Striking genetic homogeneity in the widespread South American bracken.

PREMISE: Bracken (Pteridium, Dennstaedtiaceae) is a cosmopolitan genus of aggressive disturbance colonizers that are toxic to agricultural livestock. The taxonomy of Pteridium has been treated in multiple schemes, ranging from one to six species worldwide, with numerous subspecies and varieties. Recent work has focused on the worldwide distribution and systematics of the bracken fern, but South America has been poorly represented. We present the first continent-wide sampling and analysis of Pteridium esculentum, a Southern Hemisphere diploid species. METHODS: Within South America, P. esculentum has several morphotypes, distinguished into subspecies by variation in indument and lamina architecture. We used double digest restriction site-associated DNA sequencing (ddRADSeq) to assess the phylogenetic relationships of P. esculentum subspecies. RESULTS: We found a striking genetic homogeneity in the species, being able to support only two morphotypes from molecular data: P. e. arachnoideum and P. e. campestre. We had high confidence for shallow and deep phylogenetic relationships, but less support for relationships among crown groups. CONCLUSIONS: We describe an east-west geographic pattern that would explain the relationships between populations; and, in contrast to previous studies, we detected differences with P. esculentum from Australia. These results will lay the foundations for studying variations in this species' behavior as a weed, as well as its impact on the production of agricultural livestock in South America.

Three new flavonoid glycosides isolated from Pteridium aquilinum.

Three new flavonoid glycosides, (S)-4',6,8-trihydroxyflavanone-7-C-glucoside (1), (R)-4',6,8- trihydroxyflavanone-7-C-glucoside (2) and distenin-7-O-ß-D-glucoside (3), along with nine known flavonoids (4-12) were isolated from the aerial of Pteridium acquilinum. Their structures were elucidated by the analysis of spectroscopy data and their comparison with the reported values. The two C-glycosyl flavanones (1 and 2), were isolated from this specie, which might be chemotaxonomic markers of this specie. In addition, three new flavonoids were preliminarily examined for their anti-inflammatory activity. Compounds 1-3 inhibited the NF-κB induction by 46.3%, 59.6% and 29.2%, respectively.

Cross-species oncogenomics offers insight into human muscle-invasive bladder cancer.

BACKGROUND: In humans, muscle-invasive bladder cancer (MIBC) is highly aggressive and associated with a poor prognosis. With a high mutation load and large number of altered genes, strategies to delineate key driver events are necessary. Dogs and cats develop urothelial carcinoma (UC) with histological and clinical similarities to human MIBC. Cattle that graze on bracken fern also develop UC, associated with exposure to the carcinogen ptaquiloside. These species may represent relevant animal models of spontaneous and carcinogen-induced UC that can provide insight into human MIBC. RESULTS: Whole-exome sequencing of domestic canine (n = 87) and feline (n = 23) UC, and comparative analysis with human MIBC reveals a lower mutation rate in animal cases and the absence of APOBEC mutational signatures. A convergence of driver genes (ARID1A, KDM6A, TP53, FAT1, and NRAS) is discovered, along with common focally amplified and deleted genes involved in regulation of the cell cycle and chromatin remodelling. We identify mismatch repair deficiency in a subset of canine and feline UCs with biallelic inactivation of MSH2. Bovine UC (n = 8) is distinctly different; we identify novel mutational signatures which are recapitulated in vitro in human urinary bladder UC cells treated with bracken fern extracts or purified ptaquiloside. CONCLUSION: Canine and feline urinary bladder UC represent relevant models of MIBC in humans, and cross-species analysis can identify evolutionarily conserved driver genes. We characterize mutational signatures in bovine UC associated with bracken fern and ptaquiloside exposure, a human-linked cancer exposure. Our work demonstrates the relevance of cross-species comparative analysis in understanding both human and animal UC.

Health and Environmental Hazards of the Toxic Pteridium aquilinum (L.) Kuhn (Bracken Fern).

Bracken fern (Pteridium aquilinum (L.) Kuhn) is ubiquitous and acts as a cosmopolitan weed in pastures and similar environments. Despite its historical uses, it presents risks due to toxicity. This study, conducted in the second half of 2023, aimed to assess the environmental and health hazards of P. aquilinum, primarily focusing on its carcinogenic compound, ptaquiloside. The literature was comprehensively reviewed using diverse databases, including PubMed, Web of Science, Scopus, and Google Scholar. Information was synthesized from original research articles, meta-analyses, systematic reviews, and relevant animal studies. Animals grazing on bracken fern face annual production losses due to toxin exposure. The substantial impact on biodiversity, animal health, and human well-being arises from the presence of ptaquiloside and related compounds in milk, meat, and water, along with the increasing global prevalence of P. aquilinum and its swift colonization in acidic soil and fire-damaged areas. The objectives were to identify major bioactive compounds and explore their effects at molecular, cellular, pathological, and population levels. Various cooking techniques were considered to mitigate toxin exposure, although complete elimination remains unattainable. Therefore, the findings emphasize the need for cautious consumption. In conclusion, continued research is necessary to better understand and manage its environmental and health implications.

Structure Characterization, Antioxidant and Immunomodulatory Activities of Polysaccharide from Pteridium aquilinum (L.) Kuhn.

Pteridium aquilinum (L.) Kuhn (Pteridaceae family) has been widely used as a food and medicine in China and Korea. Previous studies indicate that P. aquilinum contains a variety of bioactive chemical components such as flavonoids, phenols, terpenoids, saponins, polysaccharides, and so on. In the present study, a novel polysaccharide (named as PAP-3) with average molecular weight of 2.14 × 105 Da was obtained from P. aquilinum. The structure was studied through physicochemical and spectroscopic analysis. The results indicated that PAP-3 consists of arabinose, rhamnose, fucose, galactose, mannose, and xylose in a molar ratio of 1.58:1.00:3.26:4.57:4.81:3.33. The polysaccharide is mainly composed of (1→2)-linked xylose and (1→3,6)-linked mannose on the main chain, with (1→2)-linked xylose, (1→6)-linked mannose, and (1→6)- and (1→3,6)-linked galactose as side chains. Galactose, fucose, and xylose are located at the end of the side chains. The in vitro immunomodulatory and antioxidant activities were assayed. PAP-3 has strong free-radical scavenging activity on DPPH and ABTS radicals and significant immunomodulatory activity on RAW264.7 cells. These data provide useful information for further study on the polysaccharides of P. aquilinum and their applications in the food and medical industries.

Culture types and period impact gametophyte morphogenesis and sporophyte formation of eastern bracken.

BACKGROUND: Liquid suspension culture efficiently proliferates plant cells and can be applied to ferns because it rapidly increases the fresh weight of gametophytes. This study assessed gametophyte proliferation and sporophyte production of Pteridium aquilinum var. latiusculum using a suspension culture method. RESULTS: The growth curve linear phase of gametophyte cells was confirmed between 9 and 18 days of culture, and the subculture cycle was determined to be 2 weeks. A double-strength MS medium (fresh weight, 18.0 g) containing 2% sucrose and NH4+:NO3- (120 mM, 40:80) was found to be the optimal liquid medium. Gametophytes obtained after suspension culture for 18 days did not normally form sporophytes in an ex vitro soil environment. However, this issue was resolved after changing the culture type or extending the culture period to 6 weeks. A short suspension culture period increased the fresh weight of fragmented and homogenized gametophytes but yielded numerous relatively immature gametophytes (globular forms of branching gametophytes, BG). Furthermore, differences in gametophyte morphogenesis and development were indicated by changes in endogenous phytohormone content. BG with immature development exhibited high accumulation of zeatin, jasmonic acid, and salicylic acid, and relatively low levels of abscisic acid and indole-3-acetic acid. The immature development of gametophytes directly affected sporophyte formation. CONCLUSIONS: This study maximized the advantages of liquid suspension culture using eastern bracken gametophytes and provides data to resolve any associated issues, thus facilitating efficient bracken production.

1-Methylcyclopropene (1-MCP) retards the senescence of Pteridium aquilinum var. latiusculum by regulating the cellular energy status and membrane lipid metabolism.

1-MCP is an ethylene inhibitor which can delay the ripening and senescence of fruits and vegetables effectively. Pteridium aquilinum var. Latiusculum (PA) is one of the wild vegetables which is famous and nutrient in China. However, the mechanism of PA preservation treated with 1-MCP has not been reported. Consequently, the effects of postharvest 1-MCP treatment on the changes in quality, energy metabolism, and membrane lipid metabolism of PA were investigated in this study. The results indicated that 1-MCP treatment could effectively inhibit the decreases in firmness, titratable acid (TA) content and the increases in weight loss rate, malondialdehyde (MDA) content, membrane permeability, and membrane lipid metabolism-related enzymes in PA. The cellular energy charge (EC) and the levels of ATP, ATP/ADP, and ATP/AMP, the activities of energy metabolism-related enzymes, NAD+, and NADH were maintained, and the decreases in unsaturated fatty acids and the ratio of unsaturated-to-saturated fatty acids in the membrane of PA cells were effectively retarded by 1-MCP treatment. A positive correlation was observed between cellular ATP levels and the ratio of unsaturated-to-saturated fatty acids, while negative correlations were observed between the ratio of unsaturated-to-saturated fatty acids and both lipid peroxidation and membrane permeability. These results indicated that higher levels of energy status, unsaturated-to-saturated fatty acid ratios, and lipid metabolism in the membrane could preserve the membrane integrity of postharvest PA and effectively extend its shelf life.

Edible Far Eastern Ferns as a Dietary Source of Long-Chain Polyunsaturated Fatty Acids.

Young fronds of ferns are consumed as a vegetable in many countries. The aim of this study was to analyze three fern species that are available for sale in the Russian Far East as dietary sources in terms of fatty acids that are important for human physiology: arachidonic acid (20:4n-6, ARA), eicosapentaenoic acid (20:5n-3, EPA) and other valuable long-chain polyunsaturated fatty acids. The content of ARA and EPA was 5.5 and 0.5 mg/g dry weight, respectively, in Pteridium aquilinum, 4.1 and 1.1 in Matteuccia struthiopteris, and 2.2 and 0.8 in Osmundastrum asiaticum. Salted fronds of P. aquilinum contained less these fatty acids than the raw fronds, with a decrease of up to 49% for ARA and 65% for EPA. These losses were less pronounced or even insignificant in dried fronds. Cooked ferns preserved significant portions of the long-chain polyunsaturated fatty acids: cooked P. aquilinum contained 4.4 mg/g dry weight ARA and 0.3 mg/g dry weight EPA. The ferns may provide a supplemental dietary source of these valuable long-chain polyunsaturated fatty acids, especially for vegetarian diets.

Impact of Pteridium aquilinum on vegetation in Nyungwe Forest, Rwanda.

Pteridium aquilinum acts as an important ecological filters in dominated communities. A study to investigate the effects of its dominance in the vegetation of Nyungwe was conducted. Sampling was done in Mubuga and Uwajerome mountains. A total of 53 alternate plots measuring 10 m × 10 m were sampled along a transect at regular interval of 10 m. In each plot, the species were identified and the cover abundance measured subjectively. Plant strategies, succession, biological forms, distribution and conservation status of each species were also determined. Data on species composition and cover abundance were analyzed using MVSP software and Shannon -Weiner index was used to determine diversity of communities. Descriptive statistics were used to assess the characteristics of the species. A total of 141 species belonging to 100 genera and 54 families and distributed in four plant communities were identified. Pteridium aquilinum, Macaranga kilimandischarica, Lycopodium clavatum and Microglossa parvifolia were dominant in communities I, II and III, with average cover of 31%, 6% and 4% respectively. The primary forest was dominated by Pavetta rwandensis and Allophyllus chaunostachys in community IV, with 21% and 10% coverage respectively. Shannon- Weiner and evenness indices were 1.538, 2.925, 3.251 and 2.940 and 0.436, 0.716, 0.791 and 0.768 in communities I, II, III and IV respectively. Species richness were 34, 36, 61 and 46 in communities I, II, III and IV respectively. Ruderal, chamaephytes, secondary, Africa tropical and least concerns plant species predominated in Pteridium vegetation areas with 76% 48%, 69%, 43% and 90% of total species respectively. Competitive, phanerophytes, primary, Afromontane and least concerns plant species dominated in non-dominated area with 54%, 52%, 58%, 40% and 88% of total species respectively. Pteridium aquilinum restricted the growth of trees as exhibited by the presence of few phanerophytes and enhanced the growth of ruderal species, both of which are indicators of disturbed forest. The tree species observed in P. aquilinum cut-areas was Macaranga kilimandischarica.

Synthetic Seed Technology Development and Production Studies for Storage, Transport, and Industrialization of Bracken Spores.

Bracken fern (Pteridium aquilinum var. latiusculum (Desv.) Underw. ex A. Heller) has long been grown industrially in South Korea. Conventional propagation methods, including planting rhizomes and in vitro seedling culture, are labor intensive and expensive, and thus not commercially suitable. We aimed to develop a system to produce synthetic seeds using fern spores (SFS). Synthetic seeds were prepared by mixing bracken spores and alginate matrix. Spore germination and gametophyte and sporophyte growth and development from SFS proceeded normally. Spore density affected gametophyte and sporophyte numbers. SFS prepared using cold (4 °C) long-term storage spores (even 7-year-old spores) could effectively form sporophytes. The highest germination was observed at 25 °C. Soaking-treated SFS successfully formed sporophytes, even after 30 days of storage at 4 °C; indeed, sporophytes formed even after five days of storage at 25 °C during transport conditions. SFS were sown in plug trays for commercial use. Young sporophytes grown from plug seedlings were greenhouse cultivated, and transplanting within eight weeks was effective for root growth and growing-point formation. Developing synthetic seeds is a feasible solution for facilitating efficient transport and the handling of small-sized fern spores; furthermore, this SFS technology provides the basis for fern seedling culture and fern spore industrialization.

Ptaquiloside and Pterosin B Levels in Mature Green Fronds and Sprouts of Pteridium arachnoideum.

Pteridium arachnoideum, a fern of the Pteridium aquilinum species complex found in South America, is responsible for several different syndromes of poisoning. Cases of bovine enzootic hematuria and upper alimentary squamous cell carcinoma are both frequent occurrences in Brazil, whereas only bovine enzootic hematuria is noted with any frequency around the world. The reason for the high frequency of upper alimentary squamous cell carcinoma in Brazil is not currently known. One possible explanation may be the higher levels of ptaquiloside and pterosin B in Brazilian Pteridium than those present in the plant in other countries. However, these levels have not yet been determined in P. arachnoideum. Thus, the present study aimed to measure and compare ptaquiloside and pterosin B levels in mature green fronds and sprouts of P. arachnoideum collected from different locations in Brazil. Samples of P. arachnoideum were collected from the states of Minas Gerais and Rio Grande do Sul. A total of 28 mature leaf samples and 23 sprout samples were used. The mean concentrations of ptaquiloside and pterosin B present in the mature green fronds of P. arachnoideum ranged from 2.49 to 2.75 mg/g and 0.68 to 0.88 mg/g, respectively; in P. arachnoideum sprouts, mean concentrations of ptaquiloside and pterosin B ranged from 12.47 to 18.81 mg/g, and 4.03 to 10.42 mg/g for ptaquiloside and pterosin B, respectively. Thus, ptaquiloside and pterosin B levels in P. arachnoideum samples collected in Brazil were higher in sprouts than in mature green fronds, as observed in other countries. However, there was no variation in ptaquiloside levels among plants collected from different cities in Brazil. The high frequency of upper alimentary squamous cell carcinoma in Brazilian cattle may not be attributed to greater levels of ptaquiloside and pterosin B in P. arachnoideum than in other Pteridium species in other countries.

Essential oil composition and antibacterial activity of Pteridium aquilinum (L.) Kuhn.

INTRODUCTION: The present work aims to study the chemical composition of Pteridium aquilinum (L.) Kuhn essential oil and its antibacterial activity against three important phytopathogenic Gram-negative bacteria: Erwinia amylovora, Pectobacterium carotovorum subsp. caroto-vorum, and Pseudomonas savastanoi pv. savastanoi. METHODS: The chemical composition of P. aquilinum L. essential oil produced by hydrodistillation was determined by gas chromatography-mass spectrometry. The antibacterial activity was tested using disk diffusion method and by determination of minimum inhibitory concentration values. The major components were linalool (10.29%), carvacrol (8.15%), benzaldehyde (5.95%), 2-undecanone (5.32%), and cuminaldehyde (4.57%). RESULTS: The essential oil tested revealed a powerful antibacterial effect against all tested strains, with inhibition zone diameters ranging from 32.0 ± 0.58 to 33.7 ± 0.88 mm. DISCUSSION: P. aquilinum EO contained 32.86% of oxygenated monoterpenes, which are known for their very powerful antimicrobial activities. The minimum inhibitory concentration values showed that P. aquilinum essential oil has very strong activity against E. amylovora (0.625 ul/ml), followed by P. carotovorum subsp. carotovorum (2.50 ul/ml) and P. savastanoi pv. savastanoi (5.00 ul/ml). The results obtained could contribute to the development of new potential agents for the control of bacterial diseases.

Achados clínico-patológicos e métodos de controle da intoxicação por Pteridium (aquilinum) arachnoideum em uma propriedade do Rio Grande do Sul / Clinical and pathological findings and control methods of the poisoning by Pteridium (aquilinum) arachnoideum in a farm of Rio Grande do Sul, Brazil

A infestação de pastagens por Pteridium arachnoideum é um problema que afeta a pecuária em diversas partes do mundo, ocasionando perdas produtivas e mortalidades nos rebanhos, bem como a redução das áreas de pastagens. Devido aos prejuízos na bovinocultura, foi proposto o acompanhamento de uma propriedade no Rio Grande do Sul com perdas por Pteridium arachnoideum. Realizou-se um levantamento das mortes associadas à intoxicação por samambaia na propriedade durante o período de janeiro de 2007 a janeiro de 2015. Adicionalmente, testou-se a viabilidade de ovinos como ferramenta de controle biológico de samambaia e se acompanhou as tentativas de controle da planta através do uso dos herbicidas metilsulfuron-metil e picloram, os quais eram associados ou não à roçada prévia. Das mortes por intoxicação natural acompanhadas na propriedade, observou-se 22 casos de quadro agudo de diátese hemorrágica e seis de quadro crônico de carcinomas do trato digestório superior. Em cinco bovinos jovens com diátese hemorrágica, além de hemorragias disseminadas e infartos, observou-se acentuado edema laríngeo, que cursava, clinicamente, com dispneia e estertores respiratórios característicos. Os carcinomas do trato digestório superior, apesar de menos frequentes, causaram perdas expressivas, devido à mortalidade anual de reprodutoras. Notou-se que a introdução de bovinos jovens em áreas recentemente roçadas pode resultar no consumo de grandes quantidades de samambaia e na ocorrência de surtos da enfermidade aguda. O controle das populações de samambaia pelo pastejo por ovinos não foi eficiente, devido ao baixo consumo observado com a lotação de quatro ovinos por hectare, embora mortalidade de ovinos por consumo da planta não tenha sido registrada. Após a retirada do potreiro infestado por samambaia, ao final do experimento, oito dos ovinos foram acompanhados, clinicamente, por três anos e não apresentaram nenhuma alteração. A utilização dos herbicidas resultou em uma redução da cobertura de P. arachnoideum. No entanto, essa prática necessita de estudos adicionais, já que algumas áreas não tiveram recuperação satisfatória da pastagem e o seu uso pode causar impactos ambientais e aquisição de resistência de P. arachnoideum aos herbicidas.(AU)

The infestation of pastures by populations of Pteridium arachnoideum has been a considerable and global problem to the livestock production, due to animal mortality and pasture coverage reduction. Given the impact of P. arachnoideum on cattle production, it has been proposed to monitor a beef cattle farm in Rio Grande do Sul, to assess some of the losses associated with the plant consumption and the methods employed for the plant control. A survey of cattle deaths associated with fern poisoning on the farm was carried out from January 2007 to January 2015. In addition, the viability of sheep as a biological control tool was tested. Attempts to control the plant through the use of the herbicides methylsulfuron-methyl and picloram associated or not with previous mowing were monitored. Cases of natural poisoning observed in the farm included the acute form (22), known as hemorrhagic diathesis as well as the chronic form, consisting in digestive carcinomas (6). Five cases of hemorrhagic diathesis in young cattle went along with marked laryngeal edema, which was clinically manifested as dyspnea and roaring, in addition to the classic pathological changes of widespread hemorrhages and infarcts. Even though less frequent, upper digestive tract carcinomas caused significant losses, due to annual mortality of mature cows. In occasions, when cattle were moved to newly mowed infested areas, outbreaks of acute poisoning were observed. The attempted control by sheep introduction showed to be unpractical, due to the little consumption seen at a stocking rate of four sheep per hectare. However, sheep mortality due to P. arachnoideum consumption was not recorded. The plant control method applied at the farm is based on both, the mowing of densely populated areas and the use of herbicides, namely metylsulfuron-methyl and picloram, in all paddocks. The reduction of P. arachnoideum coverage showed to be varied in different paddocks, however allowed the recovery of some grazing areas. This practice needs further studies, as some areas have not had satisfactory pasture recovery, and its indiscriminate use could lead to environmental impacts and resistance of P. arachnoideum to herbicides.(AU)

Tree seed rain and seed removal, but not the seed bank, impede forest recovery in bracken (Pteridium aquilinum (L.) Kuhn)-dominated clearings in the African highlands.

Considerable areas dominated by bracken Pteridium aquilinum (L.) Kuhn occur worldwide and are associated with arrested forest recovery. How forest recovery is impeded in these areas remains poorly understood, especially in the African highlands. The component processes that can lead to recruitment limitation-including low seed arrival, availability and persistence-are important determinants of plant communities and offer a potential explanation for bracken persistence. We investigated key processes that can contribute to recruitment limitation in bracken-dominated clearings in the Bwindi Impenetrable National Park, Uganda. We examined if differences in seed rain (dispersal limitation), soil seed bank, or seed removal (seed viability and persistence) can, individually or in combination, explain the differences in tree regeneration found between bracken-dominated areas and the neighboring forest. These processes were assessed along ten 50-m transects crossing the forest-bracken boundary. When compared to the neighboring forest, bracken clearings had fewer seedlings (bracken 11,557 ± 5482 vs. forest 34,515 ± 6066 seedlings/ha), lower seed rain (949 ± 582 vs. 1605 ± 335 tree seeds m-2 year-1), comparable but sparse soil seed bank (304 ± 236 vs. 264 ± 99 viable tree seeds/m2), higher seed removal (70.1% ± 2.4% vs. 40.6% ± 2.4% over a 3-day interval), and markedly higher rodent densities (25.7 ± 5.4 vs. 5.0 ± 1.6 rodents per 100 trapping sessions). Camera traps revealed that rodents were the dominant animals visiting the seeds in our seed removal study. Synthesis: Recruitment limitation contributes to both the slow recovery of forest in bracken-dominated areas, and to the composition of the tree species that occur. Low seed arrival and low persistence of unburied seeds can both explain the reduced density of seedlings found in bracken versus neighboring forest. Seed removal, likely due to rodents, in particular appears sufficient to constrain forest recovery and impacts some species more severely than others.

Structural characterization and immunomodulatory activity of a novel polysaccharide from Pteridium aquilinum.

PAP1-A, a novel heteropolysaccharide with an average molecular mass of 1.35×105Da, was isolated from Pteridium aquilinum using a combination of chromatography by DEAE Sepharose Fast Flow and Sepharose 4B. The monosaccharide component of PAP1-A was L-rhamnose, L-arabinose, L-fucose, D-xylose, D-mannose, D-glucose and D-galactose in the molar ration of 1.82: 1.53: 1.42: 1.31: 5.24: 1: 12.35. The predicted structure of PAP1-A was established to be a complex polysaccharide with a main chain consisted of α-(1→4)-linked galactose partially substituted at O-6 position, with the (1→2)-linked xylose, (1→3)-linked arabinose, (1→3)-linked rhamnose, (1→3,6)-linked mannose, and (1→6)-linked mannose, as branches. Fucose, glucose, mannose, and rhamnose were located at the termini of the branches. The immunomodulatory activity assay showed that PAP1-A could significantly promote the RAW264.7 cells proliferation and induce the production of NO from RAW264.7 cells. Therefore, PAP1-A shows as a potent immunomodulator with potential applications in the medical and food industries.

Ptaquiloside from bracken in stream water at base flow and during storm events.

The bracken fern (Pteridium spp.) densely populates both open and woodland vegetation types around the globe. Bracken is toxic to livestock when consumed, and a group of potent illudane-type carcinogens have been identified, of which the compound ptaquiloside (PTA) is the most abundant. The highly water soluble PTA has been shown to be leachable from bracken fronds, and present in the soil and water below bracken stands. This has raised concerns over whether the compound might pose a risk to drinking water sources. We investigated PTA concentrations in a small stream draining a bracken-infested catchment at base flow and in response to storm events during a growth season, and included sampling of the bracken canopy throughfall. Streams in other bracken-dominated areas were also sampled at base flow for comparison, and a controlled pulse experiment was conducted in the field to study the in-stream dynamics of PTA. Ptaquiloside concentrations in the stream never exceeded 61 ng L-1 in the base flow samples, but peaked at 2.2 µg L-1 during the studied storm events. The mass of PTA in the stream, per storm event, was 7.5-93 mg from this catchment. A clear temporal connection was observed between rainfall and PTA concentration in the stream, with a reproducible time lag of approx. 1 h from onset of rain to elevated concentrations, and returning rather quickly (about 2 h) to base flow concentration levels. The concentration of PTA behaved similar to an inert tracer (Cl-) in the pulse experiment over a relative short time scale (minutes-hours) reflecting no PTA sorption, and dispersion and dilution considerably lowered the observed PTA concentrations downstream. Bracken throughfall revealed a potent and lasting source of PTA during rainfall, with concentrations up to 169 µg L-1, that did not decrease over the course of the event. In the stream, the throughfall contribution to PTA cannot be separated from a possible below-ground input from litter, rhizomes and soil. Catchment-specific factors such as the soil pH, topography, hydrology, and bracken coverage will evidently affect the level of PTA observed in the receiving stream, as well as the distance from bracken, but time since precipitation seems most important. Studying PTA loads and transport in surface streams fed by bracken-infested catchments, simply taking occasional grab samples will not capture the precipitation-linked pulses. The place and time of sampling governs the findings, and including event-based sampling is essential to provide a more complete picture of PTA loads to surface water.

UPLC-MS/MS determination of ptaquiloside and pterosin B in preserved natural water.

The naturally occurring carcinogen ptaquiloside and its degradation product pterosin B are found in water leaching from bracken stands. The objective of this work is to present a new sample preservation method and a fast UPLC-MS/MS method for quantification of ptaquiloside and pterosin B in environmental water samples, employing a novel internal standard. A faster, reliable, and efficient method was developed for isolation of high purity ptaquiloside and pterosin B from plant material for use as analytical standards, with purity verified by 1H-NMR. The chemical analysis was performed by cleanup and preconcentration of samples with solid phase extraction, before analyte quantification with UPLC-MS/MS. By including gradient elution and optimizing the liquid chromatography mobile phase buffer system, a total run cycle of 5 min was achieved, with method detection limits, including preconcentration, of 8 and 4 ng/L for ptaquiloside and pterosin B, respectively. The use of loganin as internal standard improved repeatability of the determination of both analytes, though it could not be employed for sample preparation. Buffering raw water samples in situ with ammonium acetate to pH ∼5.5 decisively increased sample integrity at realistic transportation and storing conditions prior to extraction. Groundwater samples collected in November 2015 at the shallow water table below a Danish bracken stand were preserved and analyzed using the above methods, and PTA concentrations of 3.8 ± 0.24 µg/L (±sd, n = 3) were found, much higher than previously reported. Graphical abstract Workflow overview of ptaquiloside determination.

Detection of Ptaquilosides in different phenologic stages of Bracken fern (Pteridium aquilinum) and analysis of mik samples in farms with hematuria in Tolima, Colombia / Detección de ptaquilósido en diferentes estados fenológicos de "helecho macho" (Pteridium aquilinum) y análisis de muestras de leche en granjas con hematuria en Tolima, Colombia / Detecção da ptaquilosida em diferentes estágios fenológicos da samambaia (Pteridiumaquilinum) e análise de amostras de leite em fazendas com hematúria em Tolima, Colômbia

Bracken fern or "Helecho Macho" (Pteridium aquilinum) is one of the most common weeds in the meadows of the mountain ranges of Colombia. Consumption of this palatable plant by bovines causes a disease known as bovine enzootic hematuria and esophageal neoplasia. The toxic effect of the plant in bovines is caused by the ptaquiloside, main carcinogenic toxin of this plant. In this study, eight samples of Bracken fern in different phenologic stages and 16 milk samples collected in two towns of Tolima, Líbano and Murillo, were analized. In both cases, Highperformance liquid cromatography (HPLC) coupled with Mass Spectrometry (MS) was used. In all Bracken fern samples analized, detectable levels of ptaquilosides were observed, ranging from 23 to 1194 µg/g, with a 342 µg/g mean. No detectable levels of ptaquilosides were found in the milk samples. The results show a great variation in the content of ptaquilosides depending on the phenologic stage and collection site of the plant. It is posible that the reported instability of the ptaquiloside and the processing given to the milk samples could have caused the lack of detection of the compound in milk

El "helecho macho" (Pteridium aquilinum) es una de las malezas más comunes en praderas de la región de cordillera en Colombia. El consumo de esta planta palatable por parte de los bovinos causa una enfermedad conocida como hematuria enzoótica bovina y neoplasias de esófago. El efecto tóxico de la planta en los bovinos es causado por el ptaquilósido, principal toxina carcinogénica de esta planta. En el presente estudio se analizaron ocho muestras en diferentes estados fenológicos del helecho obtenidas en dos municipios del departamento del Tolima (Líbano y Murillo) y 16 muestras de leche de la misma zona. En ambos casos se utilizó como técnica analítica cromatografía líquida de alta eficiencia (HPLC) acoplada a espectrometría de masas (MS). En todas las muestras analizadas se observaron niveles detectables de ptaquilósido, con un rango de 23 a 1.194 µg/g y un promedio de 342 µg/g. No se encontraron niveles detectables de ptaquilósido en ninguna de las muestras de leche. Los resultados del estudio muestran la gran variación en el contenido de ptaquilósido dependiendo del estado fenológico de la planta y del sitio de colecta. Es posible que la reportada inestabilidad del ptaquilósido y el tipo de procesamiento dado a las muestras de leche haya ocasionado la no detección del compuesto en las mismas.

A samambaia (Pteridium aquilinum) é uma das ervas daninhas mais comuns nas pastagens na região de cordilheira na Colômbia. O consumo desta planta palatável para os bovinos, causa uma doença conhecida como hematúria enzoótica bovina e neoplasias de esôfago. O efeito tóxico da planta nos bovinos é causado pelo ptaquilosídeo, principal toxina carcinogênica desta planta. No presente estudo foram analisadas oito amostras em diferentes estágios fenológicos da samambaia e 16 amostras de leite obtidas em dois municípios do departamento de Tolima (Líbano e Murillo). Em ambos os casos foi utilizada como técnica analítica a cromatografia líquida de alta eficiência (HPLC) acoplada a espectrometria de massas (MS). Em todas as amostras analisadas foram observados níveis detectáveis de ptaquilosídeo, com um rango de 23 a 1.194 µg/g e uma média de 342 µg/g. Não foram encontrados níveis detectáveis de ptaquilosídeo em nenhuma das amostras de leite. Os resultados do estudo mostram a grande variação no conteúdo de ptaquilosídeo dependendo do estágio fenológico da planta e do local da coleta. É possível que a reportada instabilidade do ptaquilosídeo e o tipo de processamento dado às amostras de leite seja a causa da falta de detecção do composto nas amostras de leite analisadas.

Isolation and characterisation of 13 pterosins and pterosides from bracken (Pteridium aquilinum (L.) Kuhn) rhizome.

Systematic phytochemical investigations of the underground rhizome of Pteridium aquilinum (L.) Kuhn (Dennstaedtiaceae) afforded thirty-five pterosins and pterosides. By detailed analysis of one- and two-dimensional nuclear magnetic resonance spectroscopy, circular dichroism (CD) and high-resolution mass spectrometric data, thirteen previously undescribed pterosins and pterosides have been identified. Interestingly, for the first time 12-O-ß-D-glucopyranoside substituted pterosins, rhedynosides C and D, and the sulfate-containing pterosin, rhedynosin H, alongside the two known compounds, histiopterosin A and (2S)-pteroside A2, were isolated from the rhizomes of subsp. aquilinum of bracken. In addition, six-membered cyclic ether pterosins and pterosides, rhedynosin A and rhedynoside A, are the first examples of this type of pterosin-sesquiterpenoid. Additionally, the three previously reported compounds (rhedynosin I, (2S)-2-hydroxymethylpterosin E and (2S)-12-hydroxypterosin A) were obtained for the first time from plants as opposed to mammalian metabolic products. Single crystal X-ray diffraction analysis was applied to the previously undescribed compounds (2R)-rhedynoside B, (2R)-pteroside B and (2S)-pteroside K, yielding the first crystal structures for pterosides, and three known pterosins, (2S)-pterosin A, trans-pterosin C and cis-pterosin C. Rhedynosin C is the only example of the cyclic lactone pterosins with a keto group at position C-14. Six selected pterosins ((2S)-pterosin A, (2R)-pterosin B and trans-pterosin C) and associated glycosides ((2S)-pteroside A, (2R)-pteroside B and pteroside Z) were assessed for their anti-diabetic activity using an intestinal glucose uptake assay; all were found to be inactive at 300 µM.

Land management of bracken needs to account for bracken carcinogens--a case study from Britain.

Bracken ferns are some of the most widespread ferns in the World causing immense problems for land managers, foresters and rangers. Bracken is suspected of causing cancer in Humans due to its content of the carcinogen ptaquiloside. Ingestion of bracken, or food and drinking water contaminated with ptaquiloside may be the cause. The aim of this study was to monitor the content of ptaquiloside in 20 bracken stands from Britain to obtain a better understanding of the ptaquiloside dynamics and to evaluate the environmental implications of using different cutting regimes in bracken management. The ptaquiloside content in fronds ranged between 50 and 5790 µg/g corresponding to a ptaquiloside load in the standing biomass of up to 590 mg/m(2) in mature fronds. Ptaquiloside was also found in the underground rhizome system (11-657 µg/g) and in decaying litter (0.1-5.8 µg/g). The amount of ptaquiloside present in bracken stands at any given time is difficult to predict and did not show any correlations with edaphic growth factors. The content of ptaquiloside turned out to be higher in fronds emerging after cutting compared to uncut fronds. Environmental risk assessment and bracken management must therefore be based on actual and site specific determinations of the ptaquiloside content. Care must be taken to avoid leaching from cut ferns to aquifers and other recipients and appropriate precautionary measures must be taken to protect staff from exposure to bracken dust.