Abstract
Context: This is the first comprehensive study of the acetylcholinesterase (AChE) inhibitory activity of species of the family Amaryllidaceae and 13 related families from Panama.
Objective: Exploration of the potential sources of AChE inhibitors with radical scavenging activity from Amaryllidaceae and 13 related families from Panama.
Materials and methods: The studied plants were screened with anti-acetylcholinesterase bioautographic and DPPH free radical scavenging assays.
Results: From the 57 plants studied, eight (14%) showed strong inhibition of AChE, and 29 (51%) plants showed moderate inhibition of AChE.
Discussion and conclusion: Sagittaria lancifolia L. (Alismataceae), Crinum jagus (Thomps.) (Amaryllidaceae), Crinum x amabile Donn (Amaryllidaceae), Crinum zeylanicum (L.) L. (Amaryllidaceae), Crocosmia x crocosmiiflora (Lemoine ex Anonymous) N.E. Br. (Iridaceae), Sisyrinchium tinctorium Kunth (Iridaceae), Agapanthus praecox subsp. orientalis (F.M. Leight.) F.M. Leight. (Liliaceae), and Xyris jupicai Rich. (Xyridaceae) were the most active plants, inhibiting AChE at 100 μg on the TLC bioautographic method for the detection of acetylcholinesterase inhibitors. Out of the eight most active plants, two plants, Crinum zeylanicum (L.) L. and Xyris jupicai Rich., showed antioxidant activity.
Introduction
Alzheimer’s disease (AD) is a neurodegenerative illness, principally characterized by a marked reduction of cholinergic neuronal function, resulting in a deficiency in acetylcholine (ACh) concentration in the brain. These reductions are associated with impairments in memory. Cholinergic enhancement strategies have been at the forefront of the efforts to ameliorate the cognitive symptoms (CitationLoizzo et al., 2008). Therefore, treatment approaches have been focused on the acetylcholinesterase inhibitors (AChEIs), which are the promising drugs today for the palliative treatment of AD (CitationHorgan, 2007).
The majority of the studies on natural products as inhibitors of AChE have emphasized alkaloids, such as physostigmine and galanthamine. Galanthamine, an alkaloid isolated from different Galanthus species (Amaryllidaceae), has been found to be a potent and reversible AChEI (CitationTumiatti et al., 2008). Plants of the Amaryllidaceae family, including ca. 65 genera and about 860 species, are amongst the top 20 in the most widely used medicinal plant families (CitationJin, 2009), and have been reported as a potential source of AChE inhibitory activity (CitationMukherjee et al., 2007). In Panama, Amaryllidaceae has five genera and 13 species (CitationCorrea et al., 2004), and 13 related families (Agavaceae, Alismataceae, Alstroemeriaceae, Araceae, Commelinaceae, Dioscoreaceae, Haemodoraceae, Hypoxidaceae, Iridaceae, Liliaceae, Pontederiaceae, Smilacaceae, and Xyridaceae). The lack of information on the potential of these families as a source of AChE inhibitors has prompted us to investigate the acetylcholinesterase (AChE) inhibitory and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities in tropical plant species of Amaryllidaceae and related families from Panama using a thin layer chromatography (TLC) autobiographic method.
Methods
Selection of plants
Plants were selected on chemotaxonomic and phylogenetic bases and collected mainly from the tropical forests of Panama. Detailed information on voucher number, date, and place of collection with global positioning system (GPS) coordinates is provided in . A cladistic analysis of monocotyledons supported the phylogenetic relationship among Amarillydaceae and the 13 related families (CitationJudd et al., 1999). Their taxonomic identity was established by the botanist specialists in these families: Dr. Abisaí García Mendoza, Botanical Garden of the Institute of Biology, National University of Mexico, Mexico (family Agavaceae); Dr. Alan W. Meerow, National Germplasm Repository, Miami, USA (family Amaryllidaceae); Dr. Anton Hofreiter, Bereich Biodiversitätsforschung, Systematische Botanik, München, Germany (family Alstroemeriaceae); Dr. Peter Goldblatt, Missouri Botanical Garden, St. Louis, USA (families Liliaceae, Xyridaceae, and Hypoxidaceae); Mr. Jesús Rodrigo Botina Papamija, Herbarium of the University of Valle (CUVC), Colombia (family Smilacaceae); Mr. Alex Espinosa and Prof. Mireya Correa, University of Panama (families Alismataceae, Araceae, Commelinaceae, Dioscoreaceae, Haemodoraceae, Iridaceae, and Pontederiaceae). Voucher specimens were deposited at the Herbarium of the University of Panama (PMA), Panama.
Table 1. AChE inhibitory activity of plants from Amaryllidaceae and related families.
Preparation of extracts
In general, dried plant material (30 g) was macerated with methanol or 85% ethanol for extraction (0.5 L; 3 × 24 h). The plant extracts were filtered and concentrated in vacuo at <40 °C in a rotary evaporator and stored at −80 °C until tested.
Biological assays
Anti-acetylcholinesterase bioautographic assay
The TLC bioautographic method in this study was carried out according to the procedure described previously by CitationMarston et al. (2002). The samples were dissolved in MeOH. The solutions were applied to TLC to deliver 100 µg on a plate. Plates were developed with a mixture of CHCl3–MeOH–H2O (65:35:5) for polar and EtOAc–hexane (1:1) for non-polar extracts, respectively. A change in coloration from purple to white indicated the presence of AChEI. The known inhibitor galanthamine HBr (product no. G 1660) was purchased from Sigma (St. Louis, MO, USA) and tested as positive control at 0.01 μg on TLC.
DPPH free radical scavenging assay
The chemical test to identify free radical scavenging compounds from plant extracts was carried out according to the technique developed by CitationTakao et al. (1994). The extracts (100 μg) were deposited on aluminum-backed silica gel GF254 plates and developed with the solvent system listed as above. After drying, the plates were sprayed with a solution of DPPH at 2 mg/mL in MeOH. A change in coloration from purple to yellow indicated the presence of active free radical scavenging compounds. Quercetin was tested as a reference compound at 0.1 μg on TLC.
Alkaloid test
The plant extracts were deposited (100 μg) on aluminum-backed silica gel GF254 plates and developed with the TLC solvent systems CHCl3–MeOH–H2O (65:35:5) and EtOAc–hexane (1:1). The dried TLC plates were developed with Dragendorff’s reagent (CitationStahl, 1969). Orange spots were observed on the TLC plate for a positive alkaloid test.
Results
In this study, 57 plant species belonging to 37 genera and 14 families were collected in Panama (). Out of 57 species, 39 (68%) were native, 17 (30%) were introduced, and one (2%), specifically, Crinum zeylanicum (L.) L., was naturalized.
Eighty-two extracts were prepared from 57 plants and were first tested at a concentration of 100 µg on the TLC plate for their AChE inhibitory activity. Seventy-eight extracts were prepared in 85% ethanol and four extracts from Anthurium pentaphyllum (Aubl.) G. and Anthurium propinquum Sodiro were prepared in methanol.
Sixty extracts displayed AChE inhibitory activity ranging from weakly active to strongly active at 100 µg on the TLC plate (). The acetylcholinesterase inhibitory activity of active extracts was scored according to the appearance of a white spot on the purple background of the TLC plate as: weakly active (+), moderately active (++), strongly active (+++). From the 57 plants studied, eight (14%) showed strong inhibition of AChE and 29 (51%) plants showed moderate inhibition of AChE. This is the first report of the AChE inhibitory activity of Sagittaria lancifolia L., Crinum x amabile Donn, Crinum zeylanicum (L.) L., Crocosmia x crocosmiiflora (Lemoine ex Anonymous) N.E. Br., Sisyrinchium tinctorium Kunth, Agapanthus praecox subsp. orientalis (F.M. Leight.) F.M. Leight., and Xyris jupicai Rich., but AChE inhibitory activity of the bulbs of Crinum jagus (Thomps.) Dandy has been reported by CitationHoughton et al. (2004). Moreover, the AChE inhibitory activity of other species of Hippeastrum has been studied previously by CitationGiordani et al. (2008).
Discussion
Plant extracts with strong and moderate inhibitory activity against AChE were tested for their DPPH free radical scavenging activity at 100 µg on TLC plates. For this assay, the free radical scavenging activity was scored from active to inactive. Of the eight most active plants, two plants, Crinum zeylanicum (L.) L. and Xyris jupicai Rich., showed antioxidant activity. The antioxidant activity of AChE inhibitory plant extracts is an interesting feature for a complementary mechanism to avoid a deficit of the cholinergic system and oxidative stress (CitationPratico, 2008) in neurodegenerative diseases such as Alzheimer’s disease.
From the same group of the most active plants, Agapanthus praecox subsp. orientalis (F.M. Leight.) F.M. Leight., Crinum x amabile Donn, Crinum zeylanicum (L.) L., Crinum jagus (Thomps.) Dandy, and Xyris jupicai Rich. showed the presence of alkaloids with Dragendorff’s reagent. Two plants (Hippeastrum x hybridum Hort. and Hypoxis sp.) with moderate AChE inhibitory activity showed antioxidant activity. Species of the families Alismataceae, Amaryllidaceae, Iridaceae, Liliaceae, and Xyridaceae displayed strong inhibition of AChE.
Alkaloids (crimidine, flexinine, 6-hydroxypowelline, lycorine, 3-acetylhamayne, 6-hydroxycrinamine, hamayne, 6-methoxycrinamine, and zeylamine) have been reported in bulbs of Crinum zeylanicum (L.) L. by CitationTrimino et al. (1988), CitationDoepke et al. (1986), and CitationTsuda et al. (1984), but no AChE inhibitory activity has been attributed to them. Acetylcholinesterase activity of the alkaloids hamayne, crinamine, lycorine, pseudolycorine, 6-hydroxycrinamine, and haemanthamine from Crinum jagus (Thomps.) Dandy has been reported by CitationHoughton et al. (2004).
The AChE inhibitory and antioxidant activities of Hippeastrum x hybridum Hort. have been confirmed by reports of these effects in other Hippeastrum species (CitationGiordani et al., 2008). AChE inhibitory effects of Amaryllidaceae alkaloids have been reported in the literature by CitationElgorashi et al. (2004).
There is no report in the literature on the chemistry and the presence of acetylcholinesterase inhibitors in Sagittaria lancifolia L., Crinum x amabile Donn, Crocosmia x crocosmiiflora (Lemoine ex Anonymous) N.E. Br., Sisyrinchium tinctorium Kunth, Agapanthus praecox subsp. orientalis (F.M. Leight.) F.M. Leight., Xyris jupicai Rich., Hippeastrum x hybridum Hort., and Hypoxis sp.
Conclusions
Eight promising plants have been identified for their ability to inhibit AChE, and two of them have potential antioxidant activity. From the 13 families related to Amaryllidaceae, the families Xyridaceae, Alismataceae, Iridaceae, and Liliaceae could be a potential source of natural inhibitors of AChE. Phytochemical studies on Xyris jupicai Rich. and Crinum zeylanicum (L.) L. are underway to isolate bioactive molecules.
Acknowledgements
Our gratitude goes to all taxonomists who identified the plants and to the National Environmental Authority of Panama for granting permission to collect plants in national parks.
Declaration of interest
This work was supported by grants awarded to one of the authors (A.I.C.) by the International Foundation for Science and Organization for Prohibition of Chemical Weapons (IFS grant no F3766-1) and the National Secretariat of Science, Technology and Innovation (SENACYT), Panama (grant no. P-06-0042A). Thanks are also due to the Organization of American States for the support to Project SEDI/AICD/AE/036/06-09.
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