Saccharomyces cerevisiae in co-fermentation with Lactobacillus species, which produce lactic acid as a major end-product of carbohydrate catabolism. The ability of lactobacilli to ferment beer is determined by their capacity to sustain brewing-related stresses, including hop iso-α acids, low pH and ethanol. Here, we evaluated the tolerance of Lactobacillus brevis BSO464 and Lactobacillus buchneri CD034 to beer conditions and different fermentation strategies as well as their use in the brewing process in mixed fermentation with a brewer’s yeast, S. cerevisiae US-05. Results were compared with those obtained with a commercial Lactobacillus plantarum (WildBrewTM Sour Pitch), a strain commonly used for kettle souring. In pure cultures, the three strains showed varying susceptibility to stresses, with L. brevis being the most resistant and L. plantarum displaying the lowest stress tolerance. When in co-fermentation with S. cerevisiae, both L. plantarum and L. brevis were able to generate sour beer in as little as 21 days, and their presence positively influenced the composition of flavor-active compounds. Both sour beers were sensorially different from each other and from a reference beer fermented by S. cerevisiae alone. While the beer produced with L. plantarum had an increased intensity in fruity odor and dried fruit odor, the L. brevis beer had a higher total flavor intensity, acidic taste and astringency. Remarkably, the beer generated with L. brevis was perceived as comparable to a commercial sour beer in multiple sensory attributes. Taken together, this study demonstrates the feasibility of using L. brevis BSO464 and L. plantarum in co-fermentation with S. cerevisiae for controlled sour beer production with shortened production time.Increasing popularity of sour beer urges the development of novel solutions for controlled fermentations both for fast acidification and consistency in product flavor and quality. One possible approach is the use of  L. acidophilus and L. delbrueckii), obligate heterofermentative (L. brevis and L. buchneri) or facultative heterofermentative (L. plantarum and L. sakei) (Lactic acid bacteria (LAB) are gram-positive, non-sporulating bacteria with lactic acid as their main product of carbohydrate metabolism . LAB inctic acid . Lactoba. sakei) . Lactoba. sakei) , kefir (. sakei) , sake (T. sakei) , sour do. sakei) , and bee. sakei) .Lactobacillus is strain dependent , a commercial brewing strain commonly used for kettle souring (biologic acidification of wort prior to yeast fermentation); and Lactobacillus buchneri CD034, a strain previously used in research on kettle souring ; Lactobacillus plantarum (WildBrewTM Sour Pitch), purchased from Lallemand; and Lactobacillus buchneri CD034, kindly donated by the Department of Biotechnology at the University of Natural resources and Life Sciences, Vienna, Austria. Starter cultures for all three strains were prepared by propagating the bacteria twice in MRS medium  and twice in wort medium at 30°C overnight. Cells were harvested by centrifugation , resuspended in wort medium (see below) supplemented with 15% glycerol (v/v) and stored at −80°C. The starter cultures were thawed at 4°C prior to use and inoculated directly. Viability after freezing and thawing was checked, and inoculations were made accordingly. The Saccharomyces cerevisiae US-05 was purchased from Fermentis (France). Dry yeast was rehydrated in sterile water at 1:10 (w/v) for 30 min at 22°C prior to inoculation in fermentation experiments. Unless otherwise stated, all fermentations were carried out in triplicate at 22°C under static conditions.The three The wort used for the stress experiments and small-scale co-fermentations was prepared by diluting concentrated brewer’s wort  in water at 120 g/L. The solution was autoclaved, solid material was removed after the solution had cooled down and the remaining clear solution is hereafter referred to as wort medium. The wort medium had a specific gravity of 1.033 (8.4°P). In the larger scale brewing experiment, wort was prepared using a 60L PRO pilot scale brewery vessel from CoEnCo . Crushed malt  was mashed in water at 0.25 kg/L according to the following scheme: 45 min at 65°C, 15 min at 72°C and 2 min at 78°C. The wort was separated from the spent grain and boiled for 60 min, yielding wort with specific gravity of 1.038 (9.6°P). Both the wort produced from concentrate and from malt, was prepared without hops. When iso-α acid concentration is specified, the concentration was obtained by addition of pre-isomerized hop extract  prior to inoculation.reference condition was wort medium, at 0% (v/v) ethanol, 0 mg/L iso-α acids, pH 5, inoculated with Lactobacillus (106 CFU/mL), incubated at 22°C for 7 days. The conditions for the high inoculation trial differed from the reference regarding inoculation with 108 CFU/mL. The conditions for the high temperature trial were identical to the reference except incubation of the flasks at 30°C. Wort media was supplemented with 5% (v/v) ethanol for the Ethanol trial, and 5 mg/L of iso-α acids for the Iso-α acid trial. Two different medias were prepared with reduced initial pH, one where the initial pH was reduced from pH 5 to 4 with lactic acid [Low pH (lactic acid)] and one where the initial pH was reduced from pH 5 to 4 with hydrochloric acid [Low pH (HCl)]. Finally, a multi-stressor trial was conducted with wort media containing 5% (v/v) ethanol, 5 mg/L iso-α acids and reduced initial pH adjusted with lactic acid. Sampling was done at 0, 4, 8, 12, 24, 32, 48, 72 h, and at 5 and 7 days. Growth was monitored on MRS agar  and pH was monitored using a Sentron pH-meter with SI probe . After the final sampling (7 days), remaining content in each fermentation flask was centrifuged at 7000 × g, 10 min, 4°C and the supernatants stored at −20°C prior to metabolite analysis.Fermentation bottles (50 mL) were prepared with wort media with various adjustments according to different stressors. The 7 cells/mL of lactobacilli and 106 cells/mL of yeast before incubation at 22°C for 21 days. Lactobacilli were inoculated at a higher ratio to give the bacteria an initial advantage and promote their contribution to the fermentation. The population dynamic was monitored during the fermentation process at established intervals ; samples were plated both on MRS agar supplemented with 25 mg/L cycloheximide  and Rose-Bengal Chloramphenicol agar , to be able to differentiate lactobacilli populations from Saccharomyces populations, respectively. pH was monitored as described above. After the final sampling, 50 mL from each fermentation flask was centrifuged  and the supernatant was stored at −20°C prior to metabolite analysis. The remaining content from each flask was used for ethanol analysis.The co-fermentation with lactobacilli and yeast was assessed in flasks containing 400 mL wort medium supplemented with 5 mg/L iso-α acids. Flasks were inoculated simultaneously with 10g, 10 min, 4°C) and the supernatant was kept at −20°C for analysis of amino acids, carbohydrates, metabolites and ethanol. After the final sampling, tanks were kept at 4°C for 14 days before the beer was slightly carbonated using an Aqvia sodastreamer  and transferred to 0.33 L bottles for sensory analysis. Beer fermentations were carried out in triplicate with yeast alone, yeast in co-fermentation with L. brevis and yeast in co-fermentation with L. plantarum.Fermentation tanks (10 L) were prepared with PRO pilot scale brewery wort supplemented with 5 mg/L iso-α acids. Inoculations, fermentation temperature and duration and monitoring of population dynamic were carried out as described above. Samples (80 mL) were withdrawn throughout fermentation, centrifuged  were used to seal the vials before they were placed in a 7679A automatic headspace sampler, with a headspace bath temperature of 50°C and manifold temperature of 60°C. The sampler was connected to a 6890 GC system with flame ionization detector (Agilent Technologies). Helium 6.0  at low rate 5.0 mL/min was used as carrier gas. Samples were mixed  prior to injection . Analytes were separated on a CP-SIL 5CB GC column  of 25 m × 0.53 mm I.D. with film thickness 5 μm. The system was operated by Open LAB EZChrom software  and identification and quantification were carried out according to calibration with external standards. The following temperature scheme was applied during analysis: 35°C for 5 min: increase of 10°C/min until 40°C and kept at 40°C for 2 min; increase of 30°C/min until 130°C and kept at 130°C for 4 min; increase of 30°C/min until 160°C and kept at 160°C for 4 min; increase of 10°C/min until 180°C and kept at 180°C for 2 min; increase of 10°C/min until 200°C and kept at 200°C for 2 min.Volatile compounds  operated at 30 rpm for 30 min. Samples were centrifuged for 15 min at 1470 × g using a Kubota 2010 centrifuge  prior to filtration through 0.2 μm PTFE membrane . Organic acids were separated on an Aminex HPX-87H column  with 0.05 M H2SO4 as mobile phase and a flow rate of 0.4 mL/min. The column, operated at 30°C, was connected to a 1260 Infinity HPLC instrument  with pump, autosampler, column oven, RI-detector  and diode array detector-ultra violet (DAD-UV) detector, used for the other organic acids. Openlab CDS software (Agilent Technologies) was used to operate the system and detection and quantification were done according to calibration with external standards. Maltose, sucrose and glucose were quantified by the K-MASUG enzymatic kit , used according to the instructions.Organic acids as well as fructose and maltotriose were detected and quantified by HPLC, according to the method described by p < 0.05. Variation in metabolites from the small- and larger scale co-fermentations were analyzed by ASCA and ANOVA with Tukey’s test as described above.Differences in metabolites from the stressor experiments were examined by Analysis of variance Simultaneous Component Analysis (ASCA) . MATLAB Beer characterization was carried out using a PBA-B instrument , consisting of a DMA 4500M density meter, an Alcolyzer Beer ME module, a CarboQC ME module and a PFD filling device. The instrument was operated through the Generation M software v2.42 .L-norvalin in 0.1 M HCl). The samples were mixed for 15 min  before being placed for 30 min in an ultrasonic water bath . The samples were then centrifuged for 40 min at 4°C at 2500 × g  before the supernatant was mixed 1:1 with 4% trichloroacetic acid, kept on ice for 30 min and centrifuged for 5 min at 4°C at 15600 × g . The supernatant was filtered through 0.2 μm cellulose acetate filters  and stored at −20°C prior to further preparation. Borate buffer  was mixed with 50 μL samples, and the samples were derivatized by allowing 5 μL to react for 15 s with 5 μL O-phthaldialdehyde (OPA) solution prior to injection. The samples were analyzed using an Agilent 1200 HPLC system  consisting of a serial pump, auto injector, column oven, thermostat and fluorescence detector. The instrument was operated through Open LAB CDS software (Agilent Technologies). A sample volume of 10 μL was injected and analytes separated on an XTerra RP 18 column  operated at 42°C. Two mobile phases were used at 0.7 mL/min: eluent A  and eluent B . The derivatized amino acids were separated by a stepwise linear gradient from 3.3 to 20.7% eluent B over 13 min, from 20.7 to 30% eluent B over 12 min and from 30 to 100% eluent B over 4 min. Free amino acids in the samples were identified and quantified based on a standard curve generated with external standards.Free amino acids were identified and quantified using an HPLC method described by p < 0.05) were selected based on the ANOVA combined with Tukey’s test and analyzed further by Principal Component Analysis (PCA) using PanelCheck V1.4.2 (Norway).A professional sensory panel of eight trained assessors at the Norwegian Institute of Food, Fisheries, and Aquaculture Research (Nofima), Aas, Norway was used for the sensory evaluation of produced beers. All panelists were previously screened for sensory abilities  and communication skills regarding sensory descriptions of products recommended in ISO 8586  in a senL. brevis L. plantarum, and L. buchneri were conducted to evaluate the effect of different beer-related stress factors on their growth and production of metabolites. The three investigated lactobacilli exhibited good fermentation performance in wort medium at reference conditions , the observed CFU/mL was 3.9 × 107 for L. brevis, 2.4 × 107 for L. plantarum, and 7.0 × 108 for L. buchneri. These findings are in agreement with previous studies reporting that the nutrient sources present in malt-based media are favorable to lactobacilli growth  and diacetyl (1.1 mg/L), but the lowest amount of acetic acid (208 mg/L) in the reference trial. At the same conditions L. brevis produced the lowest amount of lactic acid (1195 mg/L), no diacetyl and 382 mg/L acetic acid. L. buchneri did not produce diacetyl but generated 1822 mg/L lactic acid and the highest quantity of acetic acid (703 mg/L) in the reference trial. Higher relative production of acetic acid by L. brevis and L. buchneri was expected as these are both obligately heterofermentative, while L. plantarum is facultative heterofermentative and produces primarily lactic acid from hexose catabolism , in the multi-stressor trial, higher inoculation rate and high incubation temperature led to a decrease of diacetyl in comparison with the fermentation at reference conditions from 1.1 ± 0.1 mg/L to 0.3–0.9 ± 0.1 mg/L. The highest concentration of diacetyl across all strains and stressor conditions was generated by L. buchneri in the multi-stressor trial, yielding a final concentration of 2.1 ± 0.1 mg/L. Diacetyl is associated with caramel and buttery flavors  and co-cultures with S. cerevisiae  where the three different lactobacilli were inoculated simultaneously with revisiae . Vice verevisiae . For allrevisiae ; the finL. brevis appeared as the most influential with regards to metabolite composition (L. brevis beer was separated from the others in component 1 (74.3% of the variation in the model) and the L. buchneri beer was separated from the others in component 2 (18.9% of the variation in the model). Lactic and acetic acid were the most important drivers of component 1, while succinic acid and isoamyl acetate were important drivers of component 2 . The greater influence exerted by L. brevis was not only due to its superior stress tolerance and ability to carry out its metabolism during co-fermentation, but also due to an inhibiting effect on S. cerevisiae metabolism. This effect was evident from the reduced ethanol production  in the L. brevis beer compared to the three other beers  compared to S. cerevisiae alone (1.1 × 104 CFU/mL) or with L. brevis (0.7 × 104 CFU/mL).Based on the results from the stress experiment, where undesirable accumulation of diacetyl was produced by or trial , only L.ntations . S. cerepresence . The finL. brevis generated the largest pH-drop, with a final pH of 3.6, compared to 3.8 for L. plantarum and 4.1 for in the reference beer. Ethanol was produced all throughout the different fermentations  was found on ethanol production or ADF for the same yeast strain  compared to the L. plantarum beer (26 μmol/g) and the reference (43 μmol/g). The concentration of most amino acids was similar after 21 days in the L. plantarum beer and the reference beer; The L. brevis beer however, contained higher quantities of the amino acids alanine, glycine, histidine, tyrosine and phenylalanine. Both lactobacilli seemingly produced γ-aminobutyric acid (GABA), as this amino acid accumulated in the co-fermented beers   . Taken tbenefits ; GABA isbenefits  and may revisiae  and lactrevisiae , and amirevisiae . PredictL. plantarum contributed to a quicker depletion of maltose, glucose, fructose and maltotriose within 1 day  after 1 day was higher compared to the L. plantarum beer. Maltose and maltotriose were the most abundant carbohydrate detected in the final beer and their concentrations were higher in the L. brevis beer compared to the reference beer.The presence of both lactobacilli influenced the carbohydrate utilization during beer fermentation . L. planin 1 day  comparednce beer . In the L. plantarum contributes to the fermentation by depleting amino acids and carbohydrates quickly, its presence did not disrupt the yeast fermentation extensively, as the final ethanol concentration and ADF are similar to the reference beer. L. brevis did, however, affect the final ethanol concentration and ADF, likely interfering with the yeast ability to metabolize free amino acids and carbohydrates.Overall, these data suggest that lactobacilli influenced the beer fermentation in different ways. While L. brevis exerted more influence in co-fermentation with S. cerevisiae, compared to L. plantarum. Together component 1 and 2 accounted for 68 and 32% of the variation in the ASCA, respectively, explaining 68.7% of the variation in the metabolites. Lactic acid, acetic acid, ethyl hexanoate and ethyl octanoate were important drivers of the ASCA model, important in both component 1 and 2 . Remarkably, the current study demonstrates how controlled co-fermentation with S. cerevisiae and a stress-vulnerable L. plantarum can be used to produce sour beer within a 21-day fermentation period, resulting in a product with increased total flavor intensity, fruity odor and dried fruit odor. L. plantarum is commercially used in kettle souring where it performs best as no hops or other stress factors that could inhibit its growth are present in the wort. In addition, L. brevis was used to produce a sour beer with increased total flavor intensity that was similar to commercial Geuze sour beer in acidic taste, sweet taste and astringency. However, caution should be used in an industrial setting without differential brewing lines as L. brevis BSO464 is a common beer-spoilage microorganism. Controlled mixed fermentations, and fermentations of wort with non-traditional microbes, offer a great potential for creation of novel sour beer products with high production control and short production time. By extending the currently explored method to other mixed fermentations with multi-strain yeasts and/or bacteria combinations, it might be possible to shift more sensory properties in the direction of traditional sour beer products and create beer beverages with novel organoleptic properties.In conclusion, this study shows that the investigated strains All datasets generated for this study are included in the article/AD, GD, and HØ conceptualized the study. AD, E-OR, BW, and TW were involved in the design of the study and experimental planning. AD conducted the fermentation experiments. KM carried out the sensory analysis. KL performed the statistical analysis. AD wrote the first draft of the manuscript, with contribution of SL. AD, SL, and BW finalized the manuscript and prepared for publication. All authors contributed to manuscript revision and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


These sites were selected on the basis of the expected differences in their pesticide exposure. Two ditches were located in apple orchards managed by integrated pest management . The ditch “Jork” was located within an ecologically managed apple orchard with a distance of 5.8 m to the sprayed trees. These three ditches were monitored from 2001 to 2003. The ditch “Estebrügge-Grassland”, monitored from 2002 to 2003, served as an additional reference site as it was not located in pesticide treated plantations. Please see Lorenz et al. (2018) Seven monthly samplings were performed in 2001 (April to October) and four samplings in April, June, July, and September in 2002 and 2003. At each sampling date, ditch sediment was removed approximately 1 cm deep by a shovel sampler (sampled sediment area: 0.1 m²). This was done in five consecutive replicates with a distance of 20 m between them, resulting in a total sampled area of total: 0.5 m². Similarly, the water body including submersed macrophytes was sampled at a length of 5 m with a dip net (20 × 15 cm opening) in five consecutive replicates with a distance of 20 m between them. This resulted in a total sampled water volume of 750 L equaling a sampled ditch area of 5 m². The samples were washed in the laboratory using a sieve with 0.5 mm mesh size and fixed in 70% alcohol. The specimens were determined to the lowest taxonomic level whenever possible, only Aranea, Nematoda, Ostracoda, Hydracarina, and Oribatei remained on their higher taxonomic levels.Stefan Lorenz: Conceptualization, Formal analysis, Investigation, Writing - original draft, Writing - review & editing, Visualization. Angelika Süß: Methodology, Investigation, Writing - review & editing.The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article.


When increasing the current density to 28.24 A·g−1, 66% (193 F·g−1) of the initial capacitance was maintained for the GHE-based supercapacitors. High performance for GHE-based supercapacitors was attributed to large specific surface area and good electrical conductivity of GH, and its intimate contact with carbon paper.Current collectors are an important component of electrodes, functioning as conductive media by collecting currents from active materials and then exporting them to the external circuit. Common current collectors for graphene hydrogel (GH)-based supercapacitors are nickel foams or metal foils . Here, hydrothermally synthesized GH was directly pressed on carbon paper and used as electrodes (denoted as GHE) for supercapacitors. With a mass loading of 2.7 mg·cm At the current density of 1.18 A·g−1, a high gravimetric capacitance of 294 F·g−1 was obtained and the capacitance remained 66% (193 F·g−1) even when the current density was elevated to 28.24 A·g−1. Additionally, the influences of loading mass and area of GH on carbon paper on the performance of GHE-based supercapacitors were also systematically studied.Supercapacitors represent an important class of energy storage devices with a high power density, rapid charge/discharge rate, and long cycle life. The electrode is the core part of a supercapacitor, consisting of active materials and current collectors. The widely used active materials are transition metal compounds, conducting polymers, and carbon materials, etc. ,2,3,4. B−1 ,10,11. N−1 ,14,15,16−1 , gold [1−1 ,19, and −1  foils ar−1 . This st−1 . The pre−1 ,23. Alon−1, Nanjing XFNANO Materials Tech. Co., Ltd., Jiangsu, China) was added into a 22 mL Telfon-lined autoclave, and hydrothermally treated at 180 °C for 4 h. The prepared cylindrical GH was picked out and kept in distilled water for further use. For fabricating GH based electrodes, GH was cut into small slices and pressed onto the hydrophilic carbon paper  with the size of ca. 1 cm × 3 cm. The mass for each dried GH was measured to ca. 12 mg. The number of the used GH slices depended on the mass of GH loading on carbon paper. GHE with mass loadings of 2.7 mg·cm−2 at 0.64 cm2, 4.1 mg·cm−2 at 0.68 cm2, and 2.7 mg·cm−2 at 1.07 cm2, were referred to as GHE1, 2 and 3, respectively. The obtained GHE were immersed in 1 M KOH aqueous electrolyte overnight before carrying out electrochemical measurements.Firstly, GH was synthesized by hydrothermal treatment of graphene oxide (GO) ,25. 14 m2 and a mass loading of 2.7–4.1 mg·cm−2. The data from galvanostatic charge/discharge (GCD) curves were used to calculate gravimetric capacitances according to the following formula [−1, m is the dried mass of GH loading on each carbon paper of unit g, The electrochemical performance of GHE-based supercapacitors was measured in 1 M KOH aqueous electrolyte. A three-electrode system was used in which GHE, Pt wire, and saturated calomel electrode (SCE) were used as work, counter, and reference electrodes, respectively. Each GHE electrode had an active area of 0.64–1.07 cm formula :(1)C=I⋅Δ2 F30, FEI, Hillsboro, USA) and X-ray diffractometer , respectively. The microstructure and element analysis were recorded on a Raman spectrometer  with a 514 nm laser beam and X-ray photoelectron spectrometer  with Al Kα as the X-ray source and a pass energy of 30 eV, respectively. The specific surface area of Brunauer–Emmett–Teller (BET) and pore distribution analysis of Barret–Joner–Halenda (BJH) were conducted at 77 K using a gas adsorption surface area and pore size analyzer . The electrochemical properties were measured on an electrochemical workstation .The interior GH morphology was observed by scanning electron microscopy . The crystalline structure was acquired with transmission electron microscopy  analysis indicated that GH possessed a high C/O ratio of 6.5 after hydrothermal reduction. A few oxygenated groups remained for GH, including C–O (286.4 eV), C=O (288.5 eV) and COOH (290.6 eV), respectively  for a model supercapacitor using similar GH-pressed platinum foils as electrodes [−1 at 1.18 A·g−1 for GHE1 is higher than that of the most reports using metal materials as current collectors in GH-based electrodes, as shown in −1, the capacitance for GHE1 still kept 66% (193 F·g−1) of the initial value. However, similar GH pressed on platinum foil only kept ca. 16% (31 F·g−1) of the initial capacitance (198 F·g−1) with the current densities increasing to 20 folds [2 and a mass loading of 4.1 mg·cm−2 showed smaller CV area  adsorption method should be used to determine solvated GH samples. It can detect large pore sizes and avoid slight volume shrinkage of freeze-dried GH. For example, Zhang et al. reported hydrothermally synthesized GH with a BET specific surface area of 166 m2·g−1, while a much larger specific surface area of 964 m2·g−1 was obtained via using an MB adsorption technique [5 Hz (To get insight into the in-depth reasons for the excellent performance of GHE-based supercapacitors, further characterization was carried out. Typical IV isotherm character with a distinct hysteresis loop was presented in echnique . Large sue [5 Hz c. The di−1, the specific capacitance of GHE was up to 294 F·g−1, and the capacitance retention reached 66%. Even the current densities increased by ca. 24 fold. These results indicate that the GHE-based supercapacitors have high specific capacitances and excellent rate performance, and possess broad application prospects. However, increasing either mass loading or the active area of GH on the carbon paper resulted in the attenuation of the specific capacitances for GHE. Further devotion was needed to maintain outstanding supercapacitor performance for GHE with high mass loading and large active area.The carbon paper was used as the current collectors for the GH-based supercapacitors. With the perfect contact between GH and carbon paper, intrinsic high specific surface, and good electric conductivity for GH, the GHE-based supercapacitors exhibited excellent performance. At a current density of 1.18 A·g


Profiling and parallel computing techniques in a cluster of six embedded systems with multiprocessors are introduced herein to implement a chaotic cryptosystem for digital color images. The proposed encryption method is based on stream encryption using a pseudo-random number generator with high-precision arithmetic and data processing in parallel with collective communication. The profiling and parallel computing techniques allow discovery of the optimal number of processors that are necessary to improve the efficiency of the cryptosystem. That is, the processing speed improves the time for generating chaotic sequences and execution of the encryption algorithm. In addition, the high numerical precision reduces the digital degradation in a chaotic system and increases the security levels of the cryptosystem. The security analysis confirms that the proposed cryptosystem is secure and robust against different attacks that have been widely reported in the literature. Accordingly, we highlight that the proposed encryption method is potentially feasible to be implemented in practical applications, such as modern telecommunication devices employing multiprocessors, e.g., smart phones, tablets, and in any embedded system with multi-core hardware. Chaos theory is widely used in the encryption of information because of its particular properties ,2,3, sucOn the other hand, pseudo-random number generators (PRNGs) ,38,39,40A large number of methods that generate sequences with PRNG/TRNG and algorithms to encrypt information for the purpose of transmitting or storing information securely has been reported in the literature ,51,52,53Moreover, technology is constantly evolving with significant advances in the new generation of embedded systems with greater computing power including multi-core processors, for that reason, new threats and vulnerabilities that compromise security information in telecommunications systems are being devised . RecentlMeanwhile, parallel computing is a form of computation in which many instructions are executed simultaneously . It operFrom our knowledge and based on the reviewed literature, few works report the use of parallel computing for the encryption of information, see for example ,80,81,82The rest of this paper is organized as follows: The PRNG and dynamic keys generator that is implemented in this paper is shown in y0=−0.64 .(1)xn+1=The generated pseudo-random sequences consider two attributes: (i) the number of significant decimals is predefined to establish their level of precision 500), if  considerone gets :x1 = −0.From these values, a new level of precision and This procedure guarantees that the new values of initial conditions are within the space of the chaotic attractor and are valid in the long term for new initial conditions, that is, new dynamic keys are generated for the encryption of large amounts of information. When generating numerical values for the initial conditions very different from each other, they produce chaotic series with different dynamic behaviors without losing their properties, which makes them suitable for use in the encryption of images regardless of their level of resolution. B. In this way, a pseudo-random B string of bits is generated for each equation contained in the chaotic map.The proposed PRBG shown in Symmetric key [Original image OI (Object image) goes through a data distribution stage that is illustrated in the [Data scattering] block and that corresponds to divide the image into n blocks (sub-images), k-pixel of the plain image , as shown in n process encrypts the sub-cryptograms achieved Full cryptogram C. To execute the scheme of the The proposed system for encryption and decryption of digital color images is shown in tric key , and in tric key . In relal images .“line-by-line” profiling tool known as Line_Profiler for Python. Line_Profiler is supported by the tag @profile to identify the function of the program to be profiled and perform the corresponding analysis. The performance of the “Encrypter” with the profiling tool is done by means of the command: kernprof -l -v Encrypter.py, run from the GNU Linux console. The -v parameter of the kernprof command allows the user to observe the profiling analysis report as an immediate response to the execution of the previous command in the command terminal. Otherwise, the profiling tool stores the corresponding analysis information in a binary file with extension .lprof with the same name as the Python code file. The complete report can be observed executing the report file through: python -m line_ profiler Encrypter.py.lprof. The following report is an example that corresponds to the profiling analysis of the A profiler is a program that analyzes and collects information about the behavior of another object program during its execution. The type of information that is analyzed includes the processing times of the program’s subroutines and the number of times each subroutine is called. The above, with the purpose of allowing the developer to optimize the program code to improve the speed in the execution of the complete program, adjusting the code design implementing adjustment techniques and, if possible, the implementation of parallel processing techniques. The profiling tool used in this work is the Wrote profile results to Encrypter.py.lprofTimer unit: 3.79968e-07 s Total time: 862.992 sFile: Encrypter.pyFunction: encrypt at line 5Line #      Hits         Time  Per Hit   % Time  Line Contents==============================================================     4                                           @profile     5                                           def encrypt:     6         1       146078 146078.0      0.0          foto = Image.open(’land…     7         1       761832 761832.0      0.0          foto = np.array(foto)     8         1           63     63.0      0.0          ren = foto.shape[0]     9         1            7      7.0      0.0          col = foto.shape[1]    10         1            6      6.0      0.0          cap = foto.shape[2]    11         1            8      8.0      0.0          vector=ren*col*cap    12         1           97     97.0      0.0          foto=np.reshape    14         1           11     11.0      0.0          b= Decimal(’-0.6013’)    15         1            9      9.0      0.0          c= Decimal(’2.0’)    16         1            9      9.0      0.0          d= Decimal(’0.5’)    17         1            8      8.0      0.0          x0= Decimal(’-0.72’)    18         1            8      8.0      0.0          y0= Decimal(’-0.64’)    19         1            5      5.0      0.0          ret=0    20         1            4      4.0      0.0          npx0=8    21         1            4      4.0      0.0          np_prec=99    22         1           15     15.0      0.0          getcontext.prec=npx0    23         2           12      6.0      0.0          while (ret<1):    24         1           69     69.0      0.0              x1 = Decimal((x0*x0)…    25         1           14     14.0      0.0              y1 = Decimal((2*x0*y…    26         1            5      5.0      0.0              x0=x1    27         1            6      6.0      0.0              y0=y1    28         1            6      6.0      0.0              ret=ret+1    29         1            6      6.0      0.0          getcontext.prec=np_prec    30         1            6      6.0      0.0          j=0    31  16384001     78137141      4.8      3.4          while(j<vector):    32  16384000    439917303     26.9     19.4                  x1 = Decimal    40  16384000    185584508     11.3      8.2                  s1=int(x1%255)    41  16384000    337097363     20.6     14.8                  foto[j]= foto[j]^s1    42  16384000     93981721      5.7      4.1                  j=j+1    43         1           77     77.0      0.0          foto=np.reshape    45         1      5036187 5036187.0      0.2          foto.save(’output.png’)        The report initially displays the value of orted in , it is on process, and n process perform more than one operation per unit of time. The performance improvement factor n process is defined by S(n)=n process, if the efficiency of the system n process from which the efficiency of the system begins to decrease. In the analyzed program from the profiling report, of the total instructions, the The lowest system’s efficiency r of 0.5 . In pracn process among which the analyzed program can be established from Equation     # Read original image10: imagen=np.array(imagen)      # Convert the object image into an array11: ren=imagen.shape[0]       # Size of the array (rows)12: col=imagen.shape[1]       # Size of the array (cols)13: cap=imagen.shape[2]14: n_ren=np.int(ren/size)            # Resize the array15: foto=np.reshape)   # for scattering data 1: from mpi4py import MPIThe instruction “Algorithm 2 Second sequential time ( 2: foto=np.array(newData) 3: foto=np.reshape) 4: foto=Image.fromarray(foto) 5: foto.save(’output.png’) 6: fin=datetime.now 7: print(“Done!”) 8: print(end-brgin) 9: if __name__ == ’__main__’:10: encrypt 1: if rank == 0:mpiexec -nkernprof -l -v Encrypter.py, where the parameter The SoC Raspberry Pi 3 has a Cortex-A-53 64-bit Quad Core 1.2 GHz processor with 1 GB DDR2 RAM memory. One of the aspects considered in the study of parallel processing is the processor machine in which the algorithms are executed. The execution of the main algorithm that uses parallel processing through the use of the MPI library ,99 and tn process for program execution. When executing the previous command, the Raspberry Pi 3 system creates a machine with From the comparison shown in The FIAD cluster is composed of six Raspberry Pi 3 embedded systems and whose organization is generally described in To test the robustness and security of the proposed cryptosystem, the following security analyses were performed: Statistical tests of NIST for cryptographic modules, histogram analysis, entropy, differential attack tests (NPCR and UACI) and key space. The quality evaluation of the randomness is carried out to demonstrate the satisfactory security of the new proposed chaos-based cryptosystem. In addition, to test the efficiency of the cryptosystem, a performance analysis of parallel computing in information encryption is accomplished. Besides, with the purpose of testing the proposed method in a different hardware and with multiprocessing capability, it also was implemented in a computer with CPU AMD A6 4400M APU 2.7 GHz (Accelerated Processing Unit), which is a mobile dual-core processor based on the Trinity architecture, works with Windows 10 operating system and Python version 3.5.2.p-value is the probability that a perfect random number generator would have produced a sequence less random than the sequence that was tested, given the kind of non-randomness assessed by the test. If a p-value for a test is determined to be equal to 1, then the sequence appears to have perfect randomness. A p-value of zero indicates that the sequence appears to be completely non-random. A significance level  = 128, Non-Overlapping Template test - block length (m) = 9, Overlapping Template test - block length (m) = 9, Approximate Entropy test - block length (m) = 10, Serial test - block length (M) = 16, Linear Complexity test - block length (M) = 500. All these tests were performed using 1000 series (sequences) of stream length = 1,000,000 bit. For NIST tests, each ST suite  performen-random ,86,101. osystems .The histogram of a digital image is a graph that shows the number of pixels of each different intensity value found in the image. For an image with an 8-bit format, there are 256 different intensity levels . The disbell map  to generbell map ,93.In the works reported by Shannon ,103, theH(s) ,90,91, o(s) [(s),H(s)=∑i=0 at risk ,89,90. Fn process . Finally, it can be observed that the gain in execution time depends on the number of process involved in the execution of the program and the resource in software and hardware assigned to each processor, with Finally, it is important to mention that when implementing the proposed method in a Raspberry Pi 3, the execution times of the program implemented with parallel processing are affected by the execution of other applications and services, so the theoretical estimations of the Performance factor and system efficiency may vary from real-time execution results. In addition, by encrypting the image separately (divided), n processes used. Regarding key space, when implementing the Tinkerbell map with high precision of n processes in parallel for the encryption of the information, the key space is increased virtually up to n is the number of parallel processes , the performance is affected, as shown in sses see . Therefo


Aspergillus niger hyphae are self-sustaining in growth. This was shown by assessing the growth rate of individual hyphae before and after dissection of the second compartment. Using the same methodology, it is here demonstrated that single apical compartments of the septate fungi Penicillium chrysogenum and Schizophyllum commune as well as the 500-µm-apical region of the non-septate fungus Rhizopus stolonifer are also self-sustaining in growth. In contrast, single 2nd compartments (obtained by dissection of the first and third compartment) of the septate fungi or the region between 500 and 1000 µm from tips of R. stolonifer were severely impacted in their growth rate. In addition, it is shown that existing or newly formed branches originating from the 2nd compartments function as a backup system for hyphal growth when the apical part of the hypha of the three studied fungi is damaged. Together, it is concluded that the apical compartments/zones of the studied fungi are self-sustaining in growth. In contrast, the subapical region is not self-sustaining but functions as a backup once the apical zone is damaged. This back up system is relevant in nature because the apices of hyphae are the first to be exposed to (a)biotic stress conditions when entering an unexplored substrate.It was recently demonstrated that apical compartments of  Aspergillus oryzae  plug their pores with Woronin bodies, while hyphae of the basidiomycete Schizophyllum commune close their pores with the septal pore cap  of septate fungi or the region between 500 and 1000 µm from the tip of R. stolonifer were severely impacted in their growth rate.The fact that apical compartments of e Trinci . The perA. niger , 0.05% (v/v) Tween-20 from 7-day-old cultures grown in 9 cm Petri dishes on complete medium (CM). This medium consisted of minimal medium  placed on S. commune minimal medium . To this end, the dishes and medium were pre-warmed to 60  °C. 0.5 µL spore solution , or one S. commune micro-colony, was placed on a glass coverslip (18 mm in diameter and 0.16–0.19 mm thick) and left to dry. Spores were allowed to dry completely, while micro-colonies were allowed to dry until no more medium was visible around the micro-colony. 30 µL pre-warmed MMA  or SCMMA (SCMM with 1% agarose), was added to the middle of the glass bottom dish, and immediately brought in contact with coverslip-adhered A. niger, P. chrysogenum and R. stolonifer spores or S. commune micro-colonies, respectively. After the layer of medium had solidified, 2 ml of corresponding liquid medium (i.e. (SC)MM supplemented with 25 mM maltose) was pipetted on top of the coverslip. Laser dissection was performed after 48 h of growth.Strains N402 of Stauffer , R. stolP. chrysogenum and S. commune hyphae cut in the 2nd compartment. Growth from hyphae cut in the apex or in both the apex and 3rd compartment in A. niger and P. chrysogenum was recorded every 30 min during a 4 h period after dissection, with non-dissected hyphae serving as control. For R. stolonifer, growth was recorded every hour during a 4 h period after dissection, with non-dissected hyphae serving as control. Single 2nd compartments of S. commune were followed for > 7 h using the PALM RoboSoftware time-lapse function. Width and length of compartment pre- and post-dissection were also recorded, as well as translocation (µm) of vacuoles after cutting. From these parameters the post-dissection volume of the apical compartment was calculated  linked to an Observer.Z1 inverted microscope  and a CCD color camera . Growth rate of hyphae and branches was recorded every 5 min during a 15 min period prior to dissection. In addition, it was assessed whether septal pores, if present, were open or closed at the moment of dissection. Septa were classified as open when cytosol was leaking through the septal pore upon dissection , each with at least 7 technical replicates . Single 2nd compartments of P. chrysogenum, S. commune and R. stolonifer was 79 ± 4, 173 ± 16 and 102 ± 20 µm h−1, respectively , respectively apical compartments that halted their growth after dissection lost 11, 51 and 69% of their cytoplasm in the case of Subapical compartments assume peripheral growth after dissection of the apex.A. niger, P. chrysogenum, and S. commune were dissected in the middle of the apical compartment, while R. stolonifer was cut at the very tip. Growth from the 2nd compartment, or, in the case of R. stolonifer, sub-apical to the damaged apex was recorded after laser dissection. These compartments of A. niger, P. chrysogenum, S. commune and the 500-µm-peripheral zone of R. stolonifer had formed lateral branches before dissection in 10, 18, 50 and 100% of the cases, respectively. The mean extension rate of these branches was 14.8 ± 3.7, 14.9 ± 3.2 and 148.2 ± 20.6 µm h−1 for A. niger, P. chrysogenum and S. commune, respectively 2nd compartments continued their growth. These branches had a mean extension rate of 14.1 ± 12.4, 27.3 ± 8.9, and 11.4 ± 23.8 µm h−1 for A. niger, P. chrysogenum and S. commune, respectively 2nd compartments was recorded after dissection of hyphae both within the (pseudo-)apical and (pseudo-)3rd compartments. To this end, A. niger after dissecting the second compartment (Tegelaar and Wösten P. chrysogenum and R. stolonifer in the second compartment. S. commune was not included since only 4% of its hyphae stopped growing after dissection of the 2nd compartment (see above) hampering statistical analysis of potential predictors of continued growth. Growth rate of P. chrysogenum and R. stolonifer hyphae was distributed unimodally 2nd compartments of P. chrysogenum and R. stolonifer and also for A. niger sub-apical compartments are also not needed to support growth of (pseudo-)apical compartments of the basidiomycete S. commune, the ascomycete P. chrysogenum and the mucoromycete R. stolonifer. Like A. niger, these sub-apical compartments function as a back-up system in the case of apical injury. In addition, it is shown that in contrast to the apical compartment, the second compartment is not self-sustaining to reach the maximal growth rate of the (pseudo-)apical compartment.Fungal hyphae have been reported to require a minimal length or minimum number of compartments to maintain their growth rate Trinci . For insP. chrysogenum, S. commune and R. stolonifer stopped growing after dissection, respectively. The P. chrysogenum hyphae that continued growing after dissection of the second compartment lost only 1% of their cytoplasm, while losses of 13 and 27% were found for S. commune and R. stolonifer, respectively. Moreover, in agreement with Tegelaar and Wösten apical compartments of d Wösten  a closedR. stolonifer by cutting 500 and 1000 µm from the apex. Growth from single (pseudo-)2nd compartments took place from existing or newly formed branches. The branches were formed laterally, or grew through the apical or basal septum. Apical growth of the branches of the 2nd single (pseudo-)compartments was about fourfold (R. stolonifer), 13-fold (P. chrysogenum), 11-fold  lower when compared to single (pseudo-)apical compartments. This lower growth rate may be caused by decreased turgor pressure apical compartments to continue their growth raised the question if (pseudo-)2nd compartments also have this potential. This was assessed by cutting in both the first and third compartment or, in the case of sure Lew  or loss tial Lew , secretotial Lew  or protetial Lew . In addiR. stolonifer can continue growing after being isolated from the rest of the mycelium is remarkable. Intuitively, one would assume that the pseudo-apical and pseudo-second compartments would lose their cytoplasm after dissection. However, total loss of cytoplasm occurs with low incidence, if at all. This may be explained by the capillary force of the hyphae and by organelles like vacuoles that block the opening(s) of the dissected compartments. Clearly, the presence of septa promotes the incidence of hyphal survival after dissection. However, the finding that growth of pseudo-apical septa is still observed after loss of 27% of the cytoplasm suggests that R. stolonifer has evolved mechanisms to maintain growth after loss of a relatively high percentage of cytoplasm. Such mechanisms seem to be absent in ascomycetes and basidiomycetes and would partly compensate for the absence of septa in the aseptate fungi.The fact that pseudo-apical and even single pseudo-second compartments of


In the negative ion mode, the two analytes and internal standard (IS) were monitored by multiple reaction monitoring (MRM), and the mass transition pairs were as follows: m/z 369.1 → 119.1 for parecoxib, m/z 313.0 → 118.0 for valdecoxib, and m/z 380.0 → 316.0 for celecoxib (IS). Six beagles were designed as a double cycle self-control experiment. In the first cycle, after intramuscular injection of parecoxib 1.33 mg/kg, 1.0 mL blood samples were collected at different times (group A). In the second cycle, the same six beagles were intravenously injected with 2 μg/kg dexmedetomidine for 7 days after one week of washing period. On day 7, after intravenous injection of 2 μg/kg dexmedetomidine for 0.5 hours, 6 beagle dogs were intramuscularly injected with 1.33 mg/kg parecoxib, and blood samples were collected at different time points (group A). The concentration of parecoxib and valdecoxib was detected by UPLC-MS/MS, and the main pharmacokinetic parameters were calculated by DAS 2.0 software. Under the experimental conditions, the method has a good linear relationship for both analytes. The interday and intraday precision was less than 8.07%; the accuracy values were from -1.20% to 2.76%. Cmax of parecoxib in group A and group B was 2148.59 ± 406.13 ng/mL and 2100.49 ± 356.94 ng/mL, t1/2 was 0.85 ± 0.36 h and 0.85 ± 0.36 h, and AUCt) of valdecoxib in group A were 37.79% and 36.19% higher than those in group B, respectively, and t1/2 was increased from 2.44 h to 2.91 h. Vz/F and CLz/F were correspondingly reduced, respectively. The developed UPLC-MS/MS method for simultaneous determination of parecoxib and valdecoxib in beagle plasma was specific, accurate, rapid, and suitable for the pharmacokinetics and drug-drug interactions of parecoxib and valdecoxib. Dexmedetomidine can inhibit the metabolism of valdecoxib in beagles and increase the exposure of valdecoxib, but it does not affect the pharmacokinetics of parecoxib.A sensitive and reliable ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of parecoxib and its metabolite valdecoxib in beagles. The effects of dexmedetomidine on the pharmacokinetics of parecoxib and valdecoxib in beagles were studied. The plasma was precipitated by acetonitrile, and the two analytes were separated on an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7  In clin effects . Evidenc effects .After intravenous administration, dexmedetomidine quickly distributes beyond total body water. Dexmedetomidine is metabolized by glucuronization and CYP2A6 hydroxylation and then excreted almost completely as a metabolite in urine , 6. BodyParecoxib  is a watIn particular, parecoxib was evaluated in pre- and postoperative treatments of gynaecological, ophthalmological, and colorectal surgery and intracranial and digestive tract tumour resection –13.The perioperative use of parecoxib can significantly improve the effectiveness of postoperative pain after transcatheter arterial chemoembolization (TACE), and parecoxib is effective and well tolerated in relieving pain caused by acute renal colic , 15.Dexmedetomidine and parecoxib are commonly used assistant anesthesia drugs in the clinic, and dexmedetomidine is often used in combination with methoxyphene, midazolam, dezocine, nonsteroidal anti-inflammatory drugs (NSAIDs), and other drugs for analgesia of anesthesia , 17. DexDexmedetomidine is a strong inhibitor of cytochrome P450 enzyme . ValdecoΩ) was prepared by a Millipore Milli-Q purification system.Parecoxib (purity > 98.0%), valdecoxib (purity > 98.0%), and celecoxib  were purchased from Sigma . Methanol and acetonitrile of LC-grade were provided by Tianjin Kermel Chemical Reagent Co., Ltd. Ultrapure water  was provided by Pharmacia and Upjohn company. Dexmedetomidine hydrochloride injection (LOT NO.: 180222BP) was obtained from Hengrui Medicine Co., Ltd.The equipment used in this study includes Waters Acquity UPLC instrument , XEVO TQD triple quadrupole mass spectrometer , ultrapure water equipment , and electronic analytical balance and vortex instrument.μm) by gradient elution with the mobile phase of 0.1% formic acid (A) and acetonitrile (B) at the flow rate of 0.4 mL/min. The gradient program was as follows: 0.00-0.50 min, 90% A; 0.50-1.00 min, 90-10% A; 1.00-2.00 min, 10% A; 2.00-2.10 min, 10-90% A; and 2.10-3.00 min, 90% A. The column temperature was set at 45°C, and the autosampler was conditioned at 4°C. An injection volume of 2 μL was applied for analysis.Parecoxib, valdecoxib, and IS were separated on an Acquity BEH C18 column .The XEVO TQ-S triple quadrupole mass spectrometer was used for mass spectrometric (MS) measurement with an electrospray ionization (ESI) interface in a negative ionization mode. Under the multiple reaction monitoring (MRM) conditions, quantification was achieved with transitions of Parecoxib, valdecoxib, and celecoxib were prepared at a respective concentration of 1.0 mg/mL by dissolving in methanol as the stock solutions. Various working solutions for the calibration curve and quality control (QC) were obtained in methanol by the gradient dilution of the stock solutions. The final concentrations of the calibration curves were covered by several points as follows: 5, 10, 50, 100, 500, 1000, 2000, and 4000 ng/mL for parecoxib and valdecoxib. As for QC samples, they were made in the same way at three concentration levels , and the concentrations of QC samples in plasma were 10, 500, and 3000 ng/mL for parecoxib and valdecoxib. The internal standard (IS) working solution (1000 ng/mL) was also prepared by diluting the stock solution of celecoxib with acetonitrile. All solutions were stored in a refrigerator at -20°C.μL of the IS working solution (1000 ng/mL in acetonitrile) was added to 100 μL of a plasma sample. The tubes were vortex mixed for 1.0 min. After centrifugation at 15,000 g for 15 min, the supernatant (2 μL) was injected into the UPLC-MS/MS system for analysis.Before analysis, frozen beagle plasma samples were thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 200 UPLC-MS/MS is a common method for the detection of biological samples, which has the advantages of rapidity, sensitivity, and accurateness. The UPLC-MS/MS method has been reported for the simultaneous determination of parecoxib and its metabolite valdecoxib , 23. In The specificity was assessed by comparing chromatograms of blank beagle plasma samples; blank plasma spiked with parecoxib, valdecoxib, and IS; and a beagle plasma sample 3.0 h after injection of parecoxib.χ2). The lower limit of quantification (LLOQ) was selected as the lowest concentration used in the calibration curve. The carryover test was performed by injecting a blank plasma sample injected with parecoxib and valdecoxib (4000 ng/mL) followed by a blank sample.The calibration curves were constructed and validated by analyzing spiked calibration samples for three days in a row. The linearity of the assay was assessed by analyzing the calibration curves using least-squares linear regression of the peak area ratios of the analytes to the IS versus the nominal concentration of the calibration standard with a weighed factor  in six replicates. On the same day, the intraday precision was calculated, and the interday precision was calculated by continuous measurement within 3 days. Precision was defined as the relative standard deviation  and accuracy as the relative error .Parecoxib and valdecoxib extraction recovery was compared by comparing the peak area of the conventional pretreated QC sample pretreatment with the peak area after extracting the corresponding blank plasma concentration (after extraction), and the matrix effect (ME) was evaluated by comparing the peak area of the analyte to water extraction after the sample and the corresponding replacement sample.Stability studies in biosamples were also conducted at three QC levels in several different storage conditions: room temperature for 12 h, -20°C for at least 4 weeks, after three freeze-thaw cycles (-20 to 25°C), and processed samples at 4°C in an autosampler tray for 12 h. The stock solution stability of parecoxib, valdecoxib, and IS was observed through six repeated tests. The stock solution portion stored at room temperature for 24 hours was compared with the remaining stock solution stored in a -20°C refrigerator to evaluate room temperature stability. The freezing stability was evaluated by comparing the newly prepared stock solution with the stock solution stored in a refrigerator at -20°C for three months.Six beagles  were obtained from Hubei Yizhicheng Biological Technology Co., Ltd, and the animal certificate was SCXK(hubei)2016-0020. The institutional approval number for the preclinical study of this experiment was 2019100013. The beagles were adapted to the new environment for 7 days in laboratory conditions. Necessary approval from the Institutional Animal Ethics Committee was obtained to carry out the experiments.Before the test, the beagles had free access to water but were fasted for 12 h. Six beagles were designed as a double cycle self-control experiment. Blood samples (1.0 mL) were collected from the forelimb cephalic vein or the small saphenous vein of the hindlimb and collected into heparinized tubes at 0.17, 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 9, 12, and 24 h after intramuscular injection of parecoxib 1.33 mg/kg in the first cycle (group A). Blood samples were centrifuged for 10 min at 3000 × g, and the plasma was collected and kept frozen at -20°C until analysis, and the samples belonged to group A.μg/kg dexmedetomidine every morning in the second cycle (group B), with continuous injection for 7 days. On the seventh day, the six beagles were given 1.33 mg/kg parecoxib intravenously half an hour after intravenous injection of 2 μg/kg dexmedetomidine. The blood samples (1.0 mL) were collected into heparinized tubes at 0, 0.17, 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 9, 12, and 24 h after intramuscular injection of parecoxib. Plasma was also separated and preserved, and the samples belonged to group B.After the one-week washout period, the same six beagles were injected intravenously slowly with 2 The above-developed UPLC-MS/MS method was used to simultaneously detect parecoxib and its active metabolite valdecoxib in the beagle plasma of group A and group B.The pharmacokinetic parameters of parecoxib and valdecoxib were calculated by the noncompartmental analysis using the DAS 2.0 software. All data were expressed as the mean ± standard deviation (SD).Under the above experiment, parecoxib, valdecoxib, and IS were well separated from endogenous substances. Representative chromatograms of a blank plasma sample (a), a blank plasma sample spiked with two analytes and IS (b), and a beagle sample (c) are shown in y = 1.5 × 10−3 x − 1.34 × 10−2 (r = 0.9993) and y = 1.8 × 10−3 x–3.82 × 10−2 (r = 0.9991), respectively, where y represents the peak area ratio and x represents the plasma concentration. The lower limit of quantification (LLOQ) of parecoxib and valdecoxib was 5 ng/mL. Carryover test results show that in the UPLC-MS/MS analysis, carryover does not affect the determination of parecoxib and valdecoxib.In the concentration range of 5–4000 ng/mL, the typical regression equations of parecoxib and valdecoxib were The recovery and ME results were investigated and are shown in All results for the stability samples tested are summarized in Tables The plasma concentration-time curve of parecoxib of group A and group B in six beagles after a single intramuscular injection of parecoxib is shown in t1/2, Cmax, Tmax, Vz/F, CLz/F, AUCt). Meanwhile, The DAS 2.0 program was used to calculate the main pharmacokinetic parameters of parecoxib and valdecoxib, including Cmax and AUC(0 − ∞) of valdecoxib in group A were 37.79% and 36.19% higher than those in group B, respectively, and t1/2 was increased from 2.44 h to 2.91 h. Vz/F and CLz/F were correspondingly reduced, respectively. It was suggested that dexmedetomidine can inhibit the metabolism of valdecoxib and increase the exposure of valdecoxib in beagles.Results show that after intramuscular injection of parecoxib, parecoxib was absorbed rapidly, and then, it was transformed into valdecoxib, an active metabolite. There was no significant change in the pharmacokinetics of parecoxib in groups A and B, it was suggested that dexmedetomidine does not affect the conversion of parecoxib. This study established a new UPLC-MS/MS method that can simultaneously detect the content of beagle dog plasma parecoxib and its metabolite valdecoxib. This method fully complies with bioassay standards, with high sensitivity and short analysis time (3 min). In the choice of plasma processing methods, we directly precipitate proteins with acetonitrile. The operation method is simple and fast, and the detection results are reliable. At the same time, the endogenous substances in the beagle dog plasma sample do not affect the determination of the analyte.When the pharmacokinetics (PKs) and pharmacodynamics (PDs) of a drug are changed by one or more drugs, drug-drug interactions (DDI) will occur . It is iNonsteroidal anti-inflammatory drugs, or NSAIDs, play antipyretic, analgesic, and anti-inflammatory effects by inhibiting COX and reducing the synthesis of prostaglandins. NSAIDs are available as oral tablets and solutions . MetabolParecoxib was rapidly and almost completely converted to valdecoxib. The elimination of valdecoxib was extensively carried out in a variety of ways in the liver, including cytochrome CYP3A4 and CYP2C9 enzyme metabolism and sulfonamide glucose hydroformylation (about 20%) .α2-adrenoceptor agonist with sedative, antianxiety, sympathetic, and analgesic effects [Dexmedetomidine is a highly selective  effects . Dexmede effects , 6. Dexm effects . The com effects . Dexmede effects .Cmax and AUC(0 − ∞) of valdecoxib in group A were 37.79% and 36.19% higher than those in group B, respectively, and t1/2 was increased from 2.44 h to 2.91 h. Vz/F and CLz/F were correspondingly reduced, respectively. It was suggested that dexmedetomidine can inhibit the metabolism of valdecoxib and increase the exposure of valdecoxib in beagles.The results of this study show that there was no significant change in the pharmacokinetics of parecoxib in groups A and B; it was suggested that dexmedetomidine does not affect the conversion of parecoxib in beagles. Although there are some differences in the pharmacokinetics of different species, the results of animal experiments can provide some reference for clinical medication . TherefoThe developed UPLC-MS/MS method for simultaneous determination of parecoxib and its metabolite valdecoxib in beagle plasma was specific, accurate, rapid, and suitable for the pharmacokinetics and drug-drug interactions of parecoxib and valdecoxib. Dexmedetomidine can inhibit the metabolism of valdecoxib in beagles and increase the exposure of valdecoxib, but it does not affect the pharmacokinetics of parecoxib.


In the integrated analysis of phase III head-to-head trials in patients with advanced solid tumors, denosumab demonstrated superiority over zoledronic acid in preventing skeletal-related events (SREs). Regular and continued drug use (persistence) is a precondition of clinical efficacy; persistence in real-life is yet undetermined for denosumab.This was a single-arm, prospective, observational, non-interventional study in 598 patients with bone metastases from breast, prostate, lung, or other solid tumors treated with denosumab every four weeks in real-world clinical practice in Austria, Czech Republic, Hungary, Slovakia, and Bulgaria. Persistence was defined as denosumab administration at ≤ 35-day intervals over 24 or 48 weeks, respectively.Previous SREs were found in 10.9% of patients. 62.6% were persistent over 24 weeks and 40.1% over 48 weeks. The Kaplan-Meier median (95% CI) time to non-persistence was 274.0 days . The most frequent reason for non-persistence was delayed administration. There was a trend towards weaker analgesics over time, with approximately 60% of patients not requiring any analgesics. Serum calcium remained within the normal range throughout the study. Adjudicated osteonecrosis of the jaw was documented in three patients with an incidence per patient-year (95% CI) of 0.012 .Most patients received denosumab regularly once every four weeks over 24 weeks of treatment. Non-persistence was mainly due to delayed administration. The incidence of adverse drug reactions, especially of osteonecrosis of the jaw, was in line with expectations from previous studies.The online version of this article (10.1007/s00520-019-04988-7) contains supplementary material, which is available to authorized users. Bone metastases represent a frequent complication of cancer, with more than 1.5 million affected patients worldwide , 3 oftenDenosumab, a fully human monoclonal antibody of the IgG2 subtype, inhibits the receptor activator of nuclear factor κB ligand (RANKL) on bone cells. In its oncological formulation, denosumab is indicated in Europe for the prevention of skeletal-related events  in adults with advanced malignancies involving the bone and for the treatment of adults and skeletally mature adolescents with giant cell tumor of bone that is unresectable or where surgical resection is likely to result in severe morbidity .In the integrated analysis of three pivotal, phase III head-to-head trials, denosumab was superior in preventing SREs compared with zoledronic acid . Yet, inThe extent to which poor compliance and persistence affect clinical efficacy is a complex issue. From a payer’s perspective, low compliance and/or persistence often works in two directions: they reduce medication costs but subsequently increase health care resource utilization. Although this cannot necessarily be assumed in all settings, a relationship between bone metastasis–related SREs and additional inpatient stays and an increased use of surgical or other procedures has been demonstrated . To dateThis was a single-arm, prospective, observational, non-interventional, multi-center cohort study in patients with solid tumors and bone metastases in Austria and selected Central and Eastern European (CEE) countries, namely the Czech Republic, Hungary, Slovakia, and Bulgaria. As this was a non-interventional study, no laboratory, diagnostic, or therapeutic procedures other than those performed as part of the patient’s routine care were required. Patients were observed from enrollment (having received the first dose of denosumab as per standard of care within 28 days prior to enrollment) until the last denosumab dose administered up to a maximum of 48 weeks after the first administration plus 30 days of safety follow-up.Patients treated with the denosumab formulation XGEVA®  at a dose of 120 mg subcutaneously once every four weeks, in accordance with the—at time of enrollment—most current version of the European Medicines Agency’s summary of medicinal product characteristics (SmPC) were eligible to participate in this study. In addition, they had to meet the following criteria: adult age (≥ 18 years) at enrollment; a diagnosis of breast, prostate, lung, or other solid tumor with confirmed bone metastasis; an Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 2; administration of the first denosumab dose ever within 28 days prior to enrollment. Patients were excluded: if they had a diagnosis of multiple myeloma (not an approved indication at the time of study conduct), were previously treated for SRE prevention with bisphosphonates or other antiresorptive agents for more than 6 months, were previously treated with radionuclides , were enrolled in an investigational drug trial for the treatment and/or prevention of bone metastases and SREs, or had contraindications to denosumab. Patients in a trial related to the treatment of their underlying cancer or in long-term follow-up studies were eligible.The primary objective was to estimate the persistence with denosumab treatment as per routine clinical practice at 24 weeks. Secondary objectives were to estimate the persistence with denosumab at 48 weeks and the time to and reasons of non-persistence, to describe patient demographics, disease characteristics, concomitant anticancer therapy and medical history, and calcium and vitamin D supplementation patterns. Exploratory objectives included the description of pain medication patterns and patient-reported outcomes according to the EQ-5D questionnaire in countries where this was accepted by local authorities and reasons for the choice of denosumab over other options.Safety data related to denosumab were collected for up to 30 days after the last denosumab dose. Osteonecrosis of the jaw (ONJ) was regarded as an event of special interest. All suspected events of ONJ were documented and reported as serious adverse drug reactions (ADRs), regardless of whether serious criteria could be assigned and whether causal relationship with denosumab established. Any suspected case of ONJ was reviewed by an independent adjudication panel to confirm or reject the ONJ categorization.This study complied with all relevant national requirements on a country-by-country basis. Written informed consent was obtained from the patient or legally acceptable representative. National ethics committee approval of the protocol and informed consent form was obtained before recruitment of patients or any data collection.No formal hypothesis was tested. For continuous variables, descriptive statistics including the mean, standard deviation (SD), median, first (Q1) and third (Q3) quartiles, and minimum and maximum values (range) were presented along with 95% two-sided confidence intervals (CIs), where appropriate. Missing values of continuous variables were counted as “Missing”. For categorical variables, the number and percentage of patients in each category were reported. For binary variables, the number and percentage of patients were reported, along with exact two-sided CIs, where appropriate. Missing results were excluded from the calculation of CIs; however, the number and percentage of patients with missing results were given for categorical data. The statistical analyses were based on the full analysis set (FAS), which consisted of the enrolled patients who met eligibility criteria and received at least one dose of denosumab. The Kaplan-Meier method and Cox proportional hazards model were used to analyze the data for time to non-persistence. For statistical analysis, the SAS System 9.4 was used.“Persistence” measured the regularity and duration of continuous use of denosumab and was defined as continuous use from the first administration without exceeding a maximum permissible 35-day gap (4 weeks plus 7 days) until the last date of administration (discontinuation date), disenrollment (due to death or loss to follow-up), or end of the study period. Time to non-persistence was calculated as the time between the first injection and the last injection received during the period where the patient was still classified as persistent plus 28 days. When the predefined time window between injections of a maximum of 35 days was exceeded, a drop-down window of reasons for dose interruption opened in the electronic case report form, which were documented as reasons for non-persistence. Additionally, the reason “violation of permissible time window” was documented automatically when the predefined time window of a maximum of 35 days was exceeded based on the documented dates of administration.A total of 598 patients (FAS) were analyzed , denosumab discontinuation , and loss to follow-up . At week 48, 387 (64.7%) were still under observation and 211 (35.3%) had discontinued the study prematurely. The median  duration of study-related observation was 48 weeks . After the end of the study-related observation period, 379 patients (63.4%) continued denosumab treatment. Overall, 91 patients (15.4%) discontinued denosumab, 56 patients during observation and 35 after the end of observation. The documented reasons for discontinuation of denosumab were physician’s decision , patient’s decision , (S)ADRs , switch to other antiresorptive drugs , or other reasons . The overall number of deaths during the observation period including safety follow-up was 82 (13.7%), of which 71 patients (11.9% of FAS) died of their underlying cancer and 11 (1.8% of FAS) died of other causes not related to denosumab. Details on patient disposition by cancer type are shown in Table Of 598 patients initiated with denosumab, 451 (75.4%) completed 24 weeks of observation and 147 (24.6%) discontinued prematurely. The main reasons for premature study discontinuation were death  had breast cancer, 24.4% (n = 146) had prostate cancer, 9.9% (n = 59) had lung cancer, and 11.5% (n = 69) had cancers summarized as “other”. Figure n = 376), owing to the large number of patients with breast cancer (Table n = 312) was 65 years or older. Age differed by cancer type with the proportion of patients aged 75 or older ranging from 5.1% of patients with lung cancer (n = 3) to 32.2% of patients with prostate cancer (n = 47). ECOG performance status was 0 in 52.5% of patients (n = 314), 1 in 40.5% (n = 242), and 2 in 7.0% (n = 42).Of patients, 54.2% (4.4% n = 46 had prn = 263) receiving the diagnosis for cancer less than 1 year before enrollment. The median time since diagnosis ranged from 1.8 months in lung cancer patients to 35.9 months in breast cancer patients (Table n = 518) metastatic disease was diagnosed less than 1 year before enrollment. By metastasis site, 46.7% (n = 279) had bone metastases only and 53.3% (n = 319) had metastases in the bone and other sites. Other metastatic sites were in the liver in 21.6% of patients (n = 129), lung in 20.2% (n = 121), brain in 3.7% (n = 22), and other sites in 28.6% (n = 171). Patient could have metastases in more than one site. Bone metastases were diagnosed less than 1 year before enrollment in 93.0% of patients (n = 556) and were mostly asymptomatic and diagnosed by imaging  time since cancer diagnosis was 19 months  with 44.0% of patients : 7.5% (n = 45) had pathological fractures, 2.2% (n = 13) required radiation to the bone, 1.5% (n = 9) had surgery to the bone, and 0.5% (n = 3) had spinal cord compression. The time between diagnosis of an SRE and enrollment was less than 3 months in 7.4% of patients (n = 44) and between 3 and 6 months in 2.3% (n = 14); in 2 patients (0.3%), it was between 6 and 12 months, and in 5 patients (0.8%), it was longer than 12 months.Prior to enrollment, SREs were confirmed in 10.9% of patients (n = 211) had received chemotherapy prior to starting denosumab, 32.8% (n = 196) had received previous endocrine therapy, 14.4% (n = 86) radiotherapy, and 7.9% (n = 47) surgery. During the study observation period and concomitantly with denosumab, 52.3% of patients (n = 313) received chemotherapy, 46.3% (n = 277) endocrine therapy, 15.7% (n = 94) radiotherapy, and 3.5% (n = 21) surgery.In the metastatic setting, 35.3% of patients (n = 46) had received other antiresorptive agents. Previous antiresorptive therapies were zoledronic acid , ibandronate , pamidronate , and unspecified others . Antiresorptive agents were mainly administered intravenously ; 1.0% (n = 6) received them per os. All 46 patients received their antiresorptive therapy for 6 months or less, as per inclusion criteria. Reasons for not continuing previous antiresorptive therapies were intolerability in 1.5% (n = 9), patient’s wish , or physician decision . Physicians decided to stop the previous antiresorptive agents because of the route of administration , renal insufficiency , or unspecified other reasons .Prior to starting denosumab, 7.7% of patients , superior efficacy of denosumab , and better safety profile of denosumab .The most frequent physician-reported reasons for the choice of denosumab were the prevention of first SRE  of 11 doses  of denosumab over a period of 309 days .n = 420) received calcium supplementation and 71.4% (n = 427) received vitamin D supplementation. This proportion increased to approximately 80% at dose 2 and steadily decreased thereafter  serum calcium level at enrollment was 2.35  mmol/L. At the second dose of denosumab, the serum calcium level reached a nadir at 2.26  mmol/L. Serum calcium remained above this lowest value from the third dose onwards throughout the study. At enrollment, 70.2% of patients  overall, and ranged between 26.1% for lung cancer and 69.5% for breast cancer, and between 56.0% for Austria and 84.8% for Slovakia. Figures Persistence differed by tumor type, impacting on persistence in the countries. In Austria and Slovakia, the largest group of represented tumors was breast cancer; in Hungary, only prostate cancer patients were enrolled. In the Czech Republic and Bulgaria, breast and prostate cancer patients were almost equally represented and formed the largest groups Fig. .Persistence at 48 weeks was 40.1% . Patterns by tumor type and by country were similar to the persistence results for week 24.The Kaplan-Meier (KM) median (95% CI) time to non-persistence was 274.0  days, with 317.0  in breast cancer, 325.0  in prostate cancer, 118.0  in lung cancer, and 118.0  in other cancers.An analysis of median time to non-persistence by previous antiresorptive therapy (y/n) showed a KM median (95% CI) of 294.0 days  for 46 patients with previous antiresorptive therapy and 273.0 days  for 552 patients with no previous antiresorptive therapy.p < 0.05, Wald test).Using a Cox proportional hazards model, tumor type , previous antineoplastic therapy (y/n), and ECOG status (0 versus 2) were found to be significantly associated with time to non-persistence with denosumab , and unspecified other reasons. Figure n = 121; AQA score 1) or strong opioids at a low daily dose of < 75 mg oral morphine equivalents . When assessing the shift in analgesic use among patients with no or weak opioid analgesics (AQA score ≤ 2) at baseline, very few patients shifted to an AQA category > 2  at later timepoints  experienced an ADR. The most frequently reported ADR was hypocalcemia . Ten patients (1.7%) experienced ADRs leading to discontinuation of denosumab. Serious ADRs were documented for 1.3% of patients (n = 8). ONJ was documented in 0.7% of patients (n = 3). The median (IQR) duration to first ONJ event based on these three patients was 165.0 days . Other reported serious ADRs were costovertebral angle tenderness, pain, hypocalcemia, peripheral edema, dyspnea, swelling face, and cellulitis . No fatal ADRs occurred. Overall, the exposure-adjusted incidence rate was 0.187  for ADRs per 100 patient-years and 0.027  for serious ADRs.Only ADRs considered related to denosumab by the treating physician were collected. Overall, 10.2% of patients , with two confirmed cases of ONJ and one with unspecified location. In the German X-TREME study, 15 patients with suspected ONJ (1.3%) were reported. In randomized controlled studies of denosumab, 2% of breast cancer patients, 2.3% of prostate cancer patients, and 1.1% of patients with either a solid tumor or multiple myeloma experienced ONJ [The incidence of osteonecrosis was 0.5% in the present study


Illiciaceae. Due to morphological similarities among Illiciaceae fruits, fatal poisonings are frequent.  Anisatin is a neurotoxic sesquiterpene dilactone wildly found in plants of the family  This study is aimed at developing a rapid, simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to determine anisatin's bioavailability in mouse blood and the method's application to pharmacokinetics.  Blood samples were preprocessed by protein precipitation using acetonitrile. Salicin  and anisatin were gradient-eluted by a mobile phase of methanol and water (0.1% formic acid) in a UPLC BEH C18 column. This step involved using an electrospray ionization source of anisatin at a mass-to-charge ratio (m/z) of 327.1 → 127.0 and IS at m/z 285.1 → 122.9 in the negative ion mode with multiple reaction monitoring. r > 0.995), with the method's accuracy ranging from 86.3% to 106.9%. Intraday and interday precision were lower than 14%, and the matrix effect was between 93.9% and 103.3%. The recovery rate was higher than 67.2%.  The calibration curve ranged from 1 to 2000 ng/ml  and intravenous (0.5 mg/kg) administration of anisatin to mice—the absolute bioavailability of anisatin in the mouse blood was 22.6%. Illiciaceae family provide most shikimic acid, but substantial production is found only in few species [Illiciaceae are found around the world, many of which are toxic.Humanity has faced many grave respiratory infectious disease challenges in this century. Tamiflu (oseltamivir phosphate), the most clinically effective anti-influenza drug, is currently manufactured from shikimic acid, its key precursor , 2. Plan species –6. More Illicium verum Hook. f., Bajiao) represents a cultivated species with a high content of shikimic acid; it is famous for its use as both spice and medicine [Illicium Mangcao (I. henryi. and I. lanceolatum A. C. Smith) are morphologically similar and easily confounded [Chinese star anise  was obtained from Sigma , and salicin  was obtained from Chengdu Munster Biotechnology Co., Ltd. . High-performance liquid chromatography (HPLC) grade formic acid, acetonitrile, and methanol were obtained from Merck , and ultrapure (type 1) water was obtained through a Milli-Q water system obtained from Millipore Sigma .An ACQUITY ultra-performance liquid chromatography (UPLC) H-Class system  coupled to a XEVO TQS-micro triple quadrupole mass spectrometer and electrospray ionization (ESI) was employed for the analysis of mouse blood samples.μm, 2.1 mm ×50 mm) column at 30°C. The mobile phase was composed of methanol and water (0.1% formic acid) based on a gradient elution with a flow rate of 0.4 ml/min—the gradient elution was as follows: from 0 to 0.2 min, 10% methanol; 0.2 to 1.4 min, 10% to 75% methanol; 1.4 to 2.0 min, 75% methanol; 2.0 to 2.1 min, 75% to 10%, methanol; and 2.1 to 4.5 min, 10% methanol.Separation was performed by an UPLC BEH C18  flow of 900 l/h and capillary voltage of 2 kV; the source of ionization temperature was 150°C, and the desolvation temperature was 450°C. The quantitative analysis was performed in the ESI negative and multiple reaction monitoring mode, obtaining a mass-to-charge ratio (m/z) of 327.1 → 127.0 for anisatin  and m/z 285.1 → 122.9 for salicin . We prepared anisatin (1.0 mg/ml) and salicin (1.0 mg/ml) standard solutions in methanol and ultrapure water . Working standard solutions of anisatin were conducted by dilution of stock solutions appropriate with methanol to obtain the desired concentrations of 10, 50, 200, 500, 1000, 2000, 5000, 10.000, and 20.000 ng/ml. Also, the working standard solution of salicin (100 ng/ml) was diluted from its stored standard solution using methanol.Calibration standards were prepared by spiking blank mouse blood with proper amounts of working standard solutions of anisatin. We constructed a calibration plot for anisatin in blood within the range of 1 to 2000 ng/ml , and 3 quality control (QC) samples  were also prepared in the same way as calibration standards. All the solutions were stored at −20°C.μL) was added with 100 μL of acetonitrile containing IS at 100 ng/mL. The mixture was vortex mixed for 1 min and centrifuged at 13,000 rpm for 10 min at 4°C. Then, 2 μL supernate was transferred and injected into the UPLC-MS/MS system for analysis.Blood  against the standard nominal concentration (x) using 1/x.A calibration curve (1-2000 ng/ml) for anisatin was obtained in triplicate and generated by plotting the peak area ratio  in 6 replicates for 3 consecutive days. Precision was expressed as relative standard deviation. Accuracy was measured between the average value of QC samples and the true value. The actual concentrations determined in QC samples were calculated using the calibration curve obtained on the same day and compared with the nominal concentrations.Recoveries were evaluated by comparing the peak areas measured from QC samples to the corresponding standard peak areas. The matrix effects were evaluated by comparing the blank blood's peak areas with the standard solution after sample treatment and the corresponding standard solution's peak areas.The stability tests of anisatin in mouse blood were investigated by analyzing replicate QC samples stored under 3 conditions: short-term stability (2 h at room temperature), long-term stability , and freeze-thaw stability .n = 6). Anisatin was precisely weighed and completely dissolved in normal saline. A group was given anisatin (1 mg/kg) by the intragastric administration, and another was given anisatin (0.5 mg/kg) by the intravenous administration. This experiment was approved by the Wenzhou Medical University Animal Care Committee. Blood samples (20 μl) were collected at 0.083, 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 12 h from the caudal vein after administration, then stored at −20°C until analysis.We kept 12 mice  provided by the Laboratory Animal Center of Wenzhou Medical University  at the Institute of Cancer Research; the animal certificate number was wydw2019-0983. The animals were maintained in a 12 h light/12 h dark cycle at 25°C and 45% to 65% humidity with ad libitum food. The 12 mice were randomly divided into 2 groups (po × Div/(AUCiv × Dpo), where po stands for oral administration, and iv stands for intravenous administration.The data determined by UPLC-MS/MS was fitted by the DSA 2.0 ; bioavailability% = 100% × AUCy = 0.0161x + 0.0105 , y represents the ratio of anisatin's peak area to that of IS, and x is the concentration of anisatin. The lower limit of quantification (LLOQ) was 1 ng/ml with a signal-to-noise ratio of 10 in mouse blood. The precision and accuracy of the LLOQ were 12.0% and 86.3%, respectively.The calibration curve equation (1-2000 ng/ml) for anisatin was The stability of anisatin under varied conditions  was acceptable; the accuracy was within 85% and 112%, and the precision was lower than 14%.According to the pharmacokinetic results , the maiI. henryi—the partial death occurred at 0.5 mg/kg of anisatin from fruit decoction. About 200 kinds of sesquiterpenes have been reported as chemotaxonomic markers of the Illicium family, and the toxicity of neoanisatin and 2-oxo-6-deox yneoanisatin has been proved. Toxicity may be enhanced by transformation or synergism between structurally similar sesquiterpenes [In mice, the LD50 reported for anisatin ranges from 0.76 to 1 mg/kg (po and ip) , 28; we terpenes –31.Mass spectrometry conditions were optimized. We chose the negative mode because the response of anisatin was stronger than that in the positive ion mode; then, fragment peaks with relatively high fragments were selected as quantitative ion pairs, m/z 327.1 → 127.0 for anisatin  and m/z 285.1 → 122.9 for IS , as shown in Regarding comparisons between the UPLC BEH C18 column and HSS T3 column, the former proved a better peak shape, and a lower analysis time was obtained with the latter. Different mobile phases were compared, such as methanol, acetonitrile, 10 mmol/l ammonium acetate, and 0.1% formic acid. Methanol and 0.1% formic acid solution in water score especially well in terms of suitable retention time and achieve a better peak.Choosing a suitable sample treatment method was an essential step in the methodology. The extraction efficiencies of ethyl acetate, acetonitrile, and methanol were compared; acetonitrile extraction efficiencies (around 70%) were better than those of methanol (around 65%) and ethyl acetate (around 55%), with acceptable acetonitrile matrix effects (around 98%).Selecting the IS was also an important task during the method standardization process. Salicin, astragalin, rubiadin, and narciclasine were compared. Salicin had a better peak shape, a stabler structure, and a peak time similar to anisatin. It was also able to meet the correction function of a IS for this experiment.μl of blood samples was collected at each time point by tail vein bleeding, and we chose a one-step protein precipitation procedure for mouse blood. The AUC(0-t) (area under the plasma concentration-time curve) were 803.6 ± 214.9 and 362.5 ± 16.5 ng/ml × h for intravenous and oral administration, respectively. No quantitative analysis nor pharmacokinetic study of anisatin in mice has been previously reported as far as we know.The proposed method UPLC-MS/MS is much faster than traditional HPLC for detection of anisatin in mouse blood, more sensitive, and less cost. The total blood volume of rodents is about 7% of their body weight. The number of blood samples, which can be taken from a mouse (20 g), is limited. A mouse has less blood than a rat or rabbit, so it is not easy to get enough plasma volume after centrifuging for liquid-liquid extraction at each time point . With these factors taken into consideration, only 20 t1/2 after the oral dose was 5.1 h, and the bioavailability was 22.6%. In addition, the pharmacokinetics of anisatin may provide theoretical support and guidance for the clinical treatment of poisoning episodes.A simple UPLC-MS/MS method was developed to determine anisatin in mice with the LLOQ of 1 ng/ml. The method provides a basis pharmacokinetic for absorption and metabolism of anisatins in mouse blood; the