Hindawi Publishing Corporation Biochemistry Research International Volume 2015, Article ID 617620, 9 pages http://dx.doi.org/10.1155/2015/617620 Research Article Modulatory Effects of Eschscholzia californica Alkaloids on Recombinant GABAA Receptors Milan Fedurco,1 Jana Gregorová,2 Kristýna Šebrlová,2 Jana Kantorová,2 Ondlej Peš,2 Roland Baur,3 Erwin Sigel,3 and Eva Táborská2 1 Michelin Recherche et Technique S.A., Route Andre-Piller´ 30, 1762 Givisiez, Switzerland 2Department of Biochemistry, Faculty of Medicine, Masaryk University, 62500 Brno, Czech Republic 3Institute of Biochemistry and Molecular Medicine, University of Bern, Buhlstrasse¨ 28, 3012 Bern, Switzerland Correspondence should be addressed to Milan Fedurco; [email protected] Received 28 July 2015; Revised 5 September 2015; Accepted 15 September 2015 Academic Editor: Emanuel Strehler Copyright © 2015 Milan Fedurco et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The California poppy (Eschscholzia californica Cham.) contains a variety of natural compounds including several alkaloids found exclusively in this plant. Because of the sedative, anxiolytic, and analgesic effects, this herb is currently sold in pharmacies in many countries. However, our understanding of these biological effects at the molecular level is still lacking. Alkaloids detected in E. californica could be hypothesized to act at GABAA receptors, which are widely expressed in the brain mainly at the inhibitory interneurons. Electrophysiological studies on a recombinant 1 2 2 GABAA receptor showed no effect of N-methyllaurotetanine at concentrations lower than 30 M. However, ()-reticuline behaved as positive allosteric modulator at the 3, 5,and6 isoforms of GABAA receptors. The depressant properties of aerial parts of E. californica are assigned to chloride-current modulation by ( )- reticuline at the 3 2 2 and 5 2 2 GABAA receptors. Interestingly, 1, 3,and 5 were not significantly affected by ( )-reticuline, 1,2-tetrahydroreticuline, codeine, and morphine—suspected ()-reticuline metabolites in the rodent brain. | downloaded: 13.3.2017 1. Introduction desired biological effects. Even though the presence of NMT in this plant has been clearly established, its content in the The California poppy is known in folk medicine for its aerial parts of this perennial herb is currently unknown. sedative, anxiolytic, and antinociceptive effects [1–4]. These Furthermore, it is not known whether or not this molecule effects have been traditionally assigned to protopine and interacts with the GABAA receptor. allocryptopine (see Figure 1). Both alkaloids act as weak Inthepresentwork,wehavestudiedtotalalkaloidextrac- stimulators of the binding of GABAA receptor agonists in the tion from aerial parts of E. californica by two methods. First, rat brain [5, 6], as anti-inflammatory agents [7] and as acetyl- the standard chloroform/SDS method [11] relying on the ion cholinesterase inhibitors [7, 8]. Another aporphine alka- pairing of alkaloids with an anionic detergent (Method A) loid isolated from this plant, namely, N-methyllaurotetanine was used. Second, the dry plant was macerated for extended (NMT), was reported to act as antagonist at the serotonin period of time in methanol solutions, solution pH was =85 5HT1ARreceptor(EC50 =155nM, nM) [9]. Protopine adjusted, and alkaloids were extracted into diethylether [12] andallocryptopinewerealsofoundtoblockhumanserotonin (Method B). Alkaloids contained in different fractions were and noradrenaline transporters (hSERT and NERT) and analysed using the diode-array spectrophotometer coupled to https://doi.org/10.7892/boris.76359 possess antidepressant-like effects on animal models [10]. HPLC and with the electrospray-tandem MS/MS spectrom- However, it is not clear whether typical pharmacy prepara- etry (ESI-MS/MS). The main goal of the present study was tions (i.e., 300 mg of dry plant material per capsule) contain toidentifytheactiveprincipleinE. californica responsible sufficient quantities of these alkaloids required to induce for reported sedative and anxiolytic effects [1–4]. Finally, we source: 2 Biochemistry Research International O O N N HO H O H O O N HO N HO O O H OH OH OH O N-Methyllaurotetanine O Isoboldine O O H Scoulerine Corydine O O O N N O O O O H Protopine O Eschscholtzine O H O + O O N N O O O O H O Californidine -Allocryptopine H O O N O O O H O + O-Methylcaryachine N O O H O OH N Sanguinarine O O H O O H O Caryachine O OH O + O + HO N N N O O O O H Chelerythrine 6S,12S-Neocaryachine-7-O- (S)-Reticuline methyl ether N-metho salt Figure 1: Alkaloids identified in the aerial parts of Eschscholzia californica. wouldliketoconcludewithwhetherthismedicinalherb (99.8%) and codeine certified reference material (1 mg/mL in has a potential of replacing some commonly used synthetic methanol) were obtained from Sigma. Caryachine (98%) and sleeping drugs and antidepressants. O-methylcaryachine (98%) samples were kindly provided by Stefan Gafner (Tom’s of Maine, USA). 2. Material and Methods 2.2. Alkaloid Extraction Analysis. The alkaloid extraction 2.1. Chemicals. Protopine hydrochloride (≥98%) and - procedure (Method A) employed in the present study was allocryptopine (≥98%) were obtained from Sigma-Aldrich slightly modified as compared to the original protocol for the (Switzerland). (S)-(+)-Reticuline hydrochloride (95%, >90% alkaloid extraction from Chelidonium majus [11]. ee), (R)-(−)-reticuline (95%), and 1,2-tetrahydroreticuline iodide (95%) used in electrophysiological experiments at Method A.Fivegramsofthedryplantmaterial(Eschscholzia the GABAA receptors were obtained from Toronto Research californica, Arcopharma, AMM 57426001, Switzerland) in a Chemicals (Canada). Morphine sulphate salt pentahydrate form of green powder was extracted into 100 mL of methanol Biochemistry Research International 3 ×106 5 350 7 1.0 7 325 3 300 275 0.8 250 225 200 0.6 175 (mAU) 150 Intens. 0.4 125 6 100 4 75 2 1 0.2 5 50 4 3 6 1 25 8 9 2 0 0.0 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 4 5 6 7 8 9 10 (min) Time (min) Label compound (m/z) (1) Isoboldine (328.1541) (2) Unknown (330.1699) (3) Corydine/isocorydine (342.1696) (4) Reticuline (330.1699) (5) Scoulerine (328.1546) (6) Caryachine (326.1389) (7) NMT (342.1709) (a) (b) Figure 2: (a) LC UV chromatogram of Eschscholzia californica extract before fractionation. Peak identification: (1) reticuline; (2) caryachine; (3) N-methyllaurotetanine; (4) protopine; (5) californidine; (6) allocryptopine; (7) escholtzine; (8) sanguinarine; and (9) chelerythrine. (b) LC-MS trace of “NMT sample” containing 82% N-methyllaurotetanine, 10% reticuline, and 8% caryachine with traces of other alkaloids. ∘ (15 minutes at 60 C). Organic solvent (95 mL) was then Interdelta (Givisiez, Switzerland) under the number 132914. distilled out at the rotary evaporator at the reduced pressure. The finely ground dry aerial part of E. californica (1.872 kg) Green-coloured solution was diluted with 50 mL of distilled was macerated in methanol for 10 weeks. Methanol was water and solution pH was adjusted to 1.0 (drop-wise addition evaporated. The weight of dry extract was 412.5 g (22% of of 10% hydrochloric acid). Subsequently, 100 mg of 0.2 wt% dry material). HPLC chromatogram of the extract before sodium dodecyl sulfate (aq.) was added and alkaloids were fractionation is shown in Figure 2(a). The dry plant extract × extracted with chloroform (3 50 mL). The organic phase was dissolved in 1% aq. H2SO4, aqueous phase was adjusted to was dried using anhydrous Na2SO4 and the chloroform ∘ pH ≅ 9 and alkaloid fraction was extracted into diethylether. evaporated using the rotary evaporator at 40 C. Finally, the Fraction A (8.06 g) was obtained once diethylether was crude extract was dissolved in 10 mL MeOH and filtered distilled off. In order to isolate NMT, the plant sample was using 5.0 mfollowedby0.45m PTFE microfilter. The plant fractionated into two fractions: A1 (nonphenolic alkaloids) extract was then subjected to a thin-layer chromatography and A2 (phenolic alkaloids). Fraction A was dissolved in 1% (TLC) in methylene chloride/methanol (18 : 2) containing 0.1% trifluoroacetic acid as mobile phase. TLC spots were cut H2SO4, pH was adjusted to 13, and nonphenolic alkaloids out using the razor blade, extracted into 5 mL methanol, and were extracted into diethylether (fraction A1). The remaining subjected to ESI-MS/MS analysis. Electrospray ionization aqueous phase was acidified to pH 8 with2 H SO4 and high-resolution mass spectra were acquired with a FT/ICR extracted second time into diethylether (fraction A2, pheno- mass spectrometer Bruker 4.7T BioApex II (Germany). lic). After evaporation of diethylether the mass of fraction A1 Alkaloids were also separated and identified using the diode- contained 4.19 g and the mass of the fraction A2 contained array HPLC and commercial alkaloid standards. Figure 1 2.02 g. Fraction A2 was found to contain 82% NMT, 10% illustrates a typical alkaloid distribution in E. californica reticuline, and 8% caryachine (Figure 2(b)). The method was found in the present study using the HPLC and ESI- described in our previous study [12]. Briefly, the mobile phase MS/MS (see Supplementary material available online at was prepared from a stock solution of 0.01 M sodium 1- http://dx.doi.org/10.1155/2015/617620). heptanesulfonate and 0.1 M triethylamine in H2O, adjusted to pH 2.5 with phosphoric acid. Mobile phase A consisted of Method B. E. californica was obtained from Dixa AG (St 25 : 75 (v/v) acetonitrile (ACN) : stock solution. Mobile phase Gallen, Switzerland) and a voucher sample was deposited at B was 60 : 40 (v/v) ACN : stock solution. Phosphoric acid 4 Biochemistry Research International and sodium 1-heptanesulfonate were obtained from Sigma- previously [13]. Briefly, Xenopus laevis oocytes were injected Aldrich (Prague, Czech Republic).