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JPC.19.2006.4.2

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Investigation of Chelidonium Alkaloids by Use
of a Complex Bioautographic System
Ágnes Sárközi*, Ágnes M. Móricz, Péter G. Ott, Ernô Tyihák, and Ágnes Kéry
Key Words:
BioArena
Bioautography
Biological activity
Chelidonium majus L.
Formaldehyde
Isoquinoline alkaloids
Summary
The greater celandine (Chelidonium majus L.), a well-known source
of isoquinoline alkaloids, has a long history of use as a medicinal
plant. Although the antimicrobial activity of Chelidonium alkaloids
against pathogenic bacteria has been reported, the mechanism of
this action is almost unknown. The BioArena system, which integrates the modern method and biological results of bioautography
with TLC and/or OPLC, is especially suitable for investigating biochemical interactions in the adsorbent layer after chromatographic
separation. The antimicrobial effect of alkaloids obtained from
Chelidonium root has been demonstrated by use of this system. It
was assumed that the antibiotic activity of Chelidonium alkaloids
was a result of formation of formaldehyde. It was also assumed that
addition of endogenous HCHO-capture molecules, for example Larginine and glutathione, to the culture medium reduces the
antibacterial activity of Chelidonium alkaloids whereas Cu(II) ions
enhance the effect. The results obtained support these assumptions
and our earlier observations that HCHO and its reaction products
are very important in the antibiotic action of these compounds.
These small molecules (L-arginine and glutathione) can capture
HCHO molecules mobilized by alkaloids and possibly by pathogen
cells, and may be responsible for reduced antibacterial effect. The
HCHO-mobilizing power of Cu(II) ions dramatically enhanced the
antibiotic effect. The BioArena system is highly suitable for studying special interactions in the adsorbent layer.
Á. Sárközi and Á. Kéry, Department of Pharmacognosy, Faculty of Pharmacy,
Semmelweis University, Üllõi Str. 26, 1085 Budapest, Hungary; Á.M. Móricz,
Department of Chemical Technology and Environmental Chemistry, Eötvös
University, P.O.B. 112, 1518 Budapest, Hungary; and E. Tyihák and P. G. Ott,
Plant Protection Institute, Hungarian Academy of Sciences, P.O.B. 102, 1525
Budapest, Hungary.
Journal of Planar Chromatography
This paper was presented
at the Symposium
‘Planar Chromatography 2005’,
Siófok, Hungary,
May 29–31, 2005
1 Introduction
Chelidonium majus L., a member of the Papaveraceae family, is
widely distributed throughout Europe and Western Asia. The
aerial part of the plant has long been used in herbal medicine
and has been described in several pharmacopoeias, e.g. the
European Pharmacopoeia (Ph. Eur. 5) [1], the German Pharmacopoeia (DAB-10) [2], and the Hungarian Pharmacopoeia (Ph.
Hg. VIII.) [3]. The orange colored latex of Chelidonium majus
L. is a well-known source of benzophenanthridine alkaloids
(chelidonine, sanguinarine, and chelerythrine), protoberberine
alkaloids (berberine and coptisine) and protopine alkaloids.
Chelidonine, sanguinarine, and chelerythrine have been isolated
as major components of the roots. The aerial parts contain coptisine, berberine, minor alkaloids, and one-twentieth of the
amounts of sanguinarine and chelidonine found in the roots [4].
Chelidonium alkaloids are characteristically N and O-methylated, and benzophenanthridine compounds contain phenolic and
methoxy or methylene dioxy groups. Chelidonium majus L. has
a variety of biological activity, for example antimicrobial, antitumor, and anti-inflammatory effects [5]. Extracts of the plant
are active against the influenza virus [6, 7], against human adenoviruses types 5 and 12, and against the Herpes simplex
virus [8]. Chelerythrine and sanguinarine are active against Staphylococcus aureus, Escherichia coli, Salmonella gallinarum,
and Klebsiella pneumoniae, and berberine and chelidonine have
a weak antibacterial effect [9, 10]. The chelerythrine-containing
and sanguinarine-containing benzophenanthridine fraction are
ineffective against Gram-negative bacteria in vitro but have significant antimicrobial effect against Gram-positive bacteria
such as Staphylococcus aureus, two strains of Streptococcus,
and Candida albicans [11]. Sanguinarine has been found to be
active against 52 oral reference strains [12]. Sanguinarine, chelerythrine, and berberine inhibit the growth of Helicobacter
pylori [13]. Although preparations of Chelidonium majus L.
VOL. 19. JULY/AUGUST 2006
DOI: 10.1556/JPC.19.2006.4.2
267
Bioautographic Analysis of Chelidonium Alkaloids
have been used for centuries to treat malignant diseases, and
weak or mild tumor inhibition has been observed for chelidonine and protopine, the compounds have no therapeutic value
because of their high cytotoxicity in therapeutic doses [14].
Chelidonium plant extracts also have choleretic, colagogue [15], spasmolytic [16, 17], and weak analgesic activity.
The resistance of microbial strains, especially common
pathogens, to antibiotics is an increasing problem in human
therapy [18, 19]. Study of resistance mechanisms, their causal
relationships, and research to discover new antibiotics or antibiotic-like substances originating from microbial and plant
sources are therefore of great importance in the pharmaceutical
sciences [20]. Chromatographic techniques have been successfully used for analysis of antibiotics in fermentation media and
plant tissues, for assessment of the purity of raw materials, for
assay of pharmaceutical dosage forms, and for chemical determination of antibiotics in complex biological samples. Of the
numerous in-vitro biological methods used to study the antimicrobial activity of biological samples, direct bioautography [21,
22] is widely applied, especially for detection of novel compounds in complex biological matrices. Planar chromatographic techniques (TLC, OPLC) are compatible with bioautographic detection, because of the use of a layer of adsorbent. The
BioArena system, which integrates TLC and OPLC with biological detection by conventional bioautography, is especially
suitable for investigating biomedical interactions in the adsorbent bed after chromatographic separation. This separation and
detection system successfully exploits the possibility of interactions between microbes, antibiotic-like compounds, dye substances, and other small and large co-factor molecules [23]. The
development of the BioArena system has enabled progress in
biological detection in a planar adsorbent bed. The name
BioArena is used to distinguish this complex system from the
original, conventional, bioautography; it is, however, not a
trademark name. This distinction seems a necessary step
because in the BioArena system the possibilities of guided biological interactions (“fights”) are unlimited.
Although antimicrobial activity of Chelidonium alkaloids
against bacteria has been reported, very little is known about the
mechanism of this action. The objective of this study was to
demonstrate the advantages of BioArena as a sophisticated
bioautographic system for studying complex interactions
between microorganisms and the main Chelidonium alkaloids.
2 Experimental
2.1 Plant Material
Chelidonium majus L. samples were collected in Budapest.
They were identified in the Department of Pharmacognosy,
Semmelweis University, where specimens were also deposited.
After being harvested the plants were separated into the different tissues. The roots were rinsed with running water, to remove
all remnants of soil, then dried at room temperature.
268
VOL. 19. JULY/AUGUST 2006
2.2 Chemicals
The test substance (chelidonine chloride) was purchased from
Sigma–Aldrich (Budapest, Hungary). The dye reagent MTT (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazonium bromide),
L-arginine, and glutathione (reduced form) were also from
Sigma–Aldrich. Copper (II)-sulphate × 5 H2O and all other
chemicals and reagents were obtained from Reanal (Budapest,
Hungary). All substances used were of analytical quality.
2.3 Preparation of Extracts
The powdered root drug was extracted with dilute acetic acid
(12% v/v) in an ultrasonic device for 20 min (1:200), then filtered. Portions of the filtrate (100 mL) were made alkaline with
25% aqueous ammonia solution then extracted with dichloromethane (3 × 100 mL). The combined organic phases were
dried over anhydrous sodium sulfate. After evaporation of the
solvent the residue was dissolved in methanol and submitted for
chromatographic investigation.
2.4 Separation Method
TLC was performed on precoated aluminum foil-backed plates
(silica gel 60 F254, 5 cm × 10 cm; Merck, Darmstadt, Germany).
The extracts (chelidonine chloride 1 mg mL–1, 1.5–10 µL; Chelidonium root extract (10%), 2.5–5 µL) were applied to the layer
in bands by means of a microsyringe (Hamilton, Bonaduz,
Switzerland). The root extracts were co-chromatographed with
authentic samples of chelidonine. The mobile phase used,
dichloromethane–methanol, 97 + 3 (v/v), was suitable for the
separation of chelidonine. All chromatograms were developed
at room temperature (20–24°C) and 60% relative humidity in a
filter-paper-lined Desaga glass TLC chamber (length 20.7 cm,
width 7.3 cm, height 20.5 cm) previously saturated with mobilephase vapor for 60 min. Ascending development was performed
at to a distance of 90 mm. The plates were then dried in a fume
cupboard, the spots were inspected under UV light at
λ = 254 nm and 365 nm, and the plates were then used for biological/bioautographic detection.
2.5 Microorganism and Medium
The phytopathogen Pseudomonas savastanoi pv. phaseolicola
race 6, which causes halo blight on beans (source: Department
of Biological Sciences, Imperial College, London University,
Wye, UK) was used for the bacterial biotest. The bacterial cells
were grown in a rich medium (King’s B broth) at 28°C and at
170 rpm on an orbital shaker until they reached the late exponential phase (OD560 = 0.7, ca 1.5 × 109 cells mL–1). Before use
50% (v/v) fresh medium was added to the culture.
2.6 BioArena Assay
Dried chromatographic plates were immersed in the bacterial
suspension for 20 s then incubated for 2 h in a chamber at 100%
relative humidity and at 28°C. In the course of incubation the
Journal of Planar Chromatography
Bioautographic Analysis of Chelidonium Alkaloids
Figure 1
Thin layer chromatograms obtained from Chelidonium root extract and
Chelidonium alkaloid test mixture, with the chemical structures and UV spectra
of the separated alkaloids. The mobile phase was dichloromethane–methanol,
97 + 3 (v/v), and detection was by UV illumination at λ = 254 nm.
microorganism proliferated in the adsorbent bed. The antibacterial activity of Chelidonium alkaloids was visualized by staining
of bioautograms with an aqueous solution of MTT (80 mg MTT
and 100 mg Triton X-100 in 100 mL water). The living bacterium cells converted the yellow MTT to blue MTT-formazan. Triton X-100 is needed to disperse and to stabilize the MTT-formazan formed [24].
To study the mechanism of reaction of Chelidonium alkaloids
before inoculation we added the co-factors L-arginine
(5 mg mL–1), glutathione (5 mg mL–1) [25], and copper(II) sulfate (4, 6, and 8 mg 100 mL–1) to the cell suspension [26].
3 Results and Discussion
Use of the mobile phase recommended by Ph. Eur. 5,
propanol–water–formic acid, 90 + 9 + 1 (v/v), did not enable
satisfactory separation of Chelidonium alkaloids. Use of acids
as mobile phase components was avoided owing to their interference with bioautographic detection. A new mobile phase
developed by us was therefore used for separation of Chelidonium alkaloids. Separation of chelerythrine, chelidonine, and
sanguinarine was achieved by use of dichloromethane–
methanol, 97 + 3 (v/v). The other two alkaloids, coptisine and
berberine, remained at the origin (Figure 1).
Journal of Planar Chromatography
Figure 2
Effect of 5 mg mL–1 L-arginine (B) and 5 mg mL–1 glutathione (C) on the antibacterial activity of chelidonine. A is the control.
Figure 2 shows the effect of L-arginine (B) and glutathione (C)
on the antibacterial activity of chelidonine for incubation times
of 2 and 18 h. Control chromatograms (A) show the characteristic, strong antibacterial effect – a colorless chromatogram
zone on a blue background is indicative of the local inhibition of
the antibacterial compound.
Separate addition of L-arginine and glutathione, as HCHO-capture molecules [27, 28], to the bacterial suspension (Figure 3)
led to a substantial reductions in antibacterial activity, although
to different extents – addition of L-arginine resulted in partially
diminished antibacterial effect whereas use of glutathione eliminated the effect. L-Arginine proved to be a weaker HCHO scavenger than glutathione for chelidonine. These results support
our earlier observations, according to which formaldehyde and
its potential reaction products play a special role in antibiotic [25] and toxic effects in general [29].
The other Chelidonium alkaloids examined (berberine, coptisine, chelerythrine, and sanguinarine) resulted in similar inhibi-
VOL. 19. JULY/AUGUST 2006
269
Bioautographic Analysis of Chelidonium Alkaloids
Figure 5
Figure 3
Reactions between formaldehyde and formaldehyde-capturing molecules, for
example L-arginine and glutathione.
Effect of 4, 6, and 8 mg per 100 mL (B, C, and D, respectively) Cu(II) on the
antibacterial activity of components of the Chelidonium root extract 2.5 µL (1)
and the Chelidonium alkaloid test mixture (1.5 µL) (2). A is the control.
tion of the growth of Pseudomonas savastanoi cells, as visualized by use of the dye reagent MTT. In our system, demethylation of chelidonine apparently occurs more readily than for quaternary alkaloids, which therefore provide a limited number of
capturable HCHO molecules. This points to a correlation
between the amount of HCHO-capture agents and the antimicrobial activity of Chelidonium alkaloids. Larger amounts of Larginine and glutathione always resulted in a greater decrease in
antimicrobial activity. Our results are in good agreement with
earlier observations [30].
Chelidonium alkaloids seem to be demethylated in the BioArena system, as is also observed with 1’-methylascorbigen [28].
HCHO molecules and their potential reaction products formed
in the chromatographic spots can be affected by use of L-arginine and glutathione as HCHO-capture molecules in the culture
medium. L-Arginine diminished antibacterial activity as a function of concentration only whereas glutathione almost completely eliminated it. On the basis of these facts it has been stated that in both instances demethylation proceeds similarly and
the antibacterial activity is determined by HCHO molecules
formed by enzymatic demethylation of Chelidonium alkaloids.
Figure 4 illustrates the antibacterial effect of the root extract
containing chelidonine. A special method of detection, direct
bioautography, proved to be suitable for studying the antimicrobial activity of plant extracts by use of TLC or OPLC. Zones of
inhibition were visualized by use of a dehydrogenase-activitydetecting, tetrazole-type reagent. L-Arginine and glutathione
again reduced the antibacterial activity of the different compounds in the plant extract and the test substance chelidonine, as
described above.
Figure 4
Effect of 5 mg mL–1 L-arginine (B) and 5 mg mL–1 glutathione (C) on the antibacterial activity of the components of the Chelidonium root extract (5 µL) (1) and
the Chelidonium alkaloid test mixture (3 µL) (2). A is the control.
270
VOL. 19. JULY/AUGUST 2006
Figure 5 illustrates the effect of Cu(II) ions on the antibacterial
activity of the compounds studied. Cu(II), the oxidized form of
copper, an essential trace element found in biological systems,
can cause a dramatic (three to fourfold) increase in the antibacterial activity of Chelidonium alkaloids, depending on the dose
applied. This was also observed for the alkaloid-like com-
Journal of Planar Chromatography
Bioautographic Analysis of Chelidonium Alkaloids
Figure 6
Densitograms obtained from Chelidonium alkaloid test mixture and Chelidonium root extract before (a, c) and after (b, d) bioautography (λ = 305 nm (a, c); λ = 590 nm
(b, d)).
pounds at the origin of the plate. Our experimental results are in
agreement with this effect of Cu(II) ions on the antibacterial
activity of trans-resveratrol and aflatoxins also [31]. It is very
probable that the synergistic effect observed is because of the
HCHO-mobilizing power of Cu(II) ions [26].
Figure 6 shows that qualitative densitometric determination of
alkaloids before and after bioautography gives information
about chemical and biological changes in the chromatogram
spots. This is valid for the ‘negative’ densitograms also.
Finally, it should be emphasized that of the different beneficial
effects of Chelidonium ingredients their inhibiting/elimination
action is predominant. The mechanisms of the antimicrobial and
antitumor activity of these compounds can be assumed to be
similar, as has been observed for 1’-methylascorbigen and
trans-resveratrol [32, 33].
4 Conclusion
Although the antimicrobial activity of Chelidonium alkaloids
against pathogenic bacteria has been reported, their mechanism
of action is almost unknown. The new BioArena system is a fea-
Journal of Planar Chromatography
sible method for studying different biochemical interactions in
the adsorbent layer. As presented above, BioArena provides
more information on antibiotic-like compounds than is obtained
by conventional bioautography. By variation of the incubation
time, changes in the adsorbent layer may be observed in 5–6 or
more days and endogenous and/or exogenous substances affecting the antimicrobial action of the separated components can be
used in the culture medium.
Control chromatograms revealed characteristic, well-defined,
alkaloid-specific antibacterial effects, providing almost the
same information as is obtained by conventional bioautography.
By separate addition of L-arginine and glutathione to the bacterial suspension antibacterial activity could be reduced substantially. Addition of L-arginine resulted in a diminished antibacterial effect whereas use of glutathione totally eliminated the
effect. In our system L-arginine proved to be a weaker HCHO
reagent than glutathione. It has been stated there is a correlation
between amount of HCHO-capture agent and the antimicrobial
activity of Chelidonium alkaloids – greater concentrations of Larginine and glutathione always resulted in a larger decrease in
antimicrobial activity. The oxidized form of copper was found
to cause a dramatic increase in the antibacterial activity of alkaloids. It may be stated that Cu(II) ions and other trace elements
VOL. 19. JULY/AUGUST 2006
271
Bioautographic Analysis of Chelidonium Alkaloids
have synergistic effects on the antimicrobial and/or anticancer
activity of Chelidonium alkaloids. These novel results support
the above assumptions and earlier observations that formaldehyde and its potential reaction products are very important in
the antibiotic action.
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Ms received: February 10, 2006
Accepted by SN: May 19, 2006
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Journal of Planar Chromatography
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