Two fast screening methods (GC-MS and TLC-ChEI assay) for rapid evaluation of potential anticholinesterasic indole alkaloids in complex mixtures

The pharmacotherapy for Alzheimer’s disease (AD) includes the use of acetylcholinesterase inhibitors (AChEI). Recent investigations for novel AD therapeutic agents from plants suggested that Tabernaemontana genus is a promising source of novel anticholinesterasic indole alkaloids. In this work two fast screening techniques were combined in order to easily identify novel cholinesterase inhibitors (ChEI). Gas chromatography-mass spectrometry (GC-MS) of the less polar alkaloidic fractions obtained from the acid-base extraction of the stalk of T. laeta revealed thirteen monoindole alkaloids, four of them confirmed by co-injection with previously isolated alkaloids. The others were tentatively identified by mass fragmentation analysis. By gas chromatography with flame ionization detection (GC-FID) and using isatin as internal standard, affinisine and voachalotine were determined as major compounds. These fractions and fourteen previously isolated alkaloids, obtained from root bark of T. laeta and T. hystrix were investigated for acetyl (AChE) and butyrylcholinesterase (BuChE) inhibitory activities by the modified Ellman‘s method in thin layer chromatography (TLC-ChEI). Results showed selective inhibition of the alkaloids heyneanine and Nb-methylvoachalotine for BuChE, and 19-epi-isovoacristine for AChE, whereas olivacine, affinisine, ibogamine, affinine, conodurine and hystrixnine inhibited both enzymes. In addition to confirming that monoterpenoid indole alkaloids can be novel therapeutic agents for AD, this is the first report of the ChEI activity of olivacine, a pyridocarbazole alkaloid.


INTRODUCTION
The year 2006 marked a century since the first description of Alzheimer's disease (AD) by Alois Alzheimer.AD, considered the most common neurodegenerative disease that affects the elderly, is characterized by progressive memory loss, decline in language skills and other serious cognitive impairments (Goedert and Spillantini 2006).
The etiology of AD is not very clear and multiple factors, such as amyloid-β peptide (Aβ) and tau protein aggregation, excessive transition metals, oxidative stress and reduced acetylcholine (ACh) level, have been implicated (Ji and Zhang 2006).However, it is a cholinergic deficiency rather than other neurochemical deficits that is thought to play a pivotal role in the development of AD symptoms (Farlow 2002).For this reason, inhibition of brain acetylcholinesterase (AChE) has been the major therapeutic target of AD treatment strategies.
Whereas several new natural and synthetic compounds are reported in the scientific literature as cholinesterase inhibitors (ChEI) candidates, this research is continuously improved as better drugs for AD is still required.Within the structure diversity of the ChEI, plant alkaloids are the most studied already leading to the development of new drugs (e.g.galanthamine, huperzine and phenserines) (Houghton et al. 2006, Mukherjee et al. 2007).
Monoterpenic indole alkaloids have been extensively investigated for exhibiting numerous biological activities (such as anti-tumor, anti-microbial, anti-hypertensive or as a central nervous system stimulant) (Andrade et al. 2005, Saxton 1997).They can be found in plants of the Apocynaceae, Rubiaceae, and Loganiaceae families and a few have recently been reported as ChEI by different research groups (Hostettmann et al. 2006).Among the AChEI found are some indole glucoalkaloids from Chimarrhis turbinate (Rubiaceae) (Cardoso et al. 2004); sarpagan type alkaloids from the Haplophyton crooksii (Apocynaceae) (Mroue et al. 1996); iboga alkaloids from Tabernaemontana australis (Apocynaceae) (Andrade et al. 2005) and vobasinyl-iboga bis-indole alkaloids from T. divaricata (Ingkaninan et al. 2006).In fact, a recent article reported the inhibition of cortical AChE activity and the enhancement of neuronal activity of T. divaricata extracts in rats, suggesting that it is a reversible AChE inhibitor and could be beneficial as a novel therapeutic agent for AD (Chattipakorn et al. 2007).
In this work we associated two fast screening techniques, a chemical screening by GC-MS and an enzymatic activity screening by Ellman's TLC assay (TLC-ChEI), supported by a collection of previously isolated monoindole alkaloids, in order to suggest a rapid evaluation of potential cholinesterase inhibitors alkaloids from Apocynaceae family.

PLANT MATERIAL
The stalk of Tabernaemontana laeta Mart.was collected in July 2004 at the Botanical Garden of Rio de Janeiro, Rio de Janeiro State, Brazil.A voucher specimen is deposited at the herbarium of the same Institution under the number RB 342087.Cienc (2008) 80 (3) EXTRACTION AND ISOLATION OF ALKALOIDS Stalk of T. laeta (150 g) was dried at 25 • C for 15 days, ground and extracted on a Soxhlet apparatus with EtOH for 72 h.After reduced pressure concentration, the average yield of samples extracted in triplicate was 1.46 g of a brown residue.Dried ethanolic extracts were suspended in 100 mL of 1% HCl and extracted with EtOAc and CHCl 3 (3 × 30 mL each).The pH of the aqueous acidic fraction was adjusted to 3, 5, 7, 9 and 11 with aqueous NH 4 OH, extracted with CHCl 3 (3 × 30 mL) and dried over anhydrous Na 2 SO 4 .Triplicates of each pH extract were analyzed by silica gel TLC using CH 2 Cl 2 : MeOH (9:1) as eluent and staining with 50% H 2 SO 4 /heat or Dra-gendorff´s reagent.Under these conditions, fractions related to pH 7 and pH 9 were combined.After solvents evaporation under reduced pressure, concentration triplicate average yielded 14.5 mg (pH 3), 8.4 mg (pH 5), 17.5 mg (pH 7 and pH 9) and 3.9 mg (pH 11).

An Acad Bras
The alkaloids 1-17 (Table I) were previously isolated by the authors and the experimental procedures are described elsewhere (Andrade et al. 2005, Medeiros et al. 1999, 2001, Monnerat et al. 2005).

CHOLINESTERASE INHIBITORY ACTIVITY DETERMINATION (TLC-CHEI)
Acetylcholinesterase and butyrylcholinesterase inhibitory activity was determined using TLC assay method and staining with Ellman's reagent (DTNB), which follows the technique described by Rhee et al. (Rhee et al. 2001).Pure compounds (0.051 and 0.005 mol L -1 ), fractions (5 mg mL -1 ) or extracts (10 mg mL -1 ) were diluted in appropriate solvent, then 1.0μL of each sample was spotted on the silica gel TLC plates and developed with CH 2 Cl 2 :MeOH; (95:5); 1.0μL of 0.36 mmol L -1 physostigmine solution in methanol was also spotted as reference.After developing the TLC plate, enzyme inhibitory activities were detected by spraying the substrate, dye and enzyme.The presence of cholinesterase inhibitory activity was determined by the formation of well-defined white spots made visible by spraying with DTNB, which gives a yellow background.In these experiments the minimum amount (detection limit) of physostigmine that could be recognized as a positive result was 0.07μg.
False-positive reactions were eliminated according  to the method described by Rhee et al (Rhee et al. 2003).
Briefly, 3 U mL -1 AChE was incubated for 15 min at 37 • C with 1 mmol L -1 of ATCI in buffer solution and then used as the thiocoline spray reagent.The developed TLC sample spots together with the known false-positive p-anisaldehyde were analyzed by spraying the dye followed by the thiocholine solution.White spots, indicating false-positive reactions, on a yellow background were recorded, and then compared with the results of the enzymatic assays.Four TLC plates were processed as above for, respectively, AChE, BuChE, and false positive assays, or revealed under UV and with H 2 SO 4 50% or Dragendorff´s solutions before heating.

GC-FID AND GC-MS ANALYSES
The stalk of Tabernaemontana laeta Mart.collected in July 2004 at the Botanical Garden of Rio de Janeiro, Rio An Acad Bras Cienc (2008) 80 (3) de Janeiro State, Brazil, was extracted with ethanol and fractionated at five different pH (3, 5, 7, 9 and 11).Silica gel TLC of the fractions showed the presence of alkaloids after Dragendorff´s reagent stain.The less polar fractions observed in TLC (pH 3 and 5) were chosen for GC-MS analysis.By standard co-injection with previously isolated alkaloids from Tabernaemontana spp.and tentative identification by mass fragmentation pattern, 13 monoterpenic indole alkaloids were identified (Table II and Fig. 1).GC-MS and GC-FID using isatin as internal standard showed affinisine (2) and voachalotine (23) as major compounds in both fractions.As shown in Table II, other minor alkaloids with iboga, voacanga and sarpagan skeletons were also identified.
All the structures showed in Figure 1 were based on the more recent literature data and CAS registries.
The preliminary activity of the 17 alkaloids assayed in this work indicated that the structure-activity relationship of this class of compounds is complex and should be investigated in detail.Ingkaninan et al. suggested that substitutions at carbons 11', 12' and 16' affect the bis-indole alkaloids AChEI activity in Ellman´s assay (Ingkaninan et al. 2006).Although we have to be careful with the preliminary results obtained by TLC assays, this property was not observed in the present work.
Besides the iboga, voacanga and tetrahydro β-carboline sarpagan alkaloids, olivacine ( 12) was the only pyridocarbazole assayed and showed to be an active substance at the smallest quantity used (0, 051μmol).The indication of dual or selective inhibition of BuChE and AChE of some alkaloids also suggested that these compounds can be interesting models to study the biochemical interactions with cholinesterases as well as the pharmacological benefits of such selectivity.

CONCLUSIONS
GC-MS is a valuable tool to screen indole alkaloids such as those with iboga and sarpagan skeletons.These compounds frequently show intense molecular and characteristic diagnostic ions.Present investigation of the stalk of T. laeta revealed thirteen alkaloids by GC-MS, whereas only five have been previously described.
The combination of two techniques such as GC-MS and TLC-ChEI assay has led to a rapid chemical and biological screening of already known indole alkaloids.These results also confirm the AChE and BuChE inhibitory activities of monoterpenic indole alkaloids, and corroborate that TLC assay based on Ellman´s method is simpler, fast and inexpensive method to search AChE and BuChE inhibitors from natural sources.
In addition, this is the first report of the ChEI activity of olivacine (12), a pyridocarbazole alkaloid.(FAPERJ, Brazil), "Conselho Nacional de Desenvolvimento Científico e Tecnológico" (CNPq, Brazil) and "Coordenação de Aperfeiçoamento de Pessoal de Nível Superior" (CAPES, Brazil) are greatly appreciated.We also thank Dr. Bruno Rezende Maia (Botanical Garden of Rio de Janeiro) for the botanical identification and the student Meriane P. Carvalho for her help during some bioassays.

TABLE II Gas Chromatography-Mass Spectrometry (GC-MS) and Gas Chromatography (GC-FID) analyses of pH3 and pH5 fractions obtained from T. laeta stalk extracts
relative area corrected by the use of isatin as internal standard. f