Journal of Ethnopharmacology 145 (2013) 822–825

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Journal of Ethnopharmacology

0378-87

http://d

Abbre

serotonn Corr

E-m

journal homepage: www.elsevier.com/locate/jep

Ethnopharmacological communication

Screening of plants used in Danish folk medicine to treat depression andanxiety for affinity to the serotonin transporter and inhibition of MAO-A

Anna K. Jager n, Bente Gauguin, Jacob Andersen, Anne Adsersen, Lene Gudiksen

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, 2 Universitetsparken, 2100 Copenhagen O, Denmark

a r t i c l e i n f o

Article history:

Received 1 November 2012

Received in revised form

14 December 2012

Accepted 16 December 2012Available online 21 December 2012

Keywords:

Anxiety

Danish folk medicine

Depression

MAO

Serotonin transporter

41/$ - see front matter & 2012 Elsevier Irelan

x.doi.org/10.1016/j.jep.2012.12.021

viations: DMSO, dimethylsulfoxide; MAO, mo

in transporter; SSRI, selective serotonin reup

esponding author. Tel.: þ45 3533 6339; fax:

ail address: anna.jager@sund.ku.dk (A.K. Jage

a b s t r a c t

Ethnopharmacological relevance: A number of plant species are used in Danish folk medicine for

treatment of depression and anxiety.

Materials and methods: Aqueous and ethanolic extracts of 17 plant species were tested for affinity to

the serotonin transporter and for inhibition of MAO-A—both targets for antidepressive treatment.

Results: An ethanolic extract of aerial parts of Borago officinalis had affinity to the serotonin transporter.

Ten extracts, from eight plants, had IC50 values below 25 mg/ml extract in the MAO-A assay. The most

active extracts in the MAO-A assay were the ethanol extract of seeds of Trigonella foenum-graecum (IC50

4 mg/ml); ethanol extract of leaves of Apium graveolens (IC50 5 mg/ml) and the water extract of aerial

parts of Calluna vulgaris (IC50 8 mg/ml).

Conclusions: Besides Borago officinalis, which toxicity profile excludes it from further development as an

herbal drug, none of the plants had potential as serotonin reuptake inhibitors. Several plants had MAO-A

inhibitory activity.

& 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Depression and anxiety afflicts 350 million people worldwide,thus constituting a major health problem (WHO, 2012). Theserotonin transporter (SERT) has in recent years been a targetfor clinically used anti-depressants of the SSRI-type. If theserotonin binding site on the SERT is blocked, reuptake ofserotonin released into the synaptic space back into the cell willbe prevented, thus increasing the serotonin level in the synapticcleft (Rang et al., 2007). The increased level of serotonin leads toimprovements in the depressive state of the patient. Anothertarget for anti-depressive treatment is monoamine oxidase-A(MAO-A) inhibitors. Inhibition of MAO-A prevents the metabolismof monoamine neurotransmitters, leading to increased levels ofthese neurotransmitters in the brain, again improving the state ofa depressed patient (Rang et al., 2007).

The herbal drug Hypericum perforatum L. has in recent yearsbecome a much-used treatment for mild-to-moderate depression.In this study we investigated plants used in Danish folk medicinefor depression in the hope of finding other plants that maypossibly be used in the treatment of this disease that inflict largeparts of the population. We tested for activity at two presentlyknown targets for anti-depressive treatment: SERT and MAO-A.

d Ltd. All rights reserved.

noamineoxidase; SERT,

take inhibitor

þ45 3533 6041.

r).

2. Materials and methods

2.1. Selection of plant species for testing

Plant species, wild growing and cultivated, were selectedbased on the comprehensive work of the Danish ethnobotanist(Brøndegaard, 1978). This standard work describes the usage ofplants in Denmark for different purposes, including medical uses,from the middle age until now. Plant species used for treatment ofdepression and anxiety were identified (Table 1).

2.2. Plant material

Plant materials were collected at various locations in Denmark,or purchased from a commercial herbal drug dealer (Table 1).Voucher specimens and samples are deposited at the Departmentof Drug Design and Pharmacology, Faculty of Health and MedicalSciences, University of Copenhagen, voucher numbers are given inTable 1. The collected material was oven-dried at 40 1C.

2.3. Preparation of plant extracts

One gram of dried, ground plant material was extracted twice ineither 10 ml demineralized water or 96% ethanol for 30 min in anultrasonic bath. The extracts were filtered and evaporated todryness. The residues were redissolved in water or ethanol, respec-tively, to yield a concentration of 10 mg/ml for the citaloprambinding assay, or in DMSO at 10 mg/ml for the MAO-A assay.

Table 1Danish plants screened for affinity to the serotonin transporter and for inhibition of MAO-A.

Family Species Traditional usage Voucher

no.

Plant part

analysed

Extraction

solvent

Inhibition of MAO-A (%) MAO-A

IC50

(lg/ml)

3H-citalopram binding

to SERT (%)

0.001

mg/

ml

0.01

mg/

ml

0.1

mg/

ml

1

mg/

ml

10

mg/

ml

0.01

mg/ml

0.1

mg/ml

1

mg/ml

Apiaceae Pimpinella anisum L. Distilled water or candy of the flowers against

epilepsy. Tea of flowers for epilepsy by children.

Decoction used as a sedative

LG42 Fruits Ethanol 2 �2 4 59 84 124 9375 95713

Water 15 �15 8 68 90 113714 44742 10674

Apium graveolens L. In a drink for stroke and epilepsy. In wine for

hot temper

LG31 Tuber Ethanol �9 �11 5 35 115 8574 8873 8376

Water 9 9 16 16 25 93714 10674 9576

Leaves Ethanol �20 4 56 62 88 5 8675 7978 76711

Water 9 3 22 59 66 84 94 10873

Asteraceae Arnica montana L. The flowers are used in remedies for nervous

fever, paralysis and other nervous sufferings

LG52 Flowers Ethanol �5 0 23 77 97 8677 77710 6873

Water �11 �15 �11 56 78 4674 5377 55732

Boraginaceae Borago officinalis L. Sedative LG14 Aerial parts Ethanol �13 �8 77 80 OR 101725 7677 2777

Water �11 �30 15 74 96 9974 10271 74719

Cannabaceae Humulus lupulus L. For sleeplessness. For restlessness in the body LG23 Strobile Ethanol 2 �4 52 88 OR 85711 9273 9076

Water

Ericaceae Calluna vulgaris (L.) Hull Sedative LG20 Aerial parts Ethanol �3 2 64 92 112 14 8275 87711 8876

Water �14 3 66 101 90 8 10679 10076 8673

Euphorbiaceae Euphorbia peplus L. Part of a remedy against insanity. Roots in wine

and honey water drives out the ‘‘evil liquids’’

LG17 Aerial parts Ethanol �15 �15 �10 10 75 8778 83710 7175

Water �24 �24 55 55 69 106711 104714 8974

Fabaceae Trigonella foenum –

graecum L.

The seeds in beer against anxiety and hopelessness LG43 Seeds Ethanol 0 28 59 83 79 4 9078 8579 90713

Water �7 �28 �17 3 55

Lamiaceae Melissao fficinalis L. Against sleeplessness caused by heart break. Against

melancholy and sadness

LG24 Aerial parts Ethanol �13 �9 48 75 95 15 9175 9373 8178

Water 1 23 86 85 OR 12a

Origanum vulgare L. Juice in the nose will wake up patients with

sleeping sickness or epilepsy. Juice with castoreum

and pepper on the tongue for epilepsy

LG25 Aerial parts Ethanol �14 �14 0 71 86 11 86711 8975 8078

Water 3 9 76 88 121 11476 11673 9472

Thymus vulgaris L. Strengthens the brain to smell the plant. Placed on

the head for dizziness. In beer for sadness

LG29 Leaves Ethanol �107 �90 0 72 83 7774 72710 4473

Water 7 14 75 82 111 10 10278 100711 107721

Nymphaeaceae Nuphar lutea (L.) Sm. Eating and smelling the flowers cause agitation LG54 Leaves Ethanol �14 0 57 100 OR 7974 8374 6476

Water 3 9 74 100 100 116716 109711 108720

Paeoniaceae Paeonia sp. L. Seeds against nightmare and troubled sleep.

Roots and seeds as necklaces or in drinks for

epilepsy, fear of darkness and bad dreams in

children. Sugar extract from petals against nightmare

LG35 Seeds Ethanol �3 46 45 88 84 86712 8279 10074

Water 0 5 10 33 85 140726 112718 10976

Primulaceae Primula elatior (L.) Hill Distilled water of the flowers against headache.

Can counteract convulsions. Tea from the green

or dried plant against convulsions, paralysis,

sleeplessness and faintness. The sugar preserved

plant: strengthen and against stroke

LG48 Flowers Water �17 �14 �3 59 90 7474 8677 8176

Leaves Ethanol 2 �4 25 65 71 10877 74741 8478

Water �17 �3 41 45 90 8172 8277 51713

Roots Ethanol 6 0 6 82 92 94711 94712 7873

Water �12 3 �3 71 94 9977 105710 116712

Primula veris L. As P. elatior LG49 Flowers Ethanol 8 �21 9 17 44 10171 93711 3979

Water �7 0 21 72 100 101711 8179 6872

Leaves Ethanol �17 �14 8 44 60 8974 10277 86711

Water 6 0 �21 50 79 7171 8577 8879

Roots and

rhizomes

Ethanol 0 �12 10 83 107 102714 106723 7773

Water �14 �17 66 59 90 11 10175 8872 96711

Salicaceae Salix cinerea L. Juice in the nose for headache. Decoction of leaves:

bathing the head for fury and aberration

LG10 Leaves Ethanol �2 �7 57 91 107 12 6776 7475 6877

Water 7 7 39 86 111 8975 9073 9778

Tiliaceae Tilia europaea L. Distilled water or candy of the flowers against epilepsy.

Tea of flowers for epilepsy by children. Decoction used as a

sedative

LG13 Leaves Ethanol 4 6 55 86 106 119736 10678 77714

Water �14 0 48 72 55 11775 122712 9273

Voucher specimens numbers are L. Gudiksen collection numbers. OR: over range in the spectrophotometric measurement. IC50-values are calculated in total assay volume.a IC50-value calculated from dataset that also included inhibition data for 0.05 mg/ml. The IC50-value for the selective MAO-A inhibitor clorgyline was 13 nM. The IC50-value for the SERT inhibitor citalopram was 1.3 nM.

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eta

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Jou

rna

lo

fE

thn

op

ha

rma

colo

gy

14

5(2

01

3)

82

2–

82

58

23

0

25

50

75

100

[3 H]5

HT

upta

ke (%

)

10 100 1000 10000[B. officinalis] (µg/mL)

Fig. 1. Borago officinalis characterized in a functional [3H]5HT uptake inhibition

assay. IC50¼204722 mg/ml (mean7SEM; n¼6).

A.K. Jager et al. / Journal of Ethnopharmacology 145 (2013) 822–825824

2.4. Membrane preparation for [3H]-citalopram binding assay

All procedures were carried out at 0–4 1C. Whole rat brains,except cerebellum, were homogenised with an Ultra Turraxhomogenizer in 1:10 w/v buffer (5 mM TRIS base, 150 mM NaCland 20 mM EDTA, pH 7.5). The homogenate was centrifuged at16.000� g for 10 min and the tissue membranes washed with120 ml of the same buffer. The supernatant was discarded and thepellet was suspended in buffer (5 mM TRIS base and 5 mM EDTA,pH 7.5), left to react for 20 min and then centrifuged at 16.000� g

for 10 min. The supernatant was discarded and the pellet waswashed with 120 ml buffer (50 mM TRIS base, 120 mM NaCl and5 mM KCl, pH 7.5) and then centrifuged at 16.000� g for 10 min.The supernatant was discarded and the pellet finally suspended in1:10 (w/v) buffer (50 mM TRIS base, 120 mM NaCl and 5 mM KCl,pH 7.5). This homogenate was kept at �70 1C until use.

2.5. [3H]-citalopram binding assay

The method described by Plenge et al. (1990) was used withmodifications (Nielsen et al., 2004). Two hundred ml of testsolution were mixed with 50 ml of [3H]-citalopram (4 nM) and50 ml of tissue suspension, respectively. Paroxetin (1.5 mM) wasused for the determination of unspecific binding. The totalbinding of [3H]-citalopram was determined with a buffer blank.All samples were incubated for 2 h at 25 1C and then filteredunder vacuum using glass fibre filters. After 24 h the radioactivityon the filters was determined by liquid scintillation. Specificbinding was calculated as total binding minus unspecific binding.All experiments were done in triplicate. Citalopram was used as apositive control.

2.6. Functional SERT inhibition assay

The method is described in detail elsewhere (Kristensen et al.,2004) and used with minor modifications. In short: COS-7 cellswere cultured in Dulbecco’s modified Eagle’s medium (DMEM)with 10% fetal bovine serum, 100 U/ml pencillin, 100 mg/mlstreptomycin at 37 1C in a humidified 5% CO2 environment. Cellswere transfected with hSERT constructs with TransIT transfectionreagent (Mirus Inc., Madison, WI), following the protocol suppliedby the manufacturer. Subsequently, cells were dispensed intopoly-D-lysine coated white 96-well plates at 50% confluence.Uptake inhibition assay was performed 48 h after transfectionwhen cells were confluent. The media were removed and the cellswere washed twice with phosphate-buffered saline (in mM: NaCl,137; KCl, 2.7; Na2HPO4, 4.3; and KH2PO4, 1.4, pH 7.3) containing0.5 mM CaCl2 and 0.5 mM MgCl2 (PBSCM). After washing, cellswere incubated for 30 min in PBSCM containing 50 nM [3H]-5-HTand increasing concentrations of the Borago officinalis extract.Uptake was terminated by washing two times with PBSCM.All washing steps were carried out with an automatic platewasher (ELx50 Microplate Strip Washer from Biotek). The amountof accumulated [3H]-5-HT was determined by solubilising cells inscintillant (MicroScint-20) followed by direct counting of plates ina Packard TopCounter. Resulting counts were converted to %inhibition of control wells that lacked plant extract.

2.7. MAO-A assay

The assay was based on Holt et al. (1997) and Stafford et al.(2007), with modifications. The assay was carried out in 96-wellmicrotitre plates. For the assay, to each well was added 50 mlplant extract in DMSO, or DMSO (as blank); 50 ml chromogenicsolution (0.8 mM vanillic acid and 417 mM 4-aminoantipyrine in a0.2 M phosphate buffer pH 7.6) containing 8 U/ml horse radish

peroxidase (Sigma); 100 ml 3 mM p-tyramine in 0.2 M phosphatebuffer pH 7.6 and 50 ml 8 U/ml MAO-A (Sigma). To test forinhibition of peroxidase or oxidation of reagents of the chromo-genic solution, a parallel set of controls were set up; to each wellwas added: 50 ml plant extract in DMSO; 50 ml chromogenicsolution containing 8 U/ml horse radish peroxidase; 100 ml3 mM p-tyramine and a 50 ml 0.2 M phosphate buffer pH 7.6.The plates were incubated at 36 1C. At 5 min and 30 min, the platewas read at 492 nm in a microtitre plate reader.

The absorption read at 5 and 30 min (there was linearity inthis interval, results not shown), corrected for possible changes inabsorption due to inhibition of peroxidase or oxidation ofreagents, was plotted against time and the slope of the line wascalculated by linear regression. The percent MAO inhibition wascalculated by the formula: (1�slope test/slope blank)�100.Experiments were carried out in triplicate. IC50 values wereestimated by Grafit 5 software. Clorgyline was used as a positivecontrol.

3. Results and discussion

Aqueous and ethanolic extracts were tested for affinity to theSERT in the [3H]-citalopram binding assay binding assay (Table 1).The only active extract was an ethanolic extract of aerial parts ofBorago officinalis. The extract of Borago officinalis was then testedin a functional assay for SERT inhibition, where the extract alsoshowed activity (Fig. 1).

There are no previous reports of Borago officinalis extractshaving affinity for CNS receptors. In this study, we found aconcentration-dependent activity of an ethanol extract of theaerial parts (Table 1). Borago officinalis is known to containhepatotoxic pyrrolizidine alkaloids, besides the unsaturated fattyacids the plant is used for presently (Bruneton, 1999). Furtherinvestigations might reveal whether the alkaloids in the plant areresponsible for the activity on the SERT.

The plant extracts were also tested for inhibition of MAO-A(Table 1). Ten extracts, from eight plants, had IC50 values below0.025 mg/ml extract (in total assay volume) in the MAO-A assay(Table 1). The most active plant extracts in the MAO-A assay werethe ethanol extract of Trigonella foenum-graecum seeds (IC50 4 mg/ml);leaf ethanol extract of Apium graveolens (IC50 5 mg/ml) and thewater extract of aerial parts of Calluna vulgaris (IC50 8 mg/ml). FromCalluna vulgaris, we isolated quercetin as the MAO-A inhibitor bybioassay-guided fractionation (Saaby et al., 2009).

A.K. Jager et al. / Journal of Ethnopharmacology 145 (2013) 822–825 825

It is not known which compounds in the active plants areresponsible for the activity. Previously, alkaloids (Kong et al.,2004; Lee et al., 2005; Herraiz and Chaparro, 2006), phenols (Konget al., 2004; Tao et al., 2005), flavonoids (Sloley et al., 2000;Hwang et al., 2005; Chimenti et al., 2006), chalcones (Pan et al., 2000),naphtoquinones (Choi et al., 2005), and coumarins (Jo et al., 2002)have been shown to possess MAO-A activity.

Paeonol, a phenolic compound from Paeonia moutan andPrimula auricula, has been shown to be a MAO-A inhibitor (Konget al., 2004). Primula elatior, Primula veris and the Paeonia seedstested in this study did exhibit inhibitory activity. In a screeningof Chinese plants, Paeonia lactiflora and Paeonia oboyata did notshow MAO inhibition (Lin et al., 2003).

4. Conclusion

Besides Borago officinalis, which toxicity profile excludes itfrom further development as an herbal drug, none of the plantshad potential as serotonin reuptake inhibitors. Several plants hadMAO-A inhibitory activity. Further investigation of these plantsshould be undertaken.

Acknowledgement

The Danish Medical Research Council is thanked for financialsupport. The Copenhagen University Botanical Gardens is thankedfor the donation of certain plant materials.

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