Mentha Aquatica with Affinity to the GABA-Benzodiazepine Receptor ⁎ A.K

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South African Journal of Botany 73 (2007) 518–521 www.elsevier.com/locate/sajb

Compounds from Mentha aquatica with affinity to the GABA-benzodiazepine receptor ⁎ A.K. Jäger a, , J.P. Almqvist a,b, S.A.K. Vangsøe a,b, G.I. Stafford b, A. Adsersen a, J. Van Staden b

a Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, 2 Universitetsparken, 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 18 December 2006; received in revised form 8 March 2007; accepted 10 April 2007

Abstract

Mentha aquatica L. is used in Zulu traditional medicine for spiritual purposes and an ethanolic leaf extract has previously shown strong affinity to the GABA-benzodiazepine receptor. Viridiflorol from the essential oil and (S)-naringenin from an ethanolic extract was isolated by bioassay- guided fractionation using binding to the GABA-benzodiazepine site. Viridiflorol had an IC50 of 0.19 M and (S)-naringenin of 0.0026 M. © 2007 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Convulsions; Epilepsy; GABA-benzodiazepine receptor assay; Mentha aquatica; Naringenin; Traditional medicine; Viridiflorol

1. Introduction effect, depending on the receptor subtype the compound is binding to. Mentha aquatica L. (English: water mint, Afrikaans: In the present study we isolated the compounds from M. kruisement, Zulu names: imbozisa (-amabunu), umayime and aquatica with affinity to the GABA-benzodiazepine site by umnukani) is a perennial herb growing in marshes and damp bioassay-guided fractionation. places from the South-western Cape to tropical Africa and Europe. 2. Material and methods The essential oil from M. aquatica contains mono-and sesquiterpenes with a high degree of chemo-variation (Hefen- 2.1. Plant material and extraction dehl, 1970; Umemoto et al., 1994; Jerkovic and Mastelic, 2001; Esmaeili et al., 2006). A number of flavonoids have also been Aerial parts of M. aquatica L. (Lamiaceae) were collected at isolated from the plant (Burzanskahermann et al., 1977). Cedara Agricultural College, Hilton, KwaZulu-Natal in In South Africa, the plant is traditionally used against colds, February 2006. A voucher specimen is deposited at the respiratory problems and protection against evil spirits (Pooley, University of KwaZulu-Natal Herbarium, Pietermaritzburg 2005). In a screening of South African sedative plants for (Voucher number, Stafford 84 NU). The material was dried in affinity to the GABA-benzodiazepine receptor, an ethanolic anovenat50°C. leaf extract of M. aquatica had high activity (Stafford et al., One gram of dried, powdered plant material was extracted 2005). When a compound binds to the GABA-benzodiazepine with 10 ml petroleum ether, ethyl acetate, ethanol or water site it can lead to either anti-convulsive activity or a sedative respectively for 30 min in an ultrasound bath. The extracts were filtered through paper and taken to dryness under vacuum (40 °C) before being redissolved at 10 mg/ml in ethanol. Abbreviations: GABA, γ-aminobutyric acid. Twenty grams of dried, powdered material was steam ⁎ Corresponding author. distilled with 500 ml distilled water for 4 h. The obtained E-mail address: [email protected] (A.K. Jäger). essential oil was dissolved at 10 mg/ml in ethanol.

2.2. Isolation of viridiflorol from essential oil

Powdered dry leaf material (250 g) was steam distilled with 2500 ml distilled water for 2 h, yielding a pale yellow oil. The oil was applied to a 80 g silica 60 (0.040–0.063 mm) column (65 cm long, 2 cm i.d.) and eluted with 100 ml of each com- bination of toluene:ethyl acetate (97.5:2.5; 95:5; 90:10; 85:15; 75:25; 50:50). Collected fractions (6 ml) were combined on basis of a TLC analysis, where the plate was eluted in toluene: ethyl acetate 93:7. The active fraction was subjected to GC-MS on an Agilent 6890N Network GC system coupled to a 5973 Network Mass Selective Detector. GC conditions: injector temperature: 150 °C; temperature programme: start 50 °C, 20 °C/min to 300 °C; column: HP5MS. An NIST library was used for comparison of MS data. The active fraction was also investigated by 13C-NMR (Varian Mercury Plus 300 MHz; CDCl3). Optical rotation was measured on a Perkin-Elmer 241 polarimeter.

2.3. Isolation of naringenin from an ethanolic extract

Fifteen grams of dried, powdered plant material was extracted with 150 ml ethanol for 30 min in an ultrasound bath. The extract was filtered, taken to dryness under vacuum, Fig. 1. Structures of (A) viridiflorol and (B) (S)-naringenin. and then partitioned between ethyl acetate and water. The active ethyl acetate phase was subjected to solid-phase chromatography on a Bond Elut C2, eluted with water:methanol 3:1. This fuged at 0–4 °C for 10 min at 27,000×g. The pellet was fraction was then applied to a prep TLC plate (Merck Silica gel resuspended in Tris–citrate buffer (50 mM, pH 7.1, 2 mg 60, 0.5 mm thickness) and eluted in ethyl acetate:formic acid: original tissue per ml), and then used for the binding assay. glacial acetic acid:water (100:11:11:26). A strip at the side of Membrane suspension (500 μl) was added to 25 μlof[3H] the plate was sprayed with natural products — polyethylene flumazenil (Ro 15-1788, purchased from Perkin Elmer Life glycol reagent (Wagner and Bladt, 1996). Bands were scraped Sciences) and 25 μl test solution (0.01, 0.1, 1, and 10 mg/ml), off the plate and the silica eluted with ethanol. making a final concentration in the assay of 0.00045, 0.00445, The active compound was subjected to 1H-NMR (Varian 0.045 and 0.45 mg/ml, and was mixed and incubated for 40 min Mercury Plus 300 MHz; CDCl3) and optical rotation was in an ice-bath (0–4 °C). Total and unspecific binding was determined on a Perkin-Elmer 241 polarimeter. determined using buffer or diazepam (1 μM, final concentration in assay). After incubation, 5 ml of ice-cold buffer was added to 2.4. Membrane preparation the samples and the mixture poured directly onto Adventic glass fibre filters (GC-50) under vacuum, and immediately washed The preparation was performed according to Risa et al. with 5 ml of ice-cold buffer. The amount of radioactivity on the (2004) at 0–4 °C. Cerebral cortex from four rats was homo- filters was determined by conventional liquid scintillation genized for 5 s in 20 ml of Tris–citrate buffer (50 mM, pH 7.1) counting using Ultimo Gold XR as scintillation fluid. Specific using a small Ultra Turax. The suspension was centrifuged at binding was calculated as total binding minus unspecific 27,000×g for 15 min, and the pellet was washed three times binding. All experiments were done in triplicate. with buffer. The washed pellet was homogenized in 20 ml of buffer and the suspension was incubated in a water bath 3. Results and discussion (37 °C) for 30 min to remove endogenous GABA. The suspension was then centrifuged for 10 min at 27,000×g.Thefinal The four solvent extracts and the essential oil were tested in pellet was resuspended in 30 ml buffer and stored in aliquots at the GABA-benzodiazepine assay. The results for displacement −20 °C. of the radioactive ligand indicated that water (77%), ethanol extracts (77%) and the essential oil (64%) showed good 2.5. 3H-Ro 15-1788 (flumazenil) binding assay displacement, whereas the displacement obtained with the ethyl acetate (46%) and petroleum ether (31%) extracts was lower. The assay was carried out according to Kahnberg et al. This indicated that there were both polar and lipophilic active (2002) with modifications (Risa et al., 2004). The membrane compounds in M. aquatica, and it was therefore decided to preparation was thawed and washed with 20 ml Tris–citrate continue the isolation procedure using both the essential oil and buffer (50 mM, pH 7.1, 0–4 °C). The suspension was centri- ethanolic extracts. 520 A.K. Jäger et al. / South African Journal of Botany 73 (2007) 518–521

The sesquiterpene viridiflorol (yield: 45 mg) was isolated by Bioassay-guided isolation of the ethanol extract leads to the 1 bioassay-guided fractionation from the essential oil. The GC- isolation of the flavanone naringenin. The H-NMR data (H-3eq: MS analysis of the active compound from the essential oil 2.76, 1H, dd; H-3ax: 3.10, 1H, dd; H-2: 5.34, 1H, dd; H-6: 5.97, indicated that the compound was ledol, however not with a 1H, d; H-8: 5.99, 1H, d; H-3′, H-5′: 6.9, 2H, dd; H-2′, H-6′: 6.9, good library fit (28%). There are three naturally occurring 2H, dd) in comparison with previously published data (Du et al., isomers, ledol, viridiflorol and globulol. The optical rotation of 2004) determined the structure to be naringenin. An optical 25 the isolated compound was measured to be [α]589 =0.9°. (+/−)- rotation [α] 589 = −20.7 (EtOH) was determined, which Globulol has an optical rotation of 35–60° (Wu et al., 1996), corresponds to (S)-naringenin (Giorgio et al., 2004)(Fig. 1B). which eliminated the possibility that the isolated compound The IC50 values in the GABA-benzodiazepine assay were could be globulol. Viridiflorol and ledol both have an optical determined from dose–response curves (Fig. 2) to be 0.19 M for rotation of 2° (Wu et al., 1996), so this technique could not be viridiflorol and 0.0026 M for (S)-naringenin, giving a Ki-value used to differentiate between these two isomers. 13C-NMR (C- of 2 mM for (S)-naringenin. Compared to the clinically used 1: 58.07; C-2: 25.72; C-3: 29.00; C-4: 38.38; C-5: 39.63; C-6: benzodiazepine, diazepam, which had an IC50 value of 0.1 μM, 22.21; C-7: 28.48; C-8: 18.77) with comparison to data from the two compounds isolated in this study were not very active. In Bombarda et al. (2001), who synthesized the two isomers, a study on structure–activity relationship of flavonoids binding unequivocally determined the structure to be viridiflorol to the GABA-benzodiazepine site naringenin had a Ki-value of (Fig. 1A). Viridiflorol has previously been detected in M. 770 nM, about 100 times less active than diazepam with a Ki- aquatica (Umemoto et al., 1994; Esmaeili et al., 2006). value of 8.5 nM, but it was not stated which stereoisomer of naringenin was tested (Dekermendjian et al., 1999). It is often questioned whether plant constituents are able to reach the brain. Naringenin has been shown to pass the blood– brain barrier (Youdim et al., 2004), which means that it can exercise an effect on the CNS. Naringenin has been shown to have many effects outside the CNS, such as being a cancer chemopreventive agent, a mutagenic inhibitor, a genotoxicology inhibitor, and having antioxidant, hypocholesterolic, antibacterial, antiviral, antial- lergic, antiangionetic, apopotic and cytostatic activity (Hodek et al., 2002). This non-selectivity might limit the compound's clinical use. Viridiflorol has been shown to inhibit acetylcholinesterase, an enzyme often linked with Alzheimer's disease (Miyazawa et al., 1998). None of the isolated compounds exhibits high enough affinity to the GABA-benzodiazepine site to be candidates for drug development, but the displayed activity may contribute to the CNS-activity of M. aquatica reported employed in traditional medicine.

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Edited by JJM Meyer