A thujone-free sage spissum extract (A. Vogel Menosan®, batch 041941) and the originating ethanolic tincture (67% EtOH V/V, batch 040116) were prepared from freshly harvested outer sage leaves (Salvia officinalis Folium rec.) at a drug extractant ratio (DER) 1:17. Dry mass content of the spissum extract was 19.2% [m/m] and total (α and β) thujone was < 20 ppm. The plant material was sourced from organic cultivations in Switzerland, verified and manufactured by A.Vogel AG (Roggwil, Switzerland). A.Vogel AG also provided tinctures from fresh plants [FE 150917 (only leaves), FE 150918 (only stipes), and FE 150916 (leaves and stipes)] and from dried plants [FE 150914 (only leaves), FE 150915 (only stipes), and FE 1509163 (leaves and stipes)], which have been produced according to the same extraction procedures as stated above. Dilutions of herbal preparations were made in H2OMilli-Q. Vehicle control was 67% ethanol for Salvia officinalis. Folium rec.T.1:17 and 3.7% saccharose laurate for spissum.
The following material were used for receptor binding analysis: 1-[N-methyl-3H] scopolamine methyl chloride ([3H]-NMS, hM3) was from GE Healthcare. [3H]CGP 54626 (GABAB) was purchased from BIOTREND Chemikalien GmbH (Cologne, Germany) and stored under the recommended conditions at − 20 °C. [N-methyl-3H]-Ro-15-1788 (GABAA-Bz-site), [3H]-DAMGO (μ-opioid), [3H(G)]-MK-912 (alpha 2A), and [3H]-imipramine (hydrochloride, [benzene ring – 3H(N)]-, 5-HTT) were from PerkinElmer (Rodgau, Germany). [3H]-8-OH-DPAT ([propyl-2,3-ring-1,2,3-3H], 5-HT1A) and [N-methyl-3H]-mesulergine (5-HT2B) were from ARC Inc. (St. Louis, USA).
Recombinant human AChE was from Bio-Techne GmbH (Wiesbaden-Nordenstadt, Germany). Human adrenergic α2A receptors were from Merck Millipore. Human serotonin 5-HT1A receptors, human serotonin 5-HT2C(e) receptors, human muscarinic M3 receptors, human μ opioid receptors, and human serotonin transporters were from PerkinElmer (Rodgau, Germany). Human serotonin 5-HT2B receptors were from rent-a-lab (Reutlingen, Germany).
Diazepam-ratiopharm® (GABAA-Bz-site, non specific) was from ratiopharm GmbH (Ulm, Germany). Baclofen (GABAB, non specific), 4-Diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, M3, non specifc and reference compound), D-Ala2,N-Me-Phe4,Gly5-ol)-ENKEPHALIN (DAGO, DAMGO, μ-opioid, reference compound), Naloxone-HCl (μ-opioid, non specific), 5-Hydroxytryptamine hydrochloride (Serotonin, 5-HT1A, non specific), WAY 161503 hydrochloride (5-HT2C, reference compound), Imipramine hydrochloride (5-HTT, non specifc and reference compound), acetylthiocholine, 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) and neostigmine (reference compound: AChE) were from Sigma (Taufkirchen, Germany). Mianserin hydrochloride (5-HT2C, 5-HT2B, non specific and reference compound 5-HT2B), SR 95531 hydrobromide (GABAA-Bz-site, reference compound), CGP 54626 hydrochloride (GABAB, reference compound), (±)-8-Hydroxy-2-dipropylaminotetralin hydrobromide (8-OH-DPAT, 5-HT1A, reference compound), and Rauwolscine hydrochloride (alpha 2A, non specific and reference compound) were from BIOTREND Chemikalien GmbH (Cologne, Germany).
Human frozen hypothalami were from a healthy 51-year old and a healthy 37-year-old woman (Tissue Solutions Ltd., Glasgow, UK). The tissue was minced in 20 ml 10 mM HEPES pH 7.4, 1 mM EDTA on ice with a disperger (20 s at 28 Trpm) and centrifuged for 15 min at 31,600×g at 4 °C. The pellet was homogenized (10 strokes with a glass-teflon homogenizer, 2000 rounds per minute) at 4 °C and again centrifuged. The latter procedure was repeated once and the pellet finally resupended in 50 mM HEPES pH 7.4, 4 mM MgCl2, and 1 mM EDTA; frozen in liquid nitrogen; and stored at − 80 °C until usage.
Acetylcholinesterase enzyme assay
Inhibition of AChE was assessed by a modified version of the colorimetric method of Ellman et al. [27]. AChE and effector/inhibitor were added to 100 mM phosphate buffer (pH 7.5) and 0.05% Brij L23, and the reaction was started by the addition of acetylthiocholine and DTNB. The thiocholine formed during hydrolysis of acetylthiocholine rapidly reacts with DTNB and releases a yellow 5-thio-2-nitrobenzoic acid anion. The production of this coloured anion was read after 30 min incubation at room temperature by an absorbance microplate reader (Sunrise, Tecan Deutschland GmbH, Crailsheim, Germany) at 405 nm.
The AChE enzyme assay was validated by determination of the IC50 value of the prototypic AChE inhibitor neostigmine.
Receptor binding assays
The GABAA-benzodiazepine site binding assay was performed as described by Mehta and Shank [28], with minor modifications. Membranes were incubated for 30 min at 22 °C in 50 mM Tris-HCl (pH 7.4) and 100 mM NaCl with 1 nM [3H]-Ro-15-1788. Non-specific binding was determined with 10 μM diazepam.
The GABAB receptor binding assay was performed as described by Asay & Boyd [29] with minor modifications: On the day of the assay, membranes were thawed at room temperature and kept on ice. Membranes were then washed 3 times with 25 volumes of ice-cold assay buffer by centrifugation at 18,000×g for 15 min at 4 °C. The pellet was resuspended and incubated for 90 min at room temperature in 20 mM Tris-HCl (pH 7.4), 120 mM NaCl, 6 mM Glucose, 4.7 mM KCl, 1.8 mM CaCl2, 1.2 mM MgSO4 and 1.2 mM KH2PO4 with 2 nM [3H]-CGP54626. Non specific binding was determined with 10 μM baclofen.
The alpha2A receptor binding assay was performed according to the data sheet provided by the supplier of the receptor preparation, with modifications. The receptor preparation was incubated for 60 min at 30 °C in 50 mM HEPES (pH 7.4) and 5 mM MgCl2 with 1 nM [3H]-MK-912. Non specific binding was determined with 3.16 μM rauwolscine.
The serotonin 5-HT1A receptor binding assay was performed according to the data sheet provided by the supplier of the receptor preparation, with modifications. The receptor preparation was incubated for 120 min at 37 °C in 50 mM Tris-H2SO4 (pH 7.4) and 5 mM MgSO4 with 0.5 nM [3H]-8-OH-DPAT in the dark. Non specific binding was determined with 10 μM serotonin.
The serotonin 5-HT2B receptor binding assay was performed as described by Wainscott et al. [30] with minor modifications. The receptor preparation was incubated for 120 min at room temperature in 50 mM Tris-HCl (pH 7.4), 0.1% ascorbic acid and 10 μM pargyline with 2 nM [N6-methyl-3H]-mesulergine. Non-specific binding was determined with 10 μM mianserin.
The serotonin 5-HT2C(e) receptor binding assay was performed according to the data sheet provided by the supplier of the receptor preparation, with modifications. The receptor preparation was incubated for 120 min at 37 °C in 50 mM Tris-HCl (pH 7.4), 0.1% ascorbic acid and 10 μM pargyline with 0.5 nM [N6-methyl-3H]-mesulergine in the dark. Non-specific binding was determined with 1 μM mianserin.
The μ-opioid receptor binding assay was performed according to the data sheet provided by the supplier of the receptor preparation, with modifications. The receptor preparation was incubated for 60 min at 27 °C in 50 mM Tris-HCl (pH 7.4) and 5 mM MgCl2 with 0.6 nM [3H]-DAMGO. Non specific binding was determined with 10 μM naloxone.
The muscarinic M3 receptor binding assay was performed according to the data sheet provided by the supplier of the receptor preparation, with modifications. The receptor preparation was incubated for 90 min at 25 °C in 50 mM Tris-HCl (pH 7.4), 2.5 mM MgCl2, and 1 mM EDTA with 0.3 nM [3H]-NMS. Non specific binding was determined with 1 μM 4-DAMP.
The 5-HTT serotonin transporter binding assay was performed according to the data sheet provided by the supplier of the receptor preparation, with modifications. The receptor preparation was incubated for 30 min at 27 °C in 50 mM Tris-HCl (pH 7.4), 120 mM NaCl and 5 mM KCl with 1.5 nM [3H]-imipramine. Non-specific binding was determined with 10 μM imipramine.
All assays were terminated by transfer of the samples onto filter plates (PerkinElmer or Whatman), presoaked in assaybuffer (GABAA, GABAB, alpha2A and muscarinic M3), treated with 0.5% BSA (μ-opioid), presoaked with 0.5% polyethyleneimine (5-HT2B, 5-HT2C(e)), or presoaked with 0.1% polyethyleneimine (5-HT1A, 5-HTT). Filters were washed four times with 200 μl of ice-cold 50 mM Tris-HCl pH 7.4 (GABAA, GABAB, Alpha2A, 5-HT1A, 5-HT2B 5-HT2C(e), μ-opioid, and muscarinic M3) or 50 mM Tris-HCl pH 7.4/0.5 M NaCl (5-HTT), and filter-bound radioactivity was determined by a microplate reader (Microbeta, Wallac, Finnland).
From every data point non-specific binding was subtracted and data normalized (100% specific binding represents the specific binding of the radioligand to the binding site of the receptor in the absence of any effector).
The IC50 value (concentration causing half-maximal inhibition of specific binding) was determined by non-linear regression analysis of the competition curves using the “sigmoidal dose-response” algorithm (GraphPad Prism, San Diego, USA).
Every receptor binding assay was validated by suitable well-characterised reference compounds for the respective receptor. An assay was considered validated if obtained IC50 values for the reference compound were within ±0.5 log-units of published data and/or historical data from our laboratory.