Wild Argentinian Amaryllidaceae, a New Renewable Source of the Acetylcholinesterase Inhibitor Galanthamine and Other Alkaloids

The Amaryllidaceae family is well known for its pharmacologically active alkaloids. An important approach to treat Alzheimer’s disease involves the inhibition of the enzyme acetylcholinesterase (AChE). Galanthamine, an Amaryllidaceae alkaloid, is an effective, selective, reversible, and competitive AChE inhibitor. This work was aimed at studying the alkaloid composition of four wild Argentinian Amarillydaceae species for the first time, as well as analyzing their inhibitory activity on acetylcholinesterase. Alkaloid content was characterized by means of GC-MS analysis. Chloroform basic extracts from Habranthus jamesonii, Phycella herbertiana, Rhodophiala mendocina and Zephyranthes filifolia collected in the Argentinian Andean region all contained galanthamine, and showed a strong AChE inhibitory activity (IC50 between 1.2 and 2 µg/mL). To our knowledge, no previous reports on alkaloid profiles and AChEIs activity of wild Argentinian Amarillydaceae species have been publisihed. The demand for renewable sources of industrial products like galanthamine and the need to protect plant biodiversity creates an opportunity for Argentinian farmers to produce such crops.


Introduction
Many species of medicinal and aromatic plants are cultivated for such industrial uses, but most are still collected in the wild. The demand for renewable sources of industrial products and the need to protect plant biodiversity create an opportunity for farmers to produce such plants as crops. More than 25% of the pharmaceutical drugs used in the World today are derived from plant natural products [1]. In the conventional pharmaceutical industry, pharmaceutical companies produce drugs from compounds extracted from plant material, or use plant derived compounds as starting material to produce drugs semi-synthetically [2]. Examples of the former include the anti-cancer alkaloid paclitaxel from Pacific yew (Taxus brevifolia), vinblastine from the Madagascar periwinkle (Cataranthus roseus), and digoxin from the foxglove (Digitalis lanata) [1].
The alkaloids of the Amaryllidaceae family are extensively studied for their biological activities in several pharmaceutical areas, for example, Alzheimer's disease (AD), a neurodegenerative problem of enormous economic and social impact (15 million people, mainly in developed countries, suffer from the symptoms of this disease). The treatment is based on drugs that increase levels of acetylcholine. Galanthamine is a long-acting, selective, reversible and competitive inhibitor of acetylcholinesterase (AChE) and an allosteric modulator of the neuronal nicotinic receptor for acetylcholine. AChE is responsible for the degradation of acetylcholine at the neuromuscular junction, in peripheral and central cholinergic synapses. Galanthamine has the ability to cross the blood-brain barrier and to act within the central nervous system [3,4]. According to data presented by the Alzheimer's Association in 2007, the prevalence of Alzheimer's disease will quadruple by 2050. Galanthamine hydrobromide has superior pharmacological profiles and higher tolerance as compared to the original AChE inhibitors, physostigmine or tacrine [5]. This alkaloid galanthamine (biosynthesized exclusively by species of Amaryllidaceae family) is the treatment for mild and moderate stages of the AD. Galanthamine, approved in 2001 by FDA (Razadyne ® ), was originally isolated from Galanthus woronowii. While several total syntheses of the alkaloid galanthamine are available [6][7][8][9][10], current marketing is done mainly by the limited extraction of natural populations of Leucojum aestivum from Turkey (of varying quality and low content of active principle), or from small plantations of this species in Bulgaria, which are insufficient to meet current pharmaceutical company demand. The worldwide production of galanthamine is about 250 kg per year. Around 61 species of the Amaryllidaceae family grow in Argentina, covering a wide variety of genera (Chlidanthus, Crinum, Habranthus, Haylockia, Hieronymiella, Hippeastrum, Phycella, Rhodophiala, Stenomesson and Zephyranthes) [11]. To our knowledge, there are no reports on the chemistry and biological activity of Argentinian species belonging to the Amaryllidaceae group.
Our search for plant raw materials for medicinal products is now aimed at investigating the acetylcholinesterase inhibitory activity (AChE) of basic chloroform extracts (BCE) obtained from Habranthus jamesonii, Phycella herbertiana, Rhodophiala mendocina, and Zephyranthes filifolia (Amaryllidaceae species that grow in Argentine) to find new sources of production of galanthamine, and other potential alkaloids for treating AD. AChE inhibitory activity was determined by the spectrophotometric method by Ellman et al. [12]. Alkaloid profiles were analyzed by gas chromatography-mass spectrometry (GC-MS).

Alkaloid Extraction
Dried bulbs (100 g per each plant) were extracted under reflux three times with MeOH (300 mL) for 1 h each. The solvent was evaporated under reduced pressure to give the methanolic crude extracts (MCEs). MCEs were dissolved in H 2 SO 4 (2% v/v) and neutral material was removed with CHCl 3 (200 mL). Then, the aqueous solutions were basified with 25% NaOH up to pH 10-12 and the alkaloids were extracted with CHCl 3 (3  500 mL) to obtain the basic chloroform extract (BCE).

Gas Chromatography-Mass Spectroscopy Analyses
GC-MS analyses were performed on a Hewlett Packard 6890/MSD 5975 instrument (Hewlett Packard, Palo Alto, CA, USA) operating in EI mode at 70 eV. A DB-5 MS column (30 m  0.25 mm  0.25 μm) was used. The temperature program was: 100-180 °C at 15 °C min −1 , 1 min hold at 180 °C, 180-300 °C at 5 °C min −1 , and 1 min hold at 300 °C. Injector temperature was 280 °C. The flow rate of carrier gas (He) was 0.8 mL min −1 . The split ratio was 1:20. The results obtained were analyzed using AMDIS 2.64 software (NIST). Compounds were identified through the comparison of their mass spectral patterns and retention indexes, with the data recorded in literature.

Microplate Assay for Acetylcholinesterase Activity
AChE activity was assayed as described by Ellman et al. [12] with some modifications [13]. Fifty μL of AChE in buffer phosphate (8 mM K 2 HPO 4 , 2.3 mM NaH 2 PO 4 , 0.15 M NaCl, 0.05% Tween 20, pH 7.6) and 50 μL of the sample dissolved in the same buffer were added to the wells. The plates were incubated for 30 minutes at room temperature before the addition of 100 μL of the substrate solution (0.1 M Na 2 HPO 4 , 0.5 M DTNB, 0.6 mM ATCI in Millipore water, pH 7.5). The absorbance was read in a Labsystems microplate reader (Helsinki, Finland) at 405 nm after three minutes. Enzyme activity was calculated as a percentage compared to an assay using a buffer without any inhibitor. The AChE inhibitory data were analyzed with the software package Prism (Graph Pad Inc., San Diego, CA, USA). IC 50 values are means ± SD of three individual determinations each performed in triplicate.

TLC Analysis of BCE
TLC was carried out on Merck Silica gel 60 F254 plates, using chloroform-methanol-ammonia (99:9:1) mixtures as mobile phase. TLC plates were sprayed with Dragendorff's reagent; main alkaloids gave orange spots.

Conclusions
The findings of the present study demonstrate the potential of wild Argentinian Amaryllidaceae species collected in the central Andean region, as a new renewable source of galanthamine. The most promising species seen to be H. jamesonii MDZ and Z. filifolia SJ. The demand for renewable sources of galanthamine and the need to protect plant biodiversity create an opportunity for Argentinian farmers to produce such crops. Studies of domestication of some of these species are currently in progress in order to determine which crops can be cultivated outdoors in the particular climate and soil, and which can be grown in greenhouses. Production cost and galanthamine levels in traditional cultivars are also being analyzed.