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Antibacterial potential of the Cistus incanus L. phenolics as studied with use

of thin-layer chromatography combined with direct bioautography and in situ
hydrolysis

Article in Journal of Chromatography A · December 2017

DOI: 10.1016/j.chroma.2017.12.056

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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. Móricz1*, Dariusz Szeremeta2, Magdalena Knaś2, Ewa Długosz3, Péter G. Ott1,

Teresa Kowalska2, Mieczysław Sajewicz2

1
Department of Pathophysiology, Plant Protection Institute, Centre for Agricultural Research,

Hungarian Academy of Sciences, Herman O. Street 15, 1022 Budapest, Hungary

2
Institute of Chemistry, University of Silesia, 9 Szkolna Street, 40-006 Katowice, Poland

3
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

*Corresponding author: Á.M. Móricz, Plant Protection Institute, Centre for Agricultural

Research, Hungarian Academy of Sciences, 15 Herman Ottó St, 1022 Budapest, Hungary; Tel.:

+3614877515; E-mail address: moricz.agnes@agrar.mta.hu

1
Keywords

Hairy rockrose (Cistus incanus L.)

Thin-layer chromatography–direct bioautography

Multi-development and two-dimensional HPTLC

In situ acidic hydrolysis

Flavonoid aglycons

Acylated flavonoid glycosides

2
Abstract

The main aim of this study was to detect and identify antibacterial components of fraction I

derived from eleven commercial C. incanus herbal teas. Fraction I obtained by a well-

established phytochemical protocol of a multi-step extraction was expected to contain flavonoid

aglycons alone. Antibacterial profile of fraction I was demonstrated by means of thin-layer

chromatography – direct bioautography (TLC-DB) using the Gram positive B. subtilis and the

Gram negative A. fischeri strain. Six chromatographic zones of 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 samples. The

compounds isolated from the six zones were analyzed by HPLC-DAD-ESI-MS. High

numerical m/z values valid for certain constituents of these isolates suggested that some selected

antibacterial components are, unexpectedly, flavonoid glycosides. In order to confirm this

suggestion, three independent HPTLC methods (multi-development on amino phase and two

two-dimensional developments on silica gel phase) were devised to in situ hydrolyze flavonoid

glycosides and then separate and visualize the liberated glucose and some other building blocks

of the zones’ components. Additionally, the sensitivity of glucose detection with p-

aminobenzoic acid reagent 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.

3
1. Introduction

For the millennia, many thousand medicinal plants have been used within the framework of

traditional medicines across different cultures around the globe. Due to the very high number

of medicinal plants, so far not all herbal materials have been systematically investigated for

their beneficial effects and healing potential. 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 methodology that yields a bioprofile either of a crude plant extract or the different

fractions thereof. The method to localize antimicrobial 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 position in chemistry of natural products as a relatively

inexpensive and an easy to use tool for non-targeted screening of antimicrobial 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 developments (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 rockrose (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-

4
21], methanolic [22] and the aqueous 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 the Gram positive and Gram negative bacterial strains. It has been

established that the main activity of the essential oil can be attributed to monoterpenes 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 the tooth enamel samples. It was also established that

antibacterial potential 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]. As the aqueous methanol provided high extraction yields with the phenolic compounds

including ellagitannins, flavanols, and glycosylated 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 shortcoming, 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

5
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

compounds 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 the m/z

signals too high for flavonoid aglycons. Therefore in the further characterization strategy, 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 complex antibacterial components of a plant matrix, such as fraction I of C.

incanus, by means of TLC-DB, MD-HPTLC, and 2D-HPTLC combined 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, #5554) and fine particle

size (HPTLC, #5547), and the glass-backed amino-modified silica gel 60 F254S plates (HPTLC,

#3192) were acquired from Merck (Darmstadt, Germany). All solvents used for the TLC

separations, formic acid, 38% hydrochloric acid, aluminium chloride, ferric chloride, glucose,

o-phosphoric acid and glacial acetic acid were of analytical grade (Reanal, Budapest, Hungary).

Sodium bicarbonate, 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

Millipore system (Molsheim, France). p-Aminobenzoic acid (PABA), aniline, diphenylamine

(DPA) and the test substances apigenin (purity≥97%), kaempferol (purity≥97%), and p-

6
coumaric acid (purity≥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 β-ethylamino ester) and

polyethylene glycol 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, naturally 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 József Farkas, Central Food Research Institute, 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 to T5), Albania (samples A1 to A4), and Greece (sample G1), and one sample came from a

not specified geographical location (sample ND1).

Isolation of the phenolics from the defatted, commercial C. incanus L. samples was carried out

by liquid-solid extraction in the Soxhlet apparatus using methanol 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 by hot water. Then the water

solution was cooled for 24 h, filtered and extracted with diethyl ether. The ether fraction was

extracted with 5% sodium bicarbonate and then with 5% sodium carbonate. The aqueous

7
carbonate layer was acidified with 18% hydrochloric acid (pH=2) and then extracted with

diethyl ether. The anhydrous organic layer was filtered, desiccated and re-dissolved in

methanol. 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 µL at 8-mm distance from the lower plate edge in 7-mm bands, with 5-mm

gaps. One-dimensional TLC or HPTLC separations were achieved in a 20 cm × 10 cm twin

trough chamber (CAMAG) saturated 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 (λ = 254, or 365 nm) (CAMAG). The chromatographic spots were documented through

bioassays, or after derivatization with aluminium chloride (1% methanolic solution), ferric

chloride (0.5 g FeCl3 in 2.5 mL water and 47.5 mL ethanol), NP-PEG (0.5% methanolic 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 (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-µL aliquots of the flavonoid fractions

of A1 or T3 were applied as 80-mm long 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 µm, Phenomenex,

8
Torrance, CA, USA). Active compounds were eluted by adding 300 µL 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

isolated compounds on the adsorbent layer, upon which the plate was double developed with

acetonitrile and acetonitrile – water mixture. The Maillard reaction between the reducing sugar

and the NH2 groups of the NH2-type HPTLC plate produces a local fluorescent 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 the constituents of the compounds

present in certain antibacterial zones, a 2D-HPTLC method was developed that needed no prior

isolation 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 orthogonal development using a toluene – i-propyl acetate – formic acid mixture, or

acetonitrile and a subsequent acetonitrile – water mixture. Kaempferol or glucose were applied

at a height of 80-mm, just above the track of the fraction, for their development in the second

(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 bacteria, 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 documented instantly by a cooled low-

9
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 oC, 100% humidity),

by dipping them in an aqueous solution of the MTT vital dye (1 mg mL-1), followed by the 0.5

h incubation and documentation by a Cybershot DSC-HX60 digital camera (Sony, Neu-

Isenburg, Germany). The living active cells are the only ones to emit the 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, Shimadzu, Kyoto, Japan) equipped with a binary gradient solvent

pump, a vacuum degasser, a thermostatted autosampler, a column oven, a diode array detector

and a mass analyser with the electrospray ionization (ESI-MS). Data was acquired and

processed using the LabSolutions 5.72v programme (Shimadzu, Kyoto, Japan). The separation

was carried out at 35 oC on a Kinetex C18 column (100 mm × 3 mm, 2.6 µm 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 µL. The working ESI conditions were as follows:

temperature, 350 oC; the applied voltage, 4.5 kV; the desolvation line temperature, 250 oC; the

heat block temperature, 400 oC; the nebulizer gas (N2) flow rate, 1.5 L min-1; the drying gas

flow rate, 15 L min-1. The full mass scan spectra were recorded in the positive and negative

ionization modes over the range of m/z 100–2000.

10
Hydrolysis of the isolated compounds was carried out in Eppendorf tubes by mixing 80 µL

eluate, 20 µL water and 20 µL 38% hydrochloric acid and heating the tubes on a water bath

(Büchi B-480, Flawil, Switzerland) for 30 min at 80 oC.

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

antibacterial 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 exception of those derived from T2, T4 and

G1) showed similar TLC fingerprints, seen under UV light before and after derivatization (Fig.

1 a-c). This observation confirmed the statement emphasized by Wittpahl et al. [23] that the

contents of the C. incanus extracts originating from the 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 to 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 chromatographic zones of c1 to c6, originating from

different C. incanus samples demonstrated an antibacterial potential in the two TLC-DB

bioassays (Figs 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 minutes (Fig. 1f).

11
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 to 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 to c6 (Figs 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 to

c6 were 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 aluminium chloride

resulting in fluorescent yellow spots (Figs 3a,b). An isolate from c5 contained a compound

evidently present in the zone c6 also, that could be due to an insufficient resolution of the

components from these two zones. All isolates exhibited antibacterial 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 spectrum (Fig. 5, c1), suggesting the presence of two unknown

isomeric forms. The ESI ionization technique provided 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

12
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 molecular ion and the deprotonated dimer gave the mass

signals at m/z 593 [M-H]-and m/z 1187 [2M-H]-, respectively. Although several compounds co-

migrated in the c2, c3 and 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

originating from c1 (Fig. 5, c2(c3,c4)). In the positive ionization mode, the 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 352b were present there. Moreover,

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 isolates 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 components of c1, c5 and c6 originated from crude extracts T3 and A1, and also

those from all the eleven fractions I were scrutinized in this study. However, the components

of c2, c3 and 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 following 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

13
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 λmax at 349 nm for this

flavonoid [38,40] and the isolate from c6.

It needs to be admitted that the mass signals recorded for the compounds present in the isolates

from c1 to c4 were too high for flavonoid aglycons, so the flavonoid condensates (dimers and

trimers) or the sugar conjugates had to be taken into consideration as possible candidates.

However, the negative results achieved with the visualizing reagents PABA and DPA denied

the presence of sugar conjugates in the samples (as these tests work only with reducing sugars

in an open-chain form and not in the case of the ring opening obstruction).

Kaempferol-3-O-β-D-(6″-O-(E)-p-coumaroyl)glucopyranoside (tiliroside, 594 g mol-1) and

kaempferol-3-(3′′,6′′-dicoumaroyl)- glucopyranoside (740 g mol-1) have been identified by

Wittpahl et al. [23], and the latter compound also by Gori et al. [41], as constituents of C.

incanus. Their reported UV and mass spectra proved identical with those recorded by us as

respective components of c1 to c4. The coumaric acid bonded to the C6 atom of glucose

probably blocks the ring opening of the sugar, which could explain inactivity of these

compounds in the derivatization reaction with PABA. Interestingly, unknown compounds with

a molar weight of 740 g mol-1 were found in our fractions I, but not in the crude extracts and

therefore they were rightfully considered as artifacts. At this point one can also reflect that

working conditions employed by Wittpahl et al. [23] and Gori et al. [41] for the extraction of

the phenolics from C. incanus (accelerated solvent extraction with 50% aqueous methanol, 100
o
C and 100 bar, and 70% aqueous ethanol, pH 2.5 by HCOOH, and sonication for 30 min,

respectively) could have resulted in formation of artifacts. To further explore the constituents

14
of c1 to c4, additional HPTLC-based experiments were performed, focusing on the detection

of kaempferol and glucose as possible building blocks of the molecules of interest.

3.3. Multi-development and 2D-HPTLC combined with the in situ hydrolysis

Glucose lacks a UV chromophore, and for this reason, its detection needs special techniques

such as refractometry or mass spectrometry. A different possibility is to use a chemical reagent

able to form a chromophore-containing glucose derivative and this task is particularly easy to

accomplish with the open-bed planar chromatography. Taking into account this advantage, two

HPTLC-based procedures were developed, combining the in situ liberation of glucose from a

possible conjugate on the adsorbent layer with its separation and detection. All steps of both

procedures were carried out on one and the same chromatographic plate.

The first procedure requires the thin-layer chromatographic development be carried out on

amino-modified HPTLC layers. Prior to development, the isolates from c1 to c4, the control

sample (the eluate of the blank TLC layer) and the glucose standard underwent an acidic in situ

hydrolysis by incubating the chromatographic plate in HCl vapor for 10 min, followed by

heating at 100 oC (Table 1) for another 10 min. The liberated glucose was then separated with

acetonitrile-water, 7:3 (v/v), but its chromatographic zone was distorted (Fig. 6a). This

undesirable effect was eliminated 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 glucose. Then was the dried layer 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 oC [33]. In Fig. 6, one can see glucose liberated from the

isolates of c1 to c4.

15
To avoid the time-consuming isolation of antibacterial and possibly 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 dimension, 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

paragraph), 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

(Figs 7 a-f). When comparing the derivatization 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 to c4, and also in the sample application

spots. These results supported our assumption that there are glycosylated compounds in the

antibacterial zones c1 to 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 chromatogram 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 constituent of the isolates from c1

to c4 (Fig. 7g,h).

16
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 introduced 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 to c4 (Fig. 5). Signals obtained in the positive ionization 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 to 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 to c4 are not flavonoid aglycons, 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, and the Gram negative

Escherichia coli and Pseudomonas aeruginosa bacteria, and it also exerts an antiploriferative

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 cytotoxicity

against the human lung adenocarcinoma cell line [47], an antiprotozoal activity against

Entamoeba histolytica and Giardia 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 several human tumour cell lines [50] and the

17
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, including 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 inhibiting both B. subtilis

and A. fischeri. Comparison of the crude extract with fraction I via HPLC-DAD-MS revealed

another undesired characteristics of the well-established fractionation protocol, namely

formation of artifacts.

18
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.

19
[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 edn., 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.

20
[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)

[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.

[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.

21
[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,

Industrial Crops and Products, 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. 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.

22
[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.

23
[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.

24
Table 1 Consecutive steps of the HPTLC method on the amino-modified silica gel 60 F254S

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 oC (covered with a glass sheet)
4. Heating for 2 min at 100 oC (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 oC
10. Dipping into paraffin – n-hexane 1:2 (v/v) and drying

25
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 – Development with chloroform – methanol –
ethyl acetate 75:15:10 (v/v/v) on silica gel layer ethyl acetate 75:15:10 (v/v/v) on silica gel layer
up to 75 mm (1st dimension) up to 75mm (1st dimension)
2. Incubation in cc. HCl vapor for 10 min Incubation in cc. HCl vapor for 120 min

3. Heating for 8 min at 100 oC (covered with a Drying for 5 min by cold air stream
glass sheet)
4. Heating for 2 min at 100 oC (uncovered) Development with toluene – i-propyl acetate –
formic acid 3:2:0.5 (v/v/v) in orthogonal
direction up to 75 mm (2nd dimension)
5. Pre-development with acetonitrile in orthogonal Drying for 5 min by cold air stream
direction up to 85 mm (2nd dimension)
6. Drying for 5 min by cold air stream Aluminium chloride reagent

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 oC, 5 min), PABA(140 oC, 5 min)-

paraffin or DPA (110 oC, 5 min) reagents

26
Legend for figures:

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).

27
Fig. 2. Chemical characterization of bioactive components (from c1 to 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).

28
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 to c6 and the

apigenin standard (ap) separated by means of TLC with chloroform – 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. subtilis assay (c).

29
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 the two Cistus

incanus crude extracts (T3 and A1), their flavonoid fractions I (T3/I and A1/I), the antibacterial

components isolated from zones c1 to c6 and the apigenin standard. Each m/z value is

represented by a different color.

30
Fig. 5. The UV (left), ESI+ (middle) and ESI- (right) spectra of the main components of c1,

c2(c3,c4), c5 and c6.

31
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 to 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

32
 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).

Fig. 8. The HPLC-DAD chromatograms recorded at 314 nm and the m/z values corresponding

to the peaks obtained by the HPLC-DAD-ESI(+)-MS analysis of kaempferol, p-coumaric acid

and the acid-hydrolyzed isolates (from c1 to c4).

33

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