Coreopsis tinctoria Nutt., arousing huge interest recently for its considerable biological characteristics including antioxidant, antineurotoxic, and antidiabetic activities. An ultrahigh performance liquid chromatography triple-quadrupole tandem mass spectrometry (UPLC-MS) was successfully used to determine okanin in rat plasma after oral administration of okanin. Bavachalcone acted as an internal standard (IS). By gradient elution, IS and analyte were separated on a C18 column for 7 min at a flow rate of 0.25 mL/min with acetonitrile-0.1% acetic acid mobile phase. The stability, matrix effect, extraction recovery, accuracy, precision, linearity, and selectivity of the method were firstly demonstrated. The major pharmacokinetic parameters of okanin in rat plasma were then measured using the developed UPLC-MS method. An UPLC-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was finally established to obtain the specific and accurate mass of okanin in rat plasma after oral administration, and its proposed fragmentation was further elaborated.Okanin is a major flavonoid found in  Coreopsis tinctoria Nutt.'s (C. tinctoria) capitula is consumed as herbal tea, and its extracts exhibit various biological activities including antioxidant [C. tinctoria [C. tinctoria. Okanin had the prominent effect on DPPH scavenging acts with EC50 as 6.2 μM, which was stronger than those of butylated hydroxytoluene and ascorbic acid with EC50 as 45.8 μM and 30.4 μM, separately [50 as 11.0 μM [α-glucosidase with IC50 values about 0.02 mM, suggesting that it is a potential drug for the treatment of diabetes [50 value at 15.54 μM, suggesting that it can treat neurodegenerative diseases [In China, ioxidant –4, antihioxidant , 6, antiioxidant , antidiaioxidant –11, hepaioxidant , and antioxidant . Okanin inctoria  having aparately . Meanwhi 11.0 μM . Kil et  11.0 μM . Furtherdiabetes . Besidesdiseases .C. tinctoria become necessary. The flavonoids including okanin from C. tinctoria had been successfully analyzed by UPLC, HPLC-MS, high-performance thin-layer chromatography, and capillary zone electrophoresis methods [C. tinctoria among these methods [C. tinctoria in order to better understand their pharmacological action. Only one report emphasized the characterization of the pharmacokinetics of okanin and isookanin, the two most prominent antioxidants, in rat plasma after oral administration of 100 mg/kg ethanol extract of C. tinctoria capitula; however, the pharmacokinetic parameters of two targets only measured the time to reach Cmax (Tmax) and the maximum plasma concentration (Cmax) [Smilacis glabrae extract [Due to the significant pharmacological effects, effective methods for the identification of flavonoids in  methods , 18–21.  methods , 19. Mean (Cmax) . Neverthn (Cmax) –25. Thern (Cmax) –24, but In this work, an effective UPLC–MS/MS method coupled with multichannel segmented selected reaction monitoring program was developed for measuring the levels of okanin in rat plasma after oral monomer administration. Bavachalcone served as an internal standard (IS). The structures of the okanin and IS are shown in 18, 6 cc/500 mg) were provided by Wan Cheng Bo Da . Triple deionized water was adopted during the study and then purified with a Millipore purification system .Okanin and bavachalcone with purities over 98% were provided by Shanghai Yuanye Bio-Technology Co., Ltd. . Heparin sodium was provided by Affandi . HPLC grade acetonitrile, acetic acid, and methanol were purchased from E. Merck . The SPE cartridges  with an electrospray ionization (ESI) interface. An ACQUITYTM BEH C18 column  combined with an ACQUITYTM UPLC C18 guard column was used for the separation. The column temperature was set at 35°C. The binary mobile phase system was composed by 0.1% acetic acid (A) and acetonitrile (B). The gradient conditions of the mobile phase included: 0–2 min, 95–20% (A); 2-3 min, 20–20% (A); 3–3.5 min, 20–95% (A); 3.5–7 min, 95–95% (A). The flow rate was kept at 0.25 mL/min for an overall 7-min run time. The volume of sample injection was 5 μL. Mass spectrometer was further improved as follows: capillary voltage, 3.50 kV (+); source temperature, 120°C; and desolvation temperature 300°C. Nitrogen was served as the desolvation and cone gas, and flow rates were set at 600 and 50 L/h, respectively. Argon acted as the collision gas at 2.89 × 10−3 mbar pressure. Multiple reaction monitoring (MRM) in the positive ion mode was selected in this study. The dwell time was automatically set by MassLynx NT 4.1 software. All data obtained from the centroid mode were further processed by MassLynx NT 4.1 software. The improved MRM transitions and energy parameters including cone voltage and collision energy of okanin and IS are listed in Supplementary An UPLC-MS/MS method was carried out using an ACQUITY2 was 35 V.The full-scan mass range was set at 50–1200 for obtaining accurate mass of okanin by UPLC-Q-TOF-MS analysis. The mass spectrometer conditions were listed as follows: gas temperature 550°C, spray gas 50 psi, auxiliary gas 50 psi, curtain gas 35 psi, ion spray voltage 5500 V, declustering potential 90 V, and collision energy for MSOne mg/mL of okanin was prepared with methanol as standard stock solution. Then, the standard stock solution was diluted 10 times as working solution. In the meantime, one mg/mL of bavachalcone was prepared with methanol as an IS solution. Calibration standards, low, intermediate, and high levels of quality control (QC) samples were prepared using spiking blank rat plasma with appropriate volumes of the working solutions.Six serial concentrations were 1.956, 10.95, 40.40, 200.20, 800.24, and 1390 ng/mL for calibration standard solution. The concentrations of the low, intermediate, and high levels of QC samples were 10.01, 500.10, and 1000.20 ng/mL for okanin. All samples were incubated at −20°C before application.μL blank plasma, and 50 μL of IS solution (22.06 ng/mL) was added to respective tube.Two hundred microlitres of calibration standards, QC samples, and plasma samples were added to a 1.5 mL Eppendorf tube containing 400 μL methanol and centrifuged at 16,000 ×g for 15 min. Five microlitres of aliquot was subsequently injected into the UPLC-MS for investigation.Before loading the sample, the SPE cartridges were washed with 3 mL of methanol and 5 mL of purified water separately. The mixture in respective tube was vortexed for 1.0 min and then loaded onto a SPE cartridges. The SPE cartridges were separately eluted with 2 mL purified water and 4.5 mL of methanol. The resulting eluate was dried with nitrogen. The residue was redissolved in 50 Rt) and MRM transitions were adopted for analyzing okanin and IS. Meanwhile, the accurate mass of okanin and its proposed fragmentation mechanism in rat plasma after oral administration were obtained using UPLC-Q-TOF-MS.In order to study the possible endogenous interference on okanin and IS, the blank plasma from six rats, the corresponding blank plasma added with okanin and IS, and the plasma of rats after oral administration of okanin were analyzed by UPLC-MS/MS, respectively. The retention times (x2-weighted liner least-square regression model. The lower limit of detection (LLOD) was defined as the lowest concentration with at least three signal-to-noise ratios. The lower limit of quantification (LLOQ) was defined as the lowest concentration on the standard curve that can be quantitated with an accuracy of 80–120% and a precision value not exceeding 20%.Calibration curve for okanin was plotted for the peak area ratio of okanin/IS versus plasma concentrations by a 1/n = 6) of low, middle, and high concentrations of QC samples in the same day and over three consecutive days, respectively. The concentration of sample was calculated according to the standard curve equation prepared on the same day. The precision of the method was calculated as the relative standard deviation (RSD) and was less than 15%. Accuracy was expressed as the relative error (RE) and was within ±15%. The recovery and matrix effects were measured using low-, middle- and high-concentration QC samples with six replicates. The recovery was assessed by comparing the peak areas of the spiked samples to those of the untreated sample. The matrix effect was determined by comparing the peak areas of spiked blank plasma (A) to the peak areas of the solution standards in methanol at equal concentrations (B). The matrix factor (MF) was expressed as the ratio of (A/B × 100), and the MF values ranged from 80% to 120%. The stabilities of okanin in plasma were evaluated by analyzing the three concentrations of QC samples at different storage and processing procedures. The short-term stability was also assessed by analyzing the QC sample retained in the autosampler (4°C) for 36 h. The QC sample was stored at −20°C for 30 days in order to evaluate long-term stability. The freeze-thaw stability of QC samples was evaluated by transferring them from −20°C to room temperature for three complete freeze-thaw cycles. The stability was demonstrated if the percentage deviation was no more than 15% of the nominal concentration.Intraday and interday accuracy and precision were assessed from replicate analyses  rats (250 ± 20 g) from Animal Safety Evaluation Center of Heilongjiang University of Chinese Medicine (China). The rats were divided into control and treatment groups with six animals each. The rats were maintained under a light/dark cycle of 12 h, in the temperature range of 22°C ± 2°C to ambient temperature, and at a humidity of 50% ± 5%. Before the experiment, all rats were fasted for 12 h, but allowed to drink water freely. Each rat in the treatment and control group were treated with 1 mg/kg okanin and an equal volume of distilled water in an oral manner, separately. Blood samples (approximately 0.4 mL) were collected into 1.5 mL heparinized polythene tubes from the suborbital venous plexus before administration and at 0, 0.083, 0.167, 0.33, 0.50, 0.75, 1.0, 2.0, 4.0, 8.0, 12.0, and 24 h after administration. Then, the blood samples were immediately centrifuged at 13,000 rpm for 10 min, and samples were processed as described in Cmax) and the time to reach Cmax (Tmax) were directly determined. The elimination rate constant (Kel) was then obtained via linear regression analysis on the log-linear portion of the plasma concentration versus time curve. The other major pharmacokinetic parameters of okanin in rat plasma including half life (t1/2), the mean residence time from zero to the sampling time (MRTt0–) and to infinity (MRT∞0–), and area under the plasma concentration versus time curve from zero to the sampling time (AUCt0–) and to infinity (AUC0–∞) were determined using WinNonlin Professional Version 5.2.1 (http://www.pharsight.com). The results were expressed as arithmetic mean ± standard deviation .According to observing plasma concentration versus time curve, maximum plasma concentration  by the high-resolution Q-TOF analyses for structural confirmation. In UPLC-Q-TOF-MS/MS experiment, the ion at m/z 289.0717 yielded four fragment ions at m/z 271.0431, 163.0388, 153.0182, and 135.0439, respectively. As shown in m/z 163.0388 was produced by losing A's ring. The predominant fragmentation ion at m/z 135.0439 and 153.0182 was formed from the cleavage of position 3 in the three-carbon chain, and the charge is retained in ring A and ring B, respectively. Minor fragment m/z 271.0431 was produced directly from the parent ion of m/z 289.0717 due to the neutral loss of one molecule of water, indicating the existence of orthophenol hydroxyl in the structure. The proposed fragmentation mechanism was elaborated in Okanin in rat plasma eluted at 2.38 min showed a major molecular ion at y = 19.979x + 337.59 and r2 = 0.9980  of 1296.12 ± 60.31 ng/mL (mean ± SD), revealing quick absorption of okanin into blood circulatory system. An existing report suggested Tmax and Cmax values for okanin of 0.333 h and 10.5 μM after oral administration of 100 mg/kg ethanol extract of C. tinctoria capitula [Tmax values, okanin was absorbed into blood more quickly by orally administered monomer than the extract of C. tinctoria, indicating a possible pharmacokinetic interaction of multiple components in the extract of C. tinctoria. Our conclusion was consistent with that of literatures [As listed in Supplementary capitula . In conteratures –25.t1/2, Kel, AUCt0–, AUC 0–∞, MRTt0–, and MRT∞0– values were determined here. The concentrations of okanin in rat plasma were decreased quickly, and the shortest elimination half life (t1/2) at 0.89 ± 0.09 h was observed for okanin. The Kel values were 0.75 ± 0.08 for okanin. The AUCt0– and AUC ∞0– values of okanin were 1728.78 ± 146.64 and 1826.51 ± 149.59 ng·h/mL, respectively. MRTt0– and MRT0–∞ values for okanin were 1.14 ± 0.06 h and 1.36 ± 0.10 h, separately.To further study the pharmacokinetic behaviors of okanin, more pharmacokinetic parameters including Tmax, Cmax, t1/2, Kel, AUCt0–, AUC 0–∞, MRTt0–, and MRT∞0– values were obtained in this study. Meanwhile, target okanin had shorter Tmax by monomer administration than that of herb extract administration, suggesting a potential pharmacokinetic interaction among multiple components in herb extract. The pharmacokinetic behaviors for okanin provide the basis for further studies to elucidate the mechanisms of action and facilitate clinical application.An effective UPLC–MS/MS method was developed for analyzing okanin in rat plasma after oral administration okanin monomer for the first time. Compared with the literature report, more pharmacokinetic parameters of okanin including


N-methylamino-l-alanine (BMAA) is connected to the development of neurodegenerative diseases. BMAA has been shown to accumulate in aquatic ecosystems, and filter-feeding molluscs seem particularly susceptible to BMAA accumulation. The blue mussels farmed along the Swedish coastline in the Baltic Sea are, due to their small size, exclusively used to produce feed for chicken and fish in the agro–aqua cycle. We have investigated the possible biotransfer of BMAA from mussels, via mussel-based feed, into chickens. Chickens were divided into two groups, the control and the treatment. BMAA was extracted from the muscle, liver, brain, and eye tissues in both chicken groups; a UPLC-MS/MS method was subsequently used to quantify BMAA. The results indicate detectable concentrations of BMAA in both chicken groups. However, the BMAA concentration in chicken was 5.65 times higher in the treatment group than the control group, with the highest concentration found in muscle tissue extracted from the treatment group chickens. These data suggest that there is a BMAA transfer route within the agro-aqua cycle, so further investigation is recommended before using mussel-based feed in the chicken industry.The neurotoxic non-protein amino acid β- N-methylamino-l-alanine (BMAA) is a neurotoxic non-proteinogenic amino acid naturally produced by a range of ecologically diverse phytoplankton groups such as cyanobacteria, diatoms, and dinoflagellates [β-gellates ,2,3. BMAgellates ,4. The cgellates ,11,12,13Attention to BMAA, as an environmental neurotoxic agent, commenced in the 1940s with the discovery of an unexpectedly high frequency of ALS/PDC among the indigenous Chamorro population of Guam ,15,16. TAlthough the development of neurodegenerative diseases such as ALS and Parkinson’s disease is difficult to directly associate with BMAA exposure via contaminated aquatic food, epidemiological studies show an increased risk of algal toxin exposure not only through aquatic food production but also via other sources such as recreation and aerosolization . For insMytilus edulis and M. trossulus are today exclusively used as organic fertilizers and feed for chicken and fish in the so-called agro–aqua cycle [The use of filter-feeding organisms  is a growing industry in the Baltic Sea region and is considered one way to efficiently mitigate eutrophication and clarify the water ,38,39. Hua cycle . Previouua cycle ,27. HoweM. edulis and trossulus)  or standard feed . The tissues investigated were muscle, liver, brain, and eye tissues. The amounts of BMAA in the standard feed and the feed mixed/spiked with mussel meat were also investigated. The data show that the consumption of feed mixed with mussel meat might establish the potential for BMAA transfer within the agro-aqua cycle. In addition, the occurrence of bioaccumulated BMAA found in the eye and liver tissue of the control group chickens fed only standard feed might imply that the eye and liver is more prone to bind BMAA.In the study, two groups of chickens were fed, for 36 days, either standard feed spiked with 10% Baltic Sea blue mussel . The concentration of BMAA extracted from the feed mixed with mussel meat  was 0.1818 ± 0.0319 µg BMAA g−1 DW  displayed a higher growth rate, and on average, the slaughter weight was approximately 50 g higher than in the control group  . No abnoA g−1 DW , Table 1−1 DW.BMAA was extracted from four different tissues  in both the TG and CG; it was subsequently quantified using UPLC-MS/MS and expressed in µg BMAA g−1 DW , Tinca tinca (0.00141 to 0.00561 µg BMAA g−1 DW), and Abramis brama (0.00103 ± 0.00027 to 0.00200 ± 0.00173 µg BMAA g−1 DW) [Osmerus eperlanus (0.016 ± 0.0009 to 0.24 ± 0.003 µg BMAA g−1 DW), Scophthalmus maximus (0.0008 ± 0.0003 to 1.29 ± 0.03 µg BMAA g−1 DW), Clupea harengus (0.0007 ± 0.00008 to 0.010 ± 0.001 µg BMAA g−1 DW), and Coregonus lavaretus (0.0019 ± 0.00007 to 0.059 ± 0.004 µg BMAA g−1 DW) [−1 DW (free form) and from 0.0348 ± 0.0081 to 0.230 ± 0.0974 µg BMAA g−1 DW (protein-associated form)  , and in ed form) , Table 2P. mariannus mariannus contained the highest concentration of BMAA, followed by muscle, liver, skin, and kidney tissues, while in most fish samples collected from the Baltic Sea, the highest BMAA concentrations were detected in brain tissue followed by muscle tissue [The distribution of accumulated BMAA in vertebrates varies according to species and their environments. In this study, the highest BMAA concentration was detected in muscle tissue, the tissue mostly consumed by humans. The second highest concentration was confirmed in brain, followed by eye and liver tissues , Table 3e tissue . In conte tissue , the hige tissue . All theAnother aspect of this study is the unexpectedly large number (8/12) of individual chickens fed only SF that contained BMAA . Seven oNotably, 3H-BMAA was shown to preferentially accumulate in the pigmented parts of melanin- and neuromelanin-containing tissues such as brain, eye, hair, liver, and the melanocytes surrounding blood vessels and visceral organs in mice and frogs, further suggesting that these are possible BMAA reservoirs . ThroughM. edulis and trossulus as ingredients in the industrial production of animal feed. The trial data show that BMAA bioaccumulated in all analyzed tissues dissected from the individual chickens. Furthermore, the highest amount of bioaccumulated BMAA was found in the muscle tissue of chickens fed mussel meal. Humans commonly consume the muscle tissue of chickens, so this might lead to a higher risk of neurotoxin exposure if the chicken feed is mixed with mussels.The present data clearly demonstrate the risk of using the blue mussels n = 17) was fed MF comprising 90% SF and 10% Baltic Sea blue mussels M. edulis and M. trossulus, while the CG (n = 12) was fed 100% SF comprising wheat, barley, oats, and maize. The mussels M. edulis and M. trossulus, used in producing the MF, were collected from the mussel farm located at Hasselö in Västervik municipality . Four types of tissues, i.e., muscle, liver, brain, and eye, were dissected by the veterinarian. The dissected samples were directly stored in liquid nitrogen while being transferred to the laboratory. Chicken specimens were stored at −80 °C until BMAA was extracted.Two groups of chickens grew for 36 days, from 21 May to 25 June 2018. The experiment was conducted in collaboration with the Kalmar Sound Commission and the company Ölands Chicken AB , and was performed under controlled conditions, with the chickens being monitored by a bird specialist veterinarian. Twenty-four hours after hatching, the TG  for 3 min at 70% efficiency. The procedure was performed in an ice-water bath to prevent protein degradation. Samples were homogenized by vortexing and the proteins were incubated for 48 h at 4 °C for protein precipitation. To separate the precipitated protein pellet containing the protein-associated BMAA and the supernatant containing the free BMAA, samples were centrifuged for 10 min at 10,000× g at 4 °C. To further ensure efficient protein precipitation and separation between the free and protein-associated forms of BMAA, yet another 500 µL of 0.1 M TCA was added to the pellet and the samples were once again incubated for 1 h at 4 °C. Subsequently, samples were centrifuged at 10,000× g and 4 °C for 10 min. Both the supernatant (from the first and second rounds of centrifugation) and the pellets containing the free and protein-associated forms of BMAA, respectively, were lyophilized in a freeze dryer, and hydrolyzed in 600 µL of 6 M HCl for 20 h at 110 °C. Both fractions were filtered with a centrifugal filter unit  for 1 min at 10,000× g. Samples were frozen at −80 °C for lyophilization. The lyophilized samples were later diluted before UPLC-MS/MS analysis. Samples of free BMAA were diluted with 2 mL of 20 mM HCl. Samples of protein-associated BMAA were diluted with 20 mM HCl to obtain an optimum ratio of protein to derivatization agent [The samples of the different chicken tissues  as well as the different feeds  were lyophilized using a CoolSafe freeze-dryer  at −110 °C. The numbers of biological replicates were as follows: TG, h et al.  with minh et al. ,59. SampAliquots (10 µL) of the TCA-precipitated protein sample taken after adding TCA in the second round were used to measure the total protein contents. Three technical replicates of each biological replicate were used to determine the total amount of protein per biological replicate. The amount was quantified using the BioRad RC/DC kit  according to the manufacturer’s instructions. Spectrophotometric measurements were made using a µQuant monochromatic microplate spectrophotometer .−1. The linear gradient elution program was as follows: 1% B for 0–0.5 min, 60% B for 4 min, 99% B for 4.10 min, and 1% B for 5.20–6.20 min; injection volumes were 20 μL. Analytes were analyzed in positive ion mode using electrospray ionization and selected reaction monitoring (SRM) scan mode using the following transitions: general to all three analytes, 459.1 > 119.08; 4-diaminobutyric acid (DAB) diagnostic fragment, 459.1 > 188.1; N-(2-aminoethyl) glycine (AEG) diagnostic fragment, 459.1 > 214.1; and BMAA diagnostic fragment, 459.1 > 258.09. The instrument parameters were as follows: cone voltage, 30 V; source temperature, 150 °C; desolvation temperature, 550 °C; cone gas flow, 20 L h−1; desolvation gas flow, 1000 L h−1; collision gas flow, 0.15 mL min−1; and collision energy, 26 V. MassLynx V4.1 software  was used to analyze the acquired chromatographic data.The diluted samples were derivatized with AccQ-Tag using a WAT052880 AccQ-Tag kit , i.e., 70 µL of borate buffer and 30 µL of AQC. An Acquity UPLC system coupled to a Xevo-TQ-MS system (Waters) was used for BMAA analysis, based on the Lage et al.  method w−1) and seven for the protein-associated form . Different tissue and BMAA types displayed different limits of quantification (LOQ). Concentrations below 0.3  or 1.2 (for brain tissues for the protein-associated form and for eye tissues for the free form) ng mL−1 (0.425 nM) were below the LOQ, so the samples were considered non-quantifiable (NQ). Samples with no detected BMAA peak were considered non-detectable (ND). Most MF samples (11/12) contained BMAA, so to confirm the BMAA concentration, H3-BMAA in two concentrations (0.2 and 0.5 ng mL−1), representing the range of BMAA concentrations in the sample, was added to quantify BMAA concentration.Samples with no detected BMAA were selected for constructing a standard curve in order to quantify BMAA. Calibration curves were made for each tissue type  and BMAA type  at six concentrations for the free form (i.e., 0.3, 1.2, 3, 6, 12, and 30 ng mL


In Sri Lanka, a Polyherbal Ayurvedic Formulation (PHAF), which consists of powders of seven medicinal plants, is being trialed for use as an anti-inflammatory drug. In general, anti-inflammatory drugs have good antioxidant properties. Therefore, in the present study, an attempt was made to assess the quality and evaluate the antioxidant potential of PHAF.  Physicochemical parameters such as ash content, extractable matter, phytochemical screening for secondary metabolites, levels of heavy metals, and microbes were determined according to standard protocols. Antioxidant activity was evaluated using five in vitro assays: total polyphenolic content (TPC), total flavonoid content (TFC), ORAC , DPPH , and ABTS  diammonium salt.  PHAF contained 5.6 ± 0.2% of moisture, 6.5 ± 0.1% of total ash, 1.4 ± 0.1% of water soluble ash, 0.9 ± 0.0% of acid insoluble ash, 7.7 ± 0.2% of hot water extractable matter, 3.9 ± 0.1% of cold water extractable matter, 10.5 ± 4.1% of hot-ethanol extractable matter, and 8.4 ± 0.2% of cold-ethanol extractable matter. Phytochemical screening revealed the presence of phenolic compounds, tannins, flavonoids, coumarins, and saponins in both aqueous and ethanolic extracts of the drug. TPC, TFC, ORAC, DPPH, and ABTS of aqueous and ethanol extracts of PHAF were 103.65 ± 4.94 and 327.07 ± 9.65 mg gallic acid equivalents/g extract, 76.6 ± 5.83 and 224.6 ± 8.42 mg quercetin equivalents/g of extract, 481.11 ± 17.30 and 1481.44 ± 30.20 mg trolox equivalents/g of extract, 79.50 ± 4.42 and 227.17 ± 6.16 mg trolox equivalents/g of extract, and 198.20 ± 4.55 and 577.08 ± 5.48 mg trolox equivalents/g of extract, respectively.  Ethanolic extract of PHAF is better than aqueous extract in terms of antioxidant properties. Since prehistoric times, herbs were the basis for nearly all medicinal therapy until synthetic drugs were developed in the nineteenth century, a time when the prominence of herbal remedies gradually decreased in developed countries , 2. InteFree radicals are molecular species or atoms which contain one or more unpaired electrons. These are constantly produced in the human body as a result of cell metabolism . Free raPrevious researches have indicated that phenolic compounds and flavonoids act as excellent anti-inflammatory agents . Due to A novel Polyherbal Ayurvedic Formulation (PHAF), which consists of powders of seven medicinal plants , is beinx 79° 50′ 52.01″ E) Western province, Sri Lanka, and authenticated by the Curator of National Herbarium of Peradeniya, Sri Lanka. The plant materials were identified and authenticated by Prof. M. H. A. Tissera, Professor in Dravyaguna, Department of Dravyaguna, Gampaha Wickramarachchi Ayurveda Institute, University of Kelaniya, Yakkala, Sri Lanka. A voucher specimen from each plant (PHAF1-7) was deposited at Department of Kaumarabruthya and Stree Roga, Gampaha Wickramarachchi Ayurveda Institute, University of Kelaniya, Yakkala, Sri Lanka, for future reference.Fresh plant materials required for PHAF were collected from Colombo  and mixed well.μm filter paper, and the filtrate was concentrated using a rotary evaporator (yield: 9.2% w/w) and stored at 4°C until used.PHAF powder (50 g) was added to a round bottom flask containing 150 mL of ethanol and boiled for 4 h. Then, the extract was filtered using Whatman 0.45 μm filter paper and the filtrate was concentrated using a rotary evaporator and freeze dried (yield: 12.5% w/w) and stored at 4°C until used.PHAF powder (50 g) was added to a round bottom flask containing 150 mL of water and boiled for 4 h. Then, the extract was filtered using Whatman 0.45 Physicochemical parameters of PHAF powder were assayed according to the World Health Organization (WHO) guidelines on quality control methods for herbal materials . PhysicoPhytochemical screening was carried out for PHAF (ethanol and aqueous extracts) according to methods described by Yadav and Agarwala  with sliQuantitative determination of arsenic (As) , mercuryEscherichia coli [, Salmonella [Staphylococcus aureus [Microbial tests for aerobic plate count , coliforhia coli , Salmonelmonella , and Stas aureus  were carAntioxidant activity of the ethanol and aqueous extracts of PHAF powder was investigated using the following in vitro assays.Total polyphenol content of ethanol and aqueous extracts of PHAF was determined using the Folin–Ciocalteu reagent  in 96-weTotal ﬂavonoid content of ethanol and aqueous extracts of PHAF was determined using the aluminium chloride method in 96-well microplates  with somμg/mL) was used to construct the standard curve. Results were expressed as trolox equivalents antioxidant capacity  on a dry weight basis.The DPPH  radical scavenging assay was performed in 96-well microplates according to the method described by Blois  with som+ radical scavenging assay was performed in 96-well microplates according to the method described by Re [The ABTSed by Re  with somμg/mL), fluorescein (4.8 μM), and 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) (40 mg/mL) solutions were prepared in phosphate buffer  prior to use. Ethanol or aqueous extracts of PHAF were initially dissolved in DMSO. Reaction volumes of 200 μL containing 100 μL of 4.8 Μm fluorescein and 50 µL of 15.62 and 7.81 µg/mL of different concentrations of ethanol and aqueous extracts of PHAF were preincubated at 37°C for 10 minutes followed by addition of 50 µL of AAPH (40 mg/mL) to each well to initiate the reaction. The plate was placed on the fluorescent microplate reader  set with excitation and emission at 494 nm and 535 nm and decay of fluorescein was recorded in 1-minute intervals for 60 minutes. Results were expressed as trolox equivalents antioxidant capacity  on a dry weight basis.The ORAC radical scavenging assay was performed in 96-well microplates according to the method described by Ou  with somP < 0.05 was regarded as significant.Data were statistically analyzed using GraphPad Prism version 4.0. One-way analysis of variance (ANOVA) and Duncan's multiple range test were used to determine the differences among treatment means. Physicochemical parameters of PHAF are illustrated in Tannins, flavonoids, saponins, coumarins, and phenolic compounds were present in both hot water and hot-ethanol extracts. Alkaloids and cardiac glycosides were present only in aqueous extract.Heavy metals such as Pb, Cd, Hg, and As were not detected in PHAF. The minimum detection levels of Pb, Cd, Hg, and As were 0.5 ppm, 0.05 ppm, 0.05 ppm, and 0.05 ppm, respectively.Escherichia coli, Coliforms, and Salmonella were not present in the PHAF. Aerobic plate counts revealed few (<10 CFU/g) yeasts and moulds (<10 CFU/g) and Staphylococcus aureus (<10/g).Microbes including Antioxidant properties of ethanol and aqueous extracts of PHAF powder are given in In the present study, significantly more matter was extracted by ethanol than by water. Further, more extractable matter was present in hot extracts than in cold extracts (both water and ethanol). Very little acid insoluble ash was present in PHAF , indicatC. rotundus [C. longa [A. indica [C. fistula [C. fenestratum [S. suaveolens [G. glabra, one of the ingredients in PHAF, is rich in glycyrrhizic acid which exhibits significant antioxidant activity [The main purpose of our study was to evaluate antioxidant activity of PHAF by using in vitro assays. Antioxidant activity has been evaluated previously for each of the seven plant species present in PHAF. Examples include antioxidant activities for rotundus , 26, C. C. longa , A. indi. indica , C. fist fistula , 30, C. estratum , and S. aveolens . G. glabactivity .According to traditional Ayurveda practice, PHAF is administered to patients in the hot water extract form. We, therefore, assayed antioxidant activities for the aqueous extract. However, hot water preparations have some drawbacks, such as unpleasant taste, and have lower stability, requiring them to be prepared freshly on each occasion. To avoid this drawback, it would be better to develop pharmaceutics such as syrups and tonics. Therefore, we also assayed the ethanol extract of PHAF, which exhibited overall higher levels of antioxidant activities than that of aqueous extract . Ethanol+ assay, a blue/green ABTS.+ chromophore is generated by the oxidation of ABTS with potassium persulfate. Antioxidants which have the ability of donating hydrogen reduced the blue/green color of ABTS+ and can be measured spectrophotometrically at 745 nm [.+ assay measures both hydrophilic and lipophilic antioxidants [In ABTSt 745 nm . DPPH ist 745 nm . In DPPHt 745 nm , 37 fromoxidants .50 values of ABTS+ (for both aqueous and ethanol extracts) were significantly lower than that of DPPH  dihydrochloride (AAPH), was estimated in the ORAC assay. The ORAC value is directly proportional to the degree of antioxidant power [The capacity of a compound to scavenge peroxyl radicals, generated by spontaneous decomposition of 2,2nt power , 40.+ and DPPH than aqueous extract of the drug. Similar results have been shown with Foeniculum vulgare [Phenolic compounds and flavonoids are reported to have antioxidant and free-radical scavenging activity , 42. The vulgare . Among t vulgare , 46. TheThe ash content is a criterion to judge the identity and purity of crude drugs , 48. A hThe phytochemical constituents of many medicinal plants have been recorded by a number of researchers during the last few decades , 50, 51.We have reported the quality assessment and antioxidant properties of PHAF for the first time. PHAF has potent antioxidant properties, which may be due to the presence of high content of polyphenols and flavonoids. Even though aqueous extract of PHAF is used in traditional practice, ethanol extract can be used to formulate pharmaceuticals such as tonics and syrups due to its potent antioxidant properties.


The homogeneous CeO2-GO dispersion was coated on the surface of a glassy carbon electrode (GCE), and the CeO2/electrochemically reduced graphene oxide modified electrode (CeO2/ERGO/GCE) was obtained by potentiostatic reduction. The results of X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDS), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) showed that CeO2 nanocrystals were uniformly coated by gossamer like ERGO nanosheets. The electrochemical behavior of vanillin on the CeO2/ERGO/GCE was studied by cyclic voltammetry (CV). It was found that the CeO2/ERGO/GCE has high electrocatalytic activity and good electrochemical performance for vanillin oxidation. Using the second derivative linear sweep voltammetry (SDLSV), the CeO2/ERGO/GCE provides a wide range of 0.04–20 µM and 20 µM–100 µM for vanillin detection, and the detection limit is estimated to be 0.01 µM after 120 s accumulation. This method has been successfully applied to the vanillin detection in some commercial foods.In this paper, CeO The problem of food safety has caused public concern all over the world. Unsafe food can lead to many acute and lifelong diseases, from diarrhea to various cancers . Vanilli2-CNF) for vanillin detection. In the concentration range of 0.3–135 μM, the peak current shows a good linear response, and the detection limit is 0.15 μM  as a probe, The properties of the surface of different electrodes were characterized by CV. a), GO/GCE (b), CeO2/GCE (c), and CeO2/ERGO/GCE (d) in the potential range of −0.2~0.6 V. On bare GCE a pair of well-defined redox peaks was observed with the peak-to-peak potential separation (ΔEp) of 85 mV and the redox peak current of 14.80 μA (Ipa) and 15.87μA (Ipc) (curve a). It was shown that the electron transfer process is quasi-reversible. However, the redox peak current decreased obviously on GO/GCE , ΔEp was 167 mV (curve b). This may be due to the poor conductivity of GO and the strong negative charge repulsion force between [Fe(CN)6]3−/4− and the ionized groups such as COO− in GO. On the other hand, the current response of [Fe(CN)6]3−/4− on the CeO2/GCE (curve c) increased slightly compared with that of bare GCE. On CeO2/ERGO/GCE (curve d), the ΔEp value decreased to 78 mV and the highest redox peak current was observed, suggesting a more reversible electron transfer process of [Fe(CN)6]3−/4− occurred on CeO2/ERGO/GCE. Therefore, the electron transfer rate was greatly improved due to the coexistence of ERGO and CeO2 nanoparticles.Using 1.0 mM K2/ERGO/GCE for vanillin oxidation are confirmed in 2/ERGO/GCE, a well-defined and sensitive vanillin oxidation peak (P1) appeared at 0.942 V [2/P3  was also observed, corresponding to the redox reaction of the oxidized intermediate of vanillin. The electrochemical reaction mechanism was illustrated in Ip = 25.24 μA) appeared at 0.949 V at the ERGO/GCE compared with other electrodes, which may be related to the good catalytic activity, high conductivity, and large surface area of ERGO. The maximum oxidation peak current was obtained at CeO2/ERGO/GCE, which is about two times higher than that of ERGO/GCE, demonstrating the synergistic effect of CeO2 nanoparticles and ERGO nanosheets were existed on the electrode surface. As shown in 2/ERGO/GCE > ERGO/GCE > CeO2/GO/GCE > GO/GCE > CeO2/GCE > bare GCE. In particular, ERGO nanosheets have excellent conductivity and high surface area. Also the electron exchange between CeO2 nanoparticles and vanillin promoted the electrocatalytic reaction. In addition, ERGO is a good supporter for CeO2 nanoparticles, which can effectively prevent the agglomeration of CeO2 nanoparticles and give full play to its catalytic property. Therefore, the interface conductivity of the modified electrode was greatly improved and the sensitivity of the determination of vanillin was improved.The catalytic properties of CeO 0.942 V ,13. In a2/ERGO/GCE under different scan rates were illustrated in −1. A good linear relationship between the peak current (Ip) and scan rate (ν) was observed in Ip (μA) = 0.1929 v (V s−1) − 0.4991 (R = 0.9959), It shows that the oxidation process of vanillin on the CeO2/ERGO/GCE is controlled by adsorption. By plotting the relationship between logarithm Ip and logarithm v, the adsorption control behavior of vanillin was further confirmed: log Ip (μA) = 1.0371logv (V s−1) + 2.3049 (R = 0.9987). The obtained slope of 1.0371 is close to 1.0. In Ep of vanillin moved to a positive value with the increase of scan rate, and Ep changed linearly versus Napierian logarithm of scan rate (ln v). The linear regression equation was Ep (V) = 0.0214 ln v (V s−1) + 0.9966 (R = 0.9981). For a completely irreversible adsorption control process, Ep can be expressed by the Laviron equation [In order to further investigate the oxidation mechanism, the CVs of 10 μM vanillin recorded on the CeOequation :Ep = E0′RT/αnF, so αn was calculated as 1.196. α is approximately 0.5 in a completely irreversible electrode process, thus n is about 2, which is consistent with the results obtained on silver nanoplate/GR composite modified GCE [According to equation (1), the slope of the straight line is equal to fied GCE  and grapfied GCE .D and Faradic charge Qads of vanillin at the CeO2/ERGO/GCE were calculated. Q–t obtained in the 0.1 M HCl solution with and without 0.1 mM vanillin. After background subtraction and the plot of charge (Q) against the square root of time (t1/2) (−5 C s−1/2 and Qads of 1.679 × 10−5 C. Using Equation (2) given by Anson [D can be obtained:dlQ is double layer charge, A is the electrode surface area, and c is the substrate concentration. According to A was estimated to be 0.01478 cm2.Using chronocoulometry, the diffusion coefficient e (t1/2) B, a goodby Anson , D can bD of 2.9 × 10−5 cm2 s−1 was obtained. According to the equation Qads = nFA Γs, the adsorption capacity Γs was obtained as 5.88 × 10−10 mol cm−2.Thus, 2/ERGO/GCE, the standard heterogeneous rate constant (ks) can be obtained based on Equation (4) [For the completely irreversible oxidation of vanillin at the CeOtion (4) ,47:ks= 2Ep represents the peak potential, Ep/2 is the potential at which I = Ip/2. Other symbols have their usual meanings. In our experiment, Ep − Ep/2 =34 mV, D = 2.9 × 10−5 cm2 s−1, v = 100 mV s−1, and T = 298 K. The value of ks was obtained as 1.02 × 10−2 cm s−1, confirming a relative rapid electrode reaction process.In the formula, 2/ERGO/GCE was investigated in different types of supporting electrolytes, such as HAc–NH4Ac buffer solution (pH 4.0), phosphate buffer solution (pH 3.0 and 6.0), HAc–NaAc buffer solution (pH 4.0), HCl, H2SO4, HNO3, and H3PO4 (each 0.1 M). It was found that the peak current was the largest and the peak shape was sharp in HCl solution. In the range of 0.02–0.5 M, the effect of HCl concentration on the peak current of 10 μM vanillin was studied. It was found that the maximum peak current of vanillin was obtained when the concentration of HCl reached 0.1–0.2 M  = 3.003c (μM) + 0.7587 (R = 0.9941) and I (μA) = 0.4582c (μM) + 48.159 (R = 0.9836), respectively. The detection limit was 0.01 μM. In order to estimate the analytical characteristics of the developed sensor, the CeO2/ERGO/GCE was comprehensively compared with other published electrochemical methods for vanillin detection. From the results in 2/ERGO/GCE has better detection performance with a wider dynamic range and lower detection limit.Compared with the traditional CV method, the second-order derivative linear sweep voltammetry (SDLSV) has the advantages of low detection limit and high sensitivity, and is a more effective electrochemical technique for quantitative analysis. Therefore, SDLSV was used for the determination of vanillin in this study. 2/ERGO/GCEs were fabricated by the same method to test the reproducibility, and 10 μM vanillin solution was measured under the same conditions. The relative standard deviation (RSD) of 4.62% (n = 7) indicated excellent reproducibility. In addition, to test the repeatability, the vanillin solution was determined by a single CeO2/ERGO/GCE for 10 times. After each measurement, to regenerate the electrode surface, two successive voltammetric sweeps were carried out in 0.1 M H2SO4 solution in the range of 0.2–1.2 V. The good repeatability was reflected by the RSD of 2.74%. The storage stability of the modified electrode was also evaluated by storing the modified electrode in air. After two weeks, the response of the electrode is still 91.6% of the initial current, which showed that the electrode has long-term stability. In addition, it was found that when a 1000-fold amount of sucrose, glucose, fructose, K+, Ca2+, Na+, Mg2+, Zn2+, Al3+, and a 500-fold amount of citric acid, tartaric acid, lactic acid, and caffeine were present, the peak current change of 10 μM vanillin was less than 5%. Ascorbic acid (AA), uric acid (UA) and dopamine (DA) as three kinds of important biological substances in the human fluid. 2/ERGO/GCE in the presence of 1.0 mM AA, 10 μM DA, 10 μM UA, and 1.0 μM vanillin. As shown in Seven CeO−1 (for biscuit), 69.38 μg g−1 (for chocolate) and 132.98 μg g−1 (for pudding powder), respectively. The recoveries were 97.0–104.0%, which showed that the method can be used for the accurate and rapid detection of vanillin in commercial food samples.The determination of vanillin in commercial food products such as biscuit, chocolate and pudding powder was performed using the developed method. The sample solutions were prepared according to reference , and the2 nanoparticles were synthesized by the hydrothermal method and dispersed uniformly in graphene oxide (GO) aqueous solution by ultrasonication. The homogeneous CeO2-GO dispersion was coated on the surface of a glassy carbon electrode (GCE), and GO was converted into ERGO by potentiostatic reduction to obtain the modified electrode (CeO2/ERGO/GCE). This method offers several advantages over other techniques, including being green, efficient, inexpensive, and rapid. The electrochemical behaviors of vanillin on the CeO2/ERGO/GCE were studied carefully and the electrochemical parameters were calculated. The obtained CeO2/ERGO nanocomposite exhibited excellent performance for vanillin oxidation due to its strong electrocatalytic ability, good conductivity, and large surface area. Using the SDLSV technique, vanillin can be detected in the concentration range of 0.04–20 μM and 20–100 μM with the detection limit of 0.01 μM. Furthermore, the developed method has the advantages of high sensitivity, good selectivity, simple electrode preparation, and low cost. It has a wide application prospect in the sensitive detection of vanillin in commercial foods.In this paper, CeO


During the past years, the prevalence of sleep problems has been increasing steadily in industrial societies and represents a major social and socioeconomic burden. The situation in Austria was last evaluated in 2007 by Zeitlhofer and colleagues in a representative sample of 1000 participants. In the current study, we sought to evaluate the sleep behaviour of the Austrian population in an ongoing online survey, in which we have collected data from 986 participants  between March 2018 and May 2019. Sleep duration was appropriate in 52% of the respondents (i.e. 7–9 h per night). However, we found an alarmingly high number of self-reported sleep problems (46%), and only 31% of the participants classified themselves as “good sleepers” using a validated self-report questionnaire . Furthermore, many participants reported suffering from sleep problems for a very long time  suggesting that currently available treatment options are either ineffective or not employed. Possible reasons for sleep problems could include irregular sleep-wake cycles, increased perceived stress levels, and the use of electronic devices just before sleep.The online version of this article (10.1007/s11325-019-01982-5) contains supplementary material, which is available to authorized users. Especially in Western societies, an increasingly high number of people complain about sleep problems. Among clinically relevant sleep disorders, insomnia, which is characterized by problems initiating and maintaining sleep and the feeling of sleep not being restorative, is extremely prevalent. Subjectively, patients suffer from significant distress or impairment in social, occupational or other important areas of daytime functioning. Globally, insomnia is the most commonly reported sleep problem in industrialized countries , and numwww.sleeplounge.net and run on LimeSurvey [The survey is accessible on meSurvey , and infmeSurvey  and (ii)meSurvey . The  held a university degree  had participated in the survey on sleep habits as of May 15, 2019. While all participants lived in Austria, the sample comprised 88% Austrian citizens, and 12% were other nationalities. The sample comprised participants between 15 and 90 years with a mean age of 40.9 years ±16.4 years). About eighty-eight percent were between 18 and 64 years old [general population: 61.8% according to data from Statistik Austria from 2018; .4 years.t-test was computed. For correlations between two ordinal variables, we report Kendall’s tau, for the correlation between two interval scale variables a Pearson correlation, and for the correlation between an interval scale variable and a dichotomous variable the point biserial correlation. For the analyses of the relationships between two dichotomous variables from fourfold tables, we report results from Chi-square tests. Statistical evaluation was done in R version 3.6.1 [p values for statistical tests, all tests were two-sided.For between-group comparisons, a Welch two sample on 3.6.1 . We repot (510.47) = 7.80, p <. 001). Approximately 89% of those who took the German version of the Morningness-Eveningness Questionnaire (n = 197 men and n = 414 women) were neutral types, moderate evening or moderate morning types, 8.67% were definite morning and 2.26% definite evening types (cf. Supplementary Table S3 for details). Among respondents reporting (very) poor sleep quality, a disproportionately high number seemed to sleep less than 7 h .About 52% of the respondents slept between 7 and 9 h per night (cf. Supplementary Tables S1 and S2 for details), and the mean sleep duration was 6.7 h (±1.2 h). About 47% were reported that they usually sleep less than 7 h. Here, university students or academics slept, on average, longer than non-academics  indicated they had sleep problems when they were asked whether they currently had a sleep problem. About 40% (of n = 764 participants) moreover reported (very) poor sleep when asked to rate their sleep quality on a four-step Likert scale . While the prevalence of self-reported sleep problems generally increased with age, their prevalence was higher for women aged 60 or older compared to men (χ2(1) = 4.09, p = .043; cf. Figure χ2(1) = 4.48, p = .034; cf. Supplementary Table S7). Of those who felt burdened, 49.90% indicated this was due to daily demands at work or university and 22.81% due to multiple burdens. Other reasons were psychological or physical strains (12.22%) and family- or partnership-related issues (10.18%).A total of 45.84% (χ2(1) = 7.89, p = .005), sleep problems were more prevalent among participants with irregular sleep-wake cycles (i.e. not the same sleep times every day) than regular ones  = 49.75, p < .001).About 14% reported that they had  had an accident due to fatigue with the nature of the accident not being detailed. About 11% of the respondents reported unintended napping during the day, and 18.45% reported intended habitual naps. Also, only in the age group below 30 , and women had higher PSQI values than men .Results from the PSQI indicated that, on average, no age group irrespective of gender reported good sleep quality  said they used it until just before switching off the lights to sleep. About 19% switched it off between 5 and 10 min before sleep and 16.3% within 10–30 min before sleep. Only about 20 % of the respondents indicated they do not use electronic devices within the hour before switching off lights (cf. Supplementary Table S10 for details).From 267 participants (178 women), 77.90% indicated they did not at all or only slightly suffer from the spring clock change, for the change in autumn this was reported by 80.90%. About 12% said they suffered somewhat during spring (9.36% in autumn), and 10.11% said they suffered a lot or extremely following the spring clock change (likewise 9.74% in autumn). When asked how long they needed to adapt to the new time, 35.21% said they adapt immediately in spring (40.82% in autumn), 44.57% said they adapt within 1–3 days (40.45% in autumn), and only 20.22% said they need 4 or more days to adapt . About 58% of the participants would abolish clock change, and 57.79% of these would choose perennial daylight-saving time (or “summer” time) compared to 40.26% who would prefer standard time (or “winter” time) if they were to choose.The “How does Austria sleep?” survey provides important insights into sleep behaviour and sleep problems in a convenience sample from the Austrian population in 2018/2019. While general sleep parameters such as sleep duration generally seem to be in line with what has been reported in a previous study , we idenIn more detail, in this online survey, 73.4% of the respondents reported an average sleep duration between 6 and 8 h per night and 52.4% between 7 and 9 h. However, 46.5% also report that they usually sleep less than 7 h. This suggests that, in this sample, the average sleep duration is comparable to what has previously been reported by Zeitlhofer and colleagues in 2007 , althougχ2(1) = 70.96, p < .001). The apparent focus on work-related demands may also have arisen from the fact that the sample was, on average, highly educated with 51% holding a university degree. More specifically, a high educational level often comes with more degrees of freedom and responsibility at work, which may increase perceived strain. For university students, several recent studies have emphasized that they are a highly burdened group at risk for  health problems [Regarding sleep problems, 45.8% of the sample reported that they currently suffered from sleep problems. The results from the PSQI, a valid and reliable tool to identify sleep disorders, paint an even more alarming picture with only 31% of the participants being identified as “good sleepers”, 41% as “poor sleepers” and 28% even reporting severe sleep problems. More specifically, 23% of the sample reported problems falling asleep, 24% indicated they suffered from early awakenings, and 32% had problems maintaining sleep. A recent but still unpublished study from the Medical University of Vienna  suggestsproblems . AnotherBesides sleep habits and sleep complaints, we also sought to investigate the use of electronic devices such as smart phones, laptops and e-readers before sleep. Here, 44% indicated they used a device until just before switching off the lights to sleep, and 20% switched it off just between 5 and 10 min before sleep. Critically, on the one hand, it has been suggested that the high proportions of short-wavelength light (i.e. in the “blue” range) of light-emitting diode (LED) screens may be detrimental for sleep by suppressing melatonin and increasing alertness –30. On tLast, we were also interested in the opinion of the people living in Austria on clock change, which the EU parliament wishes to abolish in 2020. In our sample, 58% would abolish clock change, and, of these, 57.8% would favour perennial daylight-saving or “summer” time (DST). While this slight preference for DST is in line with results from a non-representative EU-wide survey from 2018 , the numOne limitation of the study is that the sample, in contrast to the study by Zeitlhofer and colleagues , was notIn conclusion, sleep duration was sufficient in about 50% of the respondents but insufficient  in 47%. Generally, sleep duration is therefore comparable to what has been reported in 2007 . HoweverESM 1(DOCX 40 kb)


A comparison with other materials revealed that all three non-Fickian parameters are able to be fitted using a power law. Nevertheless, the upper boundary for the applicability of this model was not determined in this study. The Weibull distribution plots indicate that the probability of non-Fickian moisture absorption is influenced by ϕ and α at approximately 62% within a normalized thickness range of 2–3. In regards to ot, it is 82% at a normalized thickness of 6. Therefore, the Weibull distribution is proposed for the assessment of non-Fickian moisture absorption based on the material’s thickness.Moisture absorption tests for materials that exhibit non-Fickian behavior generally require a relatively long period to reach saturation. Therefore, it would be beneficial to establish a relationship between the moisture content and the thickness to minimize the experimental time and cost. This research characterizes the moisture absorption behavior of AS4/8552 carbon/epoxy composites. Specimens were prepared at 4, 8, and 16 plies and immersed in distilled water at 60 °C. The relationship between the non-Fickian parameters (Fickian to non-Fickian maximum moisture content ratio  Carbon/epoxy composites are gaining importance as aircraft materials ,2. When Accelerated tests are usually conducted in laboratories to quantify the moisture uptake in materials. However, even with accelerated tests, a long period of time may still be required to reach saturation. For example, a 16-ply carbon/epoxy composite with a thickness of 3.2 mm, immersed in distilled water at 70 °C, took more than nine months to reach saturation . AdditioAmong the non-Fickian models that are available in the literature, one of them is Bao’s model. This model was developed to describe the long-term water uptake behavior in bismaleimide (BMI) resin immersed in distilled water at 70 °C . This moThis study aims to assess the applicability of the thickness-dependent non-Fickian moisture absorption model at a greater thickness. Continuous water absorption tests in distilled water at a constant temperature of 60 °C were conducted for AS4/8552 carbon/epoxy composites at 4, 8, and 16 plies. Subsequently, the fictitious Fickian curve was plotted. Then, the non-Fickian parameters were evaluated, and the applicability of the thickness-dependent model with a 16-ply composite was investigated. Weibull distribution plots were also modeled to evaluate the non-Fickian parameters’ sensitivity to non-Fickian behavior, in order to predict an appropriate thickness for the investigated materials.3, respectively. The nominal fiber volume fraction is 57.42%, and the nominal ply thickness is 0.15 mm. Three laminates were fabricated, with 4, 8, and 16 plies, with a nominal thickness of 0.6, 1.2, and 2.4 mm, respectively. All laminates were fabricated at Composites Technology Research Malaysia (CTRM) using an autoclave with the recommended manufacturing cycle.The material used in this study was an AS4/8552 carbon/epoxy composite. According to the manufacturer, this composite is employed in primary aerospace structures due to its good impact resistance and damage tolerance. The fiber and resin density is 1.79 and 1.30 g/cm2 size. The edges of all specimens were coated with water-resistant paint to avoid water penetration from the sides. Subsequently, the initial weight of all specimens was measured using a Shimadzu ATY224 four-decimal digital balance. They were subjected to continuous distilled water immersion in an HH-6 thermostatic water bath at a fixed temperature of 60 °C. Weight gains of all specimens were measured periodically. For each reading, three measurements were made to obtain the average moisture content, M. Each laminate was cut into six specimens with a 5 × 5 cmFor one-dimensional moisture absorption with a fixed aging temperature and the absence of the initial moisture content, this thickness-dependent non-Fickian moisture absorption model is as follows ,31:(1)M, α is the non-Fickian diffusivity per square thickness, and ot indicates the initiation time of Stage II. The Macaulay bracket, < > for the time delay term, <t − ot>, depicts that non-Fickian behavior only occurs after t ≥ ot. For this model to be applicable, Stage I of the experimentally reduced moisture absorption curves (M vs. √t/h) at all thicknesses must overlap. This behavior is observed because Stage I is assumed to exhibit Fickian diffusion behavior.In Equation (1), M versus √t/h) (From the reduced moisture absorption curves (us √t/h) , identifus √t/h) , which im,FM. From there, estimate the average m,FM for the material . For the composite used in this study, zD is estimated to be 4.25 × 10−2 mm2/day;Determine the apparent diffusivity, Fh = 0.15 mm.Plot the fictitious Fickian curve (which is the first term in Equation (1)) using Equation (3). For the AS4/8552 carbon/epoxy composite, the unit ply thickness To apply the aforementioned thickness-dependent moisture absorption model, a specific thickness that follows Fickian diffusion is required. Previously, it has been demonstrated that it is suitable to regard a single-ply composite as the thickness that exhibits Fickian behavior, even without experimenting . The sinϕ, α, and ot) are determined as follows:m,F/Mmϕ = M for each thickness;Calculate o/h√t) from the figure. From there, determine ot for each thickness;Plot the fictitious Fickian curve, together with the experimental curves . EstimatM(t) from the analytical IM(t) (which is determined in the previous section). This step gives the experimental IIM(t) term;Subtract the experimental moisture content W(t) (denoted as expW(t)) using Equation (4) below:Plot the experimental W(t) term:Apply logarithms of both sides twice for the analytical expW(t)]] versus 0.75 × ln<t − ot>, and fit the data with a straight line. From the ordinate intersection (0.75 × ln α), determine the value of α. As an example, y-intercept of the 16-ply composite.Plot the curve of ln[−ln[1 − Subsequently, the non-Fickian parameters , are obtained. Fh. For ϕ, it is already in the normalized form . Both α and ot were normalized with respect to the normalized thickness values, where h’ = 2. The value of h’ = 1 corresponds to the Fickian behavior, so α and ot (which are non-Fickian parameters) do not exist.The non-Fickian parameters were further normalized to compare them with other materials that have been studied previously ,31. The ϕ and α’ follow a trend similar to that of other materials that have been studied previously [h’ = 16) in this study. Nevertheless, for o’t  obtained from the experimental data by extracting the statistical features using the probability distribution function (PDF) and cumulative distribution function (CDF). Therefore, the Weibull distribution quantifies and control the uncertainties of the non-Fickian parameters in moisture absorption behavior based on the materials’ normalized thickness. From the non-Fickian two-parameter Weibull probability distribution shown in ϕ = 0.53, α’ = 0.41, and o’t = 11 were obtained . Specifically, ϕ = 0.91h’−0.68, α’ = 3.85h’−2.01, and o’t = 0.22h’2.17. Generalization of the non-Fickian parameters is beneficial for minimizing the amount of experimental work. Furthermore, Weibull distribution plots suggest that the probability of non-Fickian moisture absorption is mainly affected by ϕ and α at approximately 62% for a normalized thickness h’ = 2–3. Moreover, the non-Fickian initiation time ot results in approximately 82% absorption when h’ = 6. Further works are required to determine the upper limit of the thickness-dependent model.This study characterized the water uptake behavior in unidirectional AS4/8552 carbon/epoxy composites at 4, 8, and 16 plies. All specimens were continuously submerged in distilled water at 60 °C. Through fitting the fictitious Fickian curve, it was estimated that the apparent diffusivity


Aeromonas hydrophila is an important water-borne pathogen that leads to a great economic loss in aquaculture. Along with the abuse of antibiotics, drug-resistant strains rise rapidly. In addition, the biofilms formed by this bacterium limited the antibacterial effect of antibiotics. Bacteriophages have been attracting increasing attention as a potential alternative to antibiotics against bacterial infections.A. hydrophila, named N21, W3, G65, Y71 and Y81, were isolated. Morphological analysis by transmission electron microscopy revealed that phages N21, W3 and G65 belong to the family Myoviridae, while Y71 and Y81 belong to the Podoviridae. These phages were found to have broad host spectra, short latent periods and normal burst sizes. They were sensitive to high temperature but had a wide adaptability to the pH. In addition, the phages G65 and Y81 showed considerable bacterial killing effect and potential in preventing formation of A. hydrophila biofilm; and the phages G65, W3 and N21 were able to scavenge mature biofilm effectively. Phage treatments applied to the pathogenic A. hydrophila in mice model resulted in a significantly decreased bacterial loads in tissues.Five phages against pathogenic A. hydrophila phages were isolated with broad host ranges, low latent periods, and wide pH and thermal tolerance. And the phages exhibited varying abilities in controlling A. hydrophila infection. This work presents promising data supporting the future use of phage therapy.Five  Aeromonas hydrophila is a Gram-negative, rod-shaped bacterium that is ubiquitous found in natural aquatic environments. The bacterium is predominantly pathogenic to poikilothermy animals, including fish, turtles, snakes and amphibians [A.hydrophila is a very typical pathogen of human-animal-fish comorbidity [phibians . It is rphibians . The bacorbidity .A. hydrophila strains are emerging; and drug residues in aquaculture products and the environment are getting worse [A. hydrophila strains isolated from fish and prawns were resistant to methicillin, rifampicin, bacitracin, and novobiocin. Recently, De Silva et al. [Aeromonas, and found that all the isolates were multidrug resistant and 100% resistant to ampicillin, colistin, vancomycin and cephalothin. In addition, each of the 43 Aeromonas strains isolated by Hossain et al. [A. hydrophila is capable of forming biofilms on host tissues, as well as multiple biotic and abiotic aquaculture substrates [In general, the prevention and treatment of diseases depend mainly on extensive application of antimicrobial agents , 4. Anting worse –8. Vivekng worse  indicatea et al.  tested an et al.  from 46 n et al. , 13. Addn et al. . A. hydrbstrates . Bacteribstrates , 17, whiEscherichia coli (UPEC). Another study indicated that infection of methicillin-resistant Staphylococcus aureus with subsequent administration of purified phage phi MR11 effectively suppressed S.aureus-induced bacteremia and lethality in mice [Aeromonas diseases. Jun et al. [A. hydrophila. Additionally, therapeutic treatments of A. hydrophila-phage 2 and A. hydrophila-phage 5 applied to the catfish during bacterial infection resulted in a significantly enhanced survival of the tested fishes [Bacteriophages, naturally-occurring bacterial viruses that can kill specific bacteria with no chemical residues and does not affect other flora, are one of the potential alternatives. A small dose of phage can achieve a good therapeutic effect as phage proliferation via auto “dosing” results in great bacterial killing . One pre in mice . Phage tn et al.  reportedd fishes . HoweverA. hydrophila. Furthermore, we investigated the efficiency of the phages for biofilm inhibition and removal, and performed a phage therapy experiment in A. hydrophila-infected mice.In this study, we isolated and characterized five phages specific to A. hydrophila NJ-35 as an indicator host, and two phages Y71 and Y81 with A. hydrophila XY-16 as an indicator host, were isolated from fish ponds and polluted rivers in Nanjing. Clear plaques appeared after 12 h incubation at 28 °C. As shown in Fig. Three phages designated as N21, W3 and G65 with Caudovirales. Phages N21, W3 and G65 possessed a morphology typical of the Myoviridae family, displaying an icosahedral head with the diameter of (62.6 ± 1.9) nm, (64.9 ± 3.2) nm and (58.8 ± 4.1) nm, respectively, a contractile tail with the length of (153.1 ± 6.2) nm, (154.1 ± 1.4) nm and (152.3 ± 9.8) nm with six long fibers, respectively, and collar and base plate structures. Phages Y71 and Y81 morphologically belonged to the Podoviridae family, possessing an icosahedral head of (62.8 ± 1.1) nm and (54.8 ± 2.9) nm in diameter, respectively, and a short tail of (22.0 ± 0.2) nm, (20.0 ± 2.2) nm in length, respectively.Purified phages were examined by transmission electron microscopy (TEM) and classifed based on the criteria proposed by Ackermann . TEM obsAeromonas species of a total of 205 isolates, including 75 A. hydrophila, 85 A. veronii, 12 A. caviae, 1 A. bestiarum, 12 A. sobria, 10 A. media, 3 A. salmonicida, 3 A. jandaei and 4 A. aquariorum (Table Aeromonas species pathogenic in fish. As shown in Table n = 17), 21.33% (n = 16), 21.33% (n = 16), 20% (n = 15) and 22.67% (n = 17) of 75 A. hydrophila isolates were susceptible to phages N21, W3, G65, Y71 and Y81. Additionally, all of the phages could infect one strain of A. caviae; phages N21, W3 and G65 also showed infectivity to A. veronii of 1, 3, and 3 strains, respectively; and phage W3 was able to infect the only A. bestiarum isolate tested in this study, showing broad infectivity against phylogenetically distant species in Aeromonadaceae.The lytic spectrum of five phages was determined against nine A. hydrophila cultures of exponential growth phase were infected with different amount of phages as designed. The phage titers were measured after incubation for 2 h. The results indicated that the optimal MOIs of phage isolates N21, W3, G65, Y71 and Y81 were 0.01, 1, 0.001, 0.1 and 0.001, respectively, which gave the highest production of phage progeny . As shown in Fig. Thermal stability of the isolated phages was assayed at pH 7.0. All of the five phages maintained almost 100% infectivity after cultured at 4 °C or 30 °C for 1 day (data not shown). Figure A. hydrophila strains NJ-35 and XY-16 at different doses of MOIs. As shown in Fig. The bacteriolytic activities of five phages were evaluated using A. hydrophila NJ-35 and XY-16 was detected at 24 h after coculture using crystal violet method. As shown in Fig. A. hydrophila cultured alone, the bacteria cocultured with phages showed a considerably decreased biofilm formation at each MOI, and the decrease was MOI-dependent. At a MOI of 1.0, all the phages exhibited a significantly inhibited effect on the biofilm formation of their corresponding host strains (P < 0.05 or P < 0.001). Interestingly, similar to what was observed in bacteriolytic activity, phages G65 and Y81 had stronger abilities to prevent biofilm formation than the other three phages. Very few biofilm could be detected when the A. hydrophila was incubated with G65 or Y81 even at a small MOI of 0.01.The capability of the phages to prevent biofilm formation of A. hydrophila strain NJ-35 or XY-16 treated with phages with a titer of 1.0 × 108 PFU/mL was determined. As shown in Fig. A. hydrophila in a time-dependent manner, as compared with untreated controls, except for the treatment with phage Y71 which resulted in a slight decrease with no statistical difference. Phages G65, W3 and N21 showed a relatively stronger biofilm clearance efficiency after treating for 24 h, with the clearance rates of 75.12, 73.35 and 67.08%, respectively, whereas phages Y81 and Y71 only exhibited biofilm removal of 34.26 and 12.47%, respectively.Decrease of mature biofilm formed by A. hydrophila in vivo, we performed an infection and therapy assay in mice. Mice infected with A. hydrophila were sacrificed at 6 h and 24 h post-treatment with phages, and hearts, livers, spleens, lungs and kidneys were collected for determination of bacterial loads. As shown in Fig. To corroborate whether the phages can prevent the proliferation of Ackermannviridae and Herelleviridae were established in the order Caudovirales based on virion morphology and genome or proteome comparisons [Myoviridae phage, AHP-1, recently identified [Ackermannviridae and Herelleviridae have been also reported to be of myovirus morphology, the tail spikes are the morphological markers of the family Ackermannviridae [Herelleviridae always infect members of the phylum Firmicutes [Myoviridae. At present, most bacteriophages reported against Aeromonas spp. have been characterized as tailed phages, and the family Myoviridae were found to be the majority [Aeromonas phages investigated belonged to the Myoviridae family. Also, several Aeromonas phages belonging to the family Podoviridae have been isolated and characterized [Podoviridae, were tentatively classified as members of this family.Phage taxonomy has progressed from a mainly morphology-based techniques to the modern applications of a holistic approach . Recentlparisons , 26. Nevparisons . In thisentified , consistentified . Althougnviridae , 30 and rmicutes . Therefomajority , 32, 33.majority  indicatecterized –36. PhagAeromonas bacteriophages that show wide host spectrum [A. hydrophila and showed the potential to infect A. caviae, A. veronii and A. bestiarum. Short latent period and large burst size are essential features for highly effective phages serving as as therapeutic agents [A. hydrophila had a burst size of 244 phage particles per cell with approximately 10 min of latent period [A. hydrophila strain, suggesting that the phages are most likely to be lytic rather than temperate phages. However, this is not always definitive and further genome-based characterizations of the phages are needed to verify this. Additionally, our phages, being more resistant to alkali than acid condition, showed a broad pH tolerance compared to most isolated phages [A. hydrophila infection or contamination in the diverse physicochemical environments.Desired properties for efficient phage therapy may depend on phage virulence, host range, latent period, obligately lytic property and so on. Broad host range usually exhibites great advantages in combating multiple pathogen infections. In general, phage showed narrow host specificity that only infect its indicator host , 38, altspectrum . Our stuc agents . Previout period . Similart period . Traditit period , 44. Alld phages –47. BesiA. hydrophila infection. The therapy effect was found to be time-dependent, and few bacteria were detected after 24 h treatment by any of the phages. It should be noted that for phage N21, W3, or Y71, the bacteriolytic activity in vivo was different from that in vitro, which indicated that bacterial number rapidly decreased to the minimum at 6 h after coculture, but began to rise after 12 h. A reason for this might be the appearance of phage-resistant variants after phage treatment in vitro. Although the emergence of phage-resistant bacteria is typically low as phages are host-specific and self-dosing, it cannot be avoided since it is a natural mechanism of bacteria-virus co-evolution [A. hydrophila infection. In this study, we have not detected phage titres in all organs. In this regard, it would be interesting to determine if the phages localised to the high bacterial titre at infection sites.Expectively, phage therapy in mice model resulted in a decreased bacterial loads in tissues compared to the control groups after treating for 6 h, suggesting a potential role for phages in controlling volution . This phvolution , 49, 50.volution . Additiovolution . Besidesvolution –56. FurtAeromonas spp. using bacteriophages [A. hydrophila. In this study, phages G65 and Y81 showed excellent effect on preventing formation of biofilm at 24 h after coculture even at a small MOI of 0.01, while relatively poor performance was observed in the other three phages. Additionally, almost no bacterial growth was detected at 24 h after treated with phages G65 or Y81. To clear whether the biofilm decrease depends on the phage activity to digest the biofilm or the reduction of bacterial viability, we detected the activities of the five phages on the biofilm formation of A. hydrophila XX-58 (accession number of gyrB gene: JX025795.1), which was determined to be not susceptible to the infection of the phages in this study. The data indicated that the biofilm formation of XX-58 was not affected by the phage activity  from Nanjing, China in 2010 and XY-16 (accession number of gyrB gene: JX025797.1) from Xinyi, China in 2009 [Aeromonas strains, including 73 A. hydrophila, 85 A. veronii, 12 A. caviae, 1 A. bestiarum, 12 A. sobria, 10 A. media, 3 A. salmonicida, 3 A. jandaei and 4 A. aquariorum were used for host range analysis. All bacterial strains were routinely cultured in Luria Bertani (LB) broth (Difco/Becton Dickinson) at 28 °C with shaking at 180 rpm. All Aeromonas strains used in this study are listed in Table Two epidemic strains of high virulence isolated from diseased crucian carp,  in 2009 , 68, werA. hydrophila strains NJ-35 and XY-16 were used as indicator hosts for phages. Ten water samples were collected from ponds, sewage and rivers in Nanjing. The samples were centrifuged at 4000 g for 30 min and the supernatants were filtered through 0.22-μm membrane filters. A 10 mL of the supernatant was added into 20 mL of 3 × LB broth, and 1 mL cultures of NJ-35 or XY-16 (late logarithmic phase) were used to inoculate the mixture. After adding 5 M CaCl2 to a final concentration of 0.1 mM, the phages were enriched by culturing for 14 h at 28 °C. A 3 mL of the culture was added to a 5 mL LB broth, followed by the addition of chloroform with a final concentration of 3%, vigorously shaken for 2 min. After static layering, 1 mL of the supernatant was filtered through a 0.22-μm filter. To confirm the presence of the lytic phage in the fltrate, the double-layer agar method [r method  was perfAeromonas strains were spread evenly on the LB agar plate. Five microliters of the phage cultures of 108 PFU/mL were dropped onto the overlaid top agar. After cultured for 12 h at 28 °C, the presence or absence of a lysis zone was observed.The host range of the harvested phages was determined using a spot assay. 2O). The plate was shaken horizontally for 5 min to fully wash off the phages. Suspensions in the plate were centrifuged at 6000 g for 5 min. For TEM analysis, 15 μL of the phage supernatant was spotted on a carbon/Formvar-coated 200-mesh copper grid . After 3 min, the suspension was removed by filter paper and the grid was baked under incandescent light for 5–10 s. The phages were negatively stained with 2% uranyl acetate for 1 min. Excess dye was removed by the filter paper and the grid was dried under an incandescent lamp. The morphology of the phage was imaged by TEM  operated at 80 kV. Phage dimensions were calculated by measuring the dimensions of five independent phages.The phages were cultured at 28 °C for 8–10 h using the double-layer plates. A plate with plenty of plaques whose edges were faintly visible was added with 2 mL of sterilized double deionized water . The phages were allowed to absorb for 5 min and then centrifuged at 13,000 g for 1 min to remove free phage particles. After discarding the supernatants, the phage-infected bacterial pellets were resuspended in 50 mL of prewarmed LB broth and the cultures were continuously incubated at 28 °C. At an interval of 5 min, samples were harvested and phage titers were immediately determined by the double-layer agar method. The burst size of phages was calculated by dividing the final titer of released phage particles by the initial count of infected bacterial cells.One-step growth experiment was performed according to the method described by Adams . A. hydr7 PFU/mL) was used to inoculate 900 μL physiological saline adjusted to pH values of 3 ~ 12 with NaOH or HCl. The mixtures were incubated at 28 °C for 2 h and aliquots were taken to measure the titers of phages at different pH values. For thermal stability tests, 2 mL phage suspension (1.0 × 107 PFU/mL) was incubated at 30 °C, 40 °C, 50 °C, and 60 °C. At an interval of 20 min, 100-μL aliquots were collected until 100 min. Survived phages were counted and the survival rates were calculated by the PFU at each time point divided by that at the primary PFU. All tests were performed in triplicate.For pH stability tests, 100 μL phage suspension (1.0 × 10A. hydrophila NJ-35 and XY-16 suspensions (1 × 108 CFU/mL) in exponential period were mixed with an equal volume of phages at different MOIs of 0.01, 0.1, 1, and 10, respectively, followed by incubation at 28 °C for 24 h. A. hydrophila cultures mixed with LB broth served as positive controls. The absorbances (OD600) of the cultures were measured at each time point. The experiment was performed in triplicate.Bacterial killing assays were conducted for the phages as previously described . BrieflyA. hydrophila strains were cultured in LB broth to logarithmic period and then normalized to 1 × 106 CFU/mL. The suspensions were mixed with equal volume of phages with the MOI of 0.01, 0.1, 0, 1, and 10, respectively. Two hundred microliters of the mixtures were added into each well of the 96-well plates. Each treatment was performed in eight replicates. Fresh LB broth served as a blank control. Then the plates were incubated at 28 °C for 24 h. Next, the culture supernatants were discarded and the plates were washed three times with sterile PBS to remove all planktonic cells. Biofilms formed in each well were fixed with 200 μL of 99% (vol/vol) methanol for 15 min. After drying, biofilms were stained with 1% crystal violet for 10 min. Then the wells were washed with distilled water to remove unbound dye. Crystal violet was dissolved in 200 μL of 95% ethanol for 10 min and the absorbance was measured at 595 nm (OD595) using a micro-plate reader .A. hydrophila strains were cultured in LB medium to logarithmic period and then normalized to OD600 of 1.0. The suspensions were inoculated to LB media (200 μL per well) in 96-well plates at a ratio of 1:1000. The plates were incubated at 28 °C for 24 h without shaking. After removing all planktonic cells, each well were treated with 200 μL phage dilution of 1 × 108 PFU/mL with sodium chloride-magnesium sulfate (SM) buffer  for 6 h, 12 h, and 24 h. Biofilms treated with SM buffer served as the control. The microplates were then washed twice with PBS and the biofilms left were stained and measured by crystal violet method. The OD595 absorbance of each well was measured using a micro-plate reader. The percentage of biofilm left was calculated by dividing OD595 of phage treated wells and the OD595 of controls without phage treatment multiplied by 100. The percentage was subtracted from 100% in order to get the percentage of biofilm clearance.A. hydrophila used in this study was chosen on the basis of a preliminary study that the mice were expected to show obvious clinical signs but no death. A. hydrophila NJ-35 and XY-16 grown to log phase were washed three times with PBS and adjusted to 5 × 107 and 1 × 108 CFU/mL, respectively. For phage therapy assay, a total of 120 mice were randomly divided into three groups and housed in cages (5 mice per cage). Feed and water were allowed ad libitum. To evaluate treatment efficiency of phages in A. hydrophila NJ-35 infection, 40 mice were intraperitoneally inoculated with 100 μL of bacterial suspension of A. hydrophila NJ-35; after 30 min, the infection group of 10 mice were then intraperitoneally injected with 100 μL of sterile PBS, while each 10 out of the other 30 mice in the treatment group were injected with 100 μL of phages N21, W3 or G65 at the MOI of 1. The blank control group of 30 mice were intraperitoneally injected with 100 μL of sterile PBS, followed by injecting 100 μL of each of the phage suspensions. Similarly, 50 mice were used to determine the therapy effect of phages Y71 and Y81 on A. hydrophila XY-16 infection. After 6 h and 24 h, five mice in each treatment were euthanized by CO2, respectively. Hearts, livers, spleens, lungs and kidneys were aseptically removed from the mice, weighed, added to 1 mL of sterilized PBS, homogenized using a tissue homogenizer , and then 10-fold serially diluted in PBS. Bacterial loads in tissues were determined by plating the dilutions on LB plates. The results are expressed as the numbers of CFU/g tissue.The animal experiment was performed in accordance with the animal welfare standards, complied with the guidelines of the Experimental Animal Welfare Ethics Committee, Chinese Association for Laboratory Animal Sciences, and was approved by the Ethical Committee for Animal Experiments of Nanjing Agricultural University, China  2017–0007). Six-week-old female Institute of Cancer Research (ICR) mice  were purchased from the Experimental Animal Center of Yangzhou University and raised in the Experimental Animal Center of College of Veterinary Medicine, Nanjin Agricultural University under specific-pathogen-free (SPF) conditions. The dose of t-test was used to analyze the difference between the phage-treatment groups and the control groups. Error bars presented in the figures represent standard deviations of the means of three replicate experiments. A P-value < 0.05 was considered as a significant difference.Data were collected and analyzed using GraphPad Prism version 5 software. A Additional file 1. Phage activity on the biofilm formation of A. hydrophila XX-58. The biofilm formation ability of A. hydrophila XX-58, a bactreiophage-resitant strain, co-incubated with phage N21, W3, G65, Y71 or Y81 at an MOI of 0.01, 0.1, 1 and 10 was measured using crystal violet staining method. Data are shown as the mean ± SD from the triplicate experiments.


Sardinella tawilis, the only known freshwater sardinella in the world, is endemic to Taal Lake, Philippines. Previous studies found the Taiwan sardinella, S. hualiensis, to be morphologically very similar to S. tawilis and identified it as the marine sister species of S. tawilis. In this study, DNA barcoding using the mitochondrial cytochrome c oxidase I (COI) gene was carried out to analyze species demarcation in the Sardinella genus, focusing primarily on the relationship between S. tawilis and S. hualiensis. The neighbour-joining (NJ) tree that was constructed using Kimura 2-parameter (K2P) model showed a single clade for the two species with 100% bootstrap support. K2P interspecific genetic divergence ranged from 0% to 0.522%, which is clearly below the suggested 3–3.5% cutoff for species discrimination. Recombination activating gene 1 (RAG1), mitochondrial control region (CR), cytochrome b, 16S rRNA, and S7 markers were used to further validate the results. Sardinella tawilis and S. hualiensis clustered together with a bootstrap support of 99–100% in each of the NJ trees. Low interspecific genetic distances between S. tawilis and S. hualiensis for all the markers except CR could be attributed to incipient allopatric speciation. Sardinella tawilis  interspecific distances of 1.3–3.5%. They also suggested that S. tawilis and S. albella could be ecomorphs of the same species since the maximum parsimony and neighbour-joining (NJ) trees they constructed grouped the two species together. Samonte et al. (c oxidase subunit I (COI) gene, showed that S. tawilis is distinctly separate from its other marine relatives with genetic divergence ranging from 16.4% to 19.3%. It was suggested that a misidentification might have taken place since the single specimen of S. albella acquired by Samonte et al. , cytb (0.1%), and S7 (0.3%) markers.The collection of S. hualiensis with S. tawilis , cytochrome b (cytb), 16S rRNA, CR, and nDNA S7 were then used to further validate the findings.Owing to the similar and overlapping morphology and meristics of S. tawilis from Taal Lake at Talisay Fish Port, Batangas, Philippines  and 10 individuals of S. hualiensis, which were provided by the National Fisheries Research and Development Institute (NFRDI). Five of the 10 S. hualiensis came from Santa Ana, Cagayan, Philippines  and the other five from Yilan County, Taiwan . Sardinella tawilis and S. hualiensis specimens were kept as vouchers at the Institute of Biology, University of the Philippines Diliman and NFRDI, respectively.Muscle tissues were obtained from 12 fresh specimens of ® Genomic DNA Purification Kit . Polymerase chain reaction (PCR) was performed in 15 μl volumes containing 0.3 μl of dNTP (0.05 mM), 0.75 μl of each primer (0.1 mM), 1.5 μl of 10× PCR buffer, 3 μl of 5× Q buffer, 0.3 μl of MgCl (25 mM) buffer, 0.075 μl of Taq polymerase, 7.125 μl of ultrapure water, and 1.2–2.4 μl of DNA template.DNA was extracted from the muscle tissues using Promega WizardCOI (636 nucleotides) – FishF2 and FishR1  – nested PCR: first round RAG1-2510F and RAG1-4063R (Li and Orti CR (433 bp) – LCHACR04 (5′-AACTCCCAAAGCTAGGATTC-3′) and CHACR01H (5′-GGCCCATCTTAACATCTTCA-3′) designed in this studycytb (405 bp) – LCHACB02 and CHACB02H  – 16Sar and 16Sbr Palumbi S7 (708 bp) – nested PCR: first round S7RPEX1F and S7PEX2R , which is the most variable region in the mitochondrial DNA and is used more often to discriminate populations and not between species. The minimum K2P genetic divergence of all other gene regions aside from CR was zero: COI , RAG1 , 16S , cytb , S7 . Based on COI , which fall below the 3–3.5% threshold to differentiate species  based on COI data. It is possible that S. tawilis and S. hualiensis most likely shared a recent common ancestor unless they are conspecific. As Willette et al. (S. hualiensis originated from the Philippines or from Taiwan. There are factors that limit and complicate the dispersal between Taiwan and the Philippines, such as the unfavourable environment of the Luzon Strait that separates the two locations and the interacting currents that limit the dispersal of larvae. It was hypothesized that the Taiwan, Batanes, and Babuyan Islands were possibly used as stepping stones or stopovers as the S. hualiensis crossed the Luzon Strait (Willette et al. S. hualiensis has never been reported in Balayan Bay or in West Philippine Sea. What is clear is that S. albella is definitely not the closest marine relative to S. tawilis, contrasting with what Samonte et al. (S. hualiensis and other species of Sardinella, S. tawilis and S. hualiensis are the most closely related. For all others, the genetic distances ranged from 16.063% to 20.250%.A particularly noteworthy finding is the 0 genetic distance between e et al.  has poine et al.  and Samoe et al.  claimed S. tawilis and S. hualiensis as belonging to one species on the basis of low interspecific genetic distances, Willette et al. (S. tawilis and S. hualiensis as separate sister species from the perspective of the biological and evolutionary species concepts due to their geographic and reproductive isolation hindering natural interbreeding, the phylogenetic species concept due to some diagnosable morphological differences despite being more similar to each other than other Sardinella species, and the physiologic evolution of the osmoregulatory mechanism from freshwater to marine. It is possible that they are still in the process of incipient allopatric speciation, which would explain the low interspecific genetic distance between S. tawilis and S. hualiensis despite geographic separation. Geographic isolations are capable of creating species over evolutionary time scales. Incipient allopatric vicariant speciation could possibly explain the low interspecific genetic distance between S. tawilis and S. hualiensis. They could be one species that started to differentiate after isolation occurred in 1754 when the Taal Lake’s saltwater connection was shut off (Hargrove Although DNA barcoding would consider e et al.  considerHargrove .