----------------- INFORMACIÓN GENERAL ------------------- 1. Título del dataset Use of carrageenan in the screening of natural anti-inflammatory molecules in fish: in vitro effects of Chiliadenus glutinosus extracts 2. Autoría: Jose Carlos Campos-Sánchez, Francisco A. Guardiola, María Ángeles Esteban* [Rellenar la información de todos los autores siguiendo el siguiente formato. Repetir el esquema, uno para cada autor.] Nombre: Jose Carlos Campos-Sánchez Institución: Universidad de Murcia Correo electrónico: josecarlos.campos@um.es ORCID: 0000-0003-0303-5412 Nombre: Francisco A. Guardiola Institución: Universidad de Murcia Correo electrónico: faguardiola@um.es ORCID: 0000-0002-1018-5446 Nombre: María Ángeles Esteban Institución: Universidad de Murcia Correo electrónico: aesteban@um.es ORCID: 0000-0002-6264-1458 3. Fecha de recogida de los datos (fecha única o rango de fechas): [12-03-2021---01-10-2021] 4. Fecha de depósito de los datos: [27-02-2024] 5. Idioma del conjunto de datos: Inglés ------------------------ INFORMACIÓN METODOLÓGICA ------------------------ 1.Descripción de la metodología utilizada para generar el conjunto de datos. 2.1. Plant collection and preparation of plant extracts Entire plants of C. glutinosus were collected in August 2019 from Lorca (Murcia, Spain, Latitude 37°49′27.8461″N and longitude 1°40′50.5888″W) and a sample was deposited in the herbarium of the University of Murcia (MUB 71209). The plants were dried at room temperature (RT) for 15 days before being ground to a fine powder and stored in dark conditions at 4ºC until analysis. Leaves and flowers were used to prepare three extracts by maceration using water, ethanol, and methanol as solvents (due to their high polarity, low cost, and eco-friendly nature). The aqueous extraction was prepared according to the method described by Othmen et al. [38], while ethanolic and methanolic extracts were obtained according to Mekonnen et al. [39]. The dried extracts were weighed and diluted with Phosphate-Saline Buffer (PBS; 11.9 mM Phosphates, 137 mM NaCl, and 2.7 mM KCl, pH 7.4) (Fisher Bioreagents) in the case of aqueous extract, and with dimethyl sulfoxide (DMSO, Sigma) in the case of ethanol and methanol extracts. In all cases, extracts were diluted to a stock concentration of 10 mg mL-1. For all experiments, the three extracts of C. glutinosus were diluted with the correspondent culture medium (see below) and adjusted to final ranging solutions of 0 (Control; PBS for aqueous extract or DMSO for ethanolic and methanolic extracts), 0.001, 0.01, 0.125, 0.25, 0.5, and 1 mg mL-1. 2.2. Total antioxidant activity Plant extracts previously adjusted using PBS to final solutions (as above explained) were used to determine the antioxidant activity with the method of ABTS [2,2′-azino-bis-3-(ethylbenzothiazoline-6-sulphonic acid)] radical [40]. The quenching of the absorbance at 730 nm was compared with a standard curve made with ascorbic acid to determine the total antioxidant activity, and results were expressed as ascorbic acid equivalents (mM). 2.3. Bactericidal activity Bactericidal activity determination against three Gram-negative marine bacteria of the Vibrionaceae family (V. harveyi strain Lg 16/00, V. anguillarum strain CECT4344 and P. damselae subsp. piscicida strain PP3) and a Gram-positive bacterium of the Flavobacteriaceae family (Tenacibaculum maritimum) of the three extracts of C. glutinosus, was carried out based on the protocol described by Guardiola et al. with some modifications [41]. In all cases, culture medium alone and PBS were used for negative and positive controls, respectively, and the results were expressed as the percentage of surviving bacteria in relation to the number of bacteria from positive controls (100%). 2.4. Cytotoxic activity against PLHC1 tumour cell line Plant extracts at the final concentration (see the section 2.1.) were used for the determination of cytotoxic activity against a fish tumour cell line: PLHC1 (ATCC® CRL2406™, hepatocellular carcinoma from Poeciliopsis lucida), as described Garcia-Beltran et al. [42]. Culture medium alone was used for negative control, while cells in the same culture medium were used as positive control. 2.5. Head-kidney leucocytes (HKLs) isolation, viability, and immune parameters 2.5.1. Animals Nine specimens (128.49 g ± 3.84 g, 18.11 cm ± 0.25 cm) of gilthead seabream (S. aurata L.) supplied from a local farm (Mazarrón, Spain), were acclimatized for one month in the Marine Fish Facilities at the University of Murcia (Spain) in a recirculation system connected to individual tanks of 450L equipped with an aeration system, biological and mechanical filters. Water was maintained at 20 ± 2 °C, 28‰ salinity and quality parameters (ammonia, nitrate, nitrite) were assessed weekly, while the light-dark cycle photoperiod was set at 12h:12 h. A commercial diet (Skretting, Spain) was provided to the fish at a daily feeding rate of 2% of the biomass. The experimental protocols were approved by the Ethical Committee of the University of Murcia (permit number A13160416). 2.5.2. HKLs isolation and incubation Fish were anesthetized with clove oil (20 mg L-1, Guinama®) and bled from the caudal vein. Excised head-kidney was cut into small fragments and leucocytes were isolated according to Esteban et al. [43]. All extracts prepared, as was mentioned in the section 2.1., were measured in an osmometer (Wescor), resuspended in RPMI and diluted until obtain final concentrations. λ-Carrageenan (Sigma, CAS Number: 9064-57-7) was diluted into sterile PBS and a stock solution of 10 mg mL-1 was prepared. Then, the stock solution of λ-carrageenan was resuspended in sRPMI and serial dilutions for each concentration were prepared. Pryor to carrying out the assays, the osmolarity of these solutions was measured in an osmometer (Wescor) to avoid effects due to this parameter. For each fish and assay, samples of 500 μL of seabream HKLs (previously adjusted to 2 x 107 cells mL-1) were dispensed into 24-well plates (Nunc). Then, two consecutive experiments were carried out. For the first experiment, HKLs were incubated with 500 μL of each C. glutinosus extract solution to make final concentrations of 0, 0.001, 0.01, 0.125, 0.25, 0.5, and 1 mg mL-1. Cells were incubated with each extract for 24 h at 25 °C, 85 % humidity and 5% CO2. In the assay, we used HKLs from 6 independent specimens (not pooled HKLs) and assayed separately. For the second experiment, HKLs were incubated with 250 μL of λ-carrageenan at a final concentration of 0 (PBS diluted in sRPMI; control) and 1,000 μg mL-1, and 250 μL of C. glutinosus aqueous extract [at a final concentration of 0 (PBS diluted in sRPMI; control), 0.25 and 0.5 mg mL-1, at the same conditions described for the first experiment. 2.5.3. HKLs viability Viability of HKLs was assessed by flow cytometry using propidium iodide (PI) to determine the abundance of viable leucocytes, according to Cuesta et al. [44]. Positive controls consisted in cells lysed with 0.02% cetyltrimethyl ammonium bromide (CTAB, Sigma) and dead cells were estimated as the percentage of cells with PI (red-PI fluorescent cells). For each sample, the total viability of leucocytes was determined as well as the specific viability for acidophilic granulocytes (R1), monocytes (R2) and lymphocytes (R3), which were represented in dot plots according to SSC and FSC. 2.5.4. Immune cellular parameters The peroxidase activity in HKLs was measured by using the method described by Quade & Roth [45] and standard samples without HKLs were used as blanks. The respiratory burst activity of HKLs was assayed by chemiluminescence [46] and the kinetic of the reactions was analysed by calculating the slope min-1 of each curve. The phagocytic activity of HKLs was studied by flow cytometry using heat killed lyophilized Saccharomyces cerevisiae (strain S288C) labelled with fluorescein isothiocyanate (FITC, Sigma), according to Rodríguez et al. [47,48]. Standard samples of FITC-labelled S. cerevisiae or HKLs were included in each phagocytosis assay. Phagocytic ability was expressed as the percentage of cells with one or more ingested yeast (green-FITC fluorescent cells) within the phagocytic cell population whilst the phagocytic capacity was the mean fluorescence intensity. 2.5.5. HKLs ultrastructure To determine possible morphologic alterations in HKLs after being incubated with λ-carrageenan, aqueous extract of C. glutinosus, the samples were processed and studied for transmission electron microscopy (TEM) [49]. After exposure for 24 h, the HKLs were centrifuged (400 x g, 5 min, 22 °C), washed in 250 μL of sRPMI and immediately fixed with 200 μL of 2.5% glutaraldehyde in 0.1 M cacocylate buffer (pH 7.2–7.4, 5-10 min, 4°C), then post-fixed for 2 h in 1% OsO4 and embedded in Epon. Semithin and ultrathin sections were obtained with a Reichert-Jung Ultracut and stained with toluidine blue and contrasted with uranyl acetate and lead citrate, respectively. Ultrathin sections were examined with a transmission electron microscope (TEM, Zeiss EM 10C). 2.5.6. HKLs gene expression by real-time PCR The sequences of the selected genes were obtained from a gilthead seabream database [50]. The Open Reading Frames (ORF) were located using the ExPASy translation software (SIB Bioinformatics Resource Portal) and an additional check was performed using NCBI BLAST sequence alignment analysis (NIH). The primers were designed with the Thermo Fisher OligoPerfectTM tool, according to the following criteria: i) each individual oligonucleotide was composed of 20 nucleotides, ii) the size of the amplicon had between 100 and 120 nucleotides, iii) with a guanidine-cytosine (GC)% between 55 % and 60 %, iv) a semi-naturalization temperature (Melting temperature) as close as possible to 60 °C, and v) the selection of primers that self-inhibit forming hairpins was avoided as far as it was possible, in order not to hinder the amplification reaction. The primer used are presented in Table 1. After 24 h of the incubation of the HKLs with λ-carrageenan and C. glutinosus, cells were obtained by centrifugation (400 x g, 10 min, 22° C), and the pellets were stored at -80°C for gene expression analysis. Total RNA was extracted with PureLink® RNA Mini Kit (Life Technologies) as indicated by the manufacturer and the quantification and purification was assessed by using the Nanodrop® spectrophotometer; the 260:280 ratios were 1.8–2.0. The RNA was then treated with DNase I (Promega) to remove genomic DNA contamination. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the SuperScript IV reverse transcriptase (Life Technologies) with an oligo-dT18 primer. In the present study, the expression of the selected genes (see Table 1) was evaluated by real-time qPCR with QuantStudio™ Real-Time PCR System Fast (Life Technologies). Reaction mixtures [containing 5 µL of SYBR Green supermix, 2.5 µL of primers (0.6 µM each) and 2.5 µL of cDNA template] were incubated for 10 min at 95 ºC, followed by 40 cycles of 15 s at 95 ºC, 1 min at 60 ºC, and finally 15 s at 95 ºC, 1 min at 60 ºC and 15 s at 95 ºC. The gene expression was analysed using the 2−ΔCt method [51], which was performed as described elsewhere [52]. The specificity of the reactions was analysed using samples without cDNA as negative controls. For each mRNA, gene expression was normalized with the geometric mean of ribosomal protein (s18) and elongation factor 1-alfa (ef1a) RNA content in each sample. Gene names follow the accepted nomenclature for zebrafish (http://zfin.org/). In all cases, each PCR was performed on triplicate samples. 2.6. Statistical analysis The results were expressed as mean ± standard error of the mean (SEM). Data were analysed by One-way ANOVA (followed by Tukey tests) to determine differences between experimental groups, respectively, in the first experiment. In the second experiment data were analysed by Two-way ANOVA (followed by Tukey tests) to determine differences between the doses of λ-carrageenan and the increasing concentrations of C. glutinosus. Normality of the data was previously assessed using a Shapiro-Wilk test and homogeneity of variance was also verified using the Levene test. All statistical analyses were conducted using the computer package SPSS (25.0 version; SPSS Inc., Chicago, IL, USA) for Windows. The level of significance used was P < 0.05 for all statistical tests. 2. Software o instrumentos necesarios para interpretar los datos: [Incluir la versión del software. Si hace falta un software específico de acceso restringido, explicar cómo obtenerlo. Valorar si es posible cambiar el conjunto de datos a un formato abierto (recomendado).] Data analysis: Computer package SPSS (25.0 version; SPSS Inc., Chicago, IL, USA) for Windows. 3. Procedimientos seguidos para asegurar la calidad de los datos Positive and negative controls in each assay. Samples and controls in triplicates to avoid pipetting errors. ------------------------ ESCTRUCTURA DE LOS ARCHIVOS --------------------------- 1. Nombres de archivos [Mencionar todos los archivos incluidos en el conjunto de datos, con el nombre y la extensión (.csv, .pdf, etc.) de cada fichero]. EA1 - Raw data.xls 2. Formato de los archivos: Libro de Excel 97-2003 (*.xls) ------------------------ MÁS INFORMACIÓN ------------------------ [Incluir cualquier otra información sobre el conjunto de datos que no haya quedado reflejada en esta plantilla y que se considere relevante.]