Screening of Plants Used in Danish Folk Medicine to Treat Memory Dysfunction for Acetylcholinesterase Inhibitory Activity

Journal of Ethnopharmacology 104 (2006) 418–422

Ethnopharmacological communication Screening of plants used in Danish folk medicine to treat memory dysfunction for acetylcholinesterase inhibitory activity Anne Adsersen ∗, Bente Gauguin, Lene Gudiksen, Anna K. Jager¨ Department of Medicinal Chemistry, The Danish University of Pharmaceutical sciences, 2 Universitetsparken, 2100 Copenhagen O, Denmark Received 24 June 2005; received in revised form 14 September 2005; accepted 22 September 2005 Available online 8 November 2005

Abstract Aqueous and methanolic extracts of 11 plants, used in Danish folk medicine for improvement of memory and cognition, and 3 Corydalis species were tested for acetylcholinesterase inhibitory activity using the Ellman colorimetric method. Significant inhibitory activity in dose-dependent manner was observed for extracts of Corydalis cava, Corydalis intermedia, Corydalis solida ssp. laxa and Corydalis solida ssp. slivenensis. Extracts of Ruta graveolens, Lavandula angustifolia, Rosmarinus officinalis, Petroselinum crispum and Mentha spicata exhibited moderate inhibition of the enzyme, defined as more than 15% at 0.1 mg/ml. © 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: AChE; Acetylcholinesterase inhibition; Danish folk medicine; Memory dysfunction

1. Introduction 2. Material and methods

Alzheimer’s disease (AD) is the most common form of 2.1. Plant material dementia among the elderly. In AD patients there is decreased levels of acetylcholine in the brain areas related to memory and Plant materials were collected in 2003 and 2004 at vari- learning (Lahiri et al., 2002). Based on the cholinergic hypoth- ous locations in Denmark, or bought from an herbal dealer. esis that memory impairments in patients suffering from AD Voucher specimens and samples are deposited in Depart- result from a defect in the cholinergic system, an important ment of Medicinal Chemistry, The Danish University of Phar- maceutical Sciences. The collected material was dried at approach to treat this disease is to enhance the acetylcholine level ◦ in the brain by inhibition of the enzyme acetylcholinesterase 40 C. (AChE) (Shetty and Woodhouse, 1999). 2.2. Preparation of plant extracts Plant species, wild growing and cultivated, were selected based on the comprehensive work of the Danish ethnobotanist One gram of dried, powdered plant material was extracted V.J. Brøndegaard (Brøndegaard, 1978). This standard work with 2× 10 ml demineralized water or methanol 2× 30 min describes the usage of plants in Denmark for different purposes, in an ultrasonic bath. The extracts were ﬁltered and evapo- including medicinal uses, from the Middle Age until now. Plant rated to dryness. The residues were redissolved in methanol or species used as memory enhancers were identiﬁed and further- demineralized water, respectively, to yield a concentration of more Corydalis species were selected as several species of this 10 mg/ml. genera have been used in treatment of memory dysfunction in folk medicines (Orhan et al., 2004). 2.3. Acetylcholinesterase inhibition assay

AChE inhibitory activity was detected by a microtitre plate Abbreviations: AD, Alzheimer’s disease; AChE, acetylcholinesterase; assay based on Ellman’s method (Rhee et al., 2001) and a thin ATCI, acetylthiocholine iodide; DTNB, 5,5-dithio-bis(2-nitrobenzoic acid); DRG, Dragendorff reagent layer chromatography (TLC) bioautographic assay (Rhee et al., ∗ Corresponding author. Tel.: +45 3530 6295; fax: +45 3530 6041. 2001; Risa et al., 2004) was used to evaluate the plant extracts E-mail address: [email protected] (A. Adsersen). most active in the microtitre plate assay.

2.3.1. Microtitre plate assay assay and reacted as brown zones with DRG indicating alka- Acetylcholinesterase (AChE) from electric eel (type VI- loids (Fig. 1). The occurrence of alkaloids is well documented S), acetylthiocholine iodide (ATCI) and 5,5-dithio-bis(2- in the genus Corydalis and the alkaloids in tuber and herb for nitrobenzoic acid) (DTNB) was purchased from Sigma. In the the same species can be different (Hegnauer, 1969), which could 96-well plates, 25 ␮l 15 mM ATCI in Millipore water, 125 ␮l explain the observed difference in activity between herbs and 3 mM DTNB in buffer C (50 mM Tris–HCl, pH 8, 0.1 M NaCl, tubers. It is also possible that there are different levels of the 0.02 M MgCl2·6H2O), 50 ␮l buffer B (50 mM Tris–HCl, pH 8, same alkaloids in the two plant parts. The chloroform:methanol 0.1% bovine serum albumin), 25 ␮l plant extract at concentra- (1:1) extract of Corydalis solida ssp. solida has previously been tions of 1, 0.5 and 0.25 mg/ml (final concentration in assay: 0.1, shown to exhibit strong AChE inhibitory activity at 1 mg/ml but 0.05 and 0.025 mg/ml) were added and the absorbance was mea- no noticeable activity at 10 ␮g/ml (Orhan et al., 2004). Kim et al. sured five times at 405 nm every 13 s in a Labsystems Multiscan (1999) found that a methanolic extract of the tuber of Corydalis EX type 355 plate reader. Then 25 ␮l 0.22 U/ml AChE were ternata showed significant inhibition of AChE, further they iso- added to the wells and the absorbance was measured again eight lated protopine, determined the IC50 value to 50 ␮M and showed times at 405 nm every 13 s. The reaction rate was calculated by that mice treated with protopine exhibited diminished scopo- Multiscan EX software version 1.0 and Microsoft Excel. Any lamine induced dementia measured in a passive avoidance task. increase in absorbance due to the spontaneous hydrolysis of sub- Protopine has as well been isolated from the tubers of Corydalis strate was corrected by subtracting the rate of the reaction before cava (Preininger et al., 1976) and the aerial parts of Corydalis adding the enzyme. The percentage inhibition was calculated by solida ssp. tauricola (S¸ener and Temizer, 1990). Hwang et al. comparing the rates for the samples to the blank (10% methanol (1996) isolated berberine from Corydalis ternata and found it in buffer A for methanol extracts). The experiment was done in was a reversible and specific AChE inhibitor with 90% inhibitory triplicate. effect at 2.5 ␮M. Ulrichova´ et al. (1983) classified coptisine, berberine and sanguinarine as strong AChE inhibitors. Coptisine 2.3.2. TLC bioautographic assay has been isolated from Corydalis cava (Preininger et al., 1976) Extracts were applied to TLC plates and after developing, and berberine and sanguinarine from Corydalis solida ssp. tau- the TLC plate was sprayed with 5 mM ATCI and 5 mM DTNB ricola (S¸ener and Temizer, 1990). The mentioned investigations in 50 mM Tris–HCl, pH 8 until the silica was saturated with indicate that protoberberine- and protopine-type alkaloids, com- the solvent. The plate was then sprayed with 3 U/ml AChE dis- mon compounds in Corydalis spp., are potent AChE inhibitors. solved in 50 mM Tris–HCl, pH 8 at 37 ◦C. After a few minutes a The only extracts with moderate activity, defined as more than yellow background appeared, with white spots for AChE inhibit- 15% inhibition at the highest concentration tested were, apart ing compounds. False-positive reactions were eliminated by the from the extracts of Corydalis spp., aqueous and methanolic method of Rhee et al. (2003). A TLC plate was developed and extracts of Ruta graveolens and methanolic extracts of Lavan- sprayed with 5 mM DTNB in 50 mM Tris–HCl, pH 8. After dry- dula angustifolia, Rosmarinus officinalis, Petroselinum crispum ing, the plate was sprayed with 5 mM ATCI and 3 U/ml AChE and Mentha spicata. All five species contains essential oil dissolved in 50 mM Tris–HCl, pH 8 at 37 ◦C. After a few min- with terpenes, a group of compounds reported to have AChE utes a yellow background appeared; occurrence of white spots inhibitory Activity (Perry et al., 2000). Essential oil can be indicated false positive reactions. extracted with methanol, not with water, and this can explain the higher activity of the methanolic extracts. 2.3.3. TLC system—Corydalis spp. Currently no AChE inhibitory activity has been reported from Methanolic extracts, 150 ␮g, were applied to Merck Silica gel Ruta graveolens. Ruta graveolens contains in addition to essen- F254 plates (0.2 mm). Toluene:ethylacetate:methanol (30:8:1) tial oil with terpenes, coumarins and alkaloids (Stashenko et al., was used as eluent and Dragendorff reagent (DRG) as spray 2000), groups of compounds reported to have AChE inhibitory reagent. activity (Howes et al., 2003; Lee et al., 2004). In the present study some AChE inhibitory activity was 3. Results and discussion detected in extracts of Lavandula angustifolia, in contrast Perry et al. (1996) found no AChE inhibitory activity of lavender oil Fifteen plant species were selected for investigation and a in a concentration of 0.1 ␮g/ml. An aqueous extract of Lavan- total of 40 extracts were tested for AChE inhibitory activity. The dula angustifolia flowers was shown to diminish glutamate- results obtained with three concentrations of all plant extracts induced neurotoxicity in rat pups cerebellar granular cell culture in the microplate assay are shown in Table 1. All the Cory- (Buy¨ ukokuro¨ glu˘ et al., 2003). Antioxidant and relatively weak dalis extracts tested showed strong inhibitory effect on AChE AChE inhibition was reported for linalool, one of the main com- in a dose-dependent manner in the microtitre assay, methano- ponents in lavender oil (Perry et al., 2000, 2003; Savelev et al., lic extracts were the most active and tubers were more active 2003). This indicates that several targets relevant to treatment than herbs. The TLC bioautographic assay demonstrated that of AD, cholinergic, neuroprotective and antioxidant activities, the activity was due to several compounds, only few false posi- could be found in Lavandula angustifolia. tive compounds were detected (Fig. 1 ). The extracts contained In the present study Melissa officinalis exhibited no AChE several compounds with a blue or yellow-green fluorescence at inhibitory activity. Kennedy et al. (2003) tested ethanolic 365 nm, which appeared as white spots in the bioautographic extracts of eight samples of dried Melissa officinalis leaf 420 A. Adsersen et al. / Journal of Ethnopharmacology 104 (2006) 418–422

Table 1 Screening of Danish plants for acetylcholinesterase inhibitory activity Species Family Plant part analyzed Extraction solvent AChE inhibition (%)

0.1 mg/ml 0.05 mg/ml 0.025 mg/ml

Carum carvi L. Apiaceae Radix Water 0 0 0 Methanol 11 1 0 Herba Water 0 0 0 Methanol 0 0 0 Corydalis cava (L.) Schw. et K. Papaveraceae Herba Water 62 37 37 Methanol 85 70 34 Tuber Water 92 78 73 Methanol 92 83 77 Corydalis intermedia (L.) Merat´ Papaveraceae Herba Water 57 39 28 Methanol 84 75 54 Tuber Water 78 52 49 Methanol 97 86 83 Corydalis solida (L.) Swartz ssp. laxa Papaveraceae Herba Water 78 63 61 Methanol 89 71 66 Tuber Water 85 82 58 Methanol 96 71 63 Corydalis solida (L.) Swartz ssp. slivenensis Papaveraceae Herba Water 48 26 0 Methanol 82 61 53 Tuber Water 87 81 68 Methanol 97 82 75 Euphrasia nemorosa (Pers.) Wallr. Scrophulariaceae Herba Water 0 0 0 Methanol 0 6 0 Lavandula angustifolia Miller Lamiaceae Herba Water 12 0 0 Methanol 34 3 2 Melissa ofﬁcinalis L. Lamiaceae Herba Water 0 0 0 Methanol 0 0 0 Mentha spicata L. Lamiaceae Herba Water 3 10 4 Methanol 15 0 0 Origanum vulgare L. Lamiaceae Herba Water 0 3 0 Methanol 3 0 0 Petroselinum crispum (Mil.) Nym.ex A.W.Hill. Apiaceae Radix Water 0 0 0 Methanol 21 14 0 Pimpinella anisum L. Apiaceae Fructus Water 0 0 0 Methanol 3 0 0 Rosmarinus ofﬁcinalis L. Lamiaceae Herba Water 12 0 0 Methanol 17 10 0 Ruta graveolens L. Rutaceae Herba Water 22 9 2 Methanol 39 30 7 Thymus vulgaris L. Lamiaceae Herba Water 0 2 0 Methanol 0 0 0

and found no AChE inhibitory activity in a concentration of bearing acetylcholine receptors with IC50 values less than 1 mg 0.25 mg/ml. Perry et al. (1996) found inhibitory activity of two dry plant material/ml, indicating that compounds with acetyl- different sources of Melissa oil, 0.1 ␮g/ml caused 76.3 and 100% choline receptor affinities are present in the extract (Wake et inhibition, respectively, activity of an ethanolic extract of fresh al., 2000). The results indicates that compounds with potential leaves but no activity in an extract of dried leaves in a con- for use in receptor orientated therapies and enzyme orientated centration of 2.0 mg/ml. Salah and Jager¨ (2005) found weak therapies for AD and other neurodegenerative diseases associ- activity in an ethyl acetate extract, and citral, a main compo- ated with aging, could be found in Melissa officinalis, but the nent in the essential oil, is known to be a weak AChE inhibitor activity depends on the source. (Ryan and Byrne, 1988). It was reported that the ethanolic extract Rosemary oil has previously been shown to exhibit moderate of Melissa officinalis displaced [3H]-(N)-nicotine and [3H]-(N)- activity (Perry et al., 1996) but currently no AChE inhibitory scopolamine dose-dependent from human brain cell membranes activity has been reported from Petroselinum crispum, and A. Adsersen et al. / Journal of Ethnopharmacology 104 (2006) 418–422 421

Fig. 1. TLC plates of extracts from Corydalis sp. (A) Acetylcholinesterase inhibitory activity, white spots indicate inhibition. (B) False positive reactions, where the white spots are not due to enzyme inhibition. (C) TLC plate in UV-365 nm. (D) TLC plate sprayed with Dragendorff-R. 1–4 are herbs of Corydalis cava, Corydalis intermedia, Corydalis solida ssp. slivenensis and Corydalis solida ssp. laxa, respectively, 5–8 are tubers of the previously mentioned plant species.

Mentha spicata. All three species contains essential oil and Howes, M.R., Perry, N.S.L., Houghton, P.J., 2003. Plants with traditional AChE inhibitory activity has been detected for several of the uses and activities, relevant to the management of Alzheimer’s disease monoterpenoids occurring in the oils (Miyazawa et al., 1997), and other cognitive disorders. Phytotherapy Research 17, 1–18. Hwang, S.Y., Chang, Y.P., Byun, S.J., Jeon, M.H., Kim, Y.C., 1996. An this could explain the activity proven in the present study. Essen- acetylcholinesterase inhibitor isolated from Corydalis tuber and its mode tial oil, aqueous and ethanolic extracts from Pimpinella anisum of action. Korean Journal of Pharmacognosy 27, 91–95. were shown to have bronchodilatory effects on isolated guinea Kennedy, D.O., Wake, G., Savelev, S., Tildesley, N.T.J., Perry, E.K., Wesnes, pig tracheal chains (Boskabady and Ramazani-Assari, 2001). K.A., Scholey, A.B., 2003. Modulation of mood and cognitive perfor- The effect was, at least in part, due to inhibitory effects on mance following acute administration of single doses of Melissa officinalis (Lemon balm) with human CNS nicotinic and muscarinic receptor-binding muscarinic receptors that could counteract any possible AChE properties. Neuropsychopharmacology 28, 1871–1881. inhibitory effects. This, and our negative in vitro results, indi- Kim, S.R., Hwang, S.Y., Jang, Y.P., Park, M.J., Markelonis, G.J., Oh, T.H., cates that any effect of Pimpinella anisum on improving memory Kim, Y.C., 1999. Protopine from Corydalis ternata has anticholinesterase could be due to some other course of action. and antiamnesic activities. Planta Medica 65, 218–221. Lahiri, D.K., Farlow, M.R., Greig, N.H., Sambamurti, K., 2002. Current drug targets for Alzheimer’s disease treatment. Drug Development Research 56, Acknowledgements 267–281. Lee, J.H., Lee, K.T., Yang, J.H., Baek, N.I., Kim, D.K., 2004. Acetyl- The Danish Medical Research Council is thanked for finan- cholinesterase inhibitors from twigs of Vaccinium oldhami Miquel. cial support. The Copenhagen University Botanical Gardens is Archives of Pharmacal Research 27, 53–56. thanked for the donation of certain plant materials. Miyazawa, M., Watanabe, H., Kameoka, H., 1997. Inhibition of acetylcholinesterase activity by monoterpenoids with a p-menthane skeleton. Journal of Agricultural and Food Chemistry 45, 677–679. References Orhan, I., S¸ener, B., Choudhary, M.I., Khalid, A., 2004. Acetylcholinesterase and butyrylcholinesterase inhibitory activity of some Turkish medicinal Boskabady, M.H., Ramazani-Assari, M., 2001. Relaxant effect of Pimpinella plants. Journal of Ethnopharmacology 91, 57–60. anisum on isolated guinea pig tracheal chains and its possible mecha- Perry, N., Court, G., Bidet, N., Court, J., Perry, E., 1996. European herbs with nism(s). Journal of Ethnopharmacology 74, 83–88. cholinergic activities: potential in dementia therapy. International Journal Brøndegaard, V.J., 1978. Folk og Flora, Dansk Etnobotanik, vols. I–IV. of Geriatric Psychiatry 11, 1063–1069. Rosenkilde og Bagger, Copenhagen, Denmark. Perry, N.S.L., Houghton, P.J., Theobald, A., Jenner, P., Perry, E.K., 2000. Buy¨ ukokuro¨ glu,˘ M.E., Gepdiremen, A., Hacimuft¨ uo¨ glu,˘ A., Oktay, M., 2003. In-vitro inhibition of human erythrocyte acetylcholinesterase by Salvia The effects of aqueous extract of Lavandula angustifolia flowers in lavandulaefolia essential oil and constituent monoterpenes. Journal of glutamate-induced neurotoxicity of cerebellar granular cell culture of rat Pharmacy and Pharmacology 52, 895–902. pups. Journal of Ethnopharmacology 84, 91–94. Perry, N.S.L., Bollen, C., Perry, E.K., Ballard, C., 2003. Salvia for dementia Hegnauer, R., 1969. Chemotaxonomie der Pflanzen V. Birkhauser¨ Verlag, therapy: review of pharmacological activity and pilot tolerability clinical Basel und Stuttgart, 288. trial. Pharmacology, Biochemistry and Behavior 75, 651–659. 422 A. Adsersen et al. / Journal of Ethnopharmacology 104 (2006) 418–422

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