New Insights into the Acetylcholinesterase Inhibitory Activity of Lycopodium clavatum

• Abstract
• Introduction
• Materials and Methods
• Results and Discussion
• References

Abstract

Looking for acetylcholinesterase (AChE) inhibiting compounds within the plant kingdom, we came across the triterpene α-onocerin, which has recently been described as the active principle (IC₅₀ of 5.2 μM) of Lycopodium clavatum L. In order to discover related terpenoid structures with similar AChE inhibitory activity, we investigated the roots of Ononis spinosa L. using Ellman’s reagent in a microplate assay. No inhibitory effect could be measured, not even with the isolated α-onocerin (1), which is in contrast to previous findings. Bioassay-guided fractionation of L. clavatum resulted in the isolation of lyclavatol (2), showing a weak, but dose-dependent inhibitory effect on AChE. ¹H- and ¹³C NMR shift assignments for 1 and 2 are presented and discussed.

Introduction

Current discussion regarding the therapy of Alzheimer’s disease (AD) highlights the importance of the cholinergic principle for compensation of the neurotransmitter deficiency in the patient’s central nervous system [1]. The application of acetylcholinesterase (AChE) inhibitors is currently the only approved therapy for enhancement of the central cholinergic function [1], [2]. They also have been reported to affect the aggregation of amyloid β-peptide plaque, which is neuropathological to AD [3]. Some potent inhibitors of AChE are derived from natural sources, e. g., galanthamine, huperzine A, and are already used for the treatment of different forms of dementia.

Most of these secondary plant metabolites belong to the chemical class of alkaloids and are clinically known to have serious side effects that are connected to the peripheral effects of these highly bioactive compounds. From this perspective, there is a high need of improved AChE inhibitors showing low toxicity, good brain penetration and high bioavailability.

In our ongoing research to discover AChE inhibitors from the plant kingdom utilizing a bioassay-guided approach and virtual screening experiments with a pharmacophore model for AChE [4], we intend to focus on non-alkaloid natural products. In the recently published study of Orhan and co-workers [5], the triterpene α-onocerin was isolated by bioassay-guided fractionation from a chloroform:methanol extract of Lycopodium clavatum L. The authors describe α-onocerin as the active principle of the investigated plant material with an IC₅₀ of 5.2 μM. Because α-onocerin is a well known constituent of the roots of Ononis spinosa L. (0.4 %) [6], we focused on this plant in order to (i) evaluate the importance of Ononidis radix (officinal to the European Pharmacopoeia [7]) with respect to its AChE inhibitory potential and to (ii) seek for further bioactive triterpenes related structurally to α-onocerin from this plant material. Up to now, only few triterpenoid structures are known to inhibit AChE, e. g., triterpene alkaloids isolated from the genus Buxus [8], [9] or ursolic acid [10].

Materials and Methods

The roots of O. spinosa and the aerial parts of L. clavatum were purchased from Mag. Kottas, Heilkräuter (Vienna, Austria; control numbers: KLA-30 377 and KLA-30 472, respectively). Their quality was macro- and microscopically checked according to the monographs Ononidis radix and Lycopodii herba of the European Pharmacopoeia. Voucher specimens (JR-20040311A1 and JR-20040415A1, respectively) are deposited in the Herbarium of the Department of Pharmacognosy, Institute of Pharmacy, University of Innsbruck, Austria.

All chemicals were of analytical grade. Solvents were either of analytical grade or of puriss. grade and were distilled before use.

Mass spectra were acquired using an Esquire 3000plus ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with electrospray ionization (ESI) or an atmospheric pressure chemical ionization (APCI) source. In ESI experiments, the spray voltage was 4.5 kV, the sheath gas: N₂, 30 psi, the dry gas: N₂, 6 L min^-1, 350 °C and the samples were injected to the MS by a Cole-Parmer 74 900 series syringe pump (Cole-Parmer Instrument Company, Illinois, USA) at a flow rate of 3 μL min^-1. In APCI experiments the sheath gas pressure was 40 psi, the dry gas 10 L min^-1 at 250 °C, the vaporizer was set to 450 °C and the corona discharge current to 10 μA. The samples were injected (10 μL) into the MS by an Agilent HPLC 1100 Liquid chromatograph (Agilent, Waldbronn, Germany).

Optical rotation was determined on a Perkin Elmer 341 polarimeter using CHCl₃ as solvent.

The NMR spectra were recorded on a Bruker-DRX300 at 300 K and calibrated to the residual solvent signal (CHD₂OD at δ_H = 3.31 ppm and CD₃OD at δ_C = 49.0 ppm). Upon request, NMR spectra can be obtained from the corresponding author.

The acetylcholinesterase inhibitory activity was determined using a modified Ellman’s method [acetylcholinesterase, acetylthiocholine iodide, and 5,5′-dithiobis(2-nitrobenzoic acid), Sigma-Aldrich Chemie GmbH, Steinheim, Germany; positive control: galanthamine hydrobromide (GNT) Tocris; Cookson Ltd, Avonmouth, UK] in a 96-well microplate assay as previously described [4]. The percentage of enzyme inhibition was calculated by comparing the rates of the sample to the blank (containing 1 % DMSO; n = 4) and analyzed with Student’s t-test. The IC₅₀ values were determined with Probit analysis. For statistical processing, the SPSS 11.5 program package was used.

Extraction and isolation: 476 g dried roots of O. spinosa were crushed to a coarse powder and consecutively macerated at 25 °C with petroleum ether (700 mL), CH₂Cl₂ (1000 mL), and CH₃OH (1000 mL), each for two days. Then the respective solvents were removed under vacuum.

247 g dried aerial parts of Lycopodii herba were ground to a fine powder and macerated exhaustively with CH₂Cl₂:CH₃OH (1 : 1;1.4 L; 1 × 3 h, 2 × 19 h; at 25 °C). Removal of the solvent under vacuum yielded 19.0 g of the dried extract. An aqueous suspension of 3.0 g of this crude extract was first acidified with HCl (pH 2.5), and then the CH₂Cl₂-dissolvable components were removed with CH₂Cl₂. The remaining aqueous solution was basified with NaOH to pH 9,0 and extracted with CH₂Cl₂ (4 × 30 mL). Evaporation of the organic solvent resulted in 0.5 g of an alkaloid fraction.

The remaining 16.0 g of the crude extract were divided into fractions soluble in petroleum ether (E_pe), diethyl ether (E_de), ethyl acetate (E_ea), and n-butanol (E_bu). Pooled fractions E_de and E_ea (2.2 g) were fractionated by silica CC (3.2 × 50 cm, Merck silica gel 60, 0.040 - 0.063 mm, 230 - 400 mesh, 250 g) using a step gradient of CH₂Cl₂-CH₃OH-EtOAc [CH₂Cl_{2 :} 100; CH₂Cl₂-CH₃OH: 99 : 1; 100 : 2; 100 : 3; 100 : 4; 100 : 5; 100 : 7; 100 : 7.5; 100 : 8; CH₂Cl₂-CH₃OH-EtOAc: 100 : 10 : 10; 100 : 11 : 8; 90 : 10 : 5; CH₃OH-EtOAc: 10 : 8; 10 : 5; 10 : 3; EtOAc: 100; EtOAc-CH₃OH: 1 : 1; 25 : 75; CH₃OH: 1]. 281 samples were collected and monitored by TLC (toluene-EtOAc-ethanol; 50 : 38 : 12; spray reagent: anisaldehyde-sulphuric acid reagent). Similar samples were collected to 12 fractions: A₁: 1 - 88; A₂: 89 - 93; A₃: 94 - 103; A₄: 104 - 117; A₅: 118 - 122; A₆: 123 - 139; A₇: 140 - 145; A₈: 146 - 155; A₉: 156 - 194; A₁₀: 195 - 213; A₁₁: 214 - 262; A₁₂: 263 - 281. At 1 mg/mL fractions A₂ and A₃ showed inhibitory effects on AChE of 77.49 ± 15.01 % and 71.46 ± 14.19 %, respectively. Due to their similar HPLC patterns they were combined (130 mg) and further separated by Sephadex® LH 20 CC (Pharmacia Biotech, Sweden; 2.5 × 80 cm). This was eluted with CH₂Cl₂-acetone (85 : 15) yielding 9 sub-fractions (B_{1 - 9}). B₃ (8.4 mg, 384 - 408 mL elution volume) and B₉ (10.3 mg, 752 - 840 mL elution volume) were further purified by crystallization in CH₃OH to obtain 5.3 mg of white microcrystalline metabolite 1 (B₃) and 2.9 mg of white microcrystalline 2 (B₉), respectively (Fig. [1]).

Results and Discussion

Ononidis radix was consecutively extracted with petroleum ether, dichloromethane and methanol. These extracts were evaluated in a microplate enzyme test assay using Ellman’s spectrophotometric method [11], [12]. Concentrations of 100 μg extract/mL showed no significant inhibitory activity on AChE. α-Onocerin was confirmed as constituent of the extracts with investigation by TLC [13]. In addition, putative α-onocerin (1) was isolated and purified from the petroleum ether extract of Ononidis radix according to Pauli [14]. The data obtained by mass spectrometry, 1D and 2D NMR (DQF-COSY, HMQC, HMBC) experiments and optical rotation resulted in the unambiguous identification of α-onocerin [14]. The ¹H- and ¹³C-NMR data are summarized in Table [1].

Surprisingly, even at concentrations of 100 μM and 1 mM, compound 1 showed no effect on AChE in the enzyme test. Therefore, we focused on the paper of Orhan and co-workers in order to (i) check the identity of α-onocerin in L. clavatum, and to (ii) comprehend the authors’ instructions. Determination of the AChE inhibitory effect of a dichloromethane:methanol (1 : 1) extract (20 g) of L. clavatum (aerial parts) resulted in an inhibition of 55.06 ± 10.58 % (c = 1 mg/mL) which is analogous to the authors’ results. One part of this extract (3.0 g) was used for isolation of an alkaloid fraction because the related species Huperzia serrata is known to contain the AChE-inhibiting alkaloid huperzine A [15]. In the enzyme test, however, the alkaloid fraction showed no activity (c = 1 mg/mL). The remaining extract (16.0 g) was subjected to bioassay-guided fractionation. The activity was spread among some minor triterpenoids and finally led to the isolation of metabolite 2.

The structure of 2 (Fig. [1] ) was elucidated with combined mass spectrometry and NMR data. ESI-mass spectrometry resulted in a signal at m/z = 923 ([2M + Na]⁺). The molecular mass of 450 corresponds to a molecular formula of C₂₈H₅₀O₄. Due to the small sample amount (2.9 mg) no ¹³C-NMR spectrum could be recorded. The ¹³C-NMR shift values presented in Table [1] were deduced from HMQC and HMBC experiments. They showed only fourteen carbon resonances. This implicates that analyte 2 has a C ₂ symmetry axis analogous to compound 1. The ¹H-NMR spectrum was similar to that of 1; however, the exo-methylene group signals of α-onocerin were missing and an additional signal of a CH(OH) moiety was found. Signal assignments were achieved by performing homo- and heteronuclear 2D NMR experiments (DQF-COSY, HMQC, HMBC). The hydroxylated carbon centres C-3(21) and C-8(14) were discriminated by the analysis of HMBC-derived coupling networks. The proton at C-3 was found to be β oriented by the analysis of its coupling pattern and by comparison to α-onocerin and to literature data [16]. Thus, compound 2 was elucidated as 26,27-dinoronocerane-3α,8β,14α,21β-tetrol (lyclavatol). The assigned ¹H- and ¹³C-NMR data of 1 and 2 are summarized in Table [1]. To the best of our knowledge, this is the first assignment of NMR resonances for lyclavatol - only selected resonances have been reported until now [17].

In the enzyme test, lyclavatol acted as a weak, but dose-dependent inhibitor of AChE with an IC₅₀ value of 673.4 μg/mL (598.3 - 771.4 μg/mL) or 1.50 mM (CI₉₅ = 1.33 - 1.71 mM). In Fig. [2], the dose dependence of lyclavatol and galanthamine is depicted.

Additionally, α-onocerin (1) was isolated from Lycopodii herba and identified by TLC, LC-MS and ¹H-NMR experiments. This isolate was tested again for its AChE inhibitory activity. In contrast to the postulate of Orhan and co-workers, no AChE inhibitory activity could be found in the enzyme test (up to c = 1 mg/mL or 2.26 mM). They also report that α-onocerin may be a candidate compound in the treatment of AD because of a higher AChE efficacy (IC₅₀ = 5.2 μM according to [5]) than that of donepezil. This statement has definitely to be revised since donepezil is known to inhibit AChE with an IC₅₀ in the nM range [18].

In summary, α-onocerin did not exhibit any AChE inhibiting activity in our study, which is in contrast to the data described by Orhan et al. [5]. The tetracyclic triterpene lyclavatol (2) isolated by activity-guided fractionation shows a very weak effect on AChE and does not fully explain the in vitro activity of the Lycopodium crude extract. We suppose that further minor constituents, probably triterpenoids, may attribute to a synergistic AChE inhibiting effect in the plant matrix.

References

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  CrossrefPubMedSearch in Google Scholar
• 12 Ingkaninan K, de Best C M, van der Heijden R, Hofte A JP, Karabatak B, Irth H. et al . High-performance liquid chromatography with on-line coupled UV, mass spectrometric and biochemical detection for identification of acetylcholinesterase inhibitors from natural products.  J Chromatogr A. 2000;  872 61-73
  CrossrefPubMedSearch in Google Scholar
• 13 Wagner H, Bladt S. Plant Drug Analysis: A Thin Layer Chromatography Atlas. 2nd edn Berlin, Heidelberg; Springer Verlag 1996: pp 338-9
  Search in Google Scholar
• 14 Pauli G F. Comprehensive spectroscopic investigation of α-onocerin.  Planta Med. 2000;  66 299-302
  Thieme ConnectPubMedSearch in Google Scholar
• 15 Tang X C, Han Y F. Pharmacological profile of huperzine A, a novel acetylcholinesterase inhibitor from Chinese herb.  CNS Drug Rev. 1999;  5 281-300
  PubMedSearch in Google Scholar
• 16 Sano T, Fujimoto T, Tsuda Y. Clavatol: a novel triterpenoid of the bisnoronocerane type isolated from Lycopodium clavatum . Chem Commun 1970: 1274-5
  Search in Google Scholar
• 17 Sano T, Fujimoto T, Tsuda Y. Triterpenoid chemistry. XIII. Lycopodium triterpenoid. 9. Structure of lyclavatol, a novel triterpenoid of the bisnoronocerane type.  Chem Pharm Bull. 1975;  23 1784-8
  PubMedSearch in Google Scholar
• 18 Ogura H, Kosasa T, Kuriya Y, Yamanishi Y. Comparison of inhibitory activities of donepezil and other cholinesterase inhibitors on acetylcholinesterase and butyrylcholinesterase in vitro .  Method Find Exp Clin. 2000;  22 609-13
  PubMedSearch in Google Scholar

Dr. Mag. pharm. Judith Maria Rollinger

Institut für Pharmazie

Pharmakognosie

Leopold-Franzens-Universität Innsbruck

Innrain 52c

Josef-Moeller Haus

6020 Innsbruck

Austria

Phone: +43-512-507-5308

Fax: +43-512-507-2939

Email: judith.rollinger@uibk.ac.at

References

• 1 Bartus R T, Dean R L, Beer B, Lipps A S. The cholinergic hypothesis of geriatric memory dysfunction: a critical review.  Science. 1982;  217 408-17
  CrossrefPubMedSearch in Google Scholar
• 2 Benzi G, Moretti A. Is there a rationale for the use of acetylcholinesterase inhibitors in the therapy of Alzheimer’s disease?.  Eur J Pharmacol. 1998;  346 1-13
  CrossrefPubMedSearch in Google Scholar
• 3 Inestrosa N C, Alvarez A, Perez C A, Moreno R D, Vicente M, Linker C. et al . Acetylcholinesterase accelerates assembly of amyloid-β-peptides into Alzheimer’s fibrils: possible role of the peripheral site of the enzyme.  Neuron. 1996;  16 881-91
  CrossrefPubMedSearch in Google Scholar
• 4 Rollinger J M, Hornick A, Langer T, Stuppner H, Prast H. Acetylcholinesterase inhibitory activity of scopolin and scopoletin discovered by virtual screening of natural products.  J Med Chem. 2004;  47 6248-54
  CrossrefPubMedSearch in Google Scholar
• 5 Orhan I, Terzioglu S, Sener B. α-Onocerin: an acetylcholinesterase inhibitor from Lycopodium clavatum .  Planta Med. 2003;  69 265-7
  Thieme ConnectPubMedSearch in Google Scholar
• 6 Rowan M G, Dean P DG. Alpha-onocerin and sterol content of twelve species of Ononis .  Phytochemistry. 1972;  11 3263-5
  CrossrefPubMedSearch in Google Scholar
• 7 European Pharmacopoeia. 4th edn Wien; Verlag Österreich GmbH 2002: p 2009
  Search in Google Scholar
• 8 Choudhary M I, Shahnaz S, Parveen S, Khalid A, Ayatollahi S AM, Atta-Ur-Rahman . et al . New triterpenoid alkaloid cholinesterase inhibitors from Buxus hyrcana .  J Nat Prod. 2003;  66 739-42
  PubMedSearch in Google Scholar
• 9 Atta-Ur- R ahman, Parveen S, Khalid A, Farooq A, Choudhary M I. Acetyl- and butyrylcholinesterase-inhibiting triterpenoid alkaloids from Buxus papillosa .  Phytochemistry. 2001;  58 963-8
  CrossrefPubMedSearch in Google Scholar
• 10 Chung Y K, Heo H J, Kim E K, Kim H K, Huh T L, Lim Y. et al . Inhibitory effect of ursolic acid purified from Origanum majorana L on the acetylcholinesterase.  Mol Cells. 2001;  11 137-43
  PubMedSearch in Google Scholar
• 11 Ellman G L, Courtney D, Andres V, Featherstone R M. A new and rapid calorimetric determination of acetylcholinesterase activity.  Biochem Pharmacol. 1961;  7 88-95
  CrossrefPubMedSearch in Google Scholar
• 12 Ingkaninan K, de Best C M, van der Heijden R, Hofte A JP, Karabatak B, Irth H. et al . High-performance liquid chromatography with on-line coupled UV, mass spectrometric and biochemical detection for identification of acetylcholinesterase inhibitors from natural products.  J Chromatogr A. 2000;  872 61-73
  CrossrefPubMedSearch in Google Scholar
• 13 Wagner H, Bladt S. Plant Drug Analysis: A Thin Layer Chromatography Atlas. 2nd edn Berlin, Heidelberg; Springer Verlag 1996: pp 338-9
  Search in Google Scholar
• 14 Pauli G F. Comprehensive spectroscopic investigation of α-onocerin.  Planta Med. 2000;  66 299-302
  Thieme ConnectPubMedSearch in Google Scholar
• 15 Tang X C, Han Y F. Pharmacological profile of huperzine A, a novel acetylcholinesterase inhibitor from Chinese herb.  CNS Drug Rev. 1999;  5 281-300
  PubMedSearch in Google Scholar
• 16 Sano T, Fujimoto T, Tsuda Y. Clavatol: a novel triterpenoid of the bisnoronocerane type isolated from Lycopodium clavatum . Chem Commun 1970: 1274-5
  Search in Google Scholar
• 17 Sano T, Fujimoto T, Tsuda Y. Triterpenoid chemistry. XIII. Lycopodium triterpenoid. 9. Structure of lyclavatol, a novel triterpenoid of the bisnoronocerane type.  Chem Pharm Bull. 1975;  23 1784-8
  PubMedSearch in Google Scholar
• 18 Ogura H, Kosasa T, Kuriya Y, Yamanishi Y. Comparison of inhibitory activities of donepezil and other cholinesterase inhibitors on acetylcholinesterase and butyrylcholinesterase in vitro .  Method Find Exp Clin. 2000;  22 609-13
  PubMedSearch in Google Scholar

Dr. Mag. pharm. Judith Maria Rollinger

Institut für Pharmazie

Pharmakognosie

Leopold-Franzens-Universität Innsbruck

Innrain 52c

Josef-Moeller Haus

6020 Innsbruck

Austria

Phone: +43-512-507-5308

Fax: +43-512-507-2939

Email: judith.rollinger@uibk.ac.at