Journal of Ethnopharmacology 121 (2009) 178181

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Ethnopharmacological communication

MAO-A inhibitory activity of quercetin from Calluna vulgaris (L.) Hull.

Lasse Saaby , Hasse B. Rasmussen, Anna K. Jger
Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, 2 Universitetsparken, 2100 Copenhagen O, Denmark

a r t i c l e

i n f o

a b s t r a c t
Aim of the study: This study investigated MAO-A inhibitory activity of methanol extract of Calluna vulgaris (L.) Hull., which traditionally has been used as a nerve calming remedy. Materials and methods: A methanolic extract of Calluna vulgaris was partitioned against heptane, ethyl acetate and water. The three fractions were tested in a photometric peroxidase linked MAO-A bioassay. The ethyl acetate phase showed the highest MAO-A inhibitory activity. Quercetin was isolated by VLC through bioassay-guided fractionation and puried by re-crystallisation. The structure was elucidated by LCMS and 1 H NMR. Results: The IC50 -value for MAO-A inhibition by quercetin was 18 0.2 M in an assay where the IC50 -value for MAO-A inhibition by clorgylin was 0.2 0.02 M. Conclusion: The content of quercetin in Calluna vulgaris might explain the reported nerve calming effect of the plant. 2008 Elsevier Ireland Ltd. All rights reserved.

Article history: Received 18 July 2008 Received in revised form 8 September 2008 Accepted 3 October 2008 Available online 1 November 2008 Keywords: Calluna vulgaris Ericaceae Monoamine oxidase A inhibitor Quercetin Traditional medicine

1. Plant Aerial parts of Calluna vulgaris (L.) Hull. (Ericaceae) were collected at a location in the northwest part of Zealand, Denmark (55 55 N 11 31 E). A voucher specimen is deposited in the General Herbarium, University of Copenhagen (Saaby No.1 2007). 2. Uses in traditional medicine The tea of Calluna vulgaris (heather) has been used in Danish traditional medicine against the common cold and rheumatoid arthritis. The tea of heather was believed to have a nerve calming effect and to increase perspiration (Brndegaard, 1978). Various uses of Calluna vulgaris have been recorded in other European societies. These include the use as an antiseptic, cholagogue, diaphoretic, diuretic, expectorant, antirheumatic, antiinammatory and in the treatment of gout (Tunon et al., 1995; Kumarasamy et al., 2002). 3. Previously isolated classes of constituents A number of avonoid glycosides have previously been isolated from Calluna vulgaris (Jalal et al., 1982; Allais et al., 1991; Simon

et al., 1993a; Simon et al., 1993b; Allais et al., 1995; Orhan et al., 2007). Isolation of chromones and triterpenes from the plant has also been reported (Simon et al., 1992, 1994). 4. Materials and methods 4.1. Extraction For bioassay-guided isolation, 100 g dried, ground plant material was defatted by extraction with 1000 ml heptane (Riedel-de Han, HPLC grade) twice in an ultrasonic bath. After drying the plant material was extracted twice with 1000 ml methanol (LabScan, HPLC grade) in an ultrasound bath. The combined methanol extract was evaporated to dryness under reduced pressure. 4.2. Partitioning The residue from the methanol extract (16.6 g) was reresuspended in 1000 ml methanol:water 90:10. The resultant solution was partitioned three times against 300 ml heptane in a separation funnel and the three heptane extracts were combined to a total volume of 900 ml. Methanol was evaporated from the methanol:water mixture, which was made up to a total volume of 500 ml with demineralised water. The resulting water suspension was partitioned three times against 300 ml ethyl acetate (Lab-Scan, HPLC grade) and the ethyl acetate extracts were combined to a total volume of 900 ml, which was evaporated to dryness under reduced pressure. The water fraction was freeze-dried.

Abbreviations: DMSO, dimethyl sulfoxide; MAO-A, monoamine oxidase A; TLC, thin layer chromatography; VLC, vacuum liquid chromatography. Corresponding author. Tel.: +45 3533 6504; fax: +45 3530 6041. E-mail address: las@farma.ku.dk (L. Saaby). 0378-8741/$ see front matter 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.10.012

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Fig. 1. HPLC chromatogram of the methanol extract at 254 nm. UV-spectra are given for peaks at indicated retention times.

4.3. Fractionation by VLC The ethyl acetate fraction (5.5 g) was fractionated on 240 g Merck Silica Gel 60 in a vacuum liquid column (70 mm 230 mm). The column was eluted with 100 ml fractions of: Heptane (fractions 12), mixtures of heptane:dichloromethane (Lab-Scan, HPLC grade):methanol; 80:18:2 (fractions 38), 65:31.5:3.5 (fractions 914), 50:45:5 (fractions 1519), 25:67.5:7.5 (fractions 2029), 10:81:9 (fractions 3034), mixture of dichloromethane:methanol 90:10 (fractions 3540) and methanol (fractions 4152). The different fractions were spotted on a TLC plate, which was evolved in heptane:dichloromethane:methanol 10:81:9 and developed with anisaldehyde reagent. Fractions showing similar patterns on the TLC plate were combined to give ve larger fractions; 1 (fractions 2026), 2 (fractions 2731), 3 (fractions 3236), 4 (fractions 3743) and 5 (fractions 4452).

4.6. Crystallisation of active compound The evaporation residue (91 mg) of fraction 3 was dissolved in 500 l acetone (SigmaAldrich, HPLC grade) in a 50 ml pointed ask and heated until boiling. Demineralised water was slowly added to the warm solution until this became turbid and the ask was subsequently left to stand at room temperature for an hour, to facilitate crystal formation. The crystals were separated from the solvent by centrifugation and subsequent removal of the supernatant. To remove impurities, the isolated crystals were redissolved in acetone and the crystallisation process described above repeated twice.

4.7. Structure elucidation

1 H NMR spectrum was recorded at 300 MHz using a Varian Mercury NMR instrument. The spectrum was obtained from a deuterated methanol solution.

4.4. HPLC analysis of fraction 3 Fraction 3 was analysed on a Waters HPLC system (2996 photodiode array detector and 1525 binary HPLC pump) equipped with a Luna 5U C-18 column (250 mm 4.6 mm) and a 20 l loop. The analysis was performed by employing a linear gradient at a ow rate of 1 ml/min. The gradient ranged from 100% MilliQ water to 100% acetonitrile (VWR Prolabo, HPLC grade) over 30 min. After 30 min 100% acetonitrile was continued until the experiment was stopped.

4.8. MAO-bioassay The method described by Holt et al. (1997) was used with modications (Stafford et al., 2007). Fifty microliters plant extract (or DMSO as blank); 50 l chromogenic solution (0.8 mM vanilic acid and 2.5 mM 4-aminoantipyrine in 0.2 M potassium phosphate buffer, pH 7.6) containing 4 U/ml horseradish peroxidase; 100 l 3 mM tyramine in 0.2 M potassium phosphate buffer, pH 7.6 and 50 l 8 U/ml MAO-A in potassium phosphate buffer, pH 7.6 were added to each well of a 96-well, at bottom microtiter plate, which was incubated at 37 C and measured for absorbance after 5, 10, 15, 20, 25 and 30 min at 490 nm. To correct for unspecic absorbance readings, control wells were set up in the same manner as described above, but without MAO-A. For calculation of the MAO-A inhibitory activity, the slope of the linear change in absorption measured for wells with plant extracts were compared to the slope of blank controls. Data were processed in Microsoft Excel. GraFit 5 was used to calculate IC50 -values.

4.5. LCMS analysis of samples Samples were analysed on an Agilent 1200 HPLC system (G1316B C18 (75 mm 2.1 mm) column) tted with an Agilent G6410A triple quadropole mass spectrometer and a Sedex 85 evaporative light scattering detector. The analysis was performed by employing a gradient system at a ow rate of 0.75 ml/min. The gradient ranged from 100% eluent A (demineralised water:acetonitrile:formic acid (Fluka, 98%) (95:5:0.1) to 50% eluent B (demineralised water:acetonitrile:formic acid (5:95:0.043) over 5 min and from 50% eluent B to 90% eluent B over 10 s.

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5. Results and discussion MAOs are avoproteins which catalyses the oxidative deamination of primary, secondary and tertiary amines (Edmondson et al., 2004). The primary functions of MAOs are the metabolism of exogenous amines and the regulation of neurotransmitter level and intracellular amine stores. MAO-A preferentially deaminates serotonin (5-hydroxytryptamine) and norepinephrine (Billett, 2004). Thus, inhibition of MAO-A may alleviate symptoms of depression (Yamada and Yasuhara, 2004). Recent unpublished work from our department shows that the water and ethanol extracts of Calluna vulgaris (L.) Hull. posses MAOA inhibitory activity. HPLC analysis of the methanol extract showed several peaks, which seemed to be different polyphenolics from their respective UV-spectrum (Fig. 1). The ethyl acetate soluble part of the methanol extract, which showed the highest activity, was fractionated by VLC (Fig. 2). HPLC fractionation of the active VLC fraction (fraction 3) showed one major peak (MAO-A inhibition 59%) and two smaller peaks (MAO-A inhibition 22% and 7%). Repeated crystallisation of fraction 3 led to the isolation of a pure compound (14.3 mg), which showed a molecular ion at m/z 303 in LCMS (positive mode) and the following 1 H NMR signals. 1 H NMR (CD3 OD, 300 MHz): 7.73 (1H, d, J = 2.2 Hz, H-2 ), 7.63 (1H, dd, J = 8.5 and 2.1 Hz, H-6 ), 6.88 (1H, d, J = 8.5 Hz, H-5 ), 6.38 (1H, d, J = 2.1 Hz, H-8), 6.18 (1H, d, J = 2.1 Hz, H-6). The data are in accordance with previously published data for quercetin (Moon et al., 2001; Dutta et al., 2007). The IC50 -value for MAO-A inhibition by quercetin was 18 2 M (Fig. 3). The IC50 -value for clorgyline (MAO-A inhibitor) was 0.2 0.02 M. Using standard addition experiments the content of quercetin in the crude methanol extract was estimated to be 41 g/mg extract. The methanol extract had an IC50 -value of 8 g/ml. The amount of quercetin in 8 g methanol extract would be 0.3 g, which means that quercetin alone cannot account for all of the observed MAO-A inhibitory activity of the methanol extract (Fig. 3). Quercetin has previously been identied as a MAO-A inhibitor. Quercetin isolated from Hypericum hircinum was shown to be a MAO-A inhibitor with an IC50 -value of 0.01 M (Chimenti et al., 2006). In another study quercetin was isolated from Caryratia japonica which was shown to inhibit MAO-A with an IC50 -value of 2.8 M and MAO-B with an IC50 -value of 90 M (Han et al., 2007). A number of avonoids have shown MAO-A inhibitory capabilities. Kaempferol isolated from Ginko biloba exhibited MAO-A inhibition with an IC50 -value of 0.7 M. Apigenin and luteolin isolated from Carytia japonica were shown to be MAO-A inhibitors with IC50 -values of 1.7 M and 4.9 M, respectively (Han et al., 2007). Formonetin and kushenol F isolated from the roots of Sophora ava-

Fig. 3. IC50 determination of quercetin.

cens showed IC50 -values against MAO-A of 21.2 M and 103.7 M, respectively (Hwang et al., 2005). Naringenin isolated from Mentha aquatica was found to be an inhibitor of MAO-A with an IC50 value of 955 M in an assay where clorgylin had an IC50 -value of 0.0003 M (Olsen et al., 2008). It is difcult to compare the IC50 values reported for the avonoids, as assay conditions vary and not all studies give IC50 -values for standard compounds. It has been questioned whether avonoids are able to pass the blood brain barrier and enter the brain. Quercetin has been shown to pass blood brain barrier, although to a signicantly lesser degree than naringenin (Youdim et al., 2004). Furthermore, quercetin has been shown to inuence the electropharmacogram of adult rats in the same manner as moclobemide, a reversible inhibitor of MAO-A (Dimpfel, 2008). This suggests that quercetin does pass the blood brain barrier and might function as a reversible inhibitor of MAO-A. The content of quercetin in Calluna vulgaris might explain the reported nerve calming effect of the plant. References
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Fig. 2. Inhibitory activity of the VLC fractions in the MAO-bioassay. Black 0.2 mg/ml, grey 0.02 mg/ml.

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