The fibers obtained over the stepwise process were characterized through infra-red/Raman spectroscopy and cyclic voltammetry. The final fiber meshes showed enhanced electroconductivity . Fiber meshes biocompatibility was evaluated using fibroblasts and neural stem cells (NSCs) following, respectively, the ISO10993 guidelines and standard adhesion/proliferation assay. NSCs cultured on PH1000 H2SO4 fibers presented normal morphology and high proliferation rates (0.37 day−1 vs. 0.16 day−1 for culture plate), indicating high biocompatibility for NSCs. Still, the low initial NSC adhesion of 7% calls for improving seeding methodologies. PAN:PEDOT:PSS fibers, here successful produced for the first time, have potential applications in neural tissue engineering and soft electronics.This study describes, for the first time, the successful incorporation of poly(ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) in Poly(acrylonitrile) (PAN) fibers. While electroconductive PEDOT:PSS is extremely challenging to electrospun into fibers. Therefore, PAN, a polymer easy to electrospun, was chosen as a carrier due to its biocompatibility and tunable chemical stability when cross-linked, particularly using strong acids. PAN:PEDOT:PSS blends, prepared from PEDOT:PSS Clevios PH1000, were electrospun into fibers (PH1000) with a diameter of 515 ± 120 nm, which after being thermally annealed (PH1000 24H) and treated with heated sulfuric acid (PH1000 H Elect−1 . PEDOT:P−1  and Yuk −1  demonstrPEDOT:PSS has been used in the development of platforms suitable for electrical stimulation, scaffolds for neural TE ,13,14, a2+) [Electrospinning is a technique that allows the production of nanometric-sized fibers. The obtained fibers have many advantages for TE applications, including (a) structural resemblance to native extracellular matrix, (b) high surface area for cell adhesion, and (c) ability for functionalization  with bioactive motifs and/or drugs. PEDOT:PSS cannot be electrospun alone due to unfavorable chain entanglement during the solvent evaporation stage , and thu2+) . As such2+) ,24,25. H2+) ; and (b)2+) ,27, and Poly(acrylonitrile) (PAN) is a promising polymer for biomedical applications, and a candidate to be a carrier polymer for PEDOT:PSS. This material presents numerous advantages, including good mechanical properties, chemical stability, and ease of industrial production . PAN canA dehydrogenation process can then take place at 300–400 °C, leading to highly π-delocalized system (stabilized PAN fiber) and, at slightly higher temperatures, there starts to occur the lateral fusion of chains (cross-linking). Thermal treatment at higher temperatures (above 600 °C) leads to graphitization with the formation of PAN-derived carbon fibers. The process can be performed under both inert ,30 or reIn this work, we propose the development of new electroconductive blend to produce electrospun fibers of PEDOT:PSS and PAN. There are no reports in the literature for electroconductive PAN:PEDOT:PSS blend electrospun fibers, although some reports describe similar electroconductive fibers using different strategies ,40,41,42® medical adhesive silicone type A) was obtained from Biesterfeld Spezialchemie Ibérica, S.L.U. . The primary antibody Anti-(Sex determining region Y)-box 2 (Anti-SOX2) (rabbit) (orb607714) was purchased from Byorbyt . Ultra-low attachment 24-well plates (flat bottom) were obtained from Corning . Dimethylformamide (DMF) was purchased from Fisher Scientific. PEDOT:PSS dispersions CleviosTM PH 1000  and CleviosTM P VP AI 4083  were purchased from Heraeus . Sulfuric acid (95.0–97.0%) was purchased from Honeywell . 3--2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide ≥99.5% (DMSO), glucose, human recombinant insulin, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), paraformaldehyde (PFA) , phalloidin–tetramethylrhodamine B isothiocyanate and poly(L-ornithine hydrobromide) were purchased from Merck-Millipore . Human recombinant epidermal growth factor (EGF) and human recombinant fibroblast growth factor 2 (FGF-2) were purchased from Peprotech . PAN  was purchased from Polysciences Inc . The primary antibody Anti-Nestin (Anti-NES) (mouse) (MAB1259) was purchased from R&D systems . Dulbecco’s phosphate buffer saline (PBS), Dulbecco’s Modified Eagle’s Medium (DMEM:F12 + glutamax (1×), Fetal Bovine Serum (FBS), Pen/Strep mixture , Antibiotic–Antimycotic mixture  (Anti-Anti), N2-supplement (100×), B27-supplement (50×), Alamar Blue® cell viability reagent, calcein AM, ethidium bromide, triton-x-100 , secondary antibodies Alexa 488 anti-mouse IgG and Alexa 546 anti-rabbit IgG were purchased from Thermofisher . Frozen stocks of ReNCell-VM (SCC008)  (Passage 8) and L-929 mouse fibroblasts (ATCC CCL1) (Passage 6) were used in these studies.Medical glue . The obtained PEDOT:PSS pellets were then subjected to 3 consecutive cycles of heat-treatment at 130 °C for 30 min and mechanical crushing. An amount of 10 mL of DMF:DMSO (9:1) was added to the obtained solid. Complete dispersion of PEDOT:PSS pellets was achieved by constant agitation (300–350 rpm) using magnets for at least 2 days (CleviosTM AI4083) or 5 days (CleviosTM PH1000).To 10 mL of commercially available PEDOT:PSS dispersions in water CleviosPAN and PAN:PEDOT:PSS solutions were prepared by adding 1 g of PAN to 10 mL of DMF or to 10 mL of PEDOT:PSS dispersion in DMF:DMSO (9:1). Solutions were left to agitate for 1–3 h at 85 °C. If not used promptly, PAN:PEDOT:PSS solutions were stored at 4 °C for up to 24 h.−1, distance from needle to collector of 25 cm, temperature of 25 °C, relative humidity of 50%. The electrospinning setup also included a high voltage power source , a syringe pump  and a teflon tube . Samples were collected on an aluminum foil-coated static copper collector. An air-blower was used to force fiber deposition on the collector.PAN and/or PAN:PEDOT:PSS solutions were electrospun using a spinneret system  and a 21G needle  under the following conditions: voltage of 20 kV, flow rate at 1.0 mL hTM P VP AI 4083—“Samples AI4083”; and PAN:PEDOT:PSS fibers made with PEDOT:PSS dispersion CleviosTM PH 1000—“Samples PH1000”.Electrospun fiber samples were named according to their composition, in particular to the source of PEDOT:PSS used in their composition. They are: pristine PAN fibers—“Samples PAN”; PAN:PEDOT:PSS fibers made with PEDOT:PSS dispersion CleviosThe obtained electrospun fibers were subjected to thermal treatment at a steady temperature (210 °C) in a drying oven  for 1, 5, 10, and 24 h under ambient pressure. Samples were then collected and let to cool down to RT before use.PAN and PAN:PEDOT:PSS fibers were subjected to a final treatment with heated sulfuric acid. Briefly, fibers were incubated for 24 h with sulfuric acid for complete soaking. The collected fibers, soaked in sulfuric acid, were then uniformly heated for 30 min at 130 °C, cooled to RT and thoroughly washed with distilled water. Other incubation conditions are also reported as part of the optimization steps to enhance the electroconductivity of PAN:PEDOT:PSS fibers, including soaking with sulfuric acid for 1 h, and heat treatments at 100 °C or 150 °C.PEDOT:PSS films were produced by spin-coating  of PEDOT:PSS dispersions with 10% DMSO on pre-cleaned glass/quartz coverslips. The samples were dried at 125 °C. Further processing was used to match the processing conditions of PAN:PEDOT:PSS fibers, as described in The morphology of the electrospun fiber mats was evaluated using SEM  after coating with a thin layer of gold/palladium. The average diameter of the electrospun fiber samples was determined for 50–100 individual fibers using NIH ImageJ software . The diameters obtained were averaged and the histograms were plotted using Microsoft Excel.The spectra of the spin-coated PEDOT:PSS films, prepared on quartz coverslips, were obtained using a V-730 UV-Visible Spectrophotometer  in the 200–1100 nm range (resolution of 0.2 nm). The obtained spectra were normalized to the maximum peak found in the 225–235 nm region.® ATR accessory . Transmittance spectra were obtained from 400 to 4000 cm−1  at room temperature and an automatic baseline correction treatment was applied using the acquisition software. The obtained spectra were normalized to the maximum peak obtained.The obtained PAN and PAN:PEDOT:PSS fibers were analyzed by ATR—FTIR using a Spectrum Two FT-IR Spectrometer , equipped with a Pike Technologies MIRacle−1), and Renishaw software Wire 4. The obtained spectra were normalized to the maximum peak found in the 1200–1700 cm−1 region.The spectra of PAN fibers, PAN:PEDOT:PSS fibers, and PEDOT:PSS spin-coated films were obtained using a Renishaw In-Via confocal Raman microscope , using a 532 nm excitation and 10× objective .PAN fibers, PAN:PEDOT:PSS fiber mats and PEDOT:PSS spin-coated films were analyzed by the four-probe method to evaluate electroconductivity. Four 50 nm thick gold stripes were deposited by thermal evaporation ) to improve the electrical contact between the samples and the measurement equipment. The electrical resistance of 5 different films was measured by the 4-point probe method, using a current source Keithley 2400 DC power source  and a multimeter Agilent 34401A Multimeter . Finally, the thicknesses of the fiber mats and spin coated films, used to calculate the electroconductivity, were measured, respectively, using a caliper and a Bruker’s Dektak−1 to 0.2 V s−1.Cyclic voltammetry scans were run for PAN fibers and PAN:PEDOT:PSS fibers using working and auxiliary carbon screen printed electrodes (SPEs) and an Ag|AgCl reference electrode , connected to a potentiostat . Fiber samples were deposited on the working electrode surface. The electrodes were dipped in 2.5 mL of degassed PBS 0.01 M, pH 7.4, as electrolytic medium. Cycles were run in a potential window of −0.7 V to +0.7 V, with scan rates ranging from 0.01 V sn = 5 different samples) and the crosshead speed was set constant at 10 mm min−1 during the uniaxial test. Young’s modulus was calculated from the 0–15% strain linear region in the stress−strain curve. The ultimate tensile strength (UTS) and the maximum extension were measured from the highest peak of the stress−strain curve.The mechanical properties of PAN and PAN:PEDOT:PSS fibers were analyzed using a texture analyzer  equipped with 50 N load cell and tensile grips. Specimens were cut into rectangular strips  or 80,000 cells cm−2 (indirect contact). PAN fibers, PAN:PEDOT:PSS fibers, and latex (positive control) were sterilized with UV (1.5 h per sample side) before further processing. Samples were then incubated in medium composed of DMEM + 10% FBS + 1% anti-anti for 24 h (6 cm2 mL−1) for lixiviate extraction (indirect contact). For direct contact, each of the samples were placed on top of the cell’s monolayer for 24 h before image analysis on an optical microscope . For the indirect contact assay, the media of L-929 cells were switched to media containing lixiviates from processing materials and the cells were left to incubate for 24 h, after which cell viability was quantified using MTT assay. Briefly, after PBS washing, cells were incubated with MTT solution (1 mg mL−1) for 2 h at 37 °C. The resulting formazan salt was then dissolved using 0.1 M HCl in isopropanol and absorbance was quantified at 570 nm. Relative cell viability was calculated using the negative control as the reference.Cytotoxicity tests were performed following the recommendations of ISO 10993-5 and 10993-12 guidelines. Briefly, mouse fibroblast cell line L-929 (Passage 10) were cultured in Dulbecco’s modified eagle’s medium (DMEM + 10% FBS + 1% Anti-Anti) at 37 °C and 5% COc-myc) were used as a cell model for neural applications [−1) and laminin (2 h—10 µg mL−1) coated plates in supplemented N2 medium at 37 °C and 5% CO2, as recommended [−1), insulin (20 µg mL−1), and pen-strep (1:100). N2 medium was then supplemented with EGF (20 ng mL−1), FGF-2 (20 ng mL−1), and B27 (20 µL mL−1).ReNCell-VM  and laminin (2 h—10 µg mL−1) before being seeded with ReNCell-VM (Passage 10) at 50,000 cells cm−2. Supplemented N2 medium was added 1 h after seeding (37 °C and 5% CO2) to promote initial cell attachment. Medium was exchanged at day 1, when cell adhesion was calculated, at day 2 and then every 2 days thereafter until the end of the experiment (day 7). Cellular metabolic activity was assessed using Alamar Blue® by measuring fluorescence at 590 nm (excitation at 560 nm) at days 1, 4, and 7. Equivalent cell number was determined using a calibration curve. Cell adhesion (Equation (1)), growth rate, (Equation (2)) and duplication time (Equation (3)) were calculated using the following formulae [Different electrospun samples, previously immobilized on glass coverslips with medical glue, were first UV sterilized for 1.5 h per each sample side and treated with 1% Anti-Anti solution in PBS for 3 h. The samples were then coated with poly(L-ornithine) (overnight—20 µg mLformulae :Cell adh−1), and glucose (0.056 M) in PBS, for 20 min at 37 °C. Cells were then washed, kept in PBS supplemented with glucose (0.056 M), and imaged using an optical microscope  equipped with a fluorescent lamp.At the end of the proliferation assay (day 7), cells were washed once with PBS and then incubated with a staining solution composed of calcein (4 µM), ethidium bromide (2 µM mL−1). Samples were then washed with PBS twice and kept in PBS until imaging.Cell samples were fixed in PFA 4% for 10 min, washed with PBS twice and then permeabilized with staining solution (FBS 5% and Triton-X-100 0.1% in PBS) for 15 min at RT. Cells were incubated with the primary antibodies Anti-NES (1:250) and Anti-SOX2 (1:100) in staining solution, overnight at 4 °C. Next, cells were incubated with the secondary antibodies Alexa 488 IgG Anti-Mouse (1:250) and Alexa 546 IgG Anti-Rabbit (1:250), for 2 h at RT. Counter-staining was performed with DAPI  using the Savitzky–Golay method . Data are presented as mean values ± standard deviations (std). Statistical analysis was performed using Microsoft Excel : significant differences between groups were determined using ANOVA test, followed by post hoc analysis and Bonferroni correction. A PAN and PAN:PEDOT:PSS fibers were electrospun to create electroconductive scaffolds for neural applications. PEDOT:PSS, composed of the electrically conductive component PEDOT and its doping agent PSS, was re-dispersed in DMF:DMSO 9:1) following a modified protocol from Lu and colleagues . The ele:1 followWhen 10% PAN fibers are heat-treated, cyclization followed by dehydrogenation occurs, leading to the formation of a π-system that can stand electrical transport. The extent of this change can be accompanied by observable color changes of PAN fibers from white (starting fibers) to light brown (1 h and 5 h samples) and finally to dark brown (10 h and 24 h samples). SEM images  and respn = 50). Fiber diameter changed when the thermal annealing at 210 °C was performed for 1 h , 5 h , 10 h , and 24 h . When sample PAN 24H was incubated with heated sulfuric acid (PAN H2SO4), the obtained fiber diameter was 612 ± 107 nm (n = 100), which is statistically different from the values obtained for PAN, PAN 10H, and PAN 24H (without acid treatment). Further observed statistical differences found are summarized in The obtained PAN fibers were smooth, homogeneous, and bead-free. Untreated PAN fibers (PAN) had an average fiber diameter of 640 ± 159 nm (n = 100) and PAN:PEDOT:PSS PH1000 pristine  were also smooth, homogeneous, and bead-free (n = 100) and PAN:PEDOT:PSS PH1000 24 h . However, no statistically significant differences between these fibers and their pristine counterparts were found. Incubation with heated sulfuric acid led to morphological changes in samples PAN:PEDOT:PSS AI4083 24 h + H2SO4  and PAN:PEDOT:PSS PH1000 24 h + H2SO4 . The increase in fiber diameter of sample PH1000 H2SO4 is statistically significant with respect to samples PH1000 and PH1000 24H. Moreover, we also observed statistically significant differences between PH1000 H2SO4 and samples AI4083 H2SO4 and PAN H2SO4. The commercial formulations of PEDOT:PSS CleviosTM PH 1000 and CleviosTM P VP AI 4083 have different PEDOT to PSS ratios. As such, the relative amount of PEDOT present in the PH1000 H2SO4 sample is higher than in the AI4083 H2SO4 sample. This suggests that the increase in fiber diameter, observed for sample PH1000 H2SO4, is related to a higher amount of PEDOT (or to a lower content of PSS). This is a quite unexpected result. PEDOT-rich domains are hydrophobic and PSS-rich domains are hydrophilic [2SO4/DMSO = 38.50°) [n = 50) to 612 ± 107 nm (n = 100) when incubated with heated sulfuric acid. This reduction is also unexpected as, should there be a protonation of the imine nitrogens of thermally annealed PAN, we would anticipate an expansion instead. It is hard to find an explanation for these observations. Further studies are necessary to fully understand this phenomenon.The obtained fibers of PAN:PEDOT:PSS AI4083 pristine  . TherefoOur results suggest that thermal annealing of PAN and PAN:PEDOT:PSS fibers had a mixed impact on fiber diameter. We were curious whether these changes could also be associated with the chemical reactions involved in the cyclization/dehydrogenation performed to the fibers. As described in the literature, PAN fibers (control fibers) are typically turned electroconductive after four different sequential reactions, which can affect the obtained fiber diameter due to variable mass loss: cyclization; oxidation/dehydrogenation; carbonization; and graphitization B. CyclizIn our work, PAN thermal annealing was performed under a normal atmosphere. The presence of oxygen is positive for the annealing of PAN, as it can act as a catalyst for PAN dehydrogenation ,33. To s2 bending peaks at 1070 cm−1, 1249 cm−1, and 1455 cm−1, the double peaks at 2870 cm−1 and 2923 cm−1 for CH2 bonds stretching, and the characteristic nitrile , attributed to potential contaminant carbonyl/carboxyl groups in PAN, disappears completely. The changes in FTIR spectrum suggest that after 1 h, the cyclization reaction started, which is supported by the appearance of peak at 1585 cm−1, associated with C=N and C=C groups. After 5 h of thermal annealing (−1 (C=C–H), 1366 cm−1 (CH—methine group) and 1585 cm−1 (C=N and C=C), are clearly visible [−1, 1245 cm−1, and 1070 cm−1 (CH2 groups). This change seems to indicate that the PAN dehydrogenation, expected to occur above ca. 300 °C, occurs upon prolonged heating at 210 °C. After 10 h of thermal annealing (−1 (CH—methine group) and 1585 cm−1 (C=N and C=C), suggesting a progression of cyclization and dehydrogenation with the annealing time. After 24 h of thermal annealing (−1 (C=C–H), 1366 cm−1 (CH—methine group), and 1585 cm−1 (C=N and C=C), associated with aromatic rings formed by the combination of cyclization and dehydrogenation, and a peak at 1245 cm−1 (CH2 bonds), suggesting the presence of residual saturated carbons. At this stage, the majority of peaks characteristic of pristine PAN lost intensity or disappeared, including the double peaks at 2870 cm−1 and 2923 cm−1 (CH2 bonds), the peak at 2243 cm−1 (C−1 and 1070 cm−1 (CH2 groups). After treatment of sample PAN 24H with heated sulfuric acid , at 1657 cm−1 (C=O), and at 1206 cm−1, 1096 cm−1, 1048 cm−1, and 584 cm−1 (CH2). These results suggest partial hydrolysis of thermally annealed PAN with heated sulfuric acid.The spectra of the different PAN samples change with thermal annealing progression, and the majority of the peaks can be identified in the literature ,54,55,56 visible ,57. Othe−1 potentially attributed to the sulfoxide group of PSS. The intensity of peak 1030 cm−1 also decreases with the progression of thermal annealing (2SO4 and PH1000 H2SO4) , 1035 cm−1 (C-S stretching in thiophene ring), and 866 cm−1 (thiophene ring), which are associated with PEDOT:PSS. This might be associated to a higher exposure of PEDOT closer/at the surface, due to a removal of PSS [2SO4 sample, which increased the electroconductivity of the fiber mats.The effect of sulfuric acid treatment of sample PH1000 24H, aiming to improve its electroconductivity, was monitored using FTIR . The goal of PSS . Howeverl of PSS . We beli2SO4. New and unique peaks are clearly visible at 1154 cm−1 (C-O stretching), 1035 cm−1 (C-S stretching in thiophene ring), and 866 cm−1 (thiophene ring), which are associated with PEDOT:PSS. Peaks associated with partial PAN hydrolysis are also observed, similar to what was observed for PAN H2SO4. These include the broad band centered at 3193 cm−1 (O–H), at 1657 cm−1 (C=O), and at 1206 cm−1, 1096 cm−1, 1048 cm−1, and 584 cm−1 (CH2).Treatment of PH1000 24H with heated sulfuric acid (130 °C for 30 min) (−1 (D-band) and 1552 cm−1 (G-band), indicating the presence of carbon-based aromatic polycyclic structures arising from thermal annealing [−1) and a lower intensity D-band (≈1340 cm−1) [Raman spectroscopy  was perfnnealing . A simil40 cm−1) ,62. For 40 cm−1) . This ca−1, 1356 cm−1 and 1522 cm−1. In the PEDOT:PSS control sample  and 1546 cm−1 (G-band), indicating the formation of polycyclic PAN. A small peak is visible at 1450 cm−1, signaling the presence of PEDOT:PSS in PH1000 24H sample, as expected. Treatment with heated sulfuric acid (−1 (HSO4− groups), 1349 cm−1 (D-band), and 1570 cm−1 (G-band). Despite the possible influence of the PEDOT peaks for the FTIR spectrum in the same spectral region, it appears that the intensity of the G-band in PH1000 H2SO4 sample is higher than the D-band. This suggests the formation of a more ordered structure.Sample PH1000 Raman spectrum  × 10−4 S cm−1) and PH1000 H2SO4 ((3.2 ± 5.6) × 10−3 S cm−1) (−1 (1.05 × 10−3 S cm−1) for poly (PVA):PEDOT:PSS 3% electrospun fibers. The values obtained are also higher than those obtained by Abedi and colleagues [−3 S m−1  for chitosan (CS):PVA:PEDOT:PSS electrospun fibers. Finally, the electroconductivity values are higher than those obtained by Park and colleagues [−4 S cm−1) and PVA:PEDOT:PSS (≈9 × 10−6 S cm−1) electrospun fibers, using CleviosTM PH 1000 as the source of PEDOT:PSS. Among these studies, our PH1000 H2SO4 samples are the most electroconductive.The next step in our study was to investigate the consequences of the thermal annealing and sulfuric acid treatment on the electrical and electrochemical properties of PAN and PAN:PEDOT:PSS fibers. The only electroconductive samples in our study are AI4083 H S cm−1) . The elelleagues , who replleagues  for poly−1, i.e., 4.72 S cm−1) or polybenzimidazole  fibers [−1 upon that annealing, though they did not report the electroconductivity of the corresponding fiber mats. Additionally, some works use wet-spinning for the production of PEDOT:PSS-based electrospun fibers. Wang and colleagues [−1 [−1, using a solution of 10% NaSCN as the coagulation bath. The authors attribute such high value to the final tempering step, responsible for PEDOT:PSS chain alignment. The electroconductivity values obtained in both wet-spinning and PEO electrospinning studies are high, but similar studies using the same PVA and PEO-based PEDOT:PSS blends for electrospinning did not yield comparatively high values [−1. However, these fibers require extra synthesis steps before use, additional purification steps to remove impurities and also specialized equipment that make the process more complicated and troublesome for tissue engineering applications. Overall, it is possible to fabricate PEDOT:PSS fibers with higher electroconductivity using alternative production methods  and after appropriate post-processing. Comparatively to all these examples, our electrospun fibers are easily produced using commercially available reagents (PAN and PEDOT:PSS) and require simple post-processing steps to become electroconductive without affecting their morphology and/or porosity. As such, we believe our newly developed PAN:PEDOT:PSS fibers, and respective production method, to be a reliable alternative for the production of electroconductive scaffolds for  tissue engineering and soft electronic applications.There are also other works in the literature that describe the development of PEDOT:PSS-based fibers with higher electroconductivities using diverse strategies. For example, the electroconductivity values attained in the current study are lower than those obtained for PEDOT:PSS-coated electrospun fibers of cellulose (472 S m) fibers ,67. Hida) fibers  report t) fibers  reportedlleagues  reportedgues [−1 ,70. Liu gues [−1  also reph values ,64,65. Mh values  describe−1 in 0.01 M PBS, pH 7.4, are compared in We next evaluated the electrochemical responses of PAN, AI4083, and PH1000 fibers prepared under different thermal annealing conditions. We performed cyclic voltammetry using carbon SPEs, with the fibers being deposited on the working electrode. The cyclic voltammograms recorded at 0.1 V sTM P VP AI 4083 in PAN did not affect the electrochemical properties of the obtained PAN:PEDOT:PSS fibers.The increase in thermal annealing time of PAN fibers  leads toTM PH 1000 in PAN. Finally, electrochemical activity is also improved when compared to samples PH1000 1H, PH1000 5H, and PH1000 10H . At variance with PSS- chains, the sulfate ions are mobile and may improve the ionic conductivity of the fibers, complementing the increase of the PEDOT electronic conductivity [The electrochemical activity of PH1000 fibers C appears1000 10H . Sample ng agent ,75, may ng agent . Additiouctivity .TM PH 1000 films (730 ± 280 S cm−1—sample PH1000f) from the as received aqueous dispersion is higher than the corresponding sample produced using PEDOT:PSS CleviosTM P VP AI 4083 ((4.9 ± 6.3) × 10−4 S cm−1—Sample AI4083f). These results match observations from other works [PAN:PEDOT:PSS fibers can be considered a complex system. The predominance of PAN in the fibers does not allow to directly assess the changes that occur in the PEDOT:PSS sub-system. As such, we used spin-coated PEDOT:PSS films, prepared from a dispersion in DMF-DMSO containing 10% DMSO, to study the effect of processing conditions directly on PEDOT:PSS. The results obtained are summarized in 3 (4.9 ± .3 × 10−4−1 and sample AI4083f increasing to (1.7 ± 1.0) × 10−2 S cm−1. Both DMF and DMSO are solvents described as having positive effects on the electroconductivity of PEDOT:PSS, including enhancement of electroconductivity through pseudo-doping effect and promoting PEDOT segregation from PSS [The electroconductivity of films produced after redispersion of PEDOT:PSS in DMF:DMSO 9:1) changed slightly with respect to that of the films prepared with the as-received aqueous dispersions, with sample PH1000f decreasing to 297 ± 167 S cm:1 change−4 S cm−1) and sample AI4083f ((1.2 ± 0.1) × 10−4 S cm−1) (identified in Heat treatment at 210 °C for 24 h negatively impacted the electroconductivity of both sample PH1000f ((9.0 ± 8.9) × 10−1) and AI4083f ((8.2 ± 5.1) × 10−3 S cm−1). Statistical analysis was conducted to evaluate differences between the electroconductivities of the different PEDOT:PSS spin-coated films obtained. Although ANOVA test suggested the existence of statistically significant differences between samples , post hoc analysis did not evidence strong differences between the samples. Nevertheless, changes in electroconductivity of the different PEDOT:PSS films were accompanied by structural changes in the PEDOT:PSS system. We will explore more in depth such changes in the next paragraphs.Treatment with sulfuric acid allowed to partly recover the electroconductivity of both samples PH1000f  and 1594 cm−1 (attributed to Cα=Cβ asymmetric stretching and C=C of PSS), similar to what was observed by Lin and colleagues [−1 red-shifts to 1353 cm−1 and decreases in intensity. The weakening of the Cβ–Cβ bond, is consistent with an increased predominance of a benzenoid-like structure of the thiophene ring, which could be associated to a reduction of the PEDOT oxidation state. Additionally, a small peak at appears at 1426 cm−1 (Cα=Cβ symmetric stretching), which seems to be the result of a blue-shift of a shoulder seen at about 1400 cm−1 in the initial film. After treatment with heated sulfuric acid, the peak at 1353 cm−1 blue-shifts back to 1360 cm−1 (attributed to Cβ–Cβ) and increases in intensity, the peak at 1426 cm−1 (Cα=Cβ symmetric stretching) blue-shifts to 1440 cm−1 and significantly increases in intensity. The main peak at 1594 cm−1 (attributed to Cα=Cβ asymmetric stretching and C=C of PSS) remains at the same energy. In conclusion, the treatment with heated sulfuric acid, leaves unchanged the energy of the main absorption peak and appears to decrease the energy of the Cβ–Cβ bond. This is somewhat surprising as an increase of the Cβ–Cβ bond energy should be accompanied by a decrease of the Cα–Cβ bond energy. These results do not provide a clear confirmation of a change in the oxidation state of the PEDOT chains upon thermal and/or sulfuric acid treatment [Raman analysis also reveal changes on PEDOT:PSS after treatment with sulfuric acid. Sample PH1000f 0H C presentlleagues . After treatment ,84. The reatment . This is−1 (Cβ–Cβ), 1431 cm−1 (Cα=Cβ symmetric stretching), and 1590 cm−1 (Cα=Cβ asymmetric stretching and C=C of PSS). Thermal annealing induces an increase in intensity of the peak at 1349 cm−1 and the disappearance of the peak at 1431 cm−1. This profile slightly changes after treatment with sulfuric acid, and the peak at 1431 cm−1 is not visible anymore. It is possible the spectral changes observed in sample PH1000 are not so clearly visible in sample AI4083, due to the high amount of PSS (peak at ≈1590–1600 cm−1), which may mask the PEDOT spectrum.The Raman spectra of sample AI4083f presents 3 peaks at frequencies of 1349 cm2SO4 was due to a direct positive effect on PEDOT:PSS.Overall, our results with spin-coated PEDOT:PSS films show that the combined thermal annealing and heated sulfuric acid treatment affect the electroconductivity and structure of PEDOT:PSS. Sample PH1000 evidenced the most dramatic changes on electroconductivity and structure of PEDOT. The treatment with heated sulfuric acid enabled the partial rescue of sample PH1000f. As such, we deduce that the increase in electroconductivity observed for PAN:PEDOT:PSS fibers PH1000 HThe next step in our study was to determine the mechanical properties of the obtained PAN and PAN:PEDOT:PSS fibers and evaluate how they change with thermal annealing and treatment with heated sulfuric acid. The main results are summarized in 2SO4 sample. Such a trend was not followed by thermally annealed AI4083 and PH1000 samples. Overall, there was no statistically significant change on the Young’s modulus of samples AI4083 (129 ± 39 MPa) and AI4083 24H (103 ± 63 MPa). However, these values were lower than those observed for the respective PAN fibers. For PH1000 samples, from the original PH1000 fibers (78 ± 25 MPa) there was a small decrease for samples PH1000 1H (25 ± 6 MPa) and PH1000 5H (15 ± 9 MPa), only to increase for samples PH1000 10H (36 ± 18 MPa) and PH1000 24H (110 ± 49 MPa). The obtained Young’s tensile modulus for the fibers is significantly higher than that of normal neural tissue (0.4–2 kPa) [The most drastic changes in mechanical properties are observed for PAN fibers upon thermal annealing. We expected the formation of a more rigid polycyclic structure, which would make the fibers stiffer, yet brittle and fragile. Indeed, the Young’s modulus A initial4–2 kPa) ,86. The 4–2 kPa) . Other s4–2 kPa) ,89,90. A2SO4 (0.8 ± 0.3 MPa). These changes accompanied those of Young’s modulus but were not overall expected. We currently do not have an explanation to this phenomenon.Other changes in the mechanical properties of PAN and PAN:PEDOT:PSS fibers were observed. The tensile maximum strength B of PAN Changes in tensile maximum strength of PAN:PEDOT:PSS fibers are variable. There is a gradual decrease from sample AI4083 (12.1 ± 5.0 MPa) to sample AI4083 24H (3.7 ± 0.7 MPa). A similar gradual decrease is also observed from sample PH1000 (5.1 ± 1.5 MPa) to sample PH1000 24H (3.5 ± 1.6 MPa).2SO4 (9 ± 5%). Incorporation of PEDOT:PSS into PAN reduces the maximum strain for samples of AI4083 (54 ± 11%) and PH1000 (44 ± 15%). After thermal annealing, maximum strain further decreases, bottoming in samples AI4083 24H (7 ± 3%) and PH1000 24H (4 ± 1%).There is an overall decrease of the obtained maximum strain C for bot2SO4 and PH1000 H2SO4 due to quick breaking when mounted at the machine. However, the samples were easily manipulated, and we were able to conduct further assays.Overall, the mechanical properties of the obtained fibers were greatly affected by the incorporation of PEDOT:PSS and processing steps taken. Thermal annealing of PAN fibers increased their stiffness up to 10 h treatment. Incorporation of PEDOT:PSS in PAN made the obtained samples softer yet more fragile. Similar observations were made by Li and colleagues  for wet-2SO4, the most electroconductive in the study, as no work in the literature had done so.The existing measurements of biocompatibility in the literature for PAN-based biomaterials were either made of pristine PAN  or graphOur results indicate that PAN fibers are biocompatible regarding their lixiviates (77 ± 10%) , and no 2SO4 : ReNCell-VM. The biocompatibility and suitability for TE applications were evaluated using a proliferation assay, similar to what was performed for other biomaterials ,46. The 2SO4, and some cells were seeded on culture plate as controls. AlamarTM Blue assay was used to monitor cell metabolic activity on the samples , suggesting that treatment of PAN:PEDOT:PSS fibers with heated sulfuric acid had a negative impact on its surface properties. However, sample PH1000 H2SO4 had the highest growth rate (0.37 ± 0.11 day−1) and the lowest doubling time (49 ± 14 h) of the study. We observed a similar trend in our previous work [2SO4.NSCs were seeded on samples PAN 24H, AI4083 24H, PH1000 24H, and PH1000 H samples A, which  samples . From heous work  with pol2SO4 present a normal morphology at day 7. These results confirm our previous observations regarding biocompatibility. They also evidence an intimate interaction between cells and the supporting material without significant changes to the normal NSCs phenotype.Finally, we studied the morphology of NSCs at day 7. All cells showed a normal spindle-like morphology, confirming that the support material was biocompatible. Moreover, LIVE/DEAD staining B shows t2SO4, which had the highest electroconductivity in our study (3.2 × 10−3 S cm−1), is biocompatible and allowed the proliferation of NSCs at its surface. Future work should focus on fiber optimization, including further electrospinning optimization, complemented by rheological analysis of obtained solutions, and influence of production and processing parameters on fiber morphology, including fiber diameter and porosity, physico–chemical properties, and electroconductivity.In this study, we produced electrospun PAN:PEDOT:PSS blend fibers using a unique strategy not yet approached by other research groups. PEDOT:PSS incorporation in PAN was possible after its redispersion in DMF:DMSO (9:1), and electrospun fibers were successfully produced. Thermal annealing (210 °C) and treatment with heated sulfuric acid (30 min) allowed to obtain electroconductive PAN:PEDOT:PSS blend fibers. The processing conditions influenced the structure and electrochemical properties of both PAN and PEDOT:PSS. Sample PH1000 HFor the first time, we describe the development of electroconductive PAN:PEDOT:PSS electrospun blend fibers. These fibers are among the most electroconductive reported in the literature; moreover, they are electrochemically active and suitable for neural applications. Overall, the produced blend PAN:PEDOT:PSS fibers hold great promise for neural TE and other biomedical applications, including electrodes for electrical stimulation and devices for soft electronics.


Sepia officinalis) ink (CFI) was analysed in the present study. A model system consisting of minced seabream (Sparus aurata) muscle and different concentrations of an aqueous extract of CFI was subjected to a heat (50 °C) treatment for 12 days. The effects of the CFI content and the heating time on lipid oxidation (conjugated diene (CD), conjugated triene (CT), and peroxide values and fluorescent compound formation), hydrolysis (free fatty acid content) development, and changes in the fatty acid (FA) profile (polyene index (PI), unsaturated FA content, ω3/ω6 ratio) were determined. The addition of the aqueous extract of CFI led to a lower (p < 0.05) development of lipid oxidation  and to a higher (p < 0.05) retention of unsaturated FAs (PI determination). More important effects were found with increased CFI concentrations and at advanced heating times. However, a definite effect on lipid hydrolysis development  could not be inferred. A new approach for the beneficial use of cuttlefish ink is presented. According to the direct relationship between rancidity stability and nutritional and sensory values, the present study provides a new strategy for the quality enhancement of thermally treated seafood.The antioxidant effect of cuttlefish ( Seafood is reported to provide a high amount of constituents relevant for the human diet . Among sThe employment of compounds with antioxidant properties has revealed them to be valuable tools for inhibiting or partially avoiding the development of lipid oxidation in seafood. Due to human health concerns, there is currently great interest in replacing synthetic antioxidants with preservative compounds obtained from natural sources ,9. ThereCephalopods are considered a remarkable economic resource in a wide range of countries. As for other marine species, a great number of by-products  are generated during their technological processing ,15. In aAmong cephalopod by-products, ink sacs are commonly removed manually and thrown away without appropriate management. However, recent studies on this kind of by-product have revealed that they possess several properties beneficial to human health, such as a therapeutic effect against invasive pulmonary aspergillosis , suitabiSepia officinalis) ink. The basic objective was to analyse its antioxidant behaviour and properties in a heated fish muscle system. Complementary quality indices regarding the lipid damage  were measured during a heating process (50 °C for 12 days) carried out with minced seabream (Sparus aurata). The effects of the CFI concentration and the heating time were analysed.The present study is focussed on the use of cuttlefish (S. aurata)  were obtained in the fresh state from a local market and transported on ice to the laboratory. The fish specimens were then randomly divided into three (n = 3) groups (2 specimens per group). Within each group, the specimens were beheaded, eviscerated, and filleted; finally, the white muscle of the fish fillets was removed, minced, and homogenised.Seabream  ink was provided by Sepink . The ink exhibited the following proximate composition (g·100 g−1): 78.4 (moisture), 6.4 (protein), 0.1 (lipids), 9.0 (ash), and 5.6 .Commercial cuttlefish (g for 30 min at 4 °C). The resulting supernatant was then collected. This extraction process was repeated three more times. Finally, all four supernatants were pooled together and made up to 250 mL with distilled water.To prepare the cuttlefish ink (CFI) extract, a mixture of CFI (6 g) and distilled water (100 mL) was stirred (30 s), sonicated (30 s), and centrifuged (3500× Loligo formosana) [In the present study, an aqueous extract of CFI was employed. This choice was based on the results of previous studies which have shown that aqueous ink extracts obtained from different cephalopod substrates, such as splendid squid (rmosana) ,28 and crmosana) , exhibitrmosana) ,30, greermosana) , and redrmosana)  macroalgFor the preparation of the minced fish system, fish muscle portions corresponding to the above-mentioned homogenised groups were employed. In this system, 4 g portions of seabream muscle were mixed with 0, 2, 5, or 10 mL of the above-mentioned CFI extract. Next, 10, 8, 5, or 0 mL of distilled water was added to each respective mixture, resulting in Control (CTR), CFI-1 (low CFI concentration), CFI-2 (medium CFI concentration), and CFI-3 (high CFI concentration) batches.n = 3). Additionally, each chemical analysis was carried out in triplicate for each single sample.Mixtures corresponding to all batches were stirred (30 s), sonicated (30 s), and incubated (50 °C for 12 days). Sampling was carried out at 0, 2, 5, 8, and 12 days of the heating time. At each sampling time, mixtures were cooled to room temperature (18–20 °C). Then, mixtures were centrifuged, the supernatant discarded, and the solid phase  subjected to the extraction of the lipid fraction. The study was carried out by means of three independent sets that were analysed independently  and the weight (mg) of the lipid extract measured, respectively.The formation of conjugated diene (CD) compounds was determined spectrophotometrically  at 233 nm  on the lA·V·w−1, where A is the resulting spectrophotometric reading and V and w indicate the volume (mL) and the weight (mg), respectively, of the lipid extract measured.The formation of conjugated triene (CT) compounds was measured at 268 nm  on the l−1 lipids.The assessment of the peroxide value (PV) was carried out spectrophotometrically (520 nm) on the lipid fraction. For it, peroxide reduction with ferric thiocyanate was carried out . The res−1 muscle.The assessment of the free fatty acid (FFA) content was carried out on the lipid extract of the seabream muscle following the Lowry and Tinsley  method. stF/F, where F is the fluorescence measured at each excitation/emission wavelength pair and stF is the fluorescence intensity of a quinine sulphate solution (1 µg·mL−1 in 0.05 M H2SO4) at the corresponding wavelength pair. The formation of fluorescent compounds was calculated as the fluorescence ratio (FR) between the two RF values: FR = RF393/463 nm/RF327/415 nm.The determination of the interaction compound formation produced by the reaction of oxidised lipids and protein-type molecules was carried out by fluorescence spectroscopy . Fluorescent compound formation was measured at 393/463 and 327/415 nm . The relorg) and aqueous (FRaq) phases obtained from the lipid extraction [To obtain a more complete detection of the interaction compound formation, two different substrates obtained from the ink extract/minced seabream system were considered. Thus, the fluorescence ratio was analysed in the organic  by employing an acid-catalysed esterification and transesterification method  . Then, tThe results regarding FA groups (saturated FAs (STFAs), monounsaturated FAs (MUFAs), polyunsaturated FAs (PUFAs), ω3 and ω6 FAs) and FA ratios , total MUFAs + total PUFAs/total STFAs, total ω3 FAs/total ω6 FAs) were calculated considering the values obtained for individual FAs.org, FRaq, FFAs, and FA ratios) were subjected to the multifactorial ANOVA method (randomised-block design) to analyse differences as a result of two process or independent variables (the CFI concentration and the heating time) [p < 0.05). As expressed above, three independent groups  were taken into account in the present research. To carry out the comparison of means, the Fisher’s least-squares difference (LSD) method was employed. Statistical analysis was determined with the PASW Statistics 18 software for Windows . The 95% confidence intervals of each lipid parameter were calculated; for this calculation, the standard deviation of each sample and the number of replicates were considered.Values obtained for the different response or dependent variables  ; the ANOCorrelation analyses between lipid damage values and the heating times were performed according to the Pearson method. Correlation values corresponding to linear fittings are expressed; otherwise, the kind of fitting is expressed.The evolution of the CD content during the heating time is shown in p < 0.05) with heating time was detected for the CD content . A comparison among batches revealed an inhibitory effect (p < 0.05) on the formation of this kind of oxidised lipids as a result of the presence of the CFI in the heating medium. This effect was shown to increase with the CFI concentration. Thus, fish muscle corresponding to the CFI-3 batch showed lower (p < 0.05) CD levels than its counterpart CTR batch for the 2–8-day period.In most cases, a progressive formation (r = 0.87–0.93) (p < 0.05) in CTR, CFI-1, CFI-2, and CFI-3 batches was detected after 5, 8, 12, and 12 days of the heating time, respectively. As for the CD formation, the presence of the CFI in the reacting medium led to lower average CT values. Notably, differences between the CTR batch and those including the two highest CFI concentrations  were found significant for the 5–8-day period.The formation of CTs also showed a progressive increase during the whole heating time (87–0.93) . An incrp < 0.05) PV was observed after 5 days of heating time. In the case of the CFI-treated batches, the highest peroxide contents (p < 0.05) were observed after an 8-day heating time. A comparison among batches showed the highest PV in control canned fish for the 2–5-day period. Later on (8–12-day period), the highest mean values were observed in fish samples corresponding to the CFI-1 batch, and the lowest ones were detected in fish corresponding to the CFI-3 batch.Peroxide formation in heated fish lipids can be observed in p < 0.05) in the lipid extract  was detected in all batches as a result of increasing the heating time (r = 0.86–0.94) (A marked fluorescence formation (86–0.94) .p < 0.05) for the 5–8-day period when compared to fish muscle subjected to the CTR condition. Additionally, fish corresponding to the CFI-1 and CFI-3 batches showed lower (p < 0.05) values on days 2 and 15, respectively, than their counterpart CTR samples.Additionally, the highest mean levels were observed in all cases at the end of the experiment. A comparison among batches revealed lower mean values in the CFI-treated muscle than in their counterparts corresponding to the CTR condition. Thus, all tested CFI conditions led to lower values (aq) are shown in p > 0.05) after 2 days of heating treatment could be observed in any of the batches considered. However, a progressive increase (p < 0.05) with heating time was detected in all kinds of batches for the 5–12-day period (r = 0.93–0.95). A comparison among batches showed higher (p < 0.05) values in the CTR fish than in any of the CFI-treated samples during the 8–12-day heating time. The lowest mean levels were observed in fish corresponding to the CFI-1 batch for all the heating time.Data concerning the FR in the aqueous phase resulting from the lipid extraction  was detected in fish muscle corresponding to all batches. A remarkable correlation value with the heating time  was observed (p < 0.05) after a 2-day heating time. A comparison among batches showed some differences (p < 0.05); however, a definite trend in the presence and concentration of the CFI in the heating medium could not be concluded. Compared to the CTR fish, an inhibitory effect (p < 0.05) on the FFA content was observed on day 2 (CFI-1 and CFI-3 batches), day 5 (CFI-2 batch) and day 8 (CFI-3 batch). Contrarily, fish muscle corresponding to the CTR batch showed the lowest average values at the end of the experiment.A strong FFA formation : 1.96 ± 0.01 (C14:0), 0.27 ± 0.02 (C15:0), 15.39 ± 0.07 (C16:0), 3.53 ± 0.02 (C16:1ω7), 0.37 ± 0.01 (C17:0), 4.32 ± 0.05 (C18:0), 31.73 ± 0.05 (C18:1ω9), 3.60 ± 0.01 (C18:1ω7), 24.38 ± 0.08 (C18:2ω6), 1.28 ± 0.01 (C20:1ω9), 0.87 ± 0.03 (C20:2ω6), 0.83 ± 0.06 (C20:4ω6), 0.32 ± 0.02 (C22:1ω9), 2.38 ± 0.03 (C20:5ω3), 0.26 ± 0.01 (C22:4ω6), 0.39 ± 0.02 (C24:1ω9), 1.86 ± 0.02 (C22:5ω3), and 6.27 ± 0.09 (C22:6ω3). When FA groups were taken into account, the following composition was observed : 22.31 ± 0.08 (STFAs), 40.85 ± 0.11 (MUFAs), 36.84 ± 0.06 (PUFAs), 10.51 ± 0.09 , 26.34 ± 0.05 , 0.56 ± 0.01 (PI), 3.48 ± 0.09 (MUFA + PUFA/STFA ratio), and 0.40 ± 0.00 (ω3/ω6 ratio).The FA profile of the starting fish muscle showed the following composition  ,38. Meanp < 0.05) for the CTR batch. A comparison among batches showed a protective effect (p < 0.05) on PUFAs on days 5 (CFI-2 batch) and 8 (CFI-3 batch) as a result of the CFI presence.The heating time led to very scarce changes in the PI of the fish muscle corresponding to all batches . In all p > 0.05) of the heating time and the CFI presence and concentration in the heating medium. Thus, values for both ratios were included in very narrow ranges . It is worth pointing out that the CTR batch did not show differences (p > 0.05) for both ratios during the heating time. Similarly, no effect (p > 0.05) of the CFI extract could be inferred.The analysis of the two other above-mentioned FA ratios  revealed no effect  in the first stages of the heating time (0–5-day period). However, as the heating time increased, the second factor  was shown to increase its influence on the primary oxidation development. Thus, a marked peroxide content decrease was detected in the CTR batch for the 5–12-day period and in all CFI-treated batches for the 8–12-day period. This decrease was found more important in the CTR batch than in those including the CFI extracts. This difference can be justified by the presence in the ink extract of antioxidant compounds that would lead to an inhibitory effect on the breakdown of the primary oxidation compounds.org and the FRaq values, both values increasing with the heating time. Notably, this FR increase was partially inhibited by the ink extract presence in the reacting medium; indeed, an increasing inhibitory effect was proven by increasing the ink extract concentration.According to the above-mentioned third effect , the present results have shown a strong interaction between oxidised lipids and nucleophilic-type molecules present in the fish muscle. This interaction could be proved in the FRScomber scombrus) (50 °C for 11 days)  [Thunnus thynnus) and sardine (Sardina pilchardus) (low peroxide content) [S. scombrus)  [Scomber colias) (low peroxide content) [Anguilla anguilla) (low peroxide content) [Salmo salar) pasta [Most research regarding heat-treated seafood has shown low values for primary lipid oxidation compounds, indicating a marked effect of the thermal breakdown on primary compounds. Such research has concerned heated–minced mackerel (ontents)  and diffcontent) , Atlantiontents) , Atlanticontent) , and eelcontent) . Howeverr) pasta .Regarding the determination of the fluorescent compound formation during the thermal processing of marine species, a relevant increase in the FR has been proved in a wide range of studies including fish model systems ,13 and corg and the FRaq determinations  contents, a decrease in the digestibility value, and a decrease in the sensory value  have been reported to be produced ,46,47. TClupeonella cultriventris)  [S. scombrus) (PI) [S. colias) (PI) [S. salar) pasta [S. scombrus) (50 °C for 11 days) [The present results have shown scarce effects of the thermal treatment on the FA parameters measured. In agreement with this conclusion, previous canning of brine-canned sprat  , water-pus) (PI) , and brias) (PI)  did not as) (PI) , in cookr) pasta , and in 11 days) .Loligo formosana) and proved that the highest antioxidant activity was found in fractions with molecular weight lower than 3 kDa; remarkably, the radical scavenging activity remained constant if the ink extract was subjected to heat treatment (90 °C up to 30 min). Based on thin-layer chromatography analysis, Senan [Sepia pharaonis) ink extract would be a peptide compound; this study also showed that the active principle was thermally stable (40–100 °C for 30 min). Similarly, previous research had already indicated that smaller-size peptides would be more stable in response to aggregation and high temperatures than bigger ones [Sepiella maindroni) ink. These three studies [The current study has shown a strong inhibitory effect of the ink extract on the lipid oxidation development of seabream muscle. Antioxidant properties of cuttlefish ink constituents and of other cephalopod species have already been reported, both in in vitro ,53 and fs, Senan  indicateger ones . Recentlger ones  proved t studies ,54,55 deS. officinalis) [Loligo duvauceli) [Sepia esculenta) [L. formosana) had antioxidant activity according to different in vitro assays such as DPPH, ABTS, and ferric reducing power; additionally, this ink substrate showed a metal-chelating activity. Antioxidant properties  in melanin-free squid (Loligo duvaucelli) ink were also detected by Karim et al. [Taking into account all these characteristics of ink constituents, a wide range of previous in vitro studies have shown the antioxidant properties of cuttlefish and squid inks. The present results are in agreement with such inhibitory effects; however, CFI has not been employed previously with thermally treated seafood, to the best of our knowledge. Thus, based on the determination of the DPPH radical scavenging activity, a remarkable antioxidant behaviour was observed for cuttlefish  ,53, squivauceli) , and cutculenta) . Vate anculenta)  showed tm et al. .Rastrelliger kanagurta) during 15-day storage in ice by the presence of melanin-free ink obtained from splendid squid (L. formosana) [Loligo formosana) prevented lipid oxidation  in surimi gel sardine  during storage at 4 °C for 20 days. An increased shelf-life time during cold storage (15 days at 4 °C) was detected in squid (L. duvaucelli) by previous squid melanin-free ink soaking [S. officinalis) ink soaking was proven in cold-stored (12 days at 0 °C and 23 days at −2 °C) peeled shrimp (Penaeus kerathurus) [Sardinella aurita) during cold storage (4 °C for 35 days) was increased by previous soaking in cuttlefish  ink solution [Previous studies regarding the preservative effect of CFI extracts have also been focussed on non-thermally treated seafood. Thus, lipid oxidation (detection of peroxides and TBARS) was retarded in minced mackerel (rmosana) . Similarrmosana)  reported soaking ; additioathurus) . The shesolution ; additioIn the current study, the FFA content measured can be influenced by different factors. First, the thermal process can lead to the hydrolysis of higher-molecular-weight lipid classes such as triacylglycerols (TAGs) and phospholipids (PLs) ,9. SeconThunnus albacares) [S. scombrus) (50 °C for 11 days) [H. molitrix) [C. cultriventris) [T. albacares) [A. anguilla) [According to the present results, an increased FFA content has been described for thermally treated fish. This FFA formation concerns heated fish muscle systems such as cooked yellowfin tuna (bacares)  and minc11 days) . Regardiolitrix) ; sunflowventris) ; sunflowbacares) ; and sunnguilla) .Ulva lactuca or Fucus spiralis extracts to the packing medium of canned Atlantic Chub mackerel (S. colias) led to FFA values of 13.4 and 12.8 g·kg−1 lipids, respectively, that were found remarkably higher than in samples corresponding to the control condition (9.1 g·kg−1 lipids) [S. scombrus) system (50 °C for 11 days) [−1 lipids at the end of the experiment; this value was notably higher than that in the case of the control .No previous studies regarding the effect of ink extracts on the FFA content of thermally processed seafood are available, to the best of our knowledge. However, and contrary to the present results, previous research accounts for the FFA preservation in thermally treated seafood through the presence of antioxidant extracts obtained from other natural resources. Thus, the addition of macroalgae S. officinalis) ink were analysed and demonstrated in a heated fish system. The presence of an ink aqueous extract led to a lower (p < 0.05) development of lipid oxidation  and to a higher (p < 0.05) retention of unsaturated FAs (PI determination). Preservative effects were found more relevant with an increase in the CFI concentration and the heating times. Contrarily, a definite effect on the FFA content (lipid hydrolysis development) could not be concluded.The antioxidant properties of cuttlefish  and lipophilic (organic solvents) extracts is found necessary. With this purpose, the employment of advanced extraction procedures  ought to be addressed too.


The most common subtype of ovarian cancer (OC) showing immunogenic potential is represented by the high-grade serous ovarian cancer (HGSOC), which is characterized by the presence of tumor-infiltrating immune cells able to modulate immune response. Because several studies showed a close correlation between OC patient’s clinical outcome and expression of programmed cell death protein-1 or its ligand (PD-1/PD-L1), the aim of our study was to investigate if plasma levels of immunomodulatory proteins may predict prognosis of advanced HGSOC women.Through specific ELISA tests, we analyzed plasma concentrations of PD-L1, PD-1, butyrophilin sub-family 3A/CD277 receptor (BTN3A1), pan-BTN3As, butyrophilin sub-family 2 member A1 (BTN2A1), and B- and T-lymphocyte attenuator (BTLA) in one hundred patients affected by advanced HGSOC, before surgery and therapy. The Kaplan–Meier method was used to generate the survival curves, while univariate and multivariate analysis were performed using Cox proportional hazard regression models.versus short progression-free survival (PFS < 30 months). The concentration cut-offs, obtained by receiver operating characteristic (ROC) analysis, allowed to observe that poor clinical outcome and median PFS ranging between 6 and 16 months were associated with higher baseline levels of PD-L1 (> 0.42 ng/mL), PD-1 (> 2.48 ng/mL), BTN3A1 (> 4.75 ng/mL), pan-BTN3As (> 13.06 ng/mL), BTN2A1 (> 5.59 ng/mL) and BTLA (> 2.78 ng/mL). Furthermore, a lower median PFS was associated with peritoneal carcinomatosis, age at diagnosis > 60 years or Body Mass Index (BMI) > 25. A multivariate analysis also suggested that plasma concentrations of PD-L1 ≤ 0.42 ng/mL , age at diagnosis ≤ 60 years  and absence of peritoneal carcinomatosis  were significant prognostic marker for a longer PFS in advanced HGSOC patients.For each analyzed circulating biomarker, advanced HGSOC women were discriminated based on long (≥ 30 months) The identification of high-risk HGSOC women could be improved through determination of the plasma PD-L1, PD-1, BTN3A1, pan-BTN3As, BTN2A1 and BTLA levels.The online version contains supplementary material available at 10.1186/s12885-023-10911-5. Ovarian cancer (OC) is the seventh most commonly diagnosed cancer and the eighth leading cause of cancer death in women worldwide, with a 5-year relative survival of 49% .Among epithelial OCs, which represent the most common subtype, high-grade serous ovarian cancer (HGSOC) is the most frequent and is responsible for 70–80% of all OC deaths .High heterogeneity and resistance to therapy significantly contribute to the poor prognosis of HGSOC. Surgery and platinum-based chemotherapy are the standard treatment in OC . AlternaOne of the most studied immune checkpoint receptors is the programmed cell death protein 1 (PD-1), with its ligands, PD-L1 and PD-L2, which are involved in the activation, proliferation and cytotoxic secretion of T cells . PD-L1 eOther immune checkpoints involved in the interaction between cancer cells and T lymphocytes showed an interesting immunomodulatory role in different tumors . Among tIn order to investigate the potential prognostic role of these immune checkpoints in OC, we assessed whether circulating soluble forms of PD-L1 (sPD-L1), PD-1 (sPD-1), BTN3A1 (sBTN3A1), pan-BTN3As (pan-sBTN3As), BTN2A1 (sBTN2A1) and BTLA (sBTLA) may be useful to predict prognosis in advanced HGSOC patients.A prospective analysis was performed on a cohort of one hundred patients with advanced-stage HGSOC recruited at the two Sicilian hospital centers: “Sicilian Regional Center for the Prevention, Diagnosis and Treatment of Rare and Heredo-Familial Tumors” of the Section of Medical Oncology of University Hospital Policlinico “P. Giaccone” of Palermo , and Department of Gynecologic Oncology of the Hospital ARNAS Civico “Di Cristina Benfratelli” of Palermo . The information concerning the personal history of tumor, age of cancer diagnosis, International Federation of Gynecology and Obstetrics (FIGO) stages, tumor histological subtype and tumor grading were anonymously recorded and coded for all enrolled patients who had previously signed and provided a written informed consent Table S. The stuBetween May 2018 and July 2021 we prospectively collected blood samples from one hundred women with a confirmed histological diagnosis of advanced HGSOC (stage IIIB-IV) at baseline, before surgery  and starting first-line chemotherapy treatment with Carboplatin AUC (area under the curve) 5 and Paclitaxel (175 mg/m2) based on the current therapeutic strategies. In summary, patients were considered eligible for this study based on the previously reported inclusion criteria [In order to confirm the obtained results, another independent cohort of 24 advanced HGSOC patients  recruited at the Section of Medical Oncology of University Hospital Policlinico “P. Giaccone” of Palermo  was used .The peripheral blood samples from untreated patients with advanced HGSOC were collected at baseline, processed for plasma isolation and subsequently stored as previously reported , 7, 19.The plasma sPD-L1, sPD-1, sBTN3A1, pan-sBTN3As, sBTN2A1, and sBTLA concentrations were determined by means of specific enzyme-linked immunosorbent assays (ELISAs). Because other commercially available assays showed some discrepancies, we used specific ELISAs, produced by the company DYNABIO S.A. , according to the previously reported specifications , 20, 21.cut-offs for each soluble form of immune checkpoints and other examined patients characteristics (age at diagnosis and Body Mass Index), in order to discriminate HGSOC patients based on long (≥ 30 months) versus short PFS (< 30 months). The Kaplan–Meier method and log-rank test were used to calculate patient PFS. Univariate and multivariate Cox proportional hazard regression models were built to identify significant prognostic factors for PFS [An analysis by ROC curves was performed to determine the optimal concentration  for PFS .P values < 0.05 have been considered statistically significant.MedCalc software v.18.2.1 for Windows  and GraphPad Prism software v. 9.0.0  were used to generate and graphically represent data . P valueThe plasma concentrations of sPD-L1, sPD-1, sBTN3A1, pan-sBTN3As, sBTN2A1, and sBTLA were measured in peripheral blood from one hundred advanced HGSOC patients, prior to surgery and starting first-line chemotherapy treatment, using specific ELISA tests.Youden index associated criterion) able to discriminate advanced HGSOC patients based on long (≥ 30 months) versus short PFS (< 30 months). At the same time, a further ROC analysis was performed to establish the optimal cut-offs of two different parameters: age at diagnosis and BMI. The ROC curve analysis showed that the optimal concentration threshold was 0.42 ng/ml for sPD-L1 , 2.48 ng/ml for sPD-1 , 4.75 ng/ml for sBTN3A1 , 13.06 ng/ml for pan-sBTN3As , 5.59 ng/ml for sBTN2A1 , and 2.78 ng/ml for sBTLA . Furthermore, the best cut-offs for age at diagnosis and BMI were 60 years  and 25 , respectively  and 24 for BMI (range 16 to 56). Afterwards, the plasma levels of each molecule, ages at diagnosis and BMIs were graphically represented, in order to discriminate and divide the advanced HGSOC patients into 2 groups at long versus short PFS based on each investigated factor  for each soluble biomarker , whereas patients with PFS < 30 months predominantly exhibited higher circulating levels of these molecules. In addition, advanced HGSOC women with PFS less than 30 months were over 60 years of age and had a BMI > 25.The receiver operating characteristic (ROC) analysis was carried out to determine for each soluble biomarker the optimal concentration threshold (cut-offs associated with poor prognosis and shorter PFS were defined for sPD-L1 (> 0.42 ng/mL), sPD-1 (> 2.48 ng/mL), sBTN3A1 (> 4.75 ng/mL), pan-sBTN3As (> 13.06 ng/mL), sBTN2A1 (> 5.59 ng/mL) and sBTLA (> 2.78 ng/mL) ; 24 versus 30 months for sPD-1 ; 21 versus 37 months for sBTN3A1 ; 21 versus 35 months for pan-sBTN3As ; 25 versus 32 months for sBTN2A1 ; and 24 versus 32 months for sBTLA . Interestingly, baseline plasma levels of sPD-L1, sBTN3A1 and pan-sBTN3As below their respective cut-offs showed a significant benefit in terms of median PFS (14–16 months), whereas a lower advantage in median PFS (6–8 months) was associated with baseline concentrations of sPD-1, sBTN2A1 and sBTLA below their specific thresholds. Therefore, the soluble forms of all proteins investigated in this study have been shown to be potential predictive biomarkers of survival in advanced HGSOC patients.Because the clinical impact of circulating immune checkpoints as predictive biomarkers of clinical outcome has yet to be defined in advanced HGSOC patients, a Kaplan–Meier survival analysis was performed to understand the potential prognostic value of circulating sPD-L1, sPD-1, sBTN3A1, pan-sBTN3As, sBTN2A1 and sBTLA concentrations in advanced HGSOC patients, suggesting that their plasma expression levels could be useful, in the future, to predict patient survival. Therefore, we discriminated advanced HGSOC women based on low and high plasma levels for each tested biomarker, using the thresholds previously determined by ROC analysis. Then, the correlation between PFS and circulating levels of the soluble biomarkers was plotted using Kaplan–Meier curves ; 22 versus 32 months for BMI ; and 17 versus 38 months for peritoneal carcinomatosis . Interestingly, the better survival advantage was observed in the absence of peritoneal carcinomatosis at the diagnosis.In addition, we also assessed the impact on PFS of age at diagnosis, baseline BMI and peritoneal carcinomatosis at onset by Kaplan–Meier analysis Fig. g-i. Alsop = 0.024), presence of peritoneal carcinomatosis  and plasma levels of sPD-L1 > 0.42 ng/mL  were statistically significant. No statistically significant association was observed for other considered biomarkers/factors. Therefore, our analysis highlighted that age at diagnosis ≤ 60 years, absence of peritoneal carcinomatosis and expression levels of sPD-L1 ≤ 0.42 ng/mL were independent prognostic factors associated with a longer PFS in patients with advanced HGSOC.Since previous analyses showed that each circulating immune checkpoint  and other clinic-pathological factors, such as age at diagnosis, baseline BMI and peritoneal carcinomatosis, have a high predictive power of clinical outcome in patients with advanced HGSOC, a multivariate analysis for PFS was performed in order to correlate these variables between them. The prognostic relevance of each biomarker/factor was assessed by an univariate survival analysis using the Cox regression model. The results of the multivariate analysis are reported in Table cut-offs.Lastly, we used a further independent cohort of 24 peripheral blood samples from advanced HGSOC patients in order to confirm the previously observed correlations for each tested parameter. The previously determined concentration thresholds in leading cohort by the ROC curve approach were used to perform a Kaplan–Meier survival analysis. As expected, we observed a significant inverse correlation between PFS and high expression levels in plasma for each biomarker/factor Fig. . This coAdditionally, also for women from the validation cohort the impact on PFS of age at diagnosis, baseline BMI and peritoneal carcinomatosis at onset was evaluated by Kaplan–Meier curves. This further analysis showed similar results to those obtained in the leading cohort, suggesting that age at diagnosis > 60 years, BMI > 25, or presence of peritoneal carcinomatosis were associated with a shorter PFS. Finally, multivariate analysis performed on validation cohort confirmed that low plasma PD-L1 levels (≤ 0.42 ng/mL), age at diagnosis ≤ 60 years and absence of peritoneal carcinomatosis are favorable independent prognostic factors for PFS in women with advanced HGSOC Table S.In recent years, interesting results from phase II and III trials for the clinical management of patients affected by several solid tumors were obtained by the blocking of the PD-1/PD-L1 immune regulatory complex .OCs  have been shown to be potentially immunogenic tumors rich in tumor-infiltrating immune cells. The abundance of tumor-infiltrating cellsmodulates the anticancer immune response and provides the optimal condition to develop effective immunotherapy approaches , 23. HowOver the years, several studies assessed the ability of PD-1 and PD-L1 to act as a biomarkers for tumor prognosis, suggesting that their high expression is associated with poor clinical outcome in patients with different cancer types, including OC .Hamanishi et al.  reportedIn general, several technical limitations regarding tissue sampling, methodology and used antibodies were found during assessment of PD-L1 expression by IHC analysis in formalin-fixed paraffin-embedded (FFPE) tissue samples. Because PD-L1 and PD-1 are dynamic biomarkers as well as the immune system, assessing their expression in primary tumor tissue may not provide an overview of metastatic disease, which evolves during tumor progression .In the last years, circulating PD-1 and PD-L1 levels have been shown to be associated with worse survival in individuals affected by different cancers , 20. HowAdditionally, the soluble forms of other immunomodulatory molecules, such as butyrophilins and BTLA, were tested by our research group in blood from patients affected by several tumors , 20, 21.Constantly scientific research is looking for new prognostic factors which would enable predict patient survival, increasing the effectiveness of therapeutic treatments. There is today little data about immunological predictors in OC. For this purpose, our study focused on analysis of the baseline plasma expression levels of six immunoregulatory molecules, such as sPD-L1, sPD-1, sBTN3A1, pan-sBTN3As, sBTN2A1 and sBTLA, correlating them with survival data from one hundred advanced HGSOC patients. A survival analysis by Kaplan–Meier curves was performed in order to associate the plasma concentrations of these immunomodulatory proteins with PFS of advanced HGSOC patients. This investigation allowed, for each tested circulating biomarker, to discriminate advanced HGSOC patients based on long (≥ 30 months) versus short PFS (< 30 months). We surprisingly observed that circulating levels of each tested soluble protein were negatively associated with PFS in advanced HGSOC patients. Advanced HGSOC patients with plasma levels of tested immune checkpoints below the established concentration threshold showed a median PFS ranging 6 to 16 months longer compared to that of patients with concentrations above the threshold.Although several previous studies showed that the assessment of tumor PD-L1 may not be a prognostic factor for OC due to its controversial role, our study instead demonstrated that its circulating form is inversely correlated with PFS of advanced HGSOC patients.An increase in plasma levels of immune checkpoints relatively to their specific concentration thresholds has been shown to be associated with poor prognosis, therefore, these could be used in the future as potential prognostic biomarkers. Our study, for the first time, highlighted that assessing the circulating levels of some immunomodulatory molecules could concur to prognosticate survival of advanced HGSOC patients, allowing to implement optimal therapeutic strategies and discriminate those women who may take advantage from tailored therapies. Hence, the use of the plasma sPD-L1, sPD-1, sBTN3A1, pan-sBTN3As, pan-sBTN3As, sBTN2A1 and sBTLA concentrations as “inspectors” able to monitor clinical outcome of advanced HGSOC patients could be helpful to improve patient clinical management as well as prevent needless healthcare costs.OC is considered a disease of the elderly as the average age of diagnosis is around 60 years . SeveralAdditionally, our investigation also evaluated the impact of baseline BMI on survival of advanced HGSOC patients, since the incidence of obesity is increasing in the developed world and it is associated with an increased risk of malignancy, contributing to 14%-20% of cancer-related mortality . PreviouOther factors such as peritoneal carcinomatosis, which frequently occur in late-stage disease, could affect clinical outcome of OC patients , 44. OurFinally, a multivariate analysis carried out to investigate the impact of different baseline covariates on PFS showed that plasma levels of sPD-L1 ≤ 0.42 ng/mL, age at diagnosis ≤ 60 years and absence of peritoneal carcinomatosis were significant prognostic factors for a longer PFS in advanced HGSOC patients. Therefore, plasma sPD-L1 levels, age at diagnosis and presence/absence of peritoneal carcinomatosis rather than other immune checkpoints or BMI should be considered before starting the therapeutic treatment for advanced HGSOC patients.Recently, Parvathareddy et al.  demonstrFor this reason, the correlation between plasma sPD-L1 levels and peritoneal carcinomatosis with a shorter PFS observed in our study acquires greater emphasis.Several studies were performed to investigate the dynamics of sPD-L1 variations in patients receiving ICI treatment, showing controversial results depending on the type of tumor –48. In gLastly, analyses performed on independent validation cohort of 24 advanced HGSOC women confirmed the previously obtained results.The innovation of our study was to perform a serial study on plasma, a biological specimen which can be easily isolated, repeatedly, with little invasiveness, and which give us a more dynamic profile of the status of the tumor microenvironment even during therapy, thus overcoming the technical limitations of tissue biopsy . In this analysis, the soluble forms of immune checkpoints were detected in plasma rather than serum, because serum concentrations have been shown to be ten times lower than those detected in plasma from the same blood sample. However, further investigations are needed to discover the release modalities of these soluble forms from tumors.Additional file 1: Table S1. Clinical and pathological characteristics of advanced HGSOC patients. Table S2. Characteristics of ELISAs for sPD-L1, sPD-1, pan-sBTN3As, sBTN3A1, sBTN2A1, and sBTLA. Table S3. Univariate and multivariate analysis of biomarkers and other factors for PFS in the validation cohort. Figure S1. Receiver Operating Characteristic (ROC) curve analysis of age at diagnosis, BMI and plasma levels of PD-L1, PD-1, BTN3A1, pan-BTN3As, BTN2A1, BTLA.


The seed part from Kenya and Sri Lanka origins exhibited an IC50 value of 14.44 ± 3.61 (HCT116) and 17.19 ± 1.60 (MCF7). The seed part for Yemen Badami (119 ± 0.08) and epicarp part of Thailand (119 ± 0.11) mango fruit showed a significant increase in glucose utilization (50 μg/mL) as compared to the standard drug metformin (123 ± 0.07). The seed extracts of Yemen Taimoor seed (0.46 ± 0.05) and Yemen Badami (0.62 ± 0.13) produced a significant reduction in GPx activity (50 μg/mL) compared to the control cells (100 μg/mL). For α-amylase inhibition, the lowest IC50 value was observed for the endocarp part of Yemen Kalabathoor (108.8 ± 0.70 μg/mL). PCA, ANOVA, and Pearson’s statistical models revealed a significant correlation for the fruit part vs. biological activities, and seed part vs. cytotoxicity and α-amylase activity (p = 0.05). The seed of mango fruit exhibited significant biological activities; hence, further in-depth metabolomic and in vivo studies are essential to effectively utilize the seed part for the treatment of various diseases.Mango fruit is well known for its nutritional and health benefits due to the presence of a plethora of phytochemical classes. The quality of mango fruit and its biological activities may change depending upon the variation in geographical factors. For the first time, this study comprehensively screened the biological activities of all four parts of the mango fruit from twelve different origins. Various cell lines  were used to screen the extracts for their cytotoxicity, glucose uptake, glutathione peroxidase activity, and α-amylase inhibition. MTT assays were carried out to calculate the IC Mangifera indica (mango) is one of the most prevalent tropical fruits belonging to the genus Mangifera, which involves about 30 species of fruit trees in the Anacardiaceae family [Mangifera indica in the management of diverse diseases. These studies demonstrated that mango fruits have antioxidant, anticancer, and antidiabetic activities [Mangifera indica tree . Different mango cultivars exhibit a wide diversity in mango fruit characteristics  and their phytochemical profiles. Consequently, the bioactivity of each cultivar might be varied. Similarly, various parts of an individual mango fruit  possess many biological effects due to the variation in the type and quantity of chemical compounds [e family . Althouge family . Additiotivities . For exativities . Regarditivities . Anothertivities . Furthertivities . For thetivities . Additiotivities . Moreovetivities . Collecttivities . Mangifetivities . Mangifeompounds . Indeed,50 values (The cytotoxicity was observed in the range of (%) 34–91 for HCT116 and 34–98 for MCF7. The extracts with cytotoxic effect >50% were selected for further MTT assay in order to find their IC0 values : India BSix different concentrations with the addition of an MRC5 cell line were utilized for selectivity determination. The mango fruit extract from Kenya (large round red) with seed part was the most effective against HCT116 (14.44 ± 3.61), whereas the extract from Sri Lanka (large yellowish green) seed revealed a significant effect against MCF7 cell line (17.19 ± 1.60). Likewise, the extracts from Kenya (large round red) seed (14.44 ± 3.61 and 25.96 ± 1.08), Vietnam (large yellowish green) seed (26.54 ± 1.10 and 20.17 ± 1.24), and Thailand (large green) seed (20.01 ± 0.88 and 24.63 ± 2.53) showed more selective cytotoxicity towards MCF7 and HCT116 cell lines respectively, as compared to MRC5 cell line. The Sri Lankan (large yellowish green) seed extract exhibited a comparatively more significant effect against HCT116 (20.53 ± 1.56), MCF7 (17.19 ± 1.60), and MRC5 cell line (20.79 ± 1.59). The data are shown in p < 0.05) increase in glucose uptake and utilization in HepG2 cells (100 μg/mL). The extract for Yemen Badami (large yellow) seed (119 ± 0.08) showed a significant increase in glucose utilization at 50 μg/mL, whereas the results for both extracts of Yemen Badami (large yellow) seed (119 ± 0.08) and Thailand (large green) epicarp (119 ± 0.11) were comparable with the result of control (metformin = 123 ± 0.07) used in this study  on HepG2 followed by confirmation of the actual dose for glucose utilization assay. The MTT assay confirmed a lack of cytotoxicity (100 μg/mL) for all the extracts except the following: Yemen Taimoor  seed, Thailand (large green) seed, Kenya (large round red) seed, Vietnam (large yellowish green) seed, Yemen Kalabathoor (large round red) endocarp, Yemen Badami (large yellow) seed, Yemen Taimoor (large yellowish green) seed, and Yemen Kalabathoor (large round red) seed, which showed no cytotoxicity at 50 μg/mL (data not shown). The cells treated with the extracts of Thailand (large green) epicarp (119 ± 0.11), Kenya (large round red) mesocarp (118 ± 0.07), Yemen Taimoor (large yellowish green) epicarp (115 ± 0.05), Yemen Badami (large yellow) endocarp (114 ± 0.06), Indian Alphonso  mesocarp (114 ± 0.05), Kenya (large round red) endocarp (112 ± 0.06), Thailand (large green) mesocarp (110 ± 0.05), Thailand (large green) endocarp (109 ± 0.05), Yemen Badami (large yellow) epicarp (108 ± 0.04), and Kenya (large round red) epicarp (105 ± 0.02) exhibited significant (is study .p < 0.05) decrease in GPx activity was observed for cells treated with the extracts (100 μg/mL) of Egypt (reddish green) endocarp (0.25 ± 0.13), Thailand (large green) mesocarp (0.36 ± 0.12), Thailand (large green) endocarp (0.42 ± 0.11), Yemen Taimoor (large yellowish green) epicarp (0.53 ± 0.04), India Totapuri (long green) seed (0.53 ± 0.14), Sri Lanka (large yellowish green) seed (0.54 ± 0.23), Yemen Taimoor (large yellowish green) endocarp (0.57 ± 0.32), Yemen Kalabathoor (large round red) mesocarp (0.60 ± 0.17), and Sri Lanka (large yellowish green) epicarp (0.61 ± 0.11), as compared to the control cells. Extracts of Yemen Taimoor  seed (0.46 ± 0.05) and Yemen Badami (large yellow) seed (0.62 ± 0.13) showed a significant reduction for GPx activity at a dose of 50 μg/mL. The data for GPx activity are shown in For relative GPx activity, MTT-assisted cell viability was evaluated as described earlier. The tested concentration (100 μg/mL) showed a lack of cytotoxic effect for all the extracts except the following: Yemen Taimoor  seed, Thailand (large green) seed, Kenya (large round red) seed, Vietnam (large yellowish green) seed, Yemen Kalabathoor (large round red) endocarp, Yemen Badami (large yellow) seed, Yemen Taimoor (large yellowish green) seed, and Yemen Kalabathoor (large round red) seed where no sign of cytotoxicity was observed at 50 μg/mL (data not shown). A significant  endocarp showed a comparatively low IC50 value (μg/mL) of 108.8 ± 0.70, as compared to the standard drug acarbose (78.41 ± 0.67 μg/mL). The IC50 values for the tested extracts are shown in The initial screening (500 μg/mL) for α-amylase activity revealed inhibition of >50% for Yemen Kalabathoor (large round red) endocarp (60 ± 0.05), Vietnam (large yellowish green) seed (58 ± 0.07), India Badami (large green) seed (55 ± 0.13) extracts, and Yemen Taimoor  seed (54 ± 0.06), as shown in p < 0.05, whereas for correlations and component analysis, SPSS  V 22.0 was used.The results are expressed with a mean (±standard deviation (SD)) from at least three independent experiments. The data were analyzed for statistical significance between the treated and control group with the help of GraphPad Prism V-9.2.0  at The descriptive analysis showed a range with a mean (±SD) of 34–91 and 68.6 (±17.84) for HCT116, 34–98, and 64.81 (±19.28) for MCF7, 95–119, and 102.02 (±6.67) for glucose uptake, 0.25–1.30 and 0.722 (±0.21) for GPx, and 1–60 and 18.70 (±15.16) for α-amylase .p = 0.000). With regard to biological activities, a significant correlation was observed for the fruit part vs. HT116 , MCF7 , and α-amylase activity . Glucose uptake and GPx activities do not correlate with the geographical origin, fruit part, MG amount, or biological activities  the data for PCA are shown in The principal component analysis (PCA), an eigenvalue-based statistical tool, is employed to reduce the dimensions of a large dataset into principal components based on %variability. The PCA maximizes the variance for uncorrelated variables based on an individual and cumulative variance, with an impactful display of the relevant correlated variables in the same component. Herein, three components, PC1 (33.09%), PC2 (20.48%), and PC3 (14.65%), with a cumulative % variability of 68.23, were observed. For PC1 with major % variability, the variables presented were fruit part (−0.70), HCT116 (0.92), MCF7 (0.87), and α-amylase activity (−0.60), geographical origin (−0.91) and MG amount (0.83) were observed in PC2, whereas glucose uptake (0.62) and GPx activity (0.79) were observed in PC3, i.e., with the least % variability. The KMO-Bartlett’s test of sphericity showed a high A graphical presentation of the components observed with their corresponding eigenvalues is shown in p = 0.05).ANOVA with inter- and intra-correlation for the data variables also confirmed the outcomes from Pearson’s correlation and PCA analysis. The correlation of geographical origin with MG amount (−0.603), as well as fruit part with HCT116 (−0.550), MCF7 (−0.442), and α-amylase activity (0.315), was observed to be significant (0.00) with a high F-value of 627.81. The correlation for within and between groups, along with the sum of squares and mean square values, is shown in It is a well-known fact that variations in the geographical origin of a plant or fruit significantly affect its quality in terms of phytochemical profile and biological activities. The main aim of this study was to compare the quality variation among mango fruits obtained from different geographical origins. Mango fruits from eight different cultivars were collected and processed for green extraction and characterization of phytochemical profile based on MG amount in four parts of each mango fruit . The details regarding the novel green extraction and LCMSMS characterization for MG amount in these four parts of eight cultivars are described in our previous study . The ext50 values. The extract samples with <50% of cell viability were studied further at six different concentrations using an additional cell line of MRC5. In general, the seed parts of these mango cultivars were observed with considerable cytotoxic potential. The seed extract from Kenya mango (large and round) was seen to be more cytotoxic towards the tested cell lines  in general screening, whereas the MTT assay for selectivity against the three cell lines  suggested Sri Lanka (large yellowish green) seed extract with the lowest IC50 value followed by Thailand (large green) seed extract. Though various cytotoxic studies have been reported for mango fruit extract including the following: aqueous flesh extract [p < 0.05) increase in glucose uptake and utilization in HepG2 cell lines. The epicarps (Thailand (large green), Yemen Taimoor (large yellowish green), Yemen Badami (large yellow), Kenya (large round red)), endocarps (Yemen Badami (large yellow), Kenya (large round red), Thailand (large green)), and mesocarps (Kenya (large round red), Indian Alphonso , Thailand (large green)) parts were more effective as compared to the seeds of the mango fruits (100 μg/mL). In addition, the extracts of Yemen Badami (large yellow) seed and Thailand (large green) epicarp revealed a significant increase in glucose uptake at 50 μg/mL. As mentioned earlier, the MG amount was found more in the mentioned parts of these different cultivars fruit samples, epicarp, and mesocarp in particular. Mangiferin has been reported to promote glucose utilization and metabolism in a dose-dependent manner [50 value determination, where the endocarp from Yemen Kalabathoor (large round red) exhibited considerably low IC50 values when compared to the standard drug acarbose. The amount of MG in the endocarp is reported to be higher compared to seeds; hence, MG, along with some other constituents, may be responsible for the α-amylase activity. Previous studies have confirmed the α-amylase inhibitory role of mango peel-extracted MG [For cytotoxicity , a general followed by an in-depth screening was performed to determine the IC extract , peel ex extract ,18, seed extract ,20 as we extract , this is extract , along wt manner , promotet manner , enhancet manner , and dect manner . The GPxt manner ,27; howet manner ,29. An et manner ,31. One t manner ,33. In oacted MG ,35,36. Facted MG ,37. Howeacted MG .The outcomes of the biological activities suggest the presence of more than one bioactive phytochemical in mango fruit and its different parts. Albeit MG was observed with an average amount in all the 48 samples extracted for mango fruit, higher in epicarp and mesocarp parts, a systemic in-depth metabolomic analysis may be more useful to obtain a broader profile of the phytochemicals present in different varieties of the mango fruit (prone to variation based on the change in geography and other factors) vs. the effectiveness in biological activities of these samples. Moreover, the active phytochemicals  need to be isolated and tested further via in vivo studies in different concentrations. The isolated compounds might show lower activity compared to the mixture of compounds in the extract.The statistical models were applied to show the correlation among the biological activities of the different parts of the mango fruit from different cultivars. Pearson’s test revealed a significant correlation between the geographical origin vs. MG amount and fruit part vs. cytotoxicity  and α-amylase activity. Glucose uptake assay and GPx inhibition showed no correlation with the mentioned biological activities. For the fruit part, it was the seed part of the fruit that appeared as the most effective in cytotoxicity and α-amylase inhibitory activity. Likewise, the PCA analysis showed more %variability (intra-correlation) for PC1 with components of fruit part, HCT116, MCF7, and α-amylase. Geographical origin and MG amount were placed in PC2, whereas glucose uptake and GPx activities were grouped in PC3 with the least % variability, suggesting a more significant correlation for the seed fruit part with cytotoxicity and α-amylase activity. The ANOVA with inter- and intra-correlation for the dataset further confirmed an alike significant correlation with a high F-value as reported for Pearson’s and PCA analysis. The outcomes of the statistical model suggest a prominent role for the seed part of the mango fruit in various biological activities. It is noteworthy to mention that it is the first time to report the mangiferin-based characterization of the different mango fruit parts  from eight different cultivars (48 samples) with an extensive biological screening of cytotoxicity , glucose utilization (HepG2), GPx inhibition (HepG2), and α-amylase inhibition. None of the literature is available to be compared with the outcomes herein. The authors suggest a detailed metabolomic or phytochemical analysis to establish a prominent role in a series of biological activities for the seed part of the mango fruit. These in vitro studies may be followed by appropriate in vivo pharmacological experiments in order to confirm the role of mango seed, which may become a good source of phytochemicals, food products, and nutraceuticals to be utilized for the management and cure of various ailments.Fresh mango fruit varieties from different geographical origins  were collected from local markets at Khobar, Eastern Province, Kingdom of Saudi Arabia. A green ultrasonic-assisted extraction (using water as a solvent at a temperature of 40 °C) was performed for four different parts  of every individual mango fruit, as reported in our previous study .Human breast adenocarcinoma (MCF7: ATCC-HTB22); Human colorectal carcinoma (HCT116: ATCC-CCL247); Hepatocellular carcinoma (HepG2: ATCC-HB8065); normal human fetal lung fibroblast (MRC5: ATCC-CCL171).Dimethyl sulfoxide ; RPMI-1640 , DMEM (Dulbecco’s Modified Eagle Media), FBS , penicillin , and streptomycin  from Gibco, Life Technologies, Carlsbad, CA, USA; MTT assay reagent and α-amylase reagent Sigma Aldrich); Glucose uptake assay Kit (GAGO20) from Sigma Aldrich, St Louis, MO, USA; glutathione peroxidase Kit (ab102530) from Abcam, Cambridge, UK; doxorubicin (98.0–102.0% (HPLC)) from Sigma Aldrich and metformin (97%) from Merck, whereas Microplate reader used was from BIORAD, PR 4100, Hercules, CA, USA.The extracts were quantified for Mangiferin amount (MG amount) in all the mango fruit samples. UPLCMS/MS was applied to develop and validate a green, efficient, and fast analytical method for MG quantification . Though All cell lines were cultured and maintained according to the procedure reported by Ahmad et al. .The cytotoxicity of the extracts was evaluated by MTT assay, as previously reported  and the Odeyemi et al. modified method was used for glucose uptake assay ,41.The assay was performed according to the manufacturer’s protocol, and our group reported procedures ,28,39.The inhibitory activity of α-amylase was ascertained as described by Quan et al. . The extThis study investigated the quality of eight different cultivars of mango fruit in terms of the biological activities of the fruit parts. All parts of the mango fruit showed cytotoxicity, glucose uptake, GPx, and α-amylase inhibition. The statistical model suggested a significant correlation with most activities attributable to the seed part of the mango fruit. MG was correlated to the screened biological activities; however, a comprehensive phytochemical characterization with advanced in vivo studies and clinical trials may further confirm the role of mango fruit for the cure of diseases in the form of food products, nutraceuticals, or isolated medicinal compounds. The study assessed quality variation among different mango cultivars and established a correlation between phytochemistry and the biological activity of mango fruit and its parts from different geographical origins.


The lipid molecules and volatile organic compounds (VOCs) in milk are heavily influenced by diet. However, little is known about how roughage affects the lipid and VOC contents of donkey milk. Accordingly, in the present study, donkeys were fed corn straw (G1 group), wheat hulls (G2 group), or wheat straw (G3 group), and the lipid and VOC profiles of their milk were determined using LC–MS and GC–MS. Of the 1842 lipids identified in donkey milk, 153 were found to be differential, including glycerolipids, glycerophospholipids, and sphingolipids. The G1 group showed a greater variety and content of triacyclglycerol species than the G2 and G3 groups. Of 45 VOCs, 31 were identified as differential, including nitrogen compounds, esters, and alcohols. These VOCs were significantly increased in the G2 and G3 groups, with the greatest difference being between the G1 and G2 groups. Thus, our study demonstrates that dietary roughage changes the lipid and VOC profiles of donkey milk. Dairy products are among the most common foods in daily life, accounting for 14% of global agricultural trade . With onMilk lipids are not only an important indication of milk quality, they also play an important role in promoting infant brain development, regulate the cell cycle and calcium absorption, reduce inflammatory factors, and reduce skeletal muscle lipid accumulation ,7,8,9,10Previous LC–MS studies have identified 335 lipids from 13 subclasses in donkey milk, including TGs, phosphatidic acids (PAs), phosphatidylcholines (PCs), and phosphatidylethanolamines (PEs) . The basThe lipids and VOCs in milk are influenced by several factors, including metabolic differences in the animal, feed ingredients and additives, biological enzymes, microorganisms, and production processes ,19. ImpoTherefore, the present study aimed to investigate the effects of different dietary roughages on the lipid and VOC profiles of donkey milk using LC–MS and GC–MS. The results presented will increase our understanding of the effects of dietary roughage on donkey milk quality and provide valuable information for the farming of donkeys.In this study, all animal experiments were approved by the Animal Care and Use Committee of Liaocheng University  (2023022706). Thirty-six healthy pluriparous jennies  were obtained from a local farm (Shandong Province) and reared in the same semi-intensive system. They were randomly allocated to one of three groups (12 donkeys per group). Three kinds of roughage, corn straw (dry matter (DM): 93.70%, crude protein (CP): 4.28%, crude fat (CF): 1.90%, crude ash (Ash): 7.48%, neutral detergent fiber (NDF): 68.30%, acid detergent fiber (ADF): 36.60%, calcium (Ca): 0.57%, phosphorus (P): 0.18%; G1 group), wheat hulls , and wheat straw  were used as basic dietary roughage. The formulation, proximate analyses, and major FA levels of the diets are presented in v/v) at −20 °C as described previously [The process for lipid extraction was based on previous reports with minor modifications. The lipids were extracted from the milk with pre-cooled chloroform/methanol  and quantitatively analyze the samples.A donkey milk sample (15 mL) was placed in a 20 mL headspace vial . An SPME fiber (120 µm DVB/CWR/PDMS) was exposed to the headspace for 15 min at 100 °C. Then, they were subjected to GC  desorption of the VOCs at 250 °C for 5 min in splitless mode. A 30 m × 0.25 mm × 0.25 μm DB-5MS  capillary column was used for separation. The carrier gas utilized had a linear velocity of 1.2 mL/min, and the injector temperature was 250 °C. The column oven temperature was kept at 40 °C (3.5 min), then increased at 10 °C/min to 100 °C, 7 °C/min to 180 °C, and 25 °C/min to 280 °C, where the temperature was held for 5 min. Mass spectra were recorded in electron impact ionization mode at 70 eV and scanned at 1-s intervals in the n = 9) with a significant difference in variable importance in projection (VIP) > 1 and p < 0.05. The data were assessed by orthogonal partial least squares discriminant analysis (OPLS-DA). Metabolites and pathways associated with the differential lipid compounds were identified using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database . An amount of p < 0.05 was taken to indicate significant differences in the VOCs.The data were processed using SPSS version 26.0 , subjected to one-way analysis of variance (ANOVA), and compared for differences by Tukey’s test, Duncan’s multiple range test, and Fisher’s protected least significant difference (LSD) test. The lipidomic data were expressed as mean ± standard error of the mean  . Free faThere were differences between milk lipids and volatile organic compounds as a result of changes in roughage. A total of 1842 lipids were detected, among which 153 lipids were considered differential, being mainly involved in GP and SP metabolism pathways. There was significantly increased GL content in the milk of donkeys fed corn straw, while GP and SP contents were significantly increased by feeding with wheat straw. The result indicates that coarse grains mainly influence the GL, GP, and SP composition of donkey milk lipids. Furthermore, 31 out of 45 VOCs were determined to be differential, mainly esters, alcohols, and phenols, the contents of which were significantly increased for donkeys fed wheat hull and wheat straw than corn straw.