Yessotoxins in Mollusks of the Galician Coast from 2014 to 2022: Variability, Biotransformation, and Resistance to Alkaline Hydrolysis.

The presence of yessotoxins (YTXs) was analyzed in 10,757 samples of Galician bivalves from 2014 to 2022. Only YTX and 45-OH YTX were found. YTX was detected in 31% of the samples, while 45-OH YTX was found in 11.6% of them. Among the samples containing YTX, 45-OH YTX was detected in 37.3% of cases. The maximum recorded levels were 1.4 and 0.16 mg of YTX-equivalentsg-1, for YTX and 45-OH YTX, respectively, which are well below the regulatory limit of the European Union. The YTX and 45-OH YTX toxicities in the raw extracts and extracts subjected to alkaline hydrolysis were strongly and linearly related. Due to the lack of homo-YTX in Galician samples, the effect of alkaline hydrolysis on homo-YTX and 45OH-Homo-YTX was only checked in 23 additional samples, observing no negative effect but a high correlation between raw and hydrolyzed extracts. Hydrolyzed samples can be used instead of raw ones to carry out YTXs determinations in monitoring systems, which may increase the efficiency of those systems where okadaic acid episodes are very frequent and therefore a higher number of hydrolyzed samples are routinely analyzed. The presence of YTX in the studied bivalves varied with the species, with mussels and cockles having the highest percentages of YTX-detected samples. The presence of 45-OH YTX was clearly related to YTX and was detected only in mussels and cockles. Wild populations of mussels contained proportionally more 45-OH YTX than those that were raft-cultured. Spatially, toxin toxicities varied across the sampling area, with higher levels in raft-cultured mussels except those of Ría de Arousa. Ría de Ares (ARE) was the most affected geographical area, although in other northern locations, lower toxin levels were detected. Seasonally, YTX and 45-OH YTX toxicities showed similar patterns, with higher levels in late summer and autumn but lower toxicities of the 45-OH toxin in August. The relationship between the two toxins also varied seasonally, in general with a minimum proportion of 45-OH YTX in July-August but with different maximum levels for raft-cultured and wild mussel populations. Interannually, the average toxicities of YTX decreased from 2014 to 2017 and newly increased from 2018 to 2021, but decreased slightly in 2022. The relationship between 45-OH YTX and YTX also varied over the years, but neither a clear trend nor a similar trend for wild and raft mussels was observed.

Enzymatic Biotransformation of 13-desmethyl Spirolide C by Two Infaunal Mollusk Species: The Limpet Patella vulgata and the Cockle Cerastoderma edule.

The presence of a 13-desmethyl Spirolide C isomer (Iso-13-desm SPX C) is very common in some infaunal mollusks in Galicia contaminated with this toxin. Its possible origin by biological transformation was investigated by incubating homogenates of the soft tissues of limpets and cockles spiked with 13-desmethyl Spirolide C (13-desm SPX C). The involvement of an enzymatic process was also tested using a raw and boiled cockle matrix. The enzymatic biotransformation of the parent compound into its isomer was observed in the two species studied, but with different velocities. The structural similarity between 13-desm SPX C and its isomer suggests that epimerization is the most likely chemical process involved. Detoxification of marine toxins in mollusks usually implies the enzymatic biotransformation of original compounds, such as hydroxylation, demethylation, or esterification; however, this is the first time that this kind of transformation between spirolides in mollusks has been demonstrated.

Spirolides in Bivalve Mollusk of the Galician (NW Spain) Coast: Interspecific, Spatial, Temporal Variation and Presence of an Isomer of 13-Desmethyl Spirolide C.

Spirolides are cyclic imines whose risks to human health have not been sufficiently evaluated. To determine the possible impact of these compounds in Galicia (NW Spain), their presence and concentration in bivalve mollusk were studied from 2014 to 2021. Only 13-desmethyl spirolide C (13desmSPXC) and an isomer have been detected, and always at low concentrations. Mussel, Mytilus galloprovincialis, was the species which accumulated more spirolides, but the presence of its isomer was nearly restricted to cockle, Cerastoderma edule, and two clam species, Venerupis corrugata and Polititapes rhomboides. On average, the highest 13desmSPXC levels were found in autumn-winter, while those of its isomer were recorded in spring-summer. Both compounds showed decreasing trends during the study period. Geographically, the concentration tends to decrease from the southern to the north-eastern locations, but temporal variability predominates over spatial variability.

Paralytic Shellfish Poisoning (PSP) in Mussels from the Eastern Cantabrian Sea: Toxicity, Toxin Profile, and Co-Occurrence with Cyclic Imines.

In the late autumn of 2018 and 2019, some samples taken by the official monitoring systems of Cantabria and the Basque Country were found to be paralytic shellfish poisoning (PSP)-positive using a mouse bioassay. To confirm the presence of PSP toxins and to obtain their profile, these samples were analyzed using an optimized version of the Official Method AOAC 2005.06 and using LC-MS/MS (HILIC). The presence of some PSP toxins (PSTs) in that geographical area (~600 km of coast) was confirmed for the first time. The estimated toxicities ranged from 170 to 983 µg STXdiHCl eq.·kg-1 for the AOAC 2005.06 method and from 150 to 1094 µg STXdiHCl eq.·kg-1 for the LC-MS/MS method, with a good correlation between both methods (r2 = 0.94). Most samples contained STX, GTX2,3, and GTX1,4, and some also had NEO and dcGTX2. All of the PSP-positive samples also contained gymnodimine A, with the concentrations of the two groups of toxins being significantly correlated. The PSP toxin profiles suggest that a species of the genus Alexandrium was likely the causative agent. The presence of gymnodimine A suggests that A. ostenfeldii could be involved, but the contribution of a mixture of Alexandrium species cannot be ruled out.

Polyphenolic content and bioactivities of Crataegus oxyacantha L. (Rosaceae).

Solid-Liquid Extraction (SLE) using solvent of different polarities (CHCl3, EtOAc, and n-BuOH) has been applied to leaves and fruits from (Crataegus oxyacantha L.), a deciduous shrub with an expected rich phytochemical profile. The total polyphenols content and the radical scavenging activity of each extract were evaluated. These extracts were analyzed by HPLC-DAD and rutin, quercetin-3-glucoside, caftaric and caffeic acid had been positively identified. The phytochemical study of the ethyl acetate extract of C. oxyacantha, led to the isolation and structural elucidation of quercetin (1); quercetin-3-O-ß-glucoside (2); epicatechin (3); naringenin (4), reported for the first time from this species except caffeic acid and epicatechin. These compounds were identified by 1D and 2D NMR combined analysis as well as by MS and UV.The antimicrobial activity of these extracts has also been tested, showing strong antibacterial activity-solvent dependent-against Gram positive bacteria. Additionally, bactericidal power was demonstrated in fruit extracts.

Polyphenol bioavailability in nuts and seeds by an in vitro dialyzability approach.

An in vitro dialyzability approach has been undertaken to elucidate the bioavailable fraction of the total polyphenols (TPs) of edible nuts and seeds. The TP contents in samples and in dialyzates were assessed by the Folin-Ciocalteu spectrophotometric method. Antioxidant activity was determined in selected samples, using a modified method against Trolox®. TPs and antioxidant activity in nuts/seeds were determined after applying a pressurized liquid extraction sample pre-treatment. High dialyzability ratios were assessed in most nuts/seeds (TP dialyzability percentages within the 25-91% range). The highest TP dialyzability ratios were found in raw Brazil nuts (81 ±â€¯5%), toasted pistachios (88 ±â€¯9%), and fried cashews (89 ±â€¯9%), whereas TPs in pumpkin seeds were found to be very low (TPs were not detected in the dialyzable fraction). TP dialyzability was correlated with the copper content in nuts and seeds.

Matrix solid-phase dispersion as a tool for phytochemical and bioactivities characterisation: Crataegus oxyacantha L._A case study.

The use of a matrix solid-phase dispersion (MSPD) process to extract polyphenols from hawthorn (Crataegus oxyacantha L.) a deciduous shrub with an expected rich phytochemical profile, has been evaluated. MSPD extracts of fruits and leaves have an outstanding content of polyphenols, although the particular phenolic profile is solvent dependent. The extracts were analysed by HPLC-DAD for the accurate identification of the major bioactive polyphenols, some of which have never been described for this species. MSPD has proven to be a good alternative to the classic methods of obtaining natural extracts, fast and with low consumption of organic solvents, therefore, environmentally friendly. The bioactivities can be considered also very remarkable, revealing extracts with high levels of antioxidant activity.

Profiling the Fatty Acids Content of Ornamental Camellia Seeds Cultivated in Galicia by an Optimized Matrix Solid-Phase Dispersion Extraction.

Camellia (genus of flowering plants of fam. Theaceae) is one of the main crops in Asia, where tea and oil from leaves and seeds have been utilized for thousands of years. This plant is excellently adapted to the climate and soil of Galicia (northwestern Spain) and northern Portugal where it is grown not only as an ornamental plant, but to be evaluated as a source of bioactive compounds. In this work, the main fatty acids were extracted from Camellia seeds of four varieties of Camellia: sasanqua, reticulata, japonica and sinensis, by means of matrix-solid phase dispersion (MSPD), and analyzed by gas chromatography (GC) with MS detection of the corresponding methyl esters. MSPD constitutes an efficient and greener alternative to conventional extraction techniques, moreover if it is combined with the use of green solvents such as limonene. The optimization of the MSPD extraction procedure has been conducted using a multivariate approach based on strategies of experimental design, which enabled the simultaneous evaluation of the factors influencing the extraction efficiency as well as interactions between factors. The optimized method was applied to characterize the fatty acids profiles of four Camellia varieties seeds, allowing us to compare their fatty acid composition.