis, or the Gram negative A. fischeri test. The chro-
matographic zones of c1–c6, originating from different C. incanus
samples, demonstrated an antibacterial potential in the two TLC-
DB bioassays (Fig. 1d–f). At the beginning, the dark antimicrobial
zones on the A. fischeri bioautograms were poorly visible, but their
intensity perceptibly grew in the course of the next 20 min (Fig. 1f).
3.2. Characterization of active components
To confirm the presence of flavonoid aglycons in antibacterial
zones, not only aluminium chloride, but also other specific reagents
were used (Fig. 2). Derivatization of zones c1–c6 with NP-PEG and
iron(III) chloride resulted, respectively, in fluorescent yellow and
brown (under white light) spots, typical of flavonoids. Generally,
DPA and PABA are used to derivatize sugars and sugar conjugates.
The lack of blue fluorescence with PABA and the lack of visible blue
colour with DPA supported our preliminary assumption that no
sugar conjugates but only aglycons were present in zones c1–c6
(Fig. 2d,e).
Further characterization and identification of the C. incanus
flavonoids with a confirmed antibacterial activity was carried
out with the use of HPLC-DAD-ESI-MS. Active zones c1–c6 were

Please cite this article in press as: Á.M. Móricz, et al., Antibacterial potential of the Cistus incanus L. phenolics as studied
with use of thin-layer chromatography combined with direct bioautography and in situ hydrolysis, J. Chromatogr. A (2017),
https://doi.org/10.1016/j.chroma.2017.12.056
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Fig. 2. Chemical characterization of bioactive components (c1-c6) of the flavonoid

fraction I of the C. incanus samples by derivatization with aluminium chloride (a,
365 nm), NP-PEG (b, 365 nm), FeCl3 (c, white light), PABA (d, 365 nm) and DPA (e,
white light). (For interpretation of the references to colour in the text, the reader is
referred to the web version of this article.)

scraped off from the TLC layer and extracted with ethanol, as
described in Section 2.3. Then the presence of active compounds in
the isolates eluted from these scraped off zones was re-confirmed
with TLC in UV light (at 254 nm), and by derivatization with alu-
minium chloride resulting in fluorescent yellow spots (Fig. 3a,b).
An isolate from c5 contained a compound evidently present also in
the zone c6, which could be due to an insufficient resolution of the
components from these two zones. All isolates exhibited antibac-
terial activity in the TLC-B. subtilis assay, although with the isolate
from c4 this inhibiting effect was not perceptible (Fig. 3c), due to a
low amount of the respective sample applied to the TLC layer.
Then a HPLC-DAD-MS analysis was performed for isolates from
c1 to c6 derived from crude extracts A1 or T3 (Figs. 4 and 5). The
isolates from c1 contained two characteristic compounds with the
same UV and mass spectra (Fig. 5c1), suggesting the presence of
two unknown isomeric forms. The ESI ionization technique pro-
vided mass spectra registered both in the positive and the negative
ionization mode. In the positive mode, the protonated molecule at
m/z 595 [M+H]+ and the sodium adduct ion at m/z 617 [M+Na]+
predominated in the mass spectrum. Moreover, a sodium adduct
of the dimer at m/z 1211 [2M+Na]+ and four minor fragmentation
ions at m/z 309 [M−285]+ , m/z 287, m/z 165 and m/z 147 were also
detected. In the negative ionization mode, the deprotonated molec-
ular ion and the deprotonated dimer gave the mass signals at m/z
593 [M−H]− and m/z 1187 [2M−H]− , respectively. Although sev-
eral compounds co-migrated in the c2–c4 TLC zones (Fig. 4), each
of them had identical UV and mass spectra, again suggesting the
presence of isomers and molecular structures similar to those orig-
inating from c1 (Fig. 5, c2(c3,c4)). In the positive ionization mode,
intense mass signals at m/z 741 [M+H]+ , m/z 763 [M+Na]+ and m/z
455 [M−285]+ were recorded, and also low-intensity signals at m/z
1503 [2M+Na]+ , m/z 336 and m/z 352 were present there. More-
over, in the negative ionization mode a predominant mass signal at
m/z 739 [M−H]− and a low-intensity signal at m/z 1187 [2M−H]-
were acquired. Besides, the HPLC analysis confirmed that in the
isolate from c5 the major phenolic component of c6 could also be
found. In the positive ionization mode, this major component of iso-
lates from c5 and c6 gave mass signals at m/z 271 and m/z 301, and
in the negative mode at m/z 269 and m/z 299. These signals were
ascribed to the protonated [M+H]+ and the deprotonated [M−H]−
molecules, respectively (Fig. 5, c5 and c6). Obviously, the compo-
nents of c1, c5 and c6 originated from crude extracts T3 and A1,
Fig. 3. The components of the two Cistus incanus crude extracts (A4 and T3), their
flavonoid fractions I (A4/I and T3/I), the antibacterial components isolated from
zones c1-c6 and the apigenin standard (ap) separated by means of TLC with chlo-
roform – ethyl acetate – methanol, 75:10:15 (v/v/v), documented at UV 254 nm (a),
after derivatization with aluminium chloride at UV 365 nm (b) and using the B. sub-
tilis assay (c). (For interpretation of the references to colour in the text, the reader is
referred to the web version of this article.)
and also those from all the eleven fractions I were scrutinized in
this study. However, the components of c2–c4 were not detected
in the crude extracts, only in the fractions I, which suggested their
formation as artifacts in the course of the fractionation process.
Based on data taken from the literature, so far the follow-
ing flavonoid aglycons: apigenin, and kaempferol-3-methyl-ether
(isokaempferide), with molecular weights of 270 and 300 g mol−1 ,
respectively, were reported in the Cistus species. Apigenin was
found as a component of the twelve species of the Cistus genus,
including C. incanus [36–38]. Isokaempferide was identified with
three representatives of the Cistus genus, namely with C. ladanifer
[38,36], C. palhinhae [36] and C. incanus [39]. These two flavonoid
aglycons can well correspond to the main compounds of c5 and c6,
respectively. Identification of c5 as apigenin was performed with
the TLC-B. subtilis assay and the HPLC-MS analysis. In the TLC-DB
assay, the apigenin standard provided an inhibition zone at the
same hRF 67, as c5 (Fig. 3), and in the HPLC-DAD-MS experiment, the
same retention times and the UV and mass spectra were recorded
for the apigenin standard and c5 (Fig. 4). Tentative identification of
the c6 constituent as kaempferol-3-methyl-ether was supported by
the comparable UV spectra with ␭max at 349 nm for this flavonoid
[38,40] and the isolate from c6.

Please cite this article in press as: Á.M. Móricz, et al., Antibacterial potential of the Cistus incanus L. phenolics as studied
with use of thin-layer chromatography combined with direct bioautography and in situ hydrolysis, J. Chromatogr. A (2017),
https://doi.org/10.1016/j.chroma.2017.12.056
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Fig. 4. The HPLC-DAD chromatograms recorded at 314 nm (a) and the corresponding extracted ion chromatograms (b) obtained by HPLC-DAD-ESI-MS analysis of two Cistus
incanus crude extracts (T3 and A1), their flavonoid fractions I (T3/I and A1/I), the antibacterial components isolated from zones c1-c6 and the apigenin standard. Each m/z
value is represented by a different color.

Fig. 5. The UV (left), ESI+ (middle) and ESI- (right) spectra of the main components of c1, c2(c3,c4), c5 and c6.

Please cite this article in press as: Á.M. Móricz, et al., Antibacterial potential of the Cistus incanus L. phenolics as studied
with use of thin-layer chromatography combined with direct bioautography and in situ hydrolysis, J. Chromatogr. A (2017),
https://doi.org/10.1016/j.chroma.2017.12.056
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Fig. 6. Detection of glucose (G) released from the antibacterial C. incanus compounds, combined with the in situ acidic hydrolysis on the amino-modified HPTLC silica gel
layer; (a) single development with acetonitrile – water, 7:3 (v/v) and (b) pre-development of the chromatographic plate with acetonitrile, and after drying, the proper
development with acetonitrile – water, 7:3 (v/v), as given in Table 1. Zones c1–c6 (the same, as in Fig. 1) were isolated from the TLC layer and # is the control eluate of the
TLC background.

Fig. 7. Detection of glucose (G) and kaempferol (K), released from the compounds present in active zones of the C. incanus fraction I (sample A1), by means of 2D-HPTLC
on the silica gel layer, combined with in situ acidic hydrolysis (consecutive steps of these procedures are given in Table 2). Chromatoplates were documented after the first
development in the first dimension with chloroform – ethyl acetate – methanol, 75:10:15 (v/v/v) (a and g, 254 nm), after a hydrolysis followed by the second development
in the second dimension with acetonitrile and after drying with acetonitrile – water (b, 365 nm) and derivatization using DPA (c, white light), PABA (d, 365 nm) and PABA-
paraffin (e and f, 365 nm); or after the hydrolysis followed by the second development in the second dimension with toluene – i-propyl acetate – formic acid 3:2:0.5 (v/v/v)
and derivatization with aluminium chloride (b, 365 nm). Separation of glucose (the 7th step) was performed with acetonitrile – water, 35:10 (b–e) or 4:1 (f) (v/v). (For
interpretation of the references to colour in the text, the reader is referred to the web version of this article.)

It needs to be admitted that the mass signals recorded for the have resulted in formation of artifacts. To further explore the
compounds present in the isolates from c1 to c4 were too high constituents of c1–c4, additional HPTLC-based experiments were
for flavonoid aglycons, so the flavonoid condensates (dimers and performed, focusing on the detection of kaempferol and glucose as
trimers) or the sugar conjugates had to be taken into consideration possible building blocks of the molecules of interest.
as possible candidates. However, the negative results achieved with
the visualizing reagents PABA and DPA denied the presence of sugar
3.3. Multi-development and 2D-HPTLC combined with the in situ
conjugates in the samples (as these tests work only with reducing
hydrolysis
sugars in an open-chain form and not in the case of the ring opening
obstruction).
Glucose lacks a UV chromophore, and for this reason, its detec-
Kaempferol-3-O-␤-D-(6 -O-(E)-p-coumaroyl)glucopyranoside
tion needs special techniques such as refractometry or mass
(tiliroside, 594 g mol−1 ) and kaempferol-3-(3 ,6 -dicoumaroyl)-
spectrometry. A different possibility is to use a chemical reagent
glucopyranoside (740 g mol−1 ) have been identified by Wittpahl
able to form a chromophore-containing glucose derivative and this
et al. [23], and the latter compound also by Gori et al. [41], as con-
task is particularly easy to accomplish with the open-bed planar
stituents of C. incanus. Their reported UV and mass spectra proved
chromatography. Taking into account this advantage, two HPTLC-
identical with those recorded by us as respective components
based procedures were developed, combining the in situ liberation
of c1–c4. The coumaric acid bonded to the C6 atom of glucose
of glucose from a possible conjugate on the adsorbent layer with its
probably blocks the ring opening of the sugar, which could explain
separation and detection. All steps of both procedures were carried
inactivity of these compounds in the derivatization reaction with
out on one and the same chromatographic plate.
PABA. Interestingly, unknown compounds with a molar weight
The first procedure requires the thin-layer chromatographic
of 740 g mol−1 were found in our fractions I, but not in the crude
development be carried out on amino-modified HPTLC layers.
extracts and therefore they were rightfully considered as artifacts.
Prior to development, the isolates from c1 to c4, the control sam-
At this point one can also reflect that working conditions employed
ple (the eluate of the blank TLC layer) and the glucose standard
by Wittpahl et al. [23] and Gori et al. [41] for the extraction of the
underwent an acidic in situ hydrolysis by incubating the chromato-
phenolics from C. incanus (accelerated solvent extraction with 50%
graphic plate in HCl vapor for 10 min, followed by heating at 100 ◦ C
aqueous methanol, 100 ◦ C and 100 bar, and 70% aqueous ethanol,
(Table 1) for another 10 min. The liberated glucose was then sepa-
pH 2.5 by HCOOH, and sonication for 30 min, respectively) could
rated with acetonitrile-water, 7:3 (v/v), but its chromatographic

Please cite this article in press as: Á.M. Móricz, et al., Antibacterial potential of the Cistus incanus L. phenolics as studied
with use of thin-layer chromatography combined with direct bioautography and in situ hydrolysis, J. Chromatogr. A (2017),
https://doi.org/10.1016/j.chroma.2017.12.056
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Fig. 8. The HPLC-DAD chromatograms recorded at 314 nm and the m/z values corre-
sponding to the peaks obtained by the HPLC-DAD-ESI(+)-MS analysis of kaempferol,
p-coumaric acid and the acid-hydrolyzed isolates (c1-c4).
zone was distorted (Fig. 6a). This undesirable effect was elimi-
nated by developing (washing) the adsorbent layer immediately
after the hydrolysis with acetonitrile that dislocated most of the
hydrolyzates from the application zone but not the liberated glu-
cose. Then the dried layer was developed with acetonitrile – water,
7:3 (v/v) (Fig. 6b). The reaction between glucose and the amino
groups of the adsorbent was exploited to produce a fluorescent
signal of the glucose zone, by heating of the developed layer for
20 min at 170 ◦ C [33]. In Fig. 6, one can see glucose liberated from
the isolates of c1–c4.
To avoid the time-consuming isolation of antibacterial and pos-
sibly glycosylated compounds, an alternative 2D-HPTLC procedure
was devised for the detection of glucose liberated from them, which
started with the first development of fraction I in the first dimen-
sion, in order to achieve separation of the respective zones with
chloroform – methanol – ethyl acetate, 75:15:10 (v/v/v). Then the
subsequent hydrolytic liberation of glucose in the HCl vapors took
place, which was followed by the second and the third development
in the orthogonal directions, i.e., by washing the chromatoplate
with acetonitrile (for the reason explained in the preceding para-
graph), followed by the separation of the released free glucose
with the acetonitrile-water mixture (Table 2a). Visualization of
the chromatograms with DPA and PABA resulted in visible blue
and fluorescent blue zones, respectively, appearing in the presence
of free glucose only (Fig. 7a–f). When comparing the derivatiza-
tion reagents 2-naphthol sulfuric acid, o-phthalaldehyde, PABA and
DPA, PABA proved the most sensitive [42]. A subsequent dipping of
the chromatogram in paraffin – n-hexane, 1:2 (v/v), resulted in a
more uniform background and an even more sensitive detection,
(Fig. 7e,f) compared with the use of PABA alone (Fig. 7d). Based
on videodensitometric evaluation using the ImageJ program, the
intensity of the fluorescent signal was enhanced by ca. 50% due
to the aforementioned dipping. Fig. 7 shows that acidic hydrolysis
released glucose in zones c1–c4, and also in the sample applica-
tion area. These results supported our assumption that there are
glycosylated compounds in the antibacterial zones c1–c4. When
employing a similar 2D-HPTLC procedure (Table 2b), yet with a
longer incubation period in the HCl vapors, without heating (which
generates condensation of the phenolics), and developing the chro-
matogram in the orthogonal direction with toluene – i-propyl
acetate – formic acid, 3:2:0.5 (v/v/v), followed by visualization with
aluminium chloride, then kaempferol was also detected as a con-
stituent of the isolates from c1 to c4 (Fig. 7g,h).
3.4. HPLC-DAD-MS analysis of hydrolyzed isolates
The acidic hydrolysis of isolates from c1 to c4 was performed
in the bulk liquid phase as well, and the hydrolyzates were intro-
Please cite this article in press as: Á.M. Móricz, et al., Antibacterial potential of the Cistus incanus L. phenolics as studied
with use of thin-layer chromatography combined with direct bioautography and in situ hydrolysis, J. Chromatogr. A (2017),
https://doi.org/10.1016/j.chroma.2017.12.056
duced to the HPLC-DAD-MS system. All of them produced, among
other signals, also those originating from kaempferol, p-coumaric
acid and partially hydrolyzed substances (Fig. 8), which previously
appeared as fragment ions in the mass spectra of the compounds
detected in c1–c4 (Fig. 5). Signals obtained in the positive ion-
ization mode at m/z 287 and m/z 165 correspond to kaempferol
and coumaric acid, respectively. The split-off of the kaempferol
unit results in a fragment at m/z 309 present in c1 and in another
fragment at m/z 455 present in c2–c4. It is noteworthy that
the difference between c1 (594 g mol−1 ) and c2 (like c3 and c4:
740 g mol−1 ) equals to the coumaric acid unit (165 g mol−1 ) minus
one water molecule (18 g mol−1 ) that is ejected in the course of
condensation.
The results obtained with HPTLC and HPLC-MS prove that the C.
incanus antibacterial compounds in c1–c4 are not flavonoid agly-
cons, but complex molecules constituted of glucose, kaempferol
and coumaric acid units. In that way, the compounds belonging
to c1 were tentatively identified as cis- and trans-tiliroside, and
the artifactual compounds in c2–c4 as kaempferol-dicoumaroyl-
glucose isomers. Bioactivity of these compounds has been
investigated and reported in a number of studies. Apigenin exerts
antibacterial activity against the Gram positive Staphylococcus
aureus and B. subtilis bacteria, the Gram negative Escherichia coli and
Pseudomonas aeruginosa bacteria, and it also exerts an antiplorifer-
ative effect on human cancer cell lines [43]. Kaempferide was
found to inhibit the growth of B. cereus [44] and proliferation
of five human tumour cell lines [45,46]. Tiliroside shows cyto-
toxicity against the human lung adenocarcinoma cell line [47],
an antiprotozoal activity against Entamoeba histolytica and Giar-
dia lamblia [48], and it is successfully applied in the treatment of
eczema [49]. Kaempferol-3-(3 ,6 -dicoumaroyl)-glucopyranoside
displays an antiproliferative and antibacterial activity against sev-
eral human tumour cell lines [50] and Gram positive bacterial
strains (Bacillus cereus, Staphylococcus epidermidis, Staphylococcus
aureus, and Micrococcus luteus), respectively [51]. Additionally, its
isomer, kaempferol-3-(2 ,6 -dicoumaroyl)-glucopyranoside, has
proved effective against eight bacterial strains (including both
Gram positive and Gram negative ones), and fourteen fungal strains
[52].
4. Conclusions
TLC-DB was successfully applied to obtain antibacterial profile
of fraction I of eleven C. incanus herbal teas and to guide isolation of
bioactive compounds from the appropriate TLC zones. Despite the
use of an elaborate and well-established protocol claimed to yield
a fraction (fraction I) that is rich exclusively in flavonoid aglycons,
some of the antibacterial compounds found had m/z values too high
to represent flavonoids.
Multi-development HPTLC and two-dimensional HPTLC, includ-
ing an in situ acidic hydrolysis step, proved as fast and
easy-to-perform analytical methods to demonstrate the presence
of sugar conjugates in certain zones of interest with the glucose
conjugate of kaempferol among them. The use of paraffin after
the derivatization with PABA resulted in an increased sensitivity
of glucose detection by ca. 50%. The phenolic building blocks of the
antibacterial compounds were identified by HPLC-DAD-MS of the
respective hydrolizates as well.
The developed TLC/HPTLC platform allowed identification of
antibacterial components of fraction I derived from C. incanus,
namely apigenin, kaempferide, cis- and trans-tiliroside, and the
isomers of the p-coumaric acid-conjugated tiliroside: all of them
inhibited both B. subtilis and A. fischeri. Comparison of the crude
extract with fraction I via HPLC-DAD-MS revealed another unde-
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sired characteristic of the well-established fractionation protocol,
namely formation of artifacts.
Declaration
The authors declare no competing financial interest.
References
[1] A.L. Homans, A. Fuchs, Direct bioautography on thin-layer chromatograms for
detecting fungitoxic substances, J. Chromatogr. 51 (1970) 242–245.
[2] E.H. Wagman, M.J. Weinstein, Chromatography of Antibiotics, Elsevier,
Amsterdam, 1973.
[3] B.M. Lund, G.D. Lyon, Detection of inhibitions of Erwinia carotovora and E.
herbicola on thin layer chromatogram, J. Chromatogr. 110 (1975) 193–196.
[4] M.O. Hamburger, G.A. Cordell, A direct bioautographic TLC assay for
compounds possessing antibacterial activity, J. Nat. Prod. 50 (1987) 19–22.
[5] E. Tyihák, Á.M. Móricz, P.G. Ott, Biodetection and determination of biological
activity of natural compounds, in: M. Waksmundzka-Hajnos, J. Sherma, T.
Kowalska (Eds.), Thin Layer Chromatography in Phytochemistry, CRC Press,
Boca Raton, FL, 2008, pp. 193–213.
[6] C.T. Seanego, R.N. Ndip, Identification and antibacterial evaluation of
bioactive compounds from Garcinia kola (Heckel) seeds, Molecules 17 (2012)
6569–6584.
[7] G.E. Morlock, Chromatography combined with bioassays and other
hyphenations – the direct link to the compound indicating the effect, in: B.S.
Patil, G.K. Jayaprakasha, F. Pellati (Eds.), Instrumental Methods for the
Analysis and IdentiFication of Bioactive Molecules, American Chemical
Society, Washington, DC, 2014, pp. 101–121.
[8] Á.M. Móricz, P.G. Ott, Direct bioautography as a high-throughput screening
method for the detection of antibacterial components from plant sources, J.
AOAC Int. 98 (2015) 850–856.
[9] E.L. Ghisalberti, Detection and isolation of bioactive natural products, in: S.M.
Colegate, R.J. Molyneux (Eds.), Bioactive Natural Products – Detection,
Isolation and Structural Determination, 2nd edition, CRC Press, Taylor and
Francis Group, Boca Raton, FL, USA, 2008, pp. 11–76.
[10] J.C. Touchstone, Practice of Thin Layer Chromatography, 3rd ed., Wiley, New
York, 1992.
[11] H. Jork, W. Funk, W. Fischer, H. Wimmer, Thin-Layer Chromatography –
Reagents and Detection Methods, VCH, Weinheim, Germany, 1990.
[12] J. Sherma, B. Fried, Handbook of Thin Layer Chromatography, 3rd ed., Marcel
Dekker, Inc., New York, NY, 2003.
[13] G. Morlock, W. Schwack, Coupling of planar chromatography to mass
spectrometry, Trends Anal. Chem. 29 (2010) 1157–1171.
[14] Á.M. Móricz, P.G. Ott, T.T. Häbe, A. Darcsi, A. Böszörményi, Á. Alberti, D.
Krüzselyi, P. Csontos, S. Béni, G.E. Morlock, Effect-directed discovery of
bioactive compounds followed by highly targeted characterization, isolation
and identification, exemplarily shown for Solidago virgaurea, Anal. Chem. 88
(2016) 8202–8209.
[15] Á.M. Móricz, P.G. Ott, Screening and characterization of antimicrobial
components of natural products using planar chromatography coupled with
direct bioautography, spectroscopy and mass spectrometry: a review, Curr.
Org. Chem. 21 (2017) 1861–1874.
[16] N.S. Christodoulakis, M. Georgoudi, C. Fasseas, Leaf structure of Cistus creticus
L. (Rock Rose), a medicinal plant widely used in folk remedies since ancient
times, J. Herbs Spices Med. Plants 20 (2014) 103–114.
[17] P. Ellul, M. Boscaiu, O. Vicente, V. Moreno, J.A. Rossello, Intra and interspecific
variation in DNA content in Cistus (Cistaceae), Ann. Bot. 90 (2002) 345–351.
[18] P. Riehle, M. Volmer, S. Rohn, Phenolic compounds in Cistus incanus herbal
infusions-antioxidant capacity and thermal stability during the brewing
process, Food Res. Int. 53 (2013) 891–899.
[19] A. Hutschenreuther, C. Birkemeyer, K. Grötzinger, R.K. Straubinger, H.W.
Rauwald, Growth inhibiting activity of volatile oil from Cistus creticus L.
against Borrelia burgdorferi s.s, Pharmazie 65 (2010) 290–295.
[20] I. Chinou, C. Demetzos, C. Harvala, C. Roussakis, J.F. Verbist, Cytotoxic and
antibacterial labdane-type diterpenes from the aerial parts of Cistus incanus
subsp. Creticus, Planta Med. 60 (1994) 34–36.
[21] C. Demetzos, B. Stahl, T. Anastassaki, M. Gazouli, L.S. Tzouvelekis, M. Rallis,
Chemical analysis and antimicrobial activity of the resin Ladano, of its
essential oil and of the isolated compounds, Planta Med. 65 (1999) 76–78.
[22] D. Szeremeta, M. Knaś, E. Długosz, T. Kowalska, P.G. Ott, M. Sajewicz, Á.M.
Móricz, Investigation of antibacterial potential of phenolics derived from
Cistus incanus L. by means of thin-layer chromatography-direct bioautography
(TLC-DB), J. Liq. Chromatogr. Relat. Technol. (accepted for publication).
[23] G. Wittpahl, I. Kölling-Speer, S. Basche, E. Herrmann, M. Hannig, K. Speer, C.
Hannig, The polyphenolic composition of Cistus incanus herbal tea and its
antibacterial and anti-adherent activity against Streptococcus mutans, Planta
Med. 81 (2015) 1727–1735.
[24] A. Viapiana, A. Konopacka, K. Waleron, M. Wesolowski, Cistus incanus L.
commercial products as a good source of polyphenols in human diet, Ind.
Crops Prod. 107 (2017) 297–304.
Please cite this article in press as: Á.M. Móricz, et al., Antibacterial potential of the Cistus incanus L. phenolics as studied
with use of thin-layer chromatography combined with direct bioautography and in situ hydrolysis, J. Chromatogr. A (2017),
https://doi.org/10.1016/j.chroma.2017.12.056
9
[25] C. Demetzos, A. Loukis, V. Spiliotis, N. Zoakis, N. Stratigakis, H.E.
Katerinopoulos, Composition and antimicrobial activity of the essential oil of
Cistus creticus L, J. Essent. Oil Res. 7 (1995) 407–4010.
[26] C. Demetzos, H. Katerinopoulos, A. Kouvarakis, N. Stratigakis, A. Loukis, C.
Ekonomakis, V. Spiliotis, J. Tsaknis, Composition and antimicrobial activity of
the essential oil of Cistus creticus subsp. Eriocephalus, Planta Med. 63 (1997)
477–479.
[27] A. Roidaki, E. Kollia, A. Chiou, T. Varzakas, P. Markaki, C. Proestos, Superfoods
and superherbs: antioxidant and antifungal activity, Nutr. Food Sci. 4 (2016)
138–145.
[28] V. Kalli, E. Kollia, A. Roidaki, C. Proestos, P. Markaki, Cistus incanus L. extract
inhibits aflatoxin B1 production by Aspergillus parasiticus in macadamia nuts,
Ind. Crops Prod. 111 (2018) 63–68.
[29] R.K. Ibrahim, G.H. Towers, The identification by chromatography of plant
phenolic acids, Arch. Biochem. Biophys. 87 (1960) 125–127.
[30] Z. Jerzmanowska, Plant material, in: The Isolation Methods, PWN, Warszawa,
Poland, 1967, in Polish.
[31] H. Schmidtlein, K. Hermann, Quantitative analysis for phenolic acids by thin
layer chromatography, J. Chromatogr. 115 (1975) 123–128.
[32] M. Sajewicz, D. Staszek, M. Waksmundzka, T. Kowalska, Comparison of TLC
and HPLC fingerprints of phenolic acids and flavonoids derived from selected
sage (Salvia) species, J. Liq. Chromatogr. Relat. Technol. 35 (2012) 1388–1403.
[33] I. Vovk, B. Simonovska, L. Kompan, M. Prosek, TLC determination of mannitol
and lactulose on amino HPTLC plates, J. Planar Chromatogr. 16 (2003)
374–376.
[34] Á.M. Móricz, T.T. Häbe, A. Böszörményi, P.G. Ott, G.E. Morlock, Tracking and
identification of antibacterial components in the essential oil of Tanacetum
vulgare L. by the combination of high-performance thin-layer
chromatography with direct bioautography and mass spectrometry, J.
Chromatogr. A. 1422 (2015) 310–317.
[35] S. Krüger, O. Urmann, G.E. Morlock, Development of a planar
chromatographic method for quantitation of anthocyanes in pomace, feed,
juice and wine, J. Chromatogr. A 1289 (2013) 105–118.
[36] P. Proksch, P.-G. Gülz, Methylated flavonoids from cistus ladanifer and cistus
palhinhae and their taxonomic implications, Phytochemistry 23 (1984)
470–471.
[37] T. Vogt, P. Proksch, P.-G. Gülz, Epicuticular flavonoid aglycones in the genus
Cistus, Cistaceae, J. Plant Physiol. 131 (1987) 25–36.
[38] L. Barros, M. Dueñas, C.T. Alves, S. Silva, M. Henriques, C. Santos-Buelga,
I.C.F.R. Ferreira, Antifungal activity and detailed chemical characterization of
Cistus ladanifer phenolic extracts, Ind. Crops Prod. 41 (2013) 41–45.
[39] C. Demetzos, C. Harvala, S.M. Philianos, A.L. Skaltsounis, A new labdane-type
diterpene and other compounds from the leaves of Cistus incanus ssp. creticus,
J. Nat. Prod. 53 (1990) 1365–1368.
[40] S. Burapan, M. Kim, J. Han, Demethylation of polymethoxyflavones by human
gut bacterium, Blautia sp. MRG-PMF1, J. Agric. Food Chem. 65 (2017)
1620–1629.
[41] A. Gori, F. Ferrini, M. Marzano, M. Tattini, M. Centritto, M. Baratto, R. Pogni, C.
Brunetti, Characterisation and antioxidant activity of crude extract and
polyphenolic rich fractions from C. incanus leaves, Int. J. Mol. Sci. 17 (2016)
1344.
[42] G.E. Morlock, L.P. Morlock, C. Lemo, Streamlined analysis of lactose-free dairy
products, J. Chromatogr. A 1324 (2014) 215–223.
[43] R. Liu, H. Zhang, M. Yuan, J. Zhou, Q. Tu, J.-J. Liu, J. Wang, Synthesis and
biological evaluation of apigenin derivatives as antibacterial and
antiproliferative agents, Molecules 18 (2013) 11496–11511.
[44] Y. Wang, M. Hamburger, J. Gueho, K. Hostettmann, Antimicrobial flavonoids
from Psiadia trinervia and their methylated and acetylated derivatives,
Phytochemistry 28 (1989) 2323–2327.
[45] P. Forgo, I. Zupkó, J. Molnár, A. Vasas, G. Dombi, J. Hohmann,
Bioactivity-guided isolation of antiproliferative compounds from Centaurea
jacea L, Fitoterapia 83 (2012) 921–925.
[46] S. Rubio, J. Quintana, M. López, J.L. Eiroa, J. Triana, F. Estévez,
Phenylbenzopyrones structure-activity studies identify betuletol derivatives
as potential antitumoral agents, Eur. J. Pharmacol. 548 (2006) 9–20.
[47] C.-R. Liao, Y.-H. Kuo, Y.-L. Ho, C.-Y. Wang, C. Yang, C.-W. Lin, Y.-S. Chang,
Studies on cytotoxic constituents from the leaves of Elaeagnus oldhamii
Maxim. in non-small cell lung cancer A549 cells, Molecules 19 (2014)
9515–9534.
[48] F. Calzada, M. Meckes, R. Cedillo-Rivera, Antiamoebic and antigiardial activity
of plant flavonoids, Planta Med. 65 (1999) 78–80.
[49] H.D. Buchholz, C.D. Wirth, Use of Flavonoid-Derivatives for the Treatment of
Atopic Eczema, 2006 https://www.google.com/patents/EP1393733B1?cl=en.
[50] F.O. Abdullah, F.H.S. Hussain, M. Clericuzio, A. Porta, G. Vidari, A new iridoid
dimer and other constituents from the traditional Kurdish plant Pterocephalus
nestorianus Nab, Chem. Biodivers. 14 (2017) e1600281.
[51] H. Liu, J. Orjala, O. Sticher, T. Rali, Acylated flavonol glycosides from leaves of
Stenochlaena palustris, J. Nat. Prod. 62 (1999) 70–75.
[52] A. Karioti, M. Sokovic, A. Ciric, C. Koukoulitsa, A.R. Bilia, H. Skaltsa,
Antimicrobial properties of Quercus ilex L. proanthocyanidin dimers and
simple phenolics: evaluation of their synergistic activity with conventional
antimicrobials and prediction of their pharmacokinetic profile, J. Agric. Food
Chem. 59 (2011) 6412–6422.