Phytochemical and antimicrobial characterization of Macleaya cordata herb

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 Department of Biochemistry, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic 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 b c

a r t i c l e

i n f o

Article history: Received 30 March 2010 Accepted in revised form 18 June 2010 Available online 28 June 2010 Keywords: Macleaya cordata Isoquinoline alkaloids Phenolic acids Fatty acids Sanguinarine reductase Antimicrobial activity

a b s t r a c t Macleaya cordata (plume poppy) is a source of bioactive compounds, mainly isoquinoline alkaloids which are used in phytopreparations with anti-inflammatory and antimicrobial activities. In this study, the alkaloids sanguinarine, chelerythrine, their dihydro derivatives, protopine and allocryptopine and phenolics, gallic, protocatechuic, p-hydroxybenzoic, mhydroxybenzoic, gentisic, p-coumaric, caffeic, ferulic and sinapic acids were determined in extracts prepared from M. cordata aerial part, seeds, and seed capsules using HPLC with UV detection and/or LC/MS with electrospray ionization. The highest content of sanguinarine and chelerythrine was found in capsules. Protopine and allocryptopine were major alkaloids in leaves including footstalks. The seed oil contained dihydrosanguinarine, dihydrochelerythrine and twelve fatty acids of which linoleic, oleic, palmitic and stearic acids predominated. In 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 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, dihydrosanguinarine; 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: [email protected] (P. Kosina). 0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2010.06.020

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

P. Kosina et al. / Fitoterapia 81 (2010) 1006–1012

1007

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, chelidimerine, homochelidonine, cryptopine, berberine, coptisine, chelilutine, bocconarborine A, bocconarborine B, oxysanguinarine, norsanguinarine, angoline, bocconoline, 6-ethoxychelerythrine, 6-ethoxysanguinarine, protopine-N-oxide, 6-methoxy-dihydrosanguinarine, 6-acetonyl-dihyrochelerythrine and 6-acetonyldihydrosanguinarine [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 preparation 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],

the overall evaluation indicated that the substance might be regarded as safe. We previously studied the effect of the long term administration 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 specimens 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.

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 (temperature 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].

2.2. Determination of alkaloids and phenolic acids

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 evaporated 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.

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 chromatography-mass spectrometry according to a published procedure [21]. Phenolic acids were fractionated and analyzed as described 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, Phenomenex, 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

2.4. Sanguinarine reductase activity measurement

2.5. Antimicrobial activity Standard reference bacterial strains (Staphylococcus aureus 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 concentrations (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

1009

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 concentration 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 oxychelerythrine 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 environmental 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

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 insoluble esters. Benzoic acid hydroxy derivatives: gallic, protocatechuic, p-hydroxybenzoic, m-hydroxybenzoic and gentisic acids were found. Of cinnamic acid hydroxy derivatives, 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 concentrations (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.

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

Protopine

Allocryptopine

Dihydrosanguinarine

Dihydrochelerythrine

Aerial part Seeds Capsules

4.51 ± 0.23 0.07 ± 0.00 32.08 ± 0.40

2.88 ± 0.14 0.02 ± 0.00 7.36 ± 0.01

7.93 ± 0.34 0.08 ± 0.00 0.29 ± 0.02

6.77 ± 0.34 0.03 ± 0.00 0.13 ± 0.08

0.02 ± 0.00 N.D. 3.14 ± 0.20

1.11 ± 0.04 N.D. 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

Aerial part F1 Aerial part F2 Aerial part F3 Aerial part F4 Seeds F1 Seeds F2 Seeds F3 Seeds F4 Capsules F1 Capsules F2 Capsules F3 Capsules F4

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

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

0.061 ± 0.009

0.291 ± 0.058

0.584 ± 0.043

0.038 ± 0.009

0.005 ± 0.001

0.074 ± 0.012

0.411 ± 0.064

0.021 ± 0.002

0.067 ± 0.076 ± 0.020 0.185 ± 0.001 0.680 ± 0.021 0.322 ± 0.013 0.002 N.D. 0.311 ± 0.000 0.492 ± 0.039 1.057 ± 0.041 0.669 ± 0.014

N.D. 0.015 ± 0.001 N.D. 0.022 ± 0.012 N.D. 0.022 ± 0.003 N.D. 0.012 ± 0.001 0.015 ± 0.001 0.363 ± 0.034

0.229 ± 0.052 0.471 ± 0.038 0.391 ± 0.014 0.479 ± 0.068 1.368 ± 0.168

0.003 ± 0.001 0.004 ± 0.002 0.004 ± 0.000 0.002 ± 0.001 0.012 ± 0.001

N.D. N.D. N.D. N.D. N.D.

N.D. N.D. N.D. N.D. 0.021 ± 0.002

0.876 ± 0.035 0.770 ± 0.017 1.809 ± 0.156 1.071 ± 0.050

0.007 ± 0.001 0.009 ± 0.002 0.005 ± 0.001 0.004 ± 0.002 0.042 ± 0.002

Ferulic

0.106 ± 0.007 0.079 ± 0.005 0.039 ± 0.012 0.029 ± 0.014 0.515 ± 0.029

Sinapic

0.049 ± 0.013 0.082 ± 0.004 0.439 ± 0.018 0.043 ± 0.009 0.048 ± 0.004

0.036 ± 0.005

0.417 ± 0.060

2.22 ± 0.151

0.103 ± 0.013

N.D.

0.037 ± 0.001

0.573 ± 0.020

1.818 ± 0.079

0.024 ± 0.006

0.006 ± 0.002

0.062 ± 0.010

0.593 ± 0.064

0.025 ± 0.008

0.070 ± 0.111 ± 0.096 0.173 ± 0.004 0.995 ± 0.195 1.062 ± 0.031 0.009 N.D. 0.161 ± 0.052 0.158 ± 0.014 0.791 ± 0.026 1.284 ± 0.068

All results are expressed as means ± SD of three replicate analyses; N.D. = not detected.

P. Kosina et al. / Fitoterapia 81 (2010) 1006–1012 Table 3 Fatty acids determined in oil prepared from M. cordata seeds, n = 3.

1011

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

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, heptadecanoic, cis-10-heptadecanoic, stearic, elaidic, oleic, cis-11octadecenoic, 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

Antimicrobial effects of extracts from the M. cordata aerial 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 abovementioned 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 Seeds Capsules Sanguinarine Chelerythrine Protopine Allocryptopine Dihydrochelerythrine Dihydrosanguinarine

125.0 125.0 62.5 31.3 31.3 250.0 250.0 N.E. N.E.

125.0 125.0 62.5 31.3 31.3 250.0 250.0 N.E. N.E.

125.0 62.5 62.5 250.0 500.0 125.0 125.0 N.E. 500.0

125.0 250.0 125.0 31.3 125.0 125.0 125.0 N.E. N.E.

125.0 62.5 62.5 62.5 125.0 125.0 125.0 N.E. 500.0

125.0 125.0 125.0 15.6 7.8 125.0 125.0 N.E. 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 sanguinarine. 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). References [1] Xinrong Y. Encyclopedic reference of traditional Chinese medicine. Springer, ISBN 978-3-540-42846-6; 2003. p. 436. [2] Jankowski J, Zdunczyk Z, Juskiewicz J, Kozlowski K, Lecewicz A, Jeroch H. Arch Geflugelkd 2009;73:95–101. [3] Jeroch H, Kozlowski K, Jeroch J, Lipinski K, Zdunczyk Z, Jankowski J. Zuchtungskunde 2009;81:279–93. [4] Kosina P, Walterova D, Ulrichova J, Lichnovsky V, Stiborova M, Rydlova H, et al. Food Chem Toxicol 2004;42:85–91. [5] Kozlowski K, Lecewicz A, Jeroch H, Zdunczyk Z, Jankowski J. Arch Geflugelkd 2008;72:140–2. [6] Rawling MD, Merrifield DL, Davies SJ. Aquaculture 2009;294:118–22. [7] Vieira SL, Oyarzabal OA, Freitas DM, Berres J, Pena JEM, Torres CA, et al. J Appl Poult Res 2008;17:128–33. [8] Franz, C., Bauer, R., Carle, R., Tedesco, D., Tubaro, A., Zitterl-Eglseer, K., CFT/EFSA/FEEDAP/2005/01:140–152. (2005).

[9] Cool S, DeMarsh-Dodson T, Halpern A. Garden wise non-invasive plants for your garden, Eastern Washington guide. Bellingham, WA, USA: Premier Graphics; 2008. p. 7. [10] Dvorak Z, Kuban V, Klejdus B, Hlavac J, Vicar J, Ulrichova J, et al. Heterocycles 2006;68:2403–22. [11] Ye F, Feng F, Liu W. Zhongguo Zhong Yao Za Zhi 2009;34:1683–6. [12] Stiborova M, Vostalova J, Zdarilova A, Ulrichova J, Hudecek J, Tschirner K, et al. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2008;152:35–9. [13] Vicar J, Soural M, Hlavac J. Chem Listy 2010;104:51–3. [14] Zdarilova A, Malikova J, Dvorak Z, Ulrichova J, Simanek V. Chem Listy 2006;100:30–41. [15] Damm DD, Fantasia JE. Gen Dent 2002;50:466–8. [16] Damm DD, Curran A, White DK, Drummond JE. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999;87:61–6. [17] FDA Federal, Register, Vol 68, No 103, 32260–32262. [18] Zdarilova A, Vrublova E, Vostalova J, Klejdus B, Stejskal D, Proskova J, et al. Food Chem Toxicol 2008;46:3721–6. [19] Chen YZ, Liu GZ, Shen Y, Chen B, Zeng JG. J Chromatogr A 2009;1216: 2104–10. [20] Suchomelova J, Bochorakova H, Paulova H, Musil P, Taborska E. J Pharm Biomed Anal 2007;44:283–7. [21] Psotova J, Klejdus B, Vecera R, Kosina P, Kuban V, Vicar J, et al. J Chromatogr B 2006;830:165–72. [22] Ayaz FA, Hayirlioglu-Ayaz S, Gruz J, Novak O, Strnad M. J Agric Food Chem 2005;53:8116–22. [23] Cruz-Hernandez C, Destaillats F. J Liq Chromatogr Rel Technol 2009;32: 1672–88. [24] Performance standards for antimicrobial susceptibility testing, seventeenth informational supplement, M100-S17. CLSI (2007). [25] Psotova J, Kolar M, Sousek J, Svagera Z, Vicar J, Ulrichova J. Phytother Res 2003;17:1082–7. [26] Wink M. Curr Drug Metab 2008;9:996–1009. [27] Salmore AK, Hunter MD. J Chem Ecol 2001;27:1713–27. [28] Vogel M, Lawson M, Sippl W, Conrad U, Roos W. J Biol Chem 2010;285: 18397–406. [29] Manach C, Scalbert A, Morand C, Remesy C, Jimenez L. Am J Clin Nutr 2004;79:727–47. [30] Erinc H, Tekin A, Ozcan MM. Grasas Aceites 2009;60:375–81. [31] Lemordant D, Ghiglione C, Kalos Y. Adansonia 1975;14:645–54. [32] Gunstone FD, Holliday JA, Scrimgeour CM. Chem Phys Lipids 1977;20: 331–5. [33] Mani VVS, Lakshmin G. Fette Seifen Anstrichmittel Verbunden Mit Der Zeitschrift Die Ernahrungsindustrie 1972;74:268–70. [34] Rukmini C. J Am Oil Chem Soc 1975;52:171–3. [35] Das M, Khanna SK. Crit Rev Toxicol 1997;27:273–97. [36] Navarro V, Delgado G. J Ethnopharmacol 1999;66:223–6. [37] Navarro V, Villarreal ML, Rojas G, Lozoya X. J Ethnopharmacol 1996;53: 143–7. [38] Liu H, Wang JH, Zhao JL, Lu SQ, Wang JG, Jiang WB, et al. Nat Prod Commun 2009;4:1557–60. [39] Pereira NP, Cunico MM, Miguel OG, Miguel MD. J Am Oil Chem Soc 2008;85:493–4.