Searsia Species with Affinity to the N-Methyl-D-Aspartic Acid (NMDA

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South African Journal of Botany 78 (2012) 312–314 www.elsevier.com/locate/sajb

Research note Searsia species with afﬁnity to the N-methyl-D-aspartic acid (NMDA) receptor ⁎ A.K. Jäger a, , D.M. Knap a, B. Nielsen a, G.I. Stafford b, J. Van Staden b

a Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen O, Denmark b Research Centre for Plant Growth and Development, School of Biological and Conservation Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa

Received 15 March 2011; received in revised form 30 September 2011; accepted 3 October 2011

Abstract

Species of Searsia are used in traditional medicine to treat epilepsy. Previous studies on S. dentata and S. pyroides have shown that this is likely mediated via the N-methyl-D-aspartic acid (NMDA) receptor. Ethanolic extracts of leaves of six Searsia species were tested in a binding assay for affinity to the NMDA-receptor. S. dentata, S. gerrardii, S. glauca, S. natalensis, S. pentheri and S. pyroides all contained compounds with affinity to the NMDA-receptor. The species with the best affinity was S. pentheri. This might make it possible to use the Searsia species interchangeably, so the more accessible Searsia species can be used in traditional medicine. © 2011 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Epilepsy; NMDA receptor afﬁnity; Searsia

1. Introduction Further bioassay guided fractionation of the ethanolic extracts of S. pyroides, S. dentata and S. pentheri demonstrated the Species from the genus Searsia (previously Rhus) presence of three flavonoids, agathisflavone, amentoflavone and (Anacardiaceae) are used in South African traditional medi- apigenin, which showed affinity to the benzodiazepine binding cine for treatment of mental disorders (Stafford et al., 2008). site on the GABAA receptor in the nM range (Svenningsen Decoctions based on the bark from S. chirindensis are adminis- et al., 2006). Studies showed that the ethanolic extracts of S. tered for mental disturbances, roots from S. natalensis are pyroides and S. dentata inhibited the spontaneous epileptiform used for fits in children (Hutchings et al., 1996) and roots of discharge in a dose-dependent way in mouse cerebral cortical S. pyroides are used in infusions to treat delirium and epilepsy slices (Pedersen et al., 2008). However, when the three flavo- (Gelfand et al., 1985). noids were tested in the mouse cortical wedge model, none of A study by Risa et al. (2004), which investigated aqueous them had effect (Pedersen et al., 2008), indicating that the activity and ethanolic extracts of leaves and roots of Sersia species, on the brain tissue was mediated via mechanisms other than showed that ethanolic extract of S. rehmanniana and S. tridentata GABA, possibly via the N-methyl-D-aspartic acid (NMDA) had significant affinity in the [3H]-flumazenil assay, indicating receptor. The NMDA-receptor is part of the excitatory glutamate compounds binding to the gamma-amino butyric-acid (GABA) system in the brain, and is also involved in epilepsy. Binding receptor. The water extracts were not active, and the leaves studies of ethanolic extracts of S. dentata and S. pyroides con- were more active than the roots. The GABA system is the firmed the presence of NMDA-receptor antagonists in the ex- main inhibitory system in the brain, and is involved in epilepsy. tracts (Pedersen et al., 2008). In vivo studies showed that ethanolic extracts of S. pyroides protected 47% of 18-day-old ⁎ Corresponding author. Tel.: +45 35 336339; fax: +45 35 336041. rats against NMDA-induced seizures (Pedersen et al., 2010). It E-mail address: [email protected] (A.K. Jäger). thus seems likely that the anti-epileptic effect sought in traditional

0254-6299/$ - see front matter © 2011 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2011.10.002 A.K. Jäger et al. / South African Journal of Botany 78 (2012) 312–314 313 medicine is due to NMDA-receptor antagonists in the Sersia Cerebral cortices dissected from male Sprague–Dawley extracts. rats (250 g) were homogenised in 10 volumes ice-cold 0.32 M The aim of this study was to screen more species of the sucrose using an Ultra-Turrax homogeniser. The homogenate Searsia genus for NMDA-receptor antagonists. The assay is a was centrifuged at 1000 g for 10 min, and the resulting super- binding assay, there compounds in the plant extract compete natant was centrifuged at 18,000 g for 20 min. The pellet with a radiolabeled ligand for the binding site on the NMDA- was re-suspended and homogenised in 10 volumes of 5 mM receptor. If plant compounds have affinity to the binding site, Tris–HCl buffer (pH 7.4) followed by centrifugation at they will displace the radiolabeled ligand, and a lower level 8000 g for 20 min. The resulting supernatant and upper buffy of radioactivity will be measured in the brain homogenate. coat layer of the pellet were combined and centrifuged at Based on previous work (Pedersen et al., 2008, 2010), we 48,000 g for 20 min. The pellet was re-suspended in 10 volumes elected to investigate ethanolic leaf extracts. of 5 mM Tris–HCl buffer, homogenised and centrifuged at Plant material was collected in the University of KwaZulu- 48,000 g for 20 min. This wash step was repeated twice and the Natal Botanical Garden, Pietermaritzburg, and dried at 50 °C. final pellet was re-suspended in 10 volumes 5 mM Tris–HCl Vouchers are lodged at Bews Herbarium (NU), University buffer and stored at −18 °C. Before use in the assay, the homog- of KwaZulu-Natal, numbers are given in Table 1. Fifteen enate was defrosted and washed as outlined above to remove any grams of leaves was extracted three times with 200 ml ethanol glutamate naturally present in the tissue. The homogenate was (Kemetyl A/S) in an ultrasound bath for 60 min. The combined centrifuged at 48,000 g for 10 min. The pellet was re-suspended extracts were filtered before taken to dryness under vacuum. in 40–50 volumes of 50 mM Tris–HCl+2.5 mM CaCl2 The extracts were redissolved in DMSO (Sigma):Tris–HCl (Sigma) and centrifuged at 48,000 g for 10 min; this procedure buffer (Sigma) (1:9). was repeated. The pellet was re-suspended to give 0.10– 0.11 mg original membrane tissue/200 μl. The [3H]CGP 39653 binding assay was performed in a 96-well microplate. Twenty-five μl test solution, 25 μl[3H] Table 1 CGP 39653 (Perkin Elmer) (2 nM in final volume) and μ Binding of leaf extracts to the NMDA-receptor. The Ki-value for the S1 stereo- 200 l membrane homogenate were added to a well. Non- isomer of 3-(2-carboxy-pyrrolidinyl)-2-methyl acetic acid was 22 μM. specific binding was determined using 1 mM (L)-glutamate Plant species Voucher no. Concentration Speciﬁc Relative (Sigma), and total binding was determined using buffer of extract in binding standard (50 mM Tris–HCl+2.5 mM CaCl2). The S1 stereoisomer of ﬁnal assay (%) deviation 3-(2-carboxy-pyrrolidinyl)-2-methyl acetic acid (Rasmussen μ volume ( g/ml) (%) et al., 2011) was used as positive control. The plate was incu- S. dentata (Thunb.) Stafford351NU 100 26 12 bated for 60 min on ice. Then a 96 well Packard Filtermate F. A. Barkley cell harvester was used for filtration through a GF/B 50 54 7 10 99 3 filter (Perkin Elmer). To remove unbound radioactivity the 5 100 10 filter was washed three times with 250 μl ice-cold buffer. S. gerrardii (Engl.) Stafford354NU 100 54 6 The filter was dried at 50 °C. Thirty μl MicroScint 0 scintilla- Moffett tion fluid (Perkin Elmer) was added to each filter-well and 50 71 9 the filter sealed before counting, using a Packard TopCount 10 86 14 5958scintillation counter. All experiments were done in triplicate. S. glauca (Thunb.) Stafford356NU 100 25 6 The level of binding affinity to the NMDA-receptor was Moffett calculated: specific binding (%)=((DMP extract− DMP non- 50 44 6 specific binding)/(DMP total binding− DPM non-specific 10 81 5 binding))×100. 54160 S. natalensis Stafford353NU 100 35 11 The results from the screening of Searsia leaf extracts (Bernh. Ex Krauss) for affinity to the NMDA-receptor are shown in Table 1. The F. A. Barkley results show that all six species contain compounds that gave 50 44 11 a response in the bioassay. The plant species with the best ac- 10 84 5 tivity was S. pentheri, as the values were markedly lower for 5917 S. pentheri (A. Zahlbr.) Stafford352NU 100 10 9 all four test concentrations. The activities obtained in this Moffett study for S. dentata and S. pyroides were at a similar level 50 25 9 to those previously obtained for the two species (Pedersen et 10 55 13 al., 2008). The activity was dose-related for all six Searsia 5717species. In conclusion, S. dentata, S. gerrardii, S. glauca, S. pyroides (Burch.) Stafford355NU 100 23 24 Moffett S. natalensis, S. pentheri and S. pyroides all contained com- 50 52 7 pounds with affinity to the NMDA-receptor. At this stage it 10 96 11 would appear that the compounds responsible for this activity 5 101 1 are common in the genus. It would be interesting to isolate Relative standard deviation=SD/mean×100. and identify the active compounds in Searsia and investigate 314 A.K. Jäger et al. / South African Journal of Botany 78 (2012) 312–314 related genera with similar traditional use in treating epilepsy Pedersen, M.E., Baldwin, R.A., Niquet, J., Stafford, G.I., Van Staden, J., such as Lannea. Wasterlain, C.G., Jäger, A.K., 2010. Anticonvulsant effects of Searsia dentata (Anacardiaceae) leaf extract in rats. Phytotherapy Research 24, 924–927. Acknowledgement Rasmussen, J.L., Storgaard, M., Pickering, D.S., Bunch, L., 2011. Rational design, synthesis and pharmacological evaluation of the (2R)- and (2S)- G.I. Stafford was supported by the University of KwaZulu- stereoisomers of 3-(2-carboxy-pyrrolidinyl)-2-methyl acetic acid as ligands – Natal Post-doctoral Fellowship. for the ionotropic glutamate receptors. ChemMedChem 6, 498 504. Risa, J., Risa, A., Adsersen, A., Gauguin, B., Stafford, G.I., Van Staden, J., Jäger, A.K., 2004. Screening of plants used in southern Africa for epilepsy

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Edited by JN Eloff