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Phytochemical and antimicrobial characterization of Macleaya cordata herb

Article  in  Fitoterapia · December 2010

DOI: 10.1016/j.fitote.2010.06.020 · Source: PubMed

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 Fitoterapia 81 (2010) 1006–1012

Contents lists available at ScienceDirect

Fitoterapia
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e

Phytochemical and antimicrobial characterization of Macleaya cordata herb

Pavel Kosina a,⁎, Jana Gregorova b, Jiri Gruz c, Jan Vacek a, Milan Kolar d, Mathias Vogel e,
Werner Roos e, Kathrin Naumann f, Vilim Simanek a, Jitka Ulrichova a
a
Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech Republic
b
Department of Biochemistry, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic
c
Laboratory of Growth Regulators, Palacky University and Institute of Experimental Botany AS CR, Slechtitelu 11, 783 71 Olomouc, Czech Republic
d
Department of Microbiology, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, 775 15 Olomouc, Czech Republic
e
Institute of Pharmaceutical Biology and Pharmacology, Department of Molecular Cell Biology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Str. 3,
061 20 Halle (Saale), Germany
f
Phytobiotics Futterzusatzstoffe GmbH, Rossengasse 9, 65343 Eltville, Germany

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

Article history: Macleaya cordata (plume poppy) is a source of bioactive compounds, mainly isoquinoline
Received 30 March 2010 alkaloids which are used in phytopreparations with anti-inflammatory and antimicrobial
Accepted in revised form 18 June 2010 activities. In this study, the alkaloids sanguinarine, chelerythrine, their dihydro derivatives,
Available online 28 June 2010
protopine and allocryptopine and phenolics, gallic, protocatechuic, p-hydroxybenzoic, m-
hydroxybenzoic, gentisic, p-coumaric, caffeic, ferulic and sinapic acids were determined in
Keywords: extracts prepared from M. cordata aerial part, seeds, and seed capsules using HPLC with UV
Macleaya cordata detection and/or LC/MS with electrospray ionization. The highest content of sanguinarine and
Isoquinoline alkaloids
chelerythrine was found in capsules. Protopine and allocryptopine were major alkaloids in
Phenolic acids
leaves including footstalks. The seed oil contained dihydrosanguinarine, dihydrochelerythrine
Fatty acids
Sanguinarine reductase and twelve fatty acids of which linoleic, oleic, palmitic and stearic acids predominated. In
Antimicrobial activity addition, sanguinarine reductase, a key enzyme in sanguinarine/dihydrosanguinarine
equilibrium in plants, was found for the first time, in the soluble proteins of leaves. Finally,
extracts were tested for antimicrobial activity using the microdilution method on standard
reference bacterial strains.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction plants is Macleaya cordata (Willd.) R. Br. also known as plume

Widely used natural sources of prospective protective
and/or curative agents are plants of the Papaveraceae family
which contain various bioactive alkaloids. Among these
Abbreviations: SG, sanguinarine; CHE, chelerythrine; DHSG, dihydrosan-
guinarine; DHCHE, dihydrochelerythrine; PR, protopine; AL, allocryptopine;
Na-EDTA, tetrasodium-ethylenediaminetetraacetate; MIC, minimum inhibitory
concentration; CCM, the Czech Collection of Microorganisms; BHI broth, brain
heart infusion broth; QBA, Quaternary benzo[c]phenanthridine alkaloids; F1,
fractions of free phenolic acids; F2, fractions of soluble esters; F3, fractions of
glycosides; F4, fractions of insoluble esters; MOPS, morpholinoethane sulfonic
acid; DTE, dithioerythritol; PMSF, phenylmethylsulfonyl fluoride; PVP,
polyvinylpyrrolidone.
⁎ Corresponding author. Tel.: + 420 58 563 2306; fax: + 420 58 563 2302.
E-mail address: kosina@tunw.upol.cz (P. Kosina).
poppy or Bocconia cordata. In Traditional Chinese Medicine,
the aerial part of the Macleaya herb has long been used for its
analgesic and anti-inflammatory properties in humans [1]. M.
cordata is also successfully used in veterinary medicine and
agriculture [2–7]. This herb is cultivated in China and Russia as a
primary source for the production of quaternary benzo[c]
phenanthridine alkaloids (QBA) which are responsible for the
pharmacological effects [8]. M. cordata is popular ornamental
garden plant, with tall upright stems and attractive grey to
olive-green, decorative leaves [9].
The main alkaloids isolated from Macleaya spp. are
sanguinarine (SG), chelerythrine (CHE), dihydrosanguinarine
(DHSG), dihydrochelerythrine (DHCHE), protopine (PR),
and allocryptopine (AL) (Fig. 1). The following alkaloids
have also been found in minor quantities in M. cordata

0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2010.06.020
 P. Kosina et al. / Fitoterapia 81 (2010) 1006–1012
Fig. 1. The main isoquinoline alkaloids in M. cordata herb. SG, CHE, PR, and AL are shown in cationic form. For quantities of the alkaloids see Table 1.
extracts: chelirubine, macarpine, sanguidimerine, chelidimer-
ine, homochelidonine, cryptopine, berberine, coptisine, chelilu-
tine, bocconarborine A, bocconarborine B, oxysanguinarine,
norsanguinarine, angoline, bocconoline, 6-ethoxychelerythrine,
6-ethoxysanguinarine, protopine-N-oxide, 6-methoxy-dihydro-
sanguinarine, 6-acetonyl-dihyrochelerythrine and 6-acetonyl-
dihydrosanguinarine [10,11]. However, what has not been
confirmed is whether some of these alkaloids are natural
constituents of the herb or artifacts originating from native
alkaloids during the isolation steps.
M. cordata is on the European Food Safety Authority (EFSA)
list of plants exploited as a component in feed additives in
animal production [8]. Besides other constituents, the alkaloids
of this plant are of high value as an anti-inflammatory feed
component. The intact plant as a powdered mixture of leaves,
capsules and seeds, is the parent substance of the feed additive
Sangrovit® [7,12]. In Russia, a fraction of QBA from Macleaya
spp. containing an approximately equimolar mixture of SG and
CHE sulphates is known as Sanguiritrin® [10,13]. This prepa-
ration exhibits a broad spectrum of antimicrobial action,
immunomodulating, anticholinesterase activity, and it
improves animal health status in general; reviewed in [14].
Since 1998, two studies discussing a relation between
Sanguinaria (extract containing SG and CHE) and oral
leukoplakia have been published [15,16]. However, none of
these papers fully satisfy the criteria used in epidemiology for
establishing causation. Upon review of the safety and efficacy
discussion related to topical Sanguinaria extract as an
antigingivitis agent published in the Federal Register [17],
1007
the overall evaluation indicated that the substance might be
regarded as safe.
We previously studied the effect of the long term admin-
istration of the M. cordata herb and purified alkaloid extract in
feed on rat and pig metabolism and its possible genotoxicity
[4,12,18]. The results confirmed that the use of M. cordata as a
feed additive is safe under selected experimental conditions.
Recently, the roots, leaves, flowers and fruits of M. cordata
were investigated for content and distribution of SG, CHE,
their dihydro derivatives, PR, and AL [19]. However there is
still insufficient information on the other constituents of
Macleaya spp. in the current literature. In the present study,
we focused on the analysis of SG, DHSG, CHE, DHCHE, PR and
AL in the aerial part, capsules and seeds of M. cordata
cultivated in China. In addition, for the first time the phenolic
acids in individual parts and fatty acids in seed oil of this plant
were identified and quantified. The presence of sanguinarine
reductase (SGR), the key enzyme in SG/DHSG content
regulation, was confirmed in the fresh leaves of M. cordata.
The M. cordata extracts and pure alkaloids were tested for
antimicrobial activities. For this purpose, the microdilution
method with standard reference bacterial strains was used.
2. Experimental
2.1. Plant material
M. cordata (Willd.) R. Br. leaves were harvested in Central
China (Hefei, Anhui Province) during July. The fruits (seeds
1008 P. Kosina et al. / Fitoterapia 81 (2010) 1006–1012
and capsules) were collected in September 2007. The dried
plant material of i) leaves including footstalks (aerial part), ii)
seeds and iii) capsules were supplied by Phytobiotics
Futterzusatzstoffe GmbH, Eltville, Germany. Voucher speci-
mens are deposited in the Department of Medical Chemistry
and Biochemistry, Faculty of Medicine and Dentistry, Palacky
University, Olomouc, the Czech Republic. Fresh leaves of M.
cordata for sanguinarine reductase activity confirmation were
harvested from plants cultivated in the Botanical Garden of
the University of Halle (Germany) from seeds analyzed in this
study.
2.2. Determination of alkaloids and phenolic acids
The alkaloid analyses in aerial parts of herbs were carried
out using a HPLC instrument consisting of a high pressure LCP
4100.2 pump, LCD 2040 ultraviolet detector (ECOM, Czech
Republic) and Synergi™ Max-RP 80A C-12 (150 × 4.6, 4 μm)
column (Phenomenex, USA). Gradient elution of solvents (A
and B) containing: 0.01 M 1-heptanesulfonic acid, 0.1 M
triethylamine (adjusted to pH 2.5) with 25% acetonitrile
(solvent A) and/or 60% acetonitrile (solvent B), was used [20].
M. cordata virgin oil (1.5 mL) was prepared from seeds
(10 g) by solely mechanical pressing employing Hydraulic
press H62 (Trystom, the Czech Republic) with a maximal
force of 80 kN at laboratory temperature 23 °C. The oil sample
(100 μL) was resuspended in methanol (0.5 mL) using Vortex
(1250 rpm) at 25 °C for 10 min (Scientific industries, Inc., NY,
USA). After this, the sample (methanolic fraction) was diluted
50-times in methanol and analyzed using liquid chromatog-
raphy-mass spectrometry according to a published procedure
[21].
Phenolic acids were fractionated and analyzed as de-
scribed earlier [22]. Briefly, fractions of free acids (F1), soluble
esters (F2), glycosides (F3) and insoluble esters (F4) were
isolated, and subsequently analyzed by the HPLC-MS method.
The HPLC-MS system consisted of an Alliance 2690 separation
module (Waters, USA) and ZMD 2000 single-quadrupole
mass spectrometer equipped with an electrospray interface
(Micromass, U.K.). Analytes were separated on a reversed
phase column (Luna Phenyl-Hexyl, 5 μm, 250 × 2 mm, Phe-
nomenex, USA) and quantified using internal standards of
isotopically labeled [2,3,5,6-2H4] p-hydroxybenzoic and
[3,4,5,6-2H4] salicylic acids.
2.3. Determination of fatty acids
The virgin oil from M. cordata seeds was pressed by solely
mechanical procedure at room temperature. The oil (50 mg)
was diluted in isooctane (2 mL) and 0.5 M sodium methoxide
(2 mL), and sonicated for 10 min (UCC4, Powersonic Industies
Inc., IL, USA). After sonication, the samples were heated to
boiling point using an instrument with a vertical condenser
where oil fatty acids were transformed to their methyl esters.
14% boron trifluoride (2 mL) was then used for neutralization of
excess sodium methoxide and samples were diluted in
isooctane (2 mL) and saturated aqueous solution of sodium
chloride (5 mL). Finally, diluted samples were resuspended in
isooctane (2 mL) and analyzed using gas chromatography. For
this purpose, the chromatographic system HP 4890D (Hewlett
Packard) with a flame ionization detector (temperature of
detector: 280 °C), was used. The DB-23 (60 m × 0.25 mm,
0.25 μm) chromatographic column Agilent Technologies
(Santa Clara, CA, USA) and temperature program: 100 °C
(3 min), from 100 °C to 170 °C (7 min), from 170 °C to 230 °C
(15 min), 230 °C (8 min), from 230 °C to 250 °C (4 min), 250 °C
(20 min), were applied. Injection volume was 2 μL (tempera-
ture of injector: 270 °C) and nitrogen was used as carrier gas.
Pentadecanoic acid (C15:0) was used as an internal standard.
For other details see [23].
2.4. Sanguinarine reductase activity measurement
300 mg portions of leaves of M. cordata (from the
Botanical Garden of the University of Halle) were briefly
rinsed with 30% ethanol (v/v) and sterile water, frozen in
liquid nitrogen and minced in the frozen state with 2 mL
extraction buffer containing 50 mM MOPS, 5 mM Na-EDTA,
5 mM DTE, 1 mM PMSF, 5 mM ascorbic acid and 0.2% bovine
serum albumin, adjusted to pH 7.5 with KOH, and 100 mg
solid PVP (Polyclar AT©). The frozen material was collected in
Eppendorf tubes, allowed to thaw at 4 °C and at this
temperature centrifuged at 10,000 g for 20 min. The obtained
supernatant was then centrifuged for 45 min at 90,000 g. The
final supernatant which contained the soluble proteins was
dialyzed by ultracentrifugation in Centricon tubes of 10,000 D
cut off (3 times, each followed by dilution with the extraction
buffer to the original volume) and finally brought to about
40 μg protein/mL. Enzyme activity assay started by mixing
5 μL NADPH 10 mM, with 10 μL glucose-6-phosphate 10 mM,
and 2 μL glucose-6-phosphate dehydrogenase 5 U mL− 1.
After a 10 min preincubation, 20 μL of the above-mentioned
protein extract and 8 μl glutathione 30 mM were added and
the sanguinarine reduction reaction started by adding 3 μL
sanguinarine, 10 μM. After 10 min of incubation, the reaction
was stopped by adding 7 μL NaOH (1 M) and the mixture
was extracted 3 times with 1 mL ethyl acetate/chloroform
(95/5,% v/v). The solvent phases were collected and evapo-
rated under nitrogen and the remaining alkaloids dissolved in
50 μL ethyl acetate/ethanol. 5 μL were applied to HPTLC plates
and developed with the mobile phase hexane/methanol/ethyl
acetate (20/4/4, v/v/v). Alkaloid spots were identified by their
fluorescence under UV-excitation.
2.5. Antimicrobial activity
Standard reference bacterial strains (Staphylococcus aure-
us CCM 3953, S. aureus CCM 4223, Pseudomonas aeruginosa
CCM 3955, Escherichia coli CCM 4225, E. coli CCM 3954) from
the Czech Collection of Microorganisms (CCM), Faculty of
Science, Masaryk University Brno, and the Streptococcus
agalactiae strain obtained from the Teaching Hospital in
Olomouc were used. Antibacterial activities were determined
by the standard microdilution method [24,25]. Aerial part,
seeds and capsules (1 g of individual samples) were extracted
with methanol (330 ml) in Soxhlet extractor for 12 h and the
methanol extracts were evaporated using a rotary vacuum
evaporator to dryness. Evaporated samples were dissolved in
DMSO to reach a concentration 1 mg mL− 1, diluted (with BHI
broth, Becton Dickinson) to provide decreasing concentra-
tions (geometric series, with a coefficient of 2) from a
concentration C down to the concentration C:256. Maximum
 P. Kosina et al. / Fitoterapia 81 (2010) 1006–1012
tested concentration was 500 μg mL− 1, minimum tested
concentration reached 3.9 μg mL− 1. After incubation for 24–
48 h in an incubator at 37 °C, the minimum inhibitory
concentration (MIC) was evaluated as the lowest concentra-
tion of the test substance that inhibited the growth of the
bacterial strain.
3. Results and discussion
3.1. Distribution of alkaloids in M. cordata
The principal alkaloids found in all parts of the herb were
QBA as SG, CHE, and protopine type of alkaloids as PR, AL
(Table 1). Our data show that SG and CHE are major alkaloids in
the capsule but SG (32 mg g− 1) predominates. On the other
hand, the highest PR and AL content (7.9 and 6.8 mg g− 1) was
found in the aerial part without reproductive organs. Trace
amounts of the alkaloids were present in the whole seeds; up to
0.08 mg g− 1 for all compounds. Dihydro derivatives of SG and
CHE were not detectable in seeds and relatively low amounts
(0.36–3.14 mg g− 1 for both) were determined in capsules and
particularly in aerial parts. We found the highest concentration
of SG and CHE in capsules. These alkaloids probably protect the
seed against pathogens and herbivore predators [26]. The
prevalence of AL and PR in aerial parts was also found in a
previous study by Suchomelova et al. focused on determination
of alkaloids in the roots of six species of the Papaveraceae family
[20]. Recently, both compounds were determined in M. cordata
fruit [19] but without distribution between the capsules and the
seeds. In addition to the six alkaloids described, sanguilutine,
homochelidonine, norsanguinarine, oxysanguinarine and oxy-
chelerythrine were found in trace amounts, but not quantified.
The abundance of phytoallexins in plants can be affected
by different external (biotic and abiotic stressors) and
internal (health status and metabolism rate of plants) stimuli
[27]. It is clear that the factors affecting alkaloid content in
M. cordata herb should be taken into account in any study of
their distribution in plants growing under different environ-
mental conditions. The alkaloid levels in plants are directly
affected by specific enzyme systems and stress factors. In
Eschscholzia californica, another rich source of isoquinoline
alkaloids, it was discovered that SG but not DHSG is excreted
into the cell wall and this leads to plant defense. Beyond a
threshold concentration, SG is re-imported and reduced to
the less toxic DHSG by the cytoplasmic enzyme sanguinarine
reductase (SGR) [28]. We have now detected the activity of
this enzyme among the soluble proteins of M. cordata leaves
(Fig. 2). Further, PCR analysis with gene specific primers also
suggests the presence of the SGR gene in the Macleaya
genome (unpublished results). It appears therefore that SGR
is present and active in M. cordata and thus might contribute
Table 1
Alkaloid contents (mg g− 1 dry weight) in M. cordata. All results are expressed as means ± SD of three replicate analyses.
Alkaloid Sanguinarine Chelerythrine
Aerial part 4.51 ± 0.23 2.88 ± 0.14
Seeds 0.07 ± 0.00 0.02 ± 0.00
Capsules 32.08 ± 0.40 7.36 ± 0.01
1009
Fig. 2. Activity of sanguinarine reductase among the soluble proteins of
Macleaya leaves. A dialyzed fraction of soluble leaf proteins was incubated
with 6 μM sanguinarine and 1 mM NADPH for 10 min. The alkaloids were
extracted and separated by HPTLC. (Details are given in Methods). Lane 1
(left): reaction mixture, NADPH omitted; lane 2: complete reaction mixture;
lane 3: reference: sanguinarine; lane 4: reference: dihydrosanguinarine.
to protecting this and other Papaveraceae plants from the
adverse effects of the QBA, mainly SG at the cellular level.
3.2. Phenolic acids in M. cordata
Phenolics, like other bioactive natural substances, play an
important role in plants, e.g. defense against pathogens and
they are interesting sources of antioxidants for animal
consumers. The ubiquitous phytoceuticals are phenolic acids
that are usually present in higher plants in milligrams of
aglycone per gram of fresh weight, for example from 10 to
2200 μg g− 1 according to Manach's review [29]. Here,
phenolics contained in M. cordata are presented for the first
time. Using a previously developed method [22], the specific
phenolic fractions were obtained and individual phenolic
acids determined in the aerial part, seeds and capsules.
Phenolic acids were determined in four fractions: as free
acids, as constituents of soluble esters, glycosides and in-
soluble esters. Benzoic acid hydroxy derivatives: gallic,
protocatechuic, p-hydroxybenzoic, m-hydroxybenzoic and
gentisic acids were found. Of cinnamic acid hydroxy deriva-
tives, we found p-coumaric, caffeic, ferulic and sinapic acids
(Fig. 3). The major acids were p-hydroxybenzoic, ferulic and
sinapic acids in all samples (Table 2). The highest concentra-
tions (up to 2.2 mg g− 1) were determined in aerial part and
capsules. Low levels of phenolic acids compared to other parts
were found in seeds except for p-hydroxybenzoic acid.
Protopine Allocryptopine Dihydrosanguinarine Dihydrochelerythrine
7.93 ± 0.34 6.77 ± 0.34 0.02 ± 0.00 1.11 ± 0.04
0.08 ± 0.00 0.03 ± 0.00 N.D. N.D.
0.29 ± 0.02 0.13 ± 0.08 3.14 ± 0.20 0.36 ± 0.01
1010 P. Kosina et al. / Fitoterapia 81 (2010) 1006–1012

Fig. 3. Phenolic acids identified in M. cordata aerial parts, seeds, and capsules. For quantities of the phenolic acids see Table 2.

Table 2
Phenolic acid contents (mg g− 1 dry weight) in fractions of free acids (F1), soluble esters (F2), glycosides (F3) and insoluble esters (F4) from aerial parts, seeds, and
capsules of M. cordata.

Acids

Source Gallic Protocatechuic p-Hydroxybenzoic m-Hydroxybenzoic Gentisic p-Coumaric Caffeic Ferulic Sinapic

Aerial part 0.046 ± 0.007 0.215 ± 0.032 0.460 ± 0.068 0.009 ± 0.001 N.D. 0.057 ± 0.010 0.092 ± 0.003 0.315 ± 0.003 0.097 ± 0.005
F1
Aerial part 0.042 ± 0.004 0.075 ± 0.007 0.686 ± 0.017 0.030 ± 0.006 N.D. 0.412 ± 0.030 0.614 ± 0.015 1.767 ± 0.280 0.489 ± 0.050
F2
Aerial part 0.061 ± 0.009 0.291 ± 0.058 0.584 ± 0.043 0.038 ± 0.009 0.067 ± 0.076 ± 0.020 0.185 ± 0.001 0.680 ± 0.021 0.322 ± 0.013
F3 0.002
Aerial part 0.005 ± 0.001 0.074 ± 0.012 0.411 ± 0.064 0.021 ± 0.002 N.D. 0.311 ± 0.000 0.492 ± 0.039 1.057 ± 0.041 0.669 ± 0.014
F4
Seeds F1 N.D. 0.015 ± 0.001 0.229 ± 0.052 0.003 ± 0.001 N.D. N.D. 0.007 ± 0.001 0.106 ± 0.007 0.049 ± 0.013
Seeds F2 N.D. 0.022 ± 0.012 0.471 ± 0.038 0.004 ± 0.002 N.D. N.D. 0.009 ± 0.002 0.079 ± 0.005 0.082 ± 0.004
Seeds F3 N.D. 0.022 ± 0.003 0.391 ± 0.014 0.004 ± 0.000 N.D. N.D. 0.005 ± 0.001 0.039 ± 0.012 0.439 ± 0.018
Seeds F4 N.D. 0.012 ± 0.001 0.479 ± 0.068 0.002 ± 0.001 N.D. N.D. 0.004 ± 0.002 0.029 ± 0.014 0.043 ± 0.009
Capsules 0.015 ± 0.001 0.363 ± 0.034 1.368 ± 0.168 0.012 ± 0.001 N.D. 0.021 ± 0.002 0.042 ± 0.002 0.515 ± 0.029 0.048 ± 0.004
F1
Capsules 0.036 ± 0.005 0.417 ± 0.060 2.22 ± 0.151 0.103 ± 0.013 N.D. 0.876 ± 0.035 0.770 ± 0.017 1.809 ± 0.156 1.071 ± 0.050
F2
Capsules 0.037 ± 0.001 0.573 ± 0.020 1.818 ± 0.079 0.024 ± 0.006 0.070 ± 0.111 ± 0.096 0.173 ± 0.004 0.995 ± 0.195 1.062 ± 0.031
F3 0.009
Capsules 0.006 ± 0.002 0.062 ± 0.010 0.593 ± 0.064 0.025 ± 0.008 N.D. 0.161 ± 0.052 0.158 ± 0.014 0.791 ± 0.026 1.284 ± 0.068
F4

All results are expressed as means ± SD of three replicate analyses; N.D. = not detected.
 P. Kosina et al. / Fitoterapia 81 (2010) 1006–1012 1011

Table 3 (2.5%) acids in prickly poppy (Argemone mexicana) seeds. This

Fatty acids determined in oil prepared from M. cordata seeds, n = 3.
herb like M. cordata is an interesting source of QBA. Further
studies identified unusual hydroxy, epoxy, and keto long chain
fatty acids in A. mexicana seeds [32–34]. These atypical fatty
acids have not been determined in M. cordata oil.
In the oil, the alkaloids SG and CHE were not detectable
whereas levels of their dihydro derivatives were as follows:
0.67 μg g− 1 for DHSG and 0.07 μg g− 1 for DHCHE. These
results are similar to qualitative alkaloid analysis of argemone
oil where DHSG and DHCHE have been identified [35].
However, M. cordata oil contains more than 1000-times
lower levels of these alkaloids than argemone oil. The low
content of the alkaloids in M. cordata oil corresponds with
their low levels in intact seeds described above.

3.4. Antimicrobial properties of the alkaloids and

M. cordata extracts

Antimicrobial effects of extracts from the M. cordata aerial

Fraction of glycosides of phenolic acids (F3) in seeds was rich
in sinapic acid. Gallic, gentisic and p-coumaric acids were not
found. The amount of free phenolic acids in the fraction F1 was
generally lower than in fractions F2–F4. The level of individual
phenolic acids in fractions F2, F3 and F4 was variable and their
concentrations were ca. one hundred times lower than those
for alkaloids (for comparison see Tables 1 and 2).
3.3. Components of M. cordata seed oil
We focused on the study of the fatty acid profile in the virgin
oil obtained from seeds. Butyric, capronic, caprylic, capric,
undecanoic, lauric, tridecanoic, myristic, cis-10-pentadecanoic,
palmitic, palmitoleic, all-cis 7,10,13-hexadecatrienoic, hepta-
decanoic, cis-10-heptadecanoic, stearic, elaidic, oleic, cis-11-
octadecenoic, linolelaidic, linoleic, α-linolenic, arachidic, and
eicosenoic were monitored in the oil. The fatty acids, 12/23 in
total, are shown in Table 3. The oil was high in linoleic acid
(74.5%), other acids found in notable quantities were oleic
(15.5%), palmitic (6.5%), and stearic (2.2%). The fatty acid
composition of M. cordata seed oil was very similar to the seed
oils of the genus Papaver plants. For example, Erinc et al. [30]
found, as in our study, linoleic (68.8–73.9%), oleic (14.1–19.3%),
palmitic (7.7–9.3%) and stearic (2.2–2.6%) acids were the main
fatty acids in the oils of different varieties of poppy (Papaver
somniferum). Lemordant et al. [31] described linoleic (58.4%),
oleic (23.1%), linolenic (2.2%), palmitic (13.1%) and stearic
part, seeds, capsules and individual alkaloids were tested in
vitro against standard reference bacterial strains S. aureus
CCM 3953, S. aureus CCM 4223, P. aeruginosa CCM 3955, two
strains of E. coli (CCM 4225 and CCM 3954), and S. agalactiae,
representing selected human pathogens. The antimicrobial
activity of the extracts increased with SG and CHE content
and was lower than the antimicrobial activity of SG or CHE
itself on most of bacterial strains used (Table 4). PR or AL
showed lower antimicrobial activity than SG or CHE; DHSG
had a mild inhibitory effect and DHCHE was inactive. Navaro
et al. [36,37] described similar experiments; they tested the
antimicrobial activities of methanolic extracts prepared from
benzo[c]phenanthridine-rich aerial parts of Bocconia arborea,
used in Mexican traditional medicine. Recently, the effect
M. cordata alkaloids against plant microbial pathogens was
studied [38]. The most effective were found to be SG and CHE.
In our study, a weak antimicrobial activity of SG and CHE was
found against P. aeruginosa. Extracts of seeds and capsules
displayed stronger antimicrobial effect than pure alkaloids.
This may be connected with the synergic effects of other
constituents present in seeds and capsules. The antimicrobial
activity of plant oil has been published for Chamomilla recutita
seed oil on P. aeruginosa, E. coli, and Enterobacter aerogenes
bacterial strains [39]. Comparable effects of seed and capsule
extracts, higher than for extracts from the aerial part, were
found with E. coli bacterial strain CCM 3954. With the above-
mentioned exceptions, the extract from M. cordata capsules

Table 4
Minimum inhibitory concentrations (μg mL− 1) of M. cordata extracts and pure alkaloids against selected bacterial strains.

Extract/Alkaloid S. aureus CCM 3953 S. aureus CCM 4223 P. aeruginosa CCM 3955 E. coli CCM 4225 E. coli CCM 3954 S. agalactiae

Aerial part 125.0 125.0 125.0 125.0 125.0 125.0

Seeds 125.0 125.0 62.5 250.0 62.5 125.0
Capsules 62.5 62.5 62.5 125.0 62.5 125.0
Sanguinarine 31.3 31.3 250.0 31.3 62.5 15.6
Chelerythrine 31.3 31.3 500.0 125.0 125.0 7.8
Protopine 250.0 250.0 125.0 125.0 125.0 125.0
Allocryptopine 250.0 250.0 125.0 125.0 125.0 125.0
Dihydrochelerythrine N.E. N.E. N.E. N.E. N.E. N.E.
Dihydrosanguinarine N.E. N.E. 500.0 N.E. 500.0 N.E.

CCM labeling of bacterial strains; N.E. = no effect.

MICs of selected antimicrobial agents are published for CCM standard reference bacterial strains directly from standard method used.
1012 P. Kosina et al. / Fitoterapia 81 (2010) 1006–1012
had stronger antimicrobial effect than extracts from aerial
part and seeds.
4. Conclusion
In comparison with aerial part and seeds, the highest
content of QBA was found in the capsules of M. cordata while
PR and AL were the major alkaloids in the aerial part. The
main phenolics were p-hydroxybenzoic, ferulic and sinapic
acids. The spectrum of fatty acids in the seed was closely
related to the seed oils of the species genus Papaver. The
presence and activity of SGR indicates that this enzyme is
probably widespread in plants which synthesize sanguinar-
ine. The results may provide valuable data for better
characterization and further research on M. cordata.
Acknowledgements
This work was supported by the Grant Agency of the
MSMT (grant No. MSM 6198959216), by the Grant Agency of
the Czech Republic (GA CR 525/07/0871 and GA CR 525/08/
0819) and by the Joint project AV ČR–DAAD SRN (D21-CZ15/
07–08). We thank H. Müller at Martin-Luther-University
Halle-Wittenberg and A. Zdarilova at Palacky University in
Olomouc for PCR analysis confirming the presence of the SGR
gene in the Macleaya genome (unpublished results).
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