Cycad Seeds Research Articles

The cycad neurotoxic amino acid, ß- N-methylamino- l-alanine (BMAA), elevates intracellular calcium levels in dissociated rat brain cells

Seeds of the Guam cycad Cycas micronesica K.D. Hill (Cycadaceae), which contain ß-methylamino- l-alanine (BMAA), have been implicated in the etiology of the devastating neurodisease ALS–PDC that is found among the native Chamorros on Guam. The disease also occurs in the native populations on Irian Jaya and the Kii Peninsula of Japan, and in all three areas the cycad seeds are used either dietarily or medically. ALS–PDC is a complex of amyotrophic lateral sclerosis and parkinsonism dementia complex with additional symptoms of Alzheimer's. It is well known that Ca 2+ elevations in brain cells can lead to cell death and neurodiseases. Therefore, we evaluated the ability of the cycad toxin BMAA to elevate the intracellular calcium concentration ([Ca 2+] i ) in dissociated newborn rat brain cells loaded with fura-2 dye. BMAA produced an increase in intracellular calcium levels in a concentration-dependent manner. The increases were dependent not only on extracellular calcium concentrations, but also significantly on the presence of bicarbonate ion. Increasing concentrations of sodium bicarbonate resulted in a potentiation of the BMAA-induced [Ca 2+] i elevation. The bicarbonate dependence did not result from the increased sodium concentration or alkalinization of the buffer. Our results support the hypothesis that the neurotoxicity of BMAA is due to an excitotoxic mechanism, involving elevated intracellular calcium levels and bicarbonate. Furthermore, since BMAA alone produced no increase in Ca 2+ levels, these results suggest the involvement of a product of BMAA and CO 2, namely a β-carbamate, which has a structure similar to other excitatory amino acids (EAA) such as glutamate; thus, the causative agent for ALS–PDC on Guam and elsewhere may be the β-carbamate of BMAA. These findings support the theory that some forms of other neurodiseases may also involve environmental toxins.

Cycad neurotoxins, consumption of flying foxes, and ALS-PDC disease in Guam.

The Chamorro people of Guam have been afflicted with a complex of neurodegenerative diseases (now known as ALS-PDC) with similarities to ALS, AD, and PD at a far higher rate than other populations throughout the world. Chamorro consumption of flying foxes may have generated sufficiently high cumulative doses of plant neurotoxins to result in ALS-PDC neuropathologies, since the flying foxes forage on neurotoxic cycad seeds.

Identification of nonprotein amino acids from cycad seeds as N-ethoxycarbonyl ethyl ester derivatives by positive chemical-ionization gas chromatography-mass spectrometry

Nonprotein amino acids from nine species of cycad seeds were analyzed as N-ethoxycarbonyl ethyl ester (ECEE) derivatives by positive chemical-ionization gas chromatography-mass spectrometry. Based on the retention times and mass spectrometry analyses, 12 nonprotein amino acids were identified in these seeds. In addition to the excitatory and putative neurotoxin ß-N-methylamino- l-alanine (BMAA), the known neurotoxin ß-N-oxalylamino- l-alanine (BOAA) was detected from the seeds of Macrozamia moorei and M. communis, and δ-N-oxalyl-ornithine was obtained from the Cycas revoluta seeds. A novel nonprotein amino acid named cycasindene, previously reported from C. revoluta, was also found in the seeds of members of the C. angulata and C. rumphii complex. Eight additional known nonprotein amino acids were also identified. This is the first report of the neurotoxin BOAA from cycad seeds.

Mast-seeding in the cycad genus Encephalartos: a test of the predator satiation hypothesis.

Mast-seeding behaviour was monitored in 18 populations of eight species of the African cycad genus Encephalartos between 1988 and 1991. The coefficient of variation (V) in annual cone production for each population ranged between 88 and 200, indicating large fluctuations in reproductive effort between years. Data were collected to determine whether mast-seeding reduced levels of predispersal seed predation by satiating seed predators in mast years and whether it resulted in a reproductive advantage over plants which reproduced more frequently. Masting intensity was greatest in those populations in which individual plants suffered the highest levels of predispersal seed predation in years when only a few plants produced seeds. The principal seed predators were two congeneric weevil species, Antliarhinus zamiae and A. signatus, which develop exclusively on cycad seeds. The lowest intensity of mast-seeding was recorded for cycad populations with low levels of seed predation and in which A. zamiae and A. signatus occurred only in low numbers or were entirely absent. Larger seed crops appeared to result in lower levels of seed predation by A. zamiae and A. signatus in four populations of E. altensteinii, and differences in seed crop size accounted for 48-66% of variation in levels of seed predation in populations of five cycad species. In one population of E. altensteinii, lower levels of seed predation in plants reproducing periodically resulted in a reproductive advantage over plants reproducing more frequently. These results are consistent with the predator satiation hypothesis. However, in most cycad populations, numbers of seed predators did not appear to decrease significantly after a period of 2-8 years between reproductive episodes and, in two of three populations examined, periodic reproduction did not increase the number of seeds surviving to dispersal over a 4-year period. These results are interpreted to mean that periodic reproduction has not evolved in response to selection imposed by seed predators, but that selection may favour those plants which experience lower levels of seed predation by coning in synchrony with the majority of plants in the population.

Zinc, a neurotoxin to cultured neurons, contaminates cycad flour prepared by traditional guamanian methods

We have used cultured ventral mesencephalic and cerebellar granule cells to test the toxicity of extracts of cycad seeds (genus Cycas) and cycad-derived flours traditionally prepared in Guam. There was no significant difference in the toxicity of extracts prepared from the female gametophyte tissue of C. circinalis, C. revoluta, and C. media, common wheat flour, and 13 of 17 cycad flour samples. However, extracts prepared from 4 of 17 Guamanian flour samples exhibited marked dose-dependent neurotoxicity to mesencephalic and granule cell cultures. There was no correlation between toxicity and 2-amino-3-(methylamino)-propanoic acid (BMAA) content, and the concentration of BMAA in the medium arising from these extracts was far below that required to be neurotoxic. Toxicity of extracts was not blocked by the NMDA receptor antagonist MK-801 or the non-NMDA receptor antagonist 6-cyano-7-dinitroquinoxaline-2,3-dione, indicating that toxicity was not mediated by excitatory amino acid receptors. Analysis of the four toxic processed flour samples indicated high zinc content. Zinc produced a concentration-dependent neurotoxic response in mesencephalic and granule cell cultures that paralleled the calculated concentrations of zinc in the cultures derived from the four toxic flour samples. When sliced C. circinalis gametophyte tissue was "processed" in our laboratory by soaking in a galvanized container, there was a time-dependent increase in zinc content.

2‐Amino‐3‐(methylamino)‐propanoic acid (BMAA) in cycad flour

We conducted an investigation of the levels of the neurotoxin 2-amino-3-(methylamino)-propanoic acid (BMAA) in cycad flour. Analysis of 30 flour samples processed from the endosperm of Cycas circinalis seeds collected on Guam indicated that more than 87% of the total BMAA content was removed during processing. Furthermore, in 1/2 the samples almost all (greater than 99%) of the total BMAA was removed. We found no significant regional differences in the BMAA content of flour prepared from cycad seeds collected from several villages on Guam. Testing of different samples prepared by the same Chamorro woman over 2 years suggests that the washing procedure probably varies in thoroughness from preparation to preparation but is routinely efficient in removing at least 85% of the total BMAA from all batches. Analysis of a flour sample that had undergone only 24 hours of soaking indicated that this single wash removed 90% of the total BMAA. We conclude that processed cycad flour as prepared by the Chamorros of Guam and Rota contains extremely low levels of BMAA, which are in the order of only 0.005% by weight (mean values for all samples). Thus, even when cycad flour is a dietary staple and eaten regularly, it seems unlikely that these low levels could cause the delayed and widespread neurofibrillary degeneration of nerve cells observed in amyotrophic lateral sclerosis and the parkinsonism-dementia complex of Guam (ALS-PD).

Quantification of the Putative Neurotoxin 2-Amino-3-(methylamino)propanoic Acid (BMAA) in Cycadales: Analysis of the Seeds of Some Members of the Family Cycadaceae

Over the past 30 years there have been attempts to link the unusually high incidence of amyotrophic lateral sclerosis (ALS) among the Chamorros native to the island of Guam to the consumption of the seeds of Cycas circinalis L., the false sago palm. In support of this relationship it was recently shown that, when given to primates, 2-amino-3-(methylamino)-propanoic acid (BMAA), a minor cycad component, can cause selective degeneration of upper and lower motor neurons in the spinal cord and clinical features similar to those of ALS. In order to test the relationship between ALS and cycads, we have developed a sensitive and precise gas chromatographic/mass spectrometric (GC/MS) assay for BMAA which allows direct assessment of the BMAA content in foods and is directly applicable to the assay of BMAA in biologic tissues and fluids. After the addition of a deuterated isotopomer as an internal standard and transesterication with 2-methyl-1-propanol, BMAA was extracted into dichloromethane and then acylated with pentafluoropropionic anhydride before GC/MS. This method permits precise quantification of BMAA in the low picogram/sample range. Direct quantification of the BMAA content in the female gametophyte tissue (endosperm) of a range of cycad seeds collected from Guam confirmed the presence of BMAA at levels of approximately 1 g/g (dry weight). The presence of BMAA in the seed extract was confirmed after derivatization of an aliquot of the extract and GC/MS analysis in the scanning mode. BMAA was found to be present, albeit at lower levels, in the endosperm of the seeds of C. revoluta (0.32 mg/g) and C. media (0.29 mg/g).

Observations about amyotrophic lateral sclerosis and the parkinsonism-dementia complex of Guam with regard to epidemiology and etiology.

For more than 150 years, Chamorro natives of the Mariana Islands in the Western Pacific Ocean, have developed fatal paralysis in middle and later life, which we term amyotrophic lateral sclerosis/parkinsonism-dementia (ALS/PD). The cause of the disease might be exposure to seeds of the indigenous cycad. Motor system disease is induced in cynomolgus monkeys by feeding them beta-N-methylamino-L-alanine (BMAA), an amino acid present in cycad seeds. We believe that the cycad seeds which usually cause no immediate adverse symptoms when prepared and eaten as flour, or applied topically as medicine, can give rise to widespread and severe nerve cell degeneration after a latency of many decades. Furthermore, it may be that only a single exposure to this potent but silent toxin(s) can result in fatal neurological disease years later.

Formation of three new trisaccharide-azoxyglycosides by transglucosylation by cycad .BETA.-glucosidase.

transglucosylation by a β-d-glucosidase from cycad seeds. These azoxyglycosides, named neocycasin H, I, and J, were identified as O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(l→3)-O-β-d-glucopyranoside of methylazoxymethanol (MAM), O-β-d-glucopyranosyl-(1→3)-[O-β-d-glucopyranosyl-(1→6)]-O-β-d-glucopyranoside of MAM, and O-β-d-glucopyranosyl-(1→3)-[O-β-d-xylopyranosyl-(1→6)]-O-β-d-glucopyranoside of MAM, respectively. On the basis of their structures, the mechanism of the formation of these neocycasins is also discussed.

305. Biological effects of cycad seeds

305. Biological effects of cycad seeds

Studies on Cycasin, a New Toxic Glycoside, ofCycas revolutaThunb.

The polarographic behaviors and the determination of cycasin, glucosyloxyazoxymethane, are described here. In the whole pH range, cycasin shows a reduction wave, which is considered to be due to the reduction of its aliphatic azoxy group. The polarograms from pH 4 to 7 run in two steps, and two electrode reaction mechanisms may be infered. The following facts are observed on the limiting current at pH 1 or 7: It is controlled by the diffusion process, and its temperature coefficient has a value resembling that of the usual diffusion current. The wave height shows a linear proportionality to the concentration of cycasin. The contents of cycasin in cycad seeds, determined polarographically, are presented, and compared with the results obtained by the paper chromatographic method.

Studies on Cycasin, a New Toxic Glycoside, ofCycas revolutaThunb

Quantitative paper chromatography of cycasin is described, where the cycasin and sugars separated on the paper are eluted out and determined, according to the colorimetric micro analysis of sugars by Plumel. The contents of cycasin and free sugars contained in both premature and matured cycad seeds determined by this method are presented.

Studies on Cycasin, a New Toxic Glycoside, of Cycas revoluta Thunb

The polarographic behaviors and the determination of cycasin, glucosyloxyazoxymethane, are described here. In the whole pH range, cycasin shows a reduction wave, which is con-sidered to be due to the reduction of its aliphatic azoxy group. The polarograms from pH 4 to 7 run in two steps, and two electrode reaction mechanisms may be infered. The follow-ing facts are observed on the limiting current at pH 1 or 7: It is controlled by the diffusion process, and its temperature coefficient has a value resembling that of the usual diffusion current. The wave height shows a linear proportionality to the concentration of cycasin. The contents of cycasin in cycad seeds, determined polarographically, are presented, and compared with the results obtained by the paper chromatographic method.

Studies on Cycasin, a New Toxic Glycoside, of Cycas revoluta Thunb

Quantitative paper chromatography of cycasin is described, where the cycasin and sugars separated on the paper are eluted out and determined, according to the colorimetric micro analysis of sugars by Plumel. The contents of cycasin and free sugars contained in both premature and matured cycad seeds determined by this method are presented.

(295)蘇鉄種実油中の飽和炭化水素について

(295)蘇鉄種実油中の飽和炭化水素について

Occurence of a New Cyclic Fatty Acid in Cycad Seed Oil

蘇鐵種實油より組成C20H34O2と思はるる新環式脂肪酸を單離せり.該酸は眞珠様光澤を有する細き板状結晶にして,融點124.5～125°,沃素價を有せず.之より製取せるメチルエステルは100.5～101°に於て融解せり.著者は之にソテツ酸 (Sotetuic acid; Sotetusaure)なる名稱を與へたり.