Visualized sensing through fluorescence signals is a powerful method for chemical and physical detection. However, the utilization of fluorescent molecular probes still suffers from lack of precise signal self-calibration in practical use. Here we show that fluorescence and thermally activated delayed fluorescence can be simultaneously produced at the single-molecular level. The thermally activated delayed fluorescence serves as a sensing signal with its wavelength and lifetime both altered correlating to polarity, whereas the fluorescence always remains unchanged as an internal reference. Upon the establishment of a three-dimensional working curve upon the ratiometric wavelength and photoluminescence lifetime vs. polarity, disturbance factors during a relevant sensing process can be largely minimized by such a multiple self-calibration. This strategy was further applied into a precise detection of the microenvironmental polarity variation in complex phospholipid systems, towards providing new insights for convenient and accurate diagnosis of membrane lesions. New photoluminescent strategies are required to realize high-performance ratiometric sensors for bio-sensing applications. Here, the authors demonstrate a fluorescence-thermally activated delayed fluorescence dual emission strategy for ratiometric sensors with high precision. They can be applied in local microenvironments for monitoring the behavior of surrounding targets in various chemical and biological processes5, like charging cellular functional proteins6 and membrane systems7. Although the past decades have witnessed a significant progress in design and development of typical donor–acceptor based π-structures with alterable fluorescence signals and ability to sense local environmental change8, a large majority of these fluorescent sensors work simply through the responsive variation of a single emission signal9. In such cases, fluctuations in probe concentration or fluid property can easily cause erroneous signal readings during a practical sensing process10. Currently, different calibration methods, e.g., complex fluorescence with a dual-band inverse, have been developed for ratiometric calibration that can make it possible to avoid these disturbances to some degree12. However, the usage of fluorescence type-only sensors will also suffer from the overlap of background autofluorescence13. It is thus desirable to explore unique photoluminescent techniques or strategies to overcome these dilemmas.Fluorescent sensors continue to attract attention on account of their visualization ability and high sensitivity in detecting signalsA simultaneous change in emission wavelength and lifetime of photoluminescence (PL) from a molecular emitter may overcome the above-mentioned obstacles and generate a breakthrough in sensing from a multi-mode perspective. Meanwhile, a dual-emission characteristic can also be employed for ratiometric sensing, namely one emission providing an internal reference and the other acting as a sensing signal, on the basis of the spectral and temporal features. To fulfill these requirements, an organic emitter that can produce both a normal fluorescence (FL) and a thermally activated delayed fluorescence (TADF) maybe a favorable choice.14. TADF has been observed from intramolecular charge transfer (ICT) systems with a thermally accessible gap between the singlet and triplet excited states, enabling efficient up-conversion via thermally assisted reverse intersystem crossing (RISC)16. As reported for TADF molecules18, the energy difference between the singlet and triplet states (∆EST) can be minimized by adjusting the energy levels of the lowest locally excited triplet state (3LE) and CT states (both 1CT and 3CT). In addition, the hybridization of LE and CT can be observed by increasing the effective conjugation length19. Therefore, with the control of the donor as well as the conjugation length through steric or substituent patterns, LE and CT emission can be established, which could guarantee a dual-emission characteristic at the single molecular level.TADF emitters have been regarded as the third class of organic light emitting diode materials, as their harvest of both singlet and triplet excitons without noble metals facilitates smart molecular engineering with an improved internal quantum efficiency1CT state by the RISC process. The wavelength and lifetime of the TADF signal changed along with environmental polarity, whereas the FL one stays insensitive, making it possible to establish a 3-D calibration method with ratiometric wavelength and lifetime vs polarity. This strategy can be further applied for a precise detection of the microenvironmental polarity variation in complex phospholipid systems both in vitro and in vivo.In this work, a polarity sensor for building a three-dimensional (3-D) ratiometrically luminescent sensing method (change of wavelength and lifetime ratio vs polarity) was presented. It is based on the fact that the solvatoluminescence of the sensor connects to the ICT character of a molecular structure and that it can be addressed from donor–acceptor molecular engineering. To achieve a balanced contribution of LE and CT in the excited state, we present a molecular strategy to regulate the electron-donating ability of the donor group and to adjust the effective conjugation length by introducing different substituents. We designed and synthesized a series of donor–acceptor–donor (D–A–D)-type molecules with an acceptor diphenyl sulphone (DPS) group linked to three different donors ) . With mutual control studies of all these compounds, we eventually found that compound 3 can work as we envisioned. The introduction of an acetamino structure in 3 results in an FL emission from the local state, while a TADF emission is produced from the 19, and the HOMO is delocalized over the whole backbone. Meanwhile, there is a small spatial overlap between HOMO and LUMO on the phenyl ring of the phenyl sulfone moiety. These results are well matched with the experimental values and can be further illustrated by the calculated data in Supplementary Table The syntheses of D-A-D type compounds are described in Supplementary Fig. 20. Supplementary Fig. 22. For 1, the emission demonstrates a relatively small red shift along with the solvent polarity increase due to the weak CT-state character from indole to nitrobenzene. The strengthened CT transition of 2, due to the very strong electron-donating ability of the amino group, leads to a large increase in the red shift of the emission spectra and an improvement of PL quantum yield  absorption and PL spectra for 3 in toluene Fig. . The UV–1 excited state has been verified to exist, we aim next to clarify whether the emission at 401 nm in toluene is TADF. We measured the steady-state PL spectra and transient PL decay spectra in oxygen-free and saturated air conditions in toluene  for 3 equals 0.23 eV in a good agreement with calculated ΔEaTADF 0.226 eV and experimental value ~ 0.17 eV, deduced from the onsets of the fluorescence and phosphorescence spectra  between the 1CT (S1) and 3LE + 3CT (T2) states.To better understand these experimental results, natural transition orbitals (NTOs) were calculated to investigate the Sf 3 Fig. . The tratra Fig. . Such a 1LE emission (fluorescence) of the donors. Because of the small T2 → T1 gap (0.11 eV), the T2 ↔ T1 conversion should be fast and reachable by thermal activation under room-temperature conditions28. Moreover, the excitons in T2 are then able to convert to the 1CT state by the RISC process and radiatively decay to the ground state as TADF. The T2 ↔ T1 → S1 RISC is much more efficient when compared with the T1-S1 RISC because of (1) a smaller S1–T2 energy gap and (2) the admixture of LE configuration in the T2 state that provides efficient SOC between T2 and S1. Very recently, Monkman et. al29. experimentally proved such a mechanism of reverse internal conversion delayed fluorescence for the well-known TCA blue emitter with a quite large S1−T1 gap (0.21 vs 0.17 eV for our case). They postulated that reverse internal conversion (rIC) between the T1 and T2/T3 triplet states gives rise to the upper triplet-state population at room temperature, thus promoting an efficient RISC to S129. Also, we can expect in our case a strong RISC mechanism through the vibronic (nonadiabatic) coupling between the T2 state, which contains the admixture of 3LE configuration, and the lowest charge transfer triplet state (3CT)30. Such second-order coupling effects can also enhance the kRISC rate together with the RISC through the rIC step. For compound 2, the T1 and T2 states are almost pure CT states, and thus, there is no efficient second-order vibronic coupling between them and no significant SOC between them and the S1 (1CT) state. Thus, for compound 2, we observe only the prompt fluorescence, which is red shifted relative to compound 3 in agreement with time-dependent (TD)DFT calculations , toluene, DCM, tetrahydrofuran (THF) to dimethyl formamide (DMF). Such a peculiar solvatoluminescence results from the fact that the FL signal from the π-conjugated LE state, which possesses a large orbital overlap between hole and electron wave functions31, is insensitive to the solvent environment. In contrast, the TADF band from the CT excitation, showing a relatively small orbital overlap regarding the spatially hole and electron wave functions, is strongly affected by the polarity change. Figure R2 = 0.99, Supplementary Fig. Based on the demonstration of the FL and TADF dual-emission characteristic of 3, we further found an interesting photophysical phenomenon regarding the experimental polarity. Figure R2 = 0.95; Supplementary Fig. On the other hand, the lifetime of FL and TADF also exhibits another distinct sensitivity mode toward polarity. Figure Finally, we have now understood the overall molecular design represented by compound 3 with respect to ratiometric luminescent sensing. In view of taking full advantage of the wavelength and lifetime variation vs polarity, a 3-D ratiometrically luminescent sensing plot can be established based on two of the linear relationships on wavelength and lifetime modes of this molecule Fig.  show tha32. As an abnormal polarity alteration along with cholesterol-content change usually is linked to a membrane lesion, we expect that the 3-D ratiometric luminescent sensing strategy is applicable for the detection of the microenvironmental polarity change along with the alteration of the content of cholesterol in membranes. Herein, we utilized PLs systems to simulate the membranes, of which the polarity was changed by adding different Chol contents. Figure Phospholipids (PLs) are fundamental molecular components of a biological membrane and have been extensively used to determine various membrane properties. Cholesterol (Chol) plays a major role in the lateral organization of the lipid bilayer as it regulates membrane fluidity and permeabilityY = log (457/332), Z = log (4120/22)), B (Y = log (445/332), Z = log (9090/22)), and C (Y = log (466/332), Z = log (3170/22)) in YZ plane of the established 3-D plot diagram  for sensing in simulated membranes. The PL spectra and the lifetime of every Chol content were measured and are shown in Supplementary Fig. ram Fig. , the corZ-average diameter of ca. 85 nm. When the mass ratio of 3/PLs/Chol is 1/100/60, the Chol molecules are arranged perpendicular to the surface  the sensor is largely exposed to the Chol environment and (2) the presence of a higher proportion of Chol may induce a wider separation of the phospholipid headgroups, which may better surround the sensor molecules . Similarly, the combination of PLs and Chol with a content of 60 caused a decreased polarity to be 4.54 (from point B), and the excessive Chol in PLs with content 100 resulted in an increased polarity . To further explain the change of polarity with differenet Chol contents, TEM and dynamic light scattering are shown in Supplementary Fig. ace Fig. 32. The cles Fig. . MoreoveIn addition to the in vitro simulated study, compound 3 can also be used for cellular experiments. Supplementary Fig. 1CT and 3LE + 3CT states, the introduction of the LE component simultaneously caused a strong FL emission with a short lifetime. A 3-D ratiometric luminescent sensing method can be established since the wavelength and lifetime of the TADF signal both change along with polarity whereas the two features of the FL emission remain insensitive. Our present work provides a strategy to quantitatively study the polarity change in a simulated membrane system by monitoring the luminescent behavior with different Chol contents forthright in vitro and in vivo. Furthermore, we emphasize that this application is based on a well-established time-resolved sensing strategy and can pave a way to greatly reduce the measurement error and to avoid disturbance factors during practical sensing.In summary, we have demonstrated a fluorescence–thermally activated delayed fluorescence (FL–TADF) dual-emission strategy using a single molecular platform with hybridized local and CT excited states. Molecular engineering with an appropriate donor (AMID)-acceptor (DPS) linkage played a key role here. While an excellent TADF property was observed as a result of the fine-tuned energy of the p-fluorophenyl) sulfone, SnCl2·2H2O, and acetylchloride were purchased from Energy Chemical and used as received without further purification. 1H NMR and 13C NMR spectra were measured on a Bruker 400 L spectrometer in C2D6O6 at room temperature. High-resolution mass spectrometry data were measured by Matrix Assisted Laser Desorption Ionization-Time of Flight/Time of Flight Mass Spectrometer (5800). Elementary analysis was performed on a Thermo Finnigan Flash EA1112. The high-performance liquid chromatography analysis was performed on an Agilent 1260 HPLC system. The mobile phase consisted of methanol and acetonitrile with the flow rate of 1 mL/min. The absorption wavelength used was set at 330 nm. Hundred percent acetonitrile was used as the running buffer.The UV–Vis absorption spectra were recorded on a Shimadzu 1800 spectrophotometer. The emission spectra, time-resolved emission spectra, and transient PL decay characteristics of the samples were recorded on QM40 . Temperature was controlled by a cryostat (Advanced Research System Inc.). A nitrogen laser (PTI) with an excitation wavelength of 370 nm was used as the excitation source. The fluorescence quantum yields of solution were measured on QM40 with an integrating sphere (φ 150 mm).5-Nitroindole, potassium carbonate, bis(p-fluorophenyl) sulfone  in dry NMP (20 mL) was added, and then the mixture was stirred at 140 °C for an additional 4 h. After cooling, the mixture was poured into 500 ml of water, and the celadon precipitate was filtered and rinsed with water and petroleum ether. Then it was dried under high vacuum . 1H NMR  δ (ppm): 8.69 , 8.33–8.23 , 8.10 , 8.03 , 8.01–7.93 , 7.83 , 7.07 . 13C NMR : δ = 142.80, 142.39, 139.45, 138.11, 132.62, 130.04, 129.56, 125.55, 118.46, 118.41, 111.86, 107.40; MALDI-TOF MS, m/z: [M + H]+ 540.5. Elemental analysis (for C28H18N4O6S), calculated: C (62.45), H (3.37), N (10.40); found: C (62.38), H (3.40), N (10.45). The structural characterization is shown in Supplementary Fig. 5-Nitroindole  was added to a solution of potassium carbonate  in dry 1-methyl-2-pyrrolidinone (NMP) (30 mL). After the solution was stirred at room temperature for 10 min, bis in 300 mL of EtOH was heated to reflux for 20 h. After cooling, the mixture was poured into 500 mL of ice water and adjusted to pH 7 with NaOH. This mixture was then extracted with DCM (3 × 100 mL) and the extracts were dried (Na2SO4) and concentrated to be the yellow precipitate. Then it was dried under high vacuum . 1H NMR  δ (ppm): 8.14–8.09 , 7.82 , 7.59 , 7.45 , 6.79 , 6.61 , 6.51 , 4.92 . 13C NMR : δ = 144.17, 143.50, 137.25, 131.50, 129.71, 128.36, 128.02, 123.19, 113.24, 111.57, 105.13, 104.62; MALDI-TOF MS, m/z: [M + H]+ 478.2. Elemental analysis (for C28H22N4O2S), calculated: C (70.27), H (4.63), N (11.71); found: C (70.05), H (4.65), N (11.68). The structural characterization is shown in Supplementary Fig. A mixture of bis[4-(5-nitroindole)phenyl] sulfone  and SnCl2 at 0–3 °C, and then the mixture was warmed to 40 °C for 8 h. After cooling, the mixture was poured into 50 ml of ice water, and the precipitate was filtered and rinsed with water and further purified by column chromatography  to afford a brown solid . 1H NMR  δ (ppm): 9.94 , 8.18 , 8.04 , 7.89 , 7.75 , 7.65 , 7.33 , 6.74 , 2.06 . 13C NMR : δ = 168.38, 143.78, 137.96, 133.87, 131.43, 130.26, 129.83, 129.08, 124.00, 116.38, 111.54, 111.23, 105.92, 24.43; MALDI-TOF MS, m/z: [M + Na]+ 585.5. Elemental analysis (for C32H26N4O4S), calculated: C (68.31), H (4.66), N (9.96); found: C (68.12), H (4.68), N (9.93). The structural characterization is shown in Supplementary Fig. Bis[4-(5-aminoindole)phenyl] sulfone  was dissolved in 8 mL DMF and then trimethylamine (0.15 mL) was added. Acetylchloride  was added dropwise in N0) at the DFT level of theory using the B3LYP35 hybrid functional and 6-31 G(d)36 basis sets. The excitation energies were calculated by employing the specially parameterized B3LYP functional  with the 6-31 + G(d)37 basis set within the TDDFT approach38. The exchange tuning of B3LYP functional requires the correct explanation of excited states that combine both CT and local excitation LE characters and possess wide “overlapping” area for the transition-responsible orbitals. We have also tested the dispersion-containing wB97XD functional and long-range corrected CAM-B3LYP functional for the optimization of the studied systems and the subsequent TDDFT calculations. We have found that there is no significant difference in the structure of S0, S1, and T1 states compared with the B3LYP results. At the same time, the energies of excited states were found to be strongly overestimated when compared with the parameterized B3LYP functional. All the calculations were performed using the polarizable continuum model39, taking the dielectric constant for DCM (ε = 8.93) as the reference. NTO analysis was performed to examine the nature of the excited states. All the calculations were performed within Gaussian 16 program package40.The structure of molecules 1~ 3 was optimized in its ground state (SSupplementary InformationPeer Review File


The performace of the obtained internally plasticized PVC materials was also investigated with TGA, DSC and leaching tests. The results showed that grafting of propargyl ether cardanol (PEC) on PVC increased the free volume and distance of PVC chains, which efficiently decreased the glass transition temperature (Tg). No migration was found in the leaching tests for internally plasticized PVC films compared with plasticized PVC materials with commercial plasticizer dioctyl phthalate (DOP). The internal plasticization mechanism was also disscussed according to lubrication theory and free volume theory. This work provides a meaningful strategy for designing no-migration PVC materials by introducing cardanol groups as branched chains.Internally plasticized poly(vinyl chloride) (PVC) materials are investigated via grafting of propargyl ether cardanol (PEC). The chemical structure of the materials was studied by FT-IR and  Tg) of PVC materials grafting hyperbranched polyglycerol groups was kept below 0 °C. Propargyl ether triethyl citrate and castor oil-based derivative were covalently bonded to the polymer matrix as an internal plasticizer, as reported by Jia et al. [Plasticizer is one of the most important plastic additives, and is used to improve processability, plasticity and flexility of plastics. The most widely used plasticizers are phthalate esters, which account for 80% of the total consumption of plasticizer . Howevera et al. ,14. ThesCardanol is one of the most favorable biomass resources for plasticizer production due to its relatively low cost and similar chemical structure to conventional phthalate plasticizers such as dioctyl phthalate (DOP). Cardanol derives from an agricultural by-product that is abundantly available in many parts of the chemical industry ,16. TherInspired by the above-mentioned pioneer works, we herein report a strategy for developing internally plasticized PVC materials by grafting propargyl ether cardanol onto the PVC matrix. Specifically, different mass of cardanol groups were grafted onto PVC matrices to obtain no-migration, flexible PVC materials. Differential scanning calorimetry (DSC) measurements were used to detect the plasticizing efficiency of the strategy, and to compare it with pure PVC. The migration stability of the internally plasticized PVC materials in n-hexane was investigated. For comparison, the migration stability of PVC films plasticized with the traditional plasticizer DOP was also tested. In addition, the internal plasticization mechanism was discussed in terms of traditional plasticization theories, such as free volume theory and lubrication theory. N,N-dimethylformamide (DMF) and dioctyl phthalate (DOP) were kindly provided by Nanjing Chemical Reagent Co., Ltd.  Polyvinyl chloride (PVC) was supplied by Hanwha .Cardanol  was provided by Jining Hengtai Chemical Co., Ltd.  Propargyl bromide solution, tetrahydrofuran (THF), potassium carbonate, sodium azide, methanol, acetone, cuprous bromide, 5,5-dimethyl-2,2-dipyridyl, To a 100 mL flask equipped with a condenser tube was added 30.4 g (100 mmol) of cardanol, 13.08 g (110 mmol) of propargyl bromide solution and 15.2 g (110 mmol) of potassium carbonate in 50 mL of acetone. The mixture was stirred at 65 °C for 12 h. The solution was purified by evaporating under vacuum after washing the mixture with deionized water (yield: 97%).3 and 100 mL of DMF. The mixture was allowed to stir at 30 °C for 24 h and precipitated into water/methanol mixture (1/1 by volume), and dried in a vacuum to obtain the PVC–N3 [3. Elemental analysis: 35.13% C, 8.21% H, 18.47% N, and 38.19% Cl.To a 100 mL flask were added 2.0 g of PVC, 2.0 g of NaNe PVC–N3 . Figure 3, PEC, cuprous bromide and 2,2′-dipyridine in 40 mL of DMF in a three-neck flask equipped with a mechanical stirrer, nitrogen pipe and thermometer. The reaction was kept at 30 °C and stirred for 24 h. Then, the mixture was precipitated into water/methanol mixture (1/1 by volume) after filtering to remove the copper salts, and dried in a vacuum to obtain PVC-0.25PEC. Internally plasticized PVC materials were prepared by dissolving a certain amount of PVC–NA total of 1 g of internally plasticized PVC materials were dissolved in 20 mL of THF. The mixture was stirred at 40 °C for 20 min until the solution appeared transparent, and was then poured into a glass petri dish (5 cm diameter), and dried in a constant temperature drying box at 60 °C for 24 h to completely remove residual THF. PVC–DOP films were prepared by dissolving a total of 2 g of PVC and 1 g of DOP in 20 mL of THF using the same method. Elemental analysis was conducted on an elemental PE-2400 analyzer .−1 at a resolution of 4 cm−1.Fourier transform infrared (FT-IR) spectra of propargyl ether cardanol and internally plasticized PVC materials were investigated on a Nicolet iS10 FTIR measurement . The spectra were acquired in the range of 4000 to 500 cm1H NMR measurements were conducted on an AV-300 NMR spectrometer  at a frequency of 400 MHz. CDCl3 was used as solvent and tetrametnylsilane (TMS) as an internal standard.Mw/Mn) were calculated by calibrating with polystyrene standards. The molecular weights of PVC and internally plasticized PVC materials were measured using an efficient gel chromatograph made by Waters, Milford, MA, USA at 30 °C  using THF as solvent. The number-average molecular weight, weight-average molecular weight and polydispersity indices (2 atmosphere (50 mL/min) at a heating rate of 10 °C/min. The temperature was scanned from 40 to 600 °C.TGA was carried out using a TG209F1 TGA thermal analysis instrument  in NTg) was characterized using a NETZSCH DSC 200 PC analyzer ; the temperature ranged from −60 °C to 100 °C in N2 atmosphere (50 mL/min) at a heating rate of 20 °C/min. Tg values reported were taken from the second heating run in order to eliminate thermal history and to correspond to the midpoint of the DSC curves measured from the extension of the pre- and post-transition baseline. Glass transition temperature .VC films .−1 in the FT-IR spectra of the cardanol was attributed to stretch vibration of –OH groups. The absorption peak appeared at 3007 cm−1, which was assigned to the olefinic C–H stretch of cardanol. The peaks at 2924 and 2853 cm−1 were associated with the =C–H and –C–H bonds, respectively. The peak at 1586 cm−1 was attributed to the C–C bonds. The broad absorption of the C=C stretching vibration mode from the benzene ring could be observed in the range of 1454–1152 cm−1 [−1 disappeared in the FT-IR spectra of PEC. A new peak appeared at 3306 cm−1, which was attributed to alkyne C–H stretch, and the C≡C stretch characteristic absorption peak appeared at 2122 cm−1.152 cm−1 ,21. In c1H NMR of cardanol and PEC was also investigated. In 1H NMR of cardanol  [3 had been prepared. With more PEC grafted onto the PVC matrix, the peaks at 2910, 1586 and 1152–1454 cm−1, which were attributed to C–H stretch, C–C bonds, and the C=C stretching vibration mode derived from the benzenoid ring of PEC, respectively, appeared stronger than PVC–N3, indicating that the PEC had been grafted onto the PVC matrix. stretch) ,24,25, i1H NMR of PVC and internally plasticized PVC materials are presented in 1H NMR spectra of PVC, peak at δ 4.5 ppm was attributed to protons of –CHCl–(CH2)– [H2)–. The 1H NMR of PVC–N3 was similar to PVC, indicating that the chemical environment of the protons was similar. Peak a′ at δ 4.5 ppm was associated with the protons of –CHCl–(CH2)–, and peak b′ at δ 2.2 ppm was attributed to the protons of –CH(N3)–(CH2). Peaks a′′ and b′′ at δ 4.5 and 2.2 ppm were associated with the protons of –CHCl–(CH2)– and –CH(N3)–(CH2)–, respectively, which were weaker than for PVC and PVC–N3, but some new peaks appeared at δ 7.18, δ 5.5, δ 2.85 and δ 0.89 ppm, which were associated with the protons of benzene rings, olefin groups, methylene groups and methyl groups of branched chains derived from PEC [l–(CH2)– ,18,26. Pfrom PEC ,28, respMn), weight-average molecular weight (MW), and polydispersity indices are presented in Mn and MW of PVC materials increased gradually with greater numbers of cardanol groups grafted onto the PVC matrix. Mn of PVC, PVC-0.25PEC, PVC-0.50PEC and PVC-0.75PEC was 15,100, 21,200, 23,800 and 25,400 g/mol, respectively, which indicates that the internally plasticized PVC materials had been obtained.The click reaction level can be evaluated by examining the molecular weight change ,30. The Tg is observed, which indicates that the PVC and cardanol are not miscible. Additionally, none of the PVC materials exhibited any melting peaks, indicating their amorphous characteristics. Tg data for PVC, PVC-0.25PEC, PVC-0.50PEC and PVC-0.75PEC are summarized in Tg with different contents of cardanol groups was mainly caused by the internal plasticizing effect of the cardanol groups. Tg is an important evaluation criteria in Mauritz and Storey’s mathematical models [Tg can be controlled by structural features of the plasticizer, such as the branchiness of the side chains and the length of those chains. For the same molecular mass, branched plasticizers have higher plasticizing efficiency parameter values than linear structures. In this study, internally plasticized PVC macromolecules with higher branchiness side chains caused it to become a kind of internal plasticizer. The decrease in Tg illustrated that internally plasticized PVC materials induced a plasticizing effect on themselves. The thermal properties of PVC, PVC-0.25PEC, PVC-0.50PEC and PVC-0.75PEC with different cardanol group contents were investigated using DSC and TGA measurements. As shown in l models . In thisTd) and char residue are summarized in Td of internally plasticized PVC materials decreased from 278.4 to around 210 °C with an increase in the number of cardanol groups grafted onto the PVC, which was caused by the unstable properties of azide groups under high temperature [2, CO, and aromatic derivatives; all of these compounds were burned and produced char residue [The onset degradation temperature (perature . Char re residue . TherefoThe common non-attached plasticizer based on cardanol acetate , epoxidiTraditional plasticization mechanisms include lubricity theory, gel theory, free volume theory, kinetic theories and mathematical models. Internal plasticization mechanisms can also be illustrated based on these theories. Kilpatrick  and otheFree volume theory can be used to explain internal plasticization by evaluating the three kinds of movement for chain-like macromolecules: end movement, subgroup movement and crankshaft movement . As can Tg of the internally plasticized PVC materials reached 42 °C. No migration was found in leaching tests for internally plasticized PVC films, but 15.7% of DOP leached from PVC–DOP films into n-hexane. Therefore, this work provides a meaningful strategy for designing advanced PVC materials by introducing cardanol groups as branched chains.Internally plasticized PVC materials were synthesized by the grafting of PEC. The obtained PVC-0.25PEC, PVC-0.50PEC and PVC-0.75PEC exhibited efficient internal plasticization effects. Because cardanol groups grafted onto PVC chains is able to serve as a kind of molecular lubricant, the structure caused internally plasticized PVC chains to move freely, further increasing the distance and free volume of PVC chains and making them much easier to move than PVC. This will cause PVC materials to possess potentially flexible qualities, making them easy to process. The obtained internally plasticized PVC materials exhibited poorer thermal stability and more char residue than PVC. The


Electrochemical detection of amino acids is important due to their correlation with certain diseases; however, most amino acids require a catalyst to electrochemically activate. One common catalyst for electrochemical detection of amino acids are metal oxides. Metal oxide nanoparticles were electrodeposited onto glassy carbon and platinum working electrodes. Cyclic voltammetry (CV) experiments in a flow cell were performed to evaluate the sensors’ ability to detect arginine, alanine, serine, and valine at micromolar and nanomolar concentrations as high as 4 mM. Solutions were prepared in phosphate buffer saline (PBS) and then 100 mM NaOH. Specifically, NiO surfaces were responsive to amino acids but variable, especially when exposed to arginine. Polarization resistance experiments and scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) data indicated that arginine accelerated the corrosion of the NiO catalyst through the formation of a Schiff base complex. Changes in baseline amino acid concentrations are correlated with various diseases such as obesity  or major3O4-graphene oxide nanocomposite . C. C17]. Ca values, differences in relative charge and branch groups, and relevancy in various diseases as potential biomarkers [Reported in this study are metal oxide surfaces on glassy carbon and platinum working electrodes and their evaluation as sensors for the detection of alanine, arginine, serine, and valine . Alanineomarkers ,25. WherCyclic voltammetry measurements were recorded on a 600+ Reference Potentiostat . Samples were injected into a platinum counterelectrode flow cell with Ag/AgCl reference electrode using a syringe pump (flow rate of 15 μL/h). A dual 3 mm glassy carbon and dual 3 mm platinum working electrode  were used in the electrochemistry experiments.3(aq) and 2 mM Ni(NO)3(aq) solutions were prepared in acetate buffer (pH ~4); 2 mM Cu(SO4)2(aq) solution was prepared in 100 mM Na2SO4(aq); and 4000 μM, 2000 μM, 1000 μM, 400 μM, 200 μM, 100 μM, 75 μM, 50 μM, and 25 μM solutions of alanine, arginine, serine, and valine were prepared by serial dilution in 100 mM sodium hydroxide as well as 10 mM phosphate buffer saline (PBS) solution (pH ~7.4).All chemicals were purchased from Fisher Scientific , as follows: 2 mM Fe(NO)A Tescan Lyra3 GMU FIB SEM  was used to obtain the SEM and EDS measurements. A default of a 22 μm view field, at 25.2 k× magnification, and either a 6.0 or 15.0 kV beam was used, indicated on the SEM figures. For EDS measurements, only a 15.0 kV beam was used.The modification procedure followed previously published attachment methods ,17. A 2 Cyclic voltammetry was performed by sweeping the potential from 0 to +0.6 V at a rate of 100 mV/s for 40 cycles. Polarization resistance was performed by flowing 100 µM amino acid in 100 mM NaOH at a rate of 1 mL/h over a NiO modified amorphous carbon electrode.Bare platinum electrodes were tested with the four amino acids which resulted in no electrochemical activity . The worAt physiological pH, catalytic current was not measured presumably because amino acid analytes are unable to chelate at near neutral pH due to protonation of the amine group . Protona2O surface leads to delamination of the metal oxide surface [2O3 surfaces.The copper oxide catalyst on the platinum working electrode did not catalyze the electrochemical oxidation of amino acids . Interes surface . In orde2O3 surface on a glassy carbon electrode exhibited very low current measurements, below 0.6 μA  × 10−3 mpy  of the NiO surface.N-isopropylacrylamide), can be used to host the nanoparticles and inhibit the Schiff base complex.The authors encourage researchers interested in measuring arginine, and other guanidine moieties, to investigate more stable catalytic surfaces. To prevent the Schiff base formation, they propose two possible approaches. First, carbon nanotubes  and graphene, have the ability to host metal oxides with a Schiff base complex, which can impede the formation of additional Schiff base complexes from the solution . Second,


At baseline, the expression of MyD88 was significantly lower, and TRIF significantly raised in children with DS. The TLR antagonist SsnB was effective in reducing TLR2 and CD11b expression and abrogating cytokine production in both cohorts. We conclude that TLR signalling and the TLR2 pathway are dysregulated in DS, and this disparate innate immunity may contribute to chronic inflammation in DS. SsnB attenuates proinflammatory mediators and may be of therapeutic benefit.Toll-like receptors (TLRs) are the key in initiating innate immune responses. TLR2 is crucial in recognising lipopeptides from gram-positive bacteria and is implicated in chronic inflammation. Children with Down syndrome (DS) are prone to infections from these pathogens and have an increased risk of autoimmunity. Sparstolonin B (SsnB) is a TLR antagonist which attenuates cytokine production and improves outcomes in sepsis. We hypothesised that TLR signalling may be abnormal in children with DS and contribute to their clinical phenotype. We evaluated TLR pathways in 3 ways: determining the expression of TLR2 on the surface of neutrophils and monocytes by flow cytometry, examining the gene expression of key regulatory proteins involved in TLR signal propagation, MyD88, IRAK4, and TRIF, by quantitative PCR, and lastly determining the cytokine production by ELISA following immunomodulation with proinflammatory stimuli (lipopolysaccharide (LPS), Pam3Csk4) and the anti-inflammatory agent SsnB. We report TLR2 expression being significantly increased on neutrophils, total monocytes, and intermediate and nonclassical monocytes in children with DS ( Down syndrome (DS) is caused by extra genetic material from chromosome 21 and is the most common of the chromosomal abnormalities affecting 1 in 700 births in the USA , 4, alteβ (TRIF), which in turn stimulate downstream kinases , leading to nuclear translocation of an increase in the transcription factors, including nuclear factor kappa B (NF-κB) and interferon regulatory factor-3 (IRF-3), ultimately leading to the production of proinflammatory cytokines [Toll-like receptors (TLRs) and their activation are crucial in initiating the innate immune response, while also linking the adaptive response to infection . Activatytokines . Strict ytokines , 13.in vivo and in vitro experiments have demonstrated significant increases in monocyte TLR2 after LPS treatment [TLR2 is involved in detecting bacterial infection and in chronic inflammation and is located at the cell membrane, where it recognises and binds to signal molecules. These molecules are derived from microorganisms such as bacteria, viruses, or fungi exhibiting pathogen-associated molecular patterns  or from dying endogenous cells bearing damage-associated molecular patterns , Snapin, and hyaluronic acid) , 15. Altreatment .Streptococcus pneumoniae and Staphylococcus aureus are the causative organisms in many of these cases, with the former also a significant contributor to recurrent otitis media with effusion (OME), which often leads to conductive hearing loss in these children [The significance of TLR2 and its affinity for constituents of gram-positive bacteria are of particular relevance in DS as individuals with DS are more prone to infection from this group of pathogens. Children with DS are at increased risk of infections and have greater risk of admission to a hospital with respiratory tract infections (RTIs), where they are more likely to have a protracted stay and require intensive care support . Gram-pochildren . Aggresschildren . Elevatechildren , 21.Sparganium stoloniferum and Scirpus yagara and has been shown to reduce inflammation [κB activity [β (IL-1β), interleukin 6 (IL-6), and tumour necrosis factor alpha (TNF-α) [Dysregulated TLR2 signalling causes autoinflammation due to unchecked proinflammatory cytokine release. This can lead to several autoimmune and inflammatory conditions such as rheumatoid arthritis, systemic lupus erythematosus, atherosclerosis, and sepsis . Childreammation . This coactivity . SsnB ha (TNF-α) .We hypothesised that TLR signalling may be abnormal in children with DS and that altered TLR2 contributes to a clinical phenotype at increased risk of gram-positive infection, chronic inflammation, and autoimmunity. We aimed to evaluate TLR pathways in 3 ways: by determining the expression of TLR2 on the surface of neutrophils, monocytes, and their subsets, by examining the gene expression of key regulatory proteins involved in TLR signal propagation, MyD88, IRAK4, and TRIF, and lastly by determining the cytokine production at baseline and following immunomodulation with proinflammatory stimuli  and the anti-inflammatory agent SsnB.This study was approved by the Ethics committees in the National Children's Hospital, Tallaght, and Our Lady's Children's Hospital, Crumlin (OLCHC), Dublin, Ireland. All families and participants received verbal and documented information on the study, and written consent was obtained prior to recruitment. There were two patient cohorts enrolled: (a) children with Down syndrome < 16 years old attending the dedicated Down syndrome clinic, or OLCHC, and (b) age-matched patients (controls) attending phlebotomy or day case procedures; in this instance, blood sampling occurred at the induction of general anaesthetic. Both groups had no recent evidence of infection or fever.n = 8) were all clinically stable with no further cardiology intervention. Both groups were afebrile at the time of blood sampling with no recent history of infection.There were 20 children with Down syndrome (DS) with a mean ± SD age of 8.79 ± 5.27 years (y) of which 11 were female and 15 controls with a mean age of 6.22 ± 4.22 y, of which 5 were female. In the DS cohort, children with a history of significant congenital heart disease requiring surgery in infancy  10 ng/mL, Pam3Csk4  5 ng/mL, and the TLR antagonist sparstolonin B  10 μM and in combination.We utilised the same experimental methods (as outlined below), as per our previous paper , appraisμL of 1% paraformaldehyde. The final cell pellet was resuspended in 100 μL PBA buffer and analysed on a BD FACSCanto II flow cytometer.Blood samples were incubated with a dead cell stain , diluted to working concentration in phosphate-buffered saline (PBS). The following fluorochrome-labelled monoclonal antibodies (mAb) were added to each sample: CD14-PerCP, CD15-PECy7, CD16-FITC, CD66b-Pacific Blue, and TLR2-APC  and PE-labelled CD11b . PBA buffer  was used to make up the antibody cocktail. Samples were incubated in the dark for 15 minutes. Next, 1 mL of FACS lysing solution  was added to each tube, and the samples were then incubated for 15 minutes in the dark. Cells were pelleted by centrifugation at 450g for 7 minutes at room temperature, washed twice with PBA buffer, and fixed in 300 The expression of TLR2 and CD11b antigens on the surface of neutrophils and monocytes was evaluated by flow cytometry. Neutrophils were delineated based on SSC-A and CD66b+ positivity as previously described , and monμg, was reverse transcribed to single-stranded cDNA using the High-Capacity cDNA Archive Kit (Applied Biosystems) following the manufacturer's protocol and stored at -80°C until use. The settings for amplification were 10 min at 25°C and 120 min at 37°C, and 5 sec at 85°C and then holding at 4°C. The evaluation of gene expression was performed by TaqMan® RT-PCR. Commercially available TaqMan® primer and probe combinations were used to detect the expression of the following genes, MyD88 (NM 001172567.1), TRIF (NM_182919.3), and IRAK4 (NM_001114182.2). The endogenous control selected was GAPDH. All samples were assayed in triplicate. Thermal cycling conditions were as follows: 2 minutes at 50°C, 10 minutes at 95°C, and, for 40 cycles, 24 seconds at 95°C and 1 minute at 60°C, using the 7900HT Fast Real-Time PCR System. Relative quantification (RQ) values were calculated using the 2-ΔΔCt method.Following incubation of samples with proinflammatory stimulants, 1 mL of TRIzol (Thermo Fisher) solution was added to 0.3 mL of whole blood. The samples were incubated for 5 min at room temperature followed by the addition of chloroform. Following lysis, the aqueous phase was used to isolate RNA, as per the manufacturer's instructions . RNA purity and concentration were determined by using the NanoDrop ND-100 Spectrophotometer and analysed using ND-1000 ver.3.1.2 software. Total RNA, 1 α), interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 8 (IL-8), interferon gamma (IFN-γ), interleukin 18 (IL-18), vascular endothelial growth factor (VEGF), erythropoietin (Epo), interleukin 1 receptor antagonist (IL-1ra), and interleukin 10 (IL-10), were analysed using a custom-made MSD® MULTI-SPOT assay plate from Mesoscale . Extracted peripheral blood serum, as described above, was transferred to a 96-well MSD plate, and these cytokines were assessed as per the manufacturer's instructions. The plate was then analysed on the SECTOR Imager and validated . The limits of detection for the individual assays were within expected ranges.The following cytokines, tumour necrosis factor alpha  and nonclassical  subpopulations than controls, there were no statistically significant differences between them. The percentage of classical monocytes was lower in children with DS compared to controls .Regarding enumeration of monocyte subsets, although children with DS had a higher percentage of intermediate . After LPS incubation, there was no change in TLR2 expression in either cohort (DS (p = 0.25) vs. control (p = 0.75); p = 0.04). TLR2 expression post-LPS treatment increased in the DS cohort but not in controls  and nonclassical monocytes (p = 0.048) in children with DS when compared to controls at baseline. Classical monocyte TLR2 was higher in children with DS but did not quite reach significance (p = 0.06) . Nonclassical monocytes displayed the lowest mean TLR2 at baseline of the three monocyte subsets which was lower than intermediate monocyte TLR2 in both cohorts  vs. control (p = 0.73); p = 0.002) and LPS+Pam3Csk4 (p = 0.0009); control: LPS (p = 0.003) and LPS+Pam3Csk4 (p = 0.0001); p = 0.57) and control (p = 0.0004); vs. LPS: DS (p = 0.14) and control (p = 0.002); After incubation with Pam3Csk4 and Pam3Csk4 and LPS in combination, there were no rises in TLR2 expression on neutrophils in either cohort , LPS , and LPS+Pam3Csk4 ; p = 0.002; control, p = 0.0009).There were rises in CD11b after Pam3Csk4, LPS, and LPS plus Pam3Csk4 incubation on neutrophils in both cohorts , LPS ; p ≤ 0.0001; control, p = 0.0002) or LPS alone  also in both groups.Total monocyte CD11b increased after all treatments in both cohorts  and control (p = 0.78), LPS: DS (p = 0.12) and control (p = 0.73); p = 0.23; controls, p = 0.001; p = 0.15) and control (p = 0.08), Pam3Csk4+SsnB: DS (p = 0.25) and control (p = 0.18); The TLR antagonist SsnB was used to assess its potential as an immunomodulator after stimulation with LPS, Pam3Csk4, and both TLR agonists together. Regarding TLR2 expression on neutrophils, there was no reduction of TLR2 in samples treated with SsnB plus Pam3Csk4 and SsnB plus LPS in children with DS and controls (Pam3Csk4: DS (p = 0.04) and LPS+Pam3Csk4 (p = 0.02); nonclassical: LPS (p = 0.03)). Please see Supplemental Fig. Monocyte subset analysis revealed that SsnB decreased TLR2 on classical and nonclassical monocytes following LPS and LPS plus Pam3Csk4 treatments  and after incubation of LPS with Pam3Csk4 (p = 0.004) in the control group; there were also rises in CD11b following treatment with LPS (p = 0.05) and LPS and Pam3Csk4 (p = 0.02) in this group. In children with DS, there was no change in CD11b on neutrophils following SsnB treatment ; there was also a rise in CD11b post-LPS incubation (p = 0.009). In children with DS, there was a fall in CD11b after SsnB and Pam3Csk4 treatment (p = 0.04); in both cohorts, there was no decrease after treatment with SsnB and both proinflammatory stimulants ; following LPS treatment, there was a rise in MyD88 in controls (p = 0.03), but this was not seen in children with DS (p ≤ 0.0001), and there was no increase in expression following LPS treatment in either group   and only significantly in the DS cohort for IFN-γ , indicating this immunomodulator generates an inferior cytokine response compared with LPS.Following stimulation of whole blood with LPS, there were rises in both cohorts of IL-1 Figures –7. The aα, IL-18, and Epo was reduced in both cohorts  . SsnB had the most marked effect on TNF-α production, resulting in diminution after its addition to LPS, Pam3Csk, and LPS plus Pam3Csk4 in both cohorts . SsnB had no effect on IL-8 and IFN-γ production   .We have demonstrated increased TLR2 expression on neutrophils, monocytes, and monocyte subsets in children with DS compared to controls. This could be clinically important as excess TLR2 expression is associated with morbidities seen more commonly in DS, such as gram-positive infections , autoimmα. Future studies are required to determine the significance of our finding of increased TLR2 expression in DS. Children with DS are at increased risk of RTIs and admission to a hospital with this presentation [S. pneumoniae and S. aureus are the causative organisms in many of these cases, and TLR2 plays a pivotal role in defence against these pathogens. Studies examining the effects of S. aureus and S. pneumoniae infection on mice deficient in TLR2 showed a decrease in proinflammatory cytokines and increased mortality [We have previously described an increase in TLR4 expression on nonclassical monocytes at baseline and a hyperresponsiveness in neutrophil CD11b following LPS stimulation in children with DS . TLR2 anentation , 35. Graortality , 37, higS. pneumoniae is a significant contributor to this condition and can stimulate persistent low-grade inflammation via TLR2 [Children with DS are also prone to develop recurrent otitis media with effusion (OME) often resulting in conductive hearing loss . S. pneuvia TLR2 , 40. Chrvia TLR2 . There avia TLR2 , 21. Thivia TLR2 ; furtherIn DS, autoimmunity such as arthropathy is also well described , 43, witκB production and attenuating proinflammatory cytokines such as IL-1β, IL-6, and TNF-α [Taken together, the burden of evidence points to TLR2 as a possible mediator in the pathogenesis of multiple clinical conditions seen more frequently in DS. We have demonstrated that the TLR2/TLR4 antagonist sparstolonin B is effective in reducing TLR2 and CD11b expression in neutrophils and monocytes in children. It has been previously shown that SsnB mediates its anti-inflammatory properties by reducing NF-nd TNF-α . It append TNF-α , and it nd TNF-α . We haveκB and TNF production. The MyD8-independent pathway signals via TRIF inducing IRF3 and finally leading to IFN-β being produced [The TLR receptors propagate signals through two main pathways dependent on the utilisation (MyD88 dependent) or not (MyD88 independent) of MyD88. The former is employed by all TLRs except TLR3. The MyD88-dependent pathway results in NF-produced . MyD88 dproduced , 51, 52.produced . The defWhile MyD88 expression is decreased in children with DS, we found that TRIF expression is increased. Signalling via the TRIF-dependent pathway leads to a number of important immune responses to infection, enhancing type 1 interferon production and mediating apoptosis, caspase activation, and necroptosis, ultimately leading to a reduction in viral replication and increased bacterial clearance . ChildreDuring our experiments, we observed that for some cell populations, positive stimulation did not work for both cohorts, for example, nonclassical monocyte TLR2 and intermediate monocyte CD11b. We previously report similar hyporesponsiveness to LPS in TLR4 expression on nonclassical and intermediate monocytes and CD11b on nonclassical monocytes in children with DS and controls . The reaIn conclusion, we describe a dysregulation of TLR pathways in DS. There is greater expression of TLR2 on the surface of neutrophils and monocytes. Downstream signalling is altered with reduced MyD88 and increased expression of TRIF, which may represent compensatory upregulation of MyD88-independent pathways. This altered innate immunity may contribute to an increased burden of infections and chronic inflammation in DS. SsnB attenuates proinflammatory mediators and could be of potential therapeutic clinical benefit.


Ide after 18 weeks of a normal chow diet. This data article is related to a research article entitled “Knockout of insulin-degrading enzyme leads to mice testicular morphological changes and impaired sperm quality” .Insulin-degrading enzyme (IDE) degrades and inactivates bioactive peptides such as insulin. As insulin is a master regulator of glucose homeostasis, lack of IDE is expected to have a profound impact on both insulin and glucose levels. This article shares data on glucose and insulin homeostasis of control, heterozygous and knockout mice for  After heterozygous breeding, wild type (WT), heterozygous (Het) and knockout (KO) mice were generated and maintained on a 12 h light/dark cycle with standard chow diet  and water ad libitum at Instituto Gulbenkian de Ciência . Mice were monitored for body weight and blood glucose levels and all procedures followed ARRIVE guidelines and the Europeans laws (Directive 2010/63/EU) regarding the use of animals in research.Full body 2.2At 18 weeks old, and after an overnight fast, blood was collected from the mouse tail to measure blood glucose and insulin levels using a glucose meter and a mouse insulin ELISA kit , respectively. Blood glucose levels were also measured 15, 30, 60 and 120 min after oral glucose administration (1.5 g/kg).2.3At 18 weeks old, and after 5 h of fasting, blood glucose levels were measured using a glucose meter before and 10, 20, 30, 45, 60, 90 and 120 min after insulin intraperitoneal injection (0.5 UI/kg).2.4The statistical significance among the experimental groups was assessed by one-way ANOVA. Experimental data is shown as mean ± SEM. Statistical analysis was performed using GraphPad Prism 6 . p < 0.05 was considered significant.


Penicillium italicum causes blue mold disease and leads to huge economic losses in citrus production. As a natural antifungal agent, clove essential oil (CEO), which is a generally recognized as safe (GRAS) substance, shows strong in vitro activity against fungal pathogens. However, few studies on CEO for controlling postharvest blue mold disease caused by P. italicum in citrus fruit have been reported. Our aims were to investigate the control efficacy and possible mechanisms involved of CEO against P. italicum. In the present study, CEO treatment inhibited the disease development of blue mold when applied at 0.05% to 0.8% (v/v), and with the effective concentration being obtained as 0.4% (v/v). Besides its direct antifungal activity, CEO treatment also spurred a rapid accumulation of H2O2 compared with untreated fruits, which might contribute to enhancing an increase in the activities of defense-related enzymes, such as β-1,3-glucanase (β-Glu), chitinase (CHI), phenylalanine ammonia-lyase (PAL), peroxidase (POD), polyphenol oxidase (PPO), and lipoxygenase (LOX) in citrus fruit. Results of real time-quantitative polymerase chain reaction (RT-qPCR) showed that the gene expressions of β-Glu, CHI, PAL, POD and PPO were up-regulated in CEO-treated fruits. At the same time, CEO treatment led to down-regulated expression of the LOX gene in citrus fruit. Clove essential oil effectively control the disease incidence of blue mold decay in citrus fruit by motivating the host-defense responses, suppressing the malondialdehyde (MDA) accumulation while enhancing the activities and gene expressions of defense-related enzymes. Our study provides an alternative preservative applying CEO to reduce postharvest fungal decay in citrus fruit. Penicillium italicum, is a serious postharvest disease and accounts for up to 20–50% of fruit decay in China [Citrus bergamia), cinnamon (Cinnamomum zeylanicum L.), clove (Syzygium aromaticum L.), sage , oregano (Origanum vulgare L.), tea tree , and thyme (Tymus vulgaris L.) to control pathogens infection and reduce postharvest fungal rot in fresh horticultural products [Citrus fruit is an important fruit and is highly popular all over the world due to its sweet, juicy, desirable flavor as well as its abundant nutrition . Howeverin China . Currentin China . Accordiproducts ,10,11,12Penicillium spp. [Alternaria spp. [Colletotrichum spp. [Aspergillus spp. [Botrytis cinerea [Essential oils (EOs) are aromatic and volatile oily liquids extracted by hydrodistillation or supercritical fluid extraction from plants and spices, which are abundant in bioactive compounds with antimicrobial and antioxidant properties ,13,14,15ium spp. ,9,16,17,ria spp. , Colletohum spp. ,19,20, Alus spp. ,21, and  cinerea ,23,24. A cinerea . In the  cinerea ,12,26. P. italicum, Penicillium digitatum, Alternaria citri and Geotrichum citri-aurantii in citrus fruit [P. italicum was widely explored, only few published works focused on the control efficacy of CEO as a natural antifungal agent towards postharvest blue mold in citrus fruit and its mechanisms remained unknown. Therefore, the aim of this research was to evaluate the control efficacy of CEO in inhibition postharvest citrus blue mold decay caused by P. italicum, and the possible defense mechanisms involved, inducing the antifungal efficacy of CEO against P. italicum in citrus fruit wounds, the response of reactive oxygen species (ROS), the activities and gene expressions of defense-related enzymes.In our previous study, we have found that CEO has strong antifungal activity against us fruit . AlthougS. aromaticum L. was provided from Shanghai Huien international business Co., Ltd. , originated from Sri Lanka, and was stored in dark bottles at 4 °C prior to use.The commercially available CEO obtained from P. italicum was isolated from an infected citrus fruit with the typical blue mold symptoms and grown on potato dextrose agar  medium at 25 °C. The spore suspension was prepared by scraping a 7-day-old pure culture of P. italicum using a sterile loop and adjusted to 5 × 104 spore/mL using a hemocytometer .The tested pathogen of Citrus reticulata Blanco cv. Xinyu tangerines) was harvested at commercial maturity from a local orchard located in Xinyu City, China and those that were healthy, had a consistent size (72–84 g), a uniform color , and were free of bruises or disease were chosen as the experimental material. The in vivo test was carried out according to our previous research [v/v) sodium hypochlorite solution for 2 min and were then washed with running tap water to remove the residual disinfectant and air-dried in a sterilized bechtop  before wounding.The citrus fruit  was reinjected into each wound. Both CEO-treated and control fruits were placed in containers (35 cm × 26 cm polyethylene-lined plastic boxes) at 25 °C. The lesion diameter was measured with a vernier caliper at 3, 5, and 7 days post inoculation. The disease severity was observed by evaluating the disease development of blue mold using the following scale: scale 0, lesion diameter = 0 mm (no decay); scale 1, 1 mm ≤ lesion diameter ≤ 10 mm; scale 2, 10 mm < lesion diameter ≤ 20 mm; scale 3, 20 mm < lesion diameter ≤ 40 mm; scale 4, lesion diameter > 40 mm. The disease severity was calculated using the following formula:A uniform wound  was made with a sterile puncher at the equatorial side per fruit. Then, 20 μL of CEO emulsion at different concentrations  was injected into each wound. After 30 min, 20 μL of P. italicum spore suspension (5 × 104 spore/mL), the CEO treated and control fruits were maintained at 25 °C. Tissue samples of healthy peel at 5–15 mm from the edge of the inoculated lesion from ten fruits in each replicate were collected at 0, 12, 24, 48, 72 and 96 hours post inoculation (hpi), immediately frozen in liquid nitrogen for 3 min, and kept at −80 °C for the biochemical analysis.The sterilized fruits were wounded  with two wounds symmetrically made at the equatorial side per fruit. Then, 20 μL of CEO emulsion (0.4%) or emulsion without CEO as the control was injected into each wound. After reinjecting 20 μL of 2O2 content. H2O2 content was determined by applying a H2O2 assay kit  following the manufacturer’s instructions and expressed as mmol per gram of frozen weight (FW).Frozen peel samples  were homogenized in 5 mL of ice-cold acetone and then centrifuged at 12,000 × g for 30 min at 4 °C . The supernatants were collected and used for the assay of H‒1 FW (frozen weight).Lipid peroxidation was determined by monitoring the malondialdehyde (MDA) content in both CEO treated or control peel samples following the method of Chen et al. , and expFrozen peel samples  were homogenized with 5 mL of different ice-cold extraction buffers to assay of the following defense-related enzymes. 100 mM ice-cold sodium acetate buffer  for β-1,3-glucanase  and chitinase  assays; 50 mM ice-cold Tris-HCl buffer  for phenylalanine ammonia-lyase  assay; 100 mM ice-cold sodium acetate buffer  for peroxidase  and polyphenol oxidase  assays; 100 mM ice-cold phosphate buffer  for lipoxygenase  assay. All homogenates were centrifuged at 12,000 × g for 30 min , and then supernatants obtained were used for assay of defense-related enzymes activities. w/v) laminarin  at 37 °C for 40 min and then quickly add 1.8 mL distilled water and 1.5 mL 3, 5-dinitrosalicilate to each tube. Subsequently, the solution was boiled in water for 5 min to terminate the reaction, after which it was diluted to 25 mL with distilled water. The absorbance was measured at 540 nm  using glucose (1 mg/mL) as the standard solution. One unit of β-Glu activity was defined as the amount of enzyme that produced 1 μM of glucose per hour and expressed as U·h−1·g−1 FW.The β-Glu activity was determined referring to the method of Abeles et al.  with sliw/v) chitinase was incubated at 37 °C for 1 h. Then, 100 μL of 0.3 (w/v) desalting snailase was added to the above reaction solution and further incubated at 37 °C for 1 h. The enzyme reaction was terminated by adding 200 μL of 0.6 M potassium tetraborate and boiling in water for 3 min. The absorbance was measured at 585 nm  using N-acetylglucosamine (0.1 mM) as the standard solution. One unit of CHI activity was defined as the amount of enzyme that produced 1 μM of N-acetylglucosamine per hour and expressed as U·h−1·g−1 FW.Chitinase (CHI) activity was assayed using chitinase (Sigma-Aldrich) as the substrate. To perform CHI activity assay, the reaction mixture consisted of 0.5 mL of enzyme solution, 0.5 mL of 50 mM sodium acetate buffer (pH 5.2) and 0.1%  activity was detected by a commercial PAL assay kit (Jiancheng Bioeng. Inst.) in accordance with manufacturer’s instructions. One unit of PAL activity was defined as an increment in absorbance of 0.01 at 290 nm  per hour, and expressed as U·h2O2 in sodium acetate buffer . One unit of POD activity was defined as an increment of 0.01 in absorbance at 470 nm due to guaiacol oxidation in the presence of H2O2, and expressed as U·min−1·g−1 FW.Peroxidase (POD) activity was determined following the method of guaiacol oxidation at 470 nm by monitoring the increase in absorbance at 470 nm (Shimadzu UV-2600) in the reaction mixture consisting of 200 μL of enzyme solution and 3.0 mL of 25 mM guaiacol in sodium acetate buffer , which was activated by addition of 200 μL of 0.5 M H−1·g−1 FW.Polyphenol oxidase (PPO) activity was determined based on the decomposition of catechol. To achieve this, 0.5 mL of enzyme solution and 1.0 mL of 50 mM catechol in sodium acetate buffer  was added to 4.0 mL of sodium acetate buffer . After 15 s, the absorbance was recorded at 420 nm every 1 min for 5 min. One unit of PPO activity was defined as an increment of 0.01 in absorbance at 420 nm per hour due to decomposition of catechol, and expressed as U·hv/v) sodium soyate (Sigma-Aldrich), and incubated at 30 °C for 15 min. The enzyme reaction was activated by adding 200 μL of enzyme solution, and the absorbance at 234 nm (Shimadzu UV-2600) was recorded every 15 sec for 3 min. One unit of LOX activity was defined as an increment in absorbance of 0.01 at 234 nm, and expressed as U·min−1·g−1 FW.Lipoxygenase (LOX) activity was determined following the method of Surrey Kenneth . FirstlyHigh quality total RNA was independently extracted from peel samples stemmed from the CEO treated or control citrus fruits according to the method described by Ballester et al.  and dissBased on the target genes  sequences from the GENBANK database, the primers for the chosen genes were designed with the primer premier 6.0 software  and listed in −ΔΔCt method [The RT-qPCR was performed in 96-well PCR plates using BIO-RAD CFX96 Real-Time PCR Detection System  under the following procedures: pre-denaturation at 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s, 60 °C for 10 s and 72 °C for 15 s. Polymerase chain reaction amplification was carried out in a total volume of 25 μL, which contained 12.5 μL of SYBR (TaKaRa), 1.0 μL of each primer (10 μmol/L), 2.0 μL of cDNA and 9.5 μL of RNase-free water. The melting curve analysis was performed at over the end of each PCR reaction from 60 °C to 95 °C. The relative RNA expression was calculated using the 2t method . p < 0.05.The experimental data were calculated and expressed as the means of three replicated samples ± standard errors (S.E.). The differences between CEO-treated and control group were analyzed by Tukey honest significant difference (HSD) test using the SPSS version 17.0  to estimate if differences were considered significant at levels of v/v) during post inoculation is shown in p < 0.05) reduced the lesion diameter  and disease severity  in citrus fruit inoculated with P. italicum. At the end of inoculation (7 dpi), the lesion diameters were 56.1 mm, 47.8 mm, 38.4 mm, 29.2 mm and 26.8 mm, and the disease severity rates were 98.8%, 95.0%, 80.0%, 66.3% and 63.1% compared with those of control fruit after CEO treatment at 0.05, 0.1, 0.2, 0.4 and 0.8%, respectively (p < 0.05).A disease development of blue mold infection in CEO-treated fruits at different concentrations  effectively lessened the lesion diameter and observably reduced the disease severity of blue mold rot. Limited data has reported the antifungal activity of CEO in citrus fruit and other horticultural products, while pioneering results of the current study showed a prominent control efficacy of blue mold caused by P. italicum in C. reticulate. This control efficacy of CEO in controlling postharvest blue mold was also observed by Regnier et al. [P. digitatum infection. These results were highly in agreement with the previous reports that the use of plant essential oils could reduce postharvest various diseases of citrus and other fruits [The presence of tender peels and nutritious flesh in italicum . Recentlt manner . In vivontration . This inr et al.  and Jhalr et al.  applyingr fruits ,23,34,352O2 content in both CEO-treated and control fruits exhibited a peak level initially at 12 hpi and then declined towards the end of incubation  disrupted the rise and delayed the decline in H2O2 levels, which remained significantly higher (p < 0.05) in CEO-treated fruits than in control fruits during the entire incubation period.Hcubation . The CEO2O2 as a signaling molecule for inducing host-defense responses in fruit hosts was infected by postharvest pathogenic fungus [2O2 production  delayed the accumulation of MDA content in citrus fruit. The MDA content in CEO-treated fruit was 0.356 mmol g−1, which was 40.01% lower than that in the control group at the end of incubation. Malondialdehyde (MDA) is used as a marker to evaluate the level of lipid peroxidation and cell membrane damage [P. italicum in citrus fruit, as has been reported in raspberry [As shown in e damage . Clove easpberry  and sweeaspberry  treated P. italicum of incubation at 25 °C  than the control group in the initial and interim time of incubation, and reached a peak value of 434.18 U·h−1·g−1 at 24 hpi, which shown 1.75 times as the control group (248.27 U·h−1·g−1) at the same time point. The CHI activity increased in both the CEO-treated and control fruit and reached its highest value at 48 hpi, but the CHI activity in the CEO-treated fruit was higher than the control fruit before 72 h of incubation (p < 0.05) promoted the increase in the activities of PAL and POD during the entire incubation period  higher than the control group throughout the entire incubation period except the first 12 hpi and peaked at 72 hpi, which was 1.55 times higher than that in the control group  proteins are a large group of proteins that have important functions in plant defense responses to pathogen attacks and other physiological stresses  . Both β-nfection , along ws fruits . It worts fruits . Many ines Penz. . Tea tre and CHI . Likewisus fruit . Thus, CThe transcript levels of six defense-related genes encoding β-Glu, CHI, PPO, POD, PAL and LOX were analyzed by using RT-qPCR and shown in P. italicum. In the present study, there is an interesting phenomenon where the expression level of β-Glu occurred at 12 hpi, which was earlier than the up-regulations of inducible resistance genes, including CHI, PAL, POD, PPO and LOX. In addition, the activities and expression levels of these defense-related enzymes were more enhanced or reinforced in the CEO-treated fruit after P. italicum infection, when compared with the control group (Aloe vera gel-treated ’Shogun’ mandarins [To further illuminate the possible defense mechanism involved, this study investigated the transcriptomics responses of CEO treatment on induced PR genes of citrus fruit infected with ol group . The traandarins , as wellandarins  subjecteP. italicum during incubation at 25°C. The possible mechanism of CEO treatment in controlling blue mold in postharvest citrus fruit was thought to be linked with the rise in H2O2 levels, the suppression of MDA accumulation, and the induction of built-in cellular defense-associated enzymes. Taken together, the results of the current study will provide an alternative strategy using CEO for citrus fruits protection against postharvest fungal decay problems.The current study suggested that CEO treatment showed a significant impact on the growth of blue mold in citrus fruits caused by


Leishmania infections. As a part of our ongoing study on the phytochemical characterization and biological investigation of plants used in the traditional medicine of western and central Asia, in the present study, we focused on Eremurus persicus root extract in order to evaluate its potential in the treatment of leishmaniosis. As a result of our study, aloesaponol III 8-methyl ether (ASME) was isolated for the first time from Eremurus persicus root extract, its chemical structure elucidated by means of IR and NMR experiments and the (R) configuration assigned by optical activity measurements: chiroptical aspects were investigated with vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) spectroscopies and DFT  quantum mechanical calculations. Concerning biological investigations, our results clearly proved that (R)-ASME inhibits Leishmania infantum promastigotes viability (IC50 73 µg/mL), inducing morphological alterations and mitochondrial potential deregulation. Moreover, it is not toxic on macrophages at the concentration tested, thus representing a promising molecule against Leishmania infections.Leishmaniosis is a neglected tropical disease which affects several millions of people worldwide. The current drug therapies are expensive and often lack efficacy, mainly due to the development of parasite resistance. Hence, there is an urgent need for new drugs effective against  Leishmania species, and is generally classified into three different clinical forms: visceral leishmaniosis (VL), cutaneous leishmaniosis (CL) and mucocutaneous leishmaniosis (MCL). These forms differ in the pattern and clinical manifestations of the infection. VL can be fatal if left untreated, CL is localized and frequently self-heals within 3–18 months, while MCL leaves disfiguring scars. Although new drugs, such as paromycin, miltefosine and liposomal amphotericin B, are available as antileishmanial therapy in several countries, currently pentavalent antimonials and amphotericin B are the most used drugs for such purpose. Still, they induce toxic effects, produce development of parasite resistance and, no less important, they are expensive +, 295 [M + Na]+, 271 [M − H]−.1H-NMR : δ 2.01 , 2.28 , 2.48 , 2.69 , 3.05 , 4.02 , 4.94 , 6.69 , 7.06 , 7.14 .13C-NMR (CDCl3): δ 203.3 , 165.9 , 159.5 , 142.0 , 140.3 , 139.9 , 119.9 , 115.4 , 113.8 , 109.2 , 108.3 , 68.0 , 56.0 , 34.0 , 30.7 , 22.1 .2 cell and 0.056 M/CDCl3 solutions. Five thousand scans were taken and solvent subtraction was made with VCD spectra in the same condition. DFT calculations ancillary to chiroptical spectroscopies were run by use of Gaussian09 set of programs , where L1 is the absorbance of control cells and L2 is the absorbance of treated cells. Three separate experiments were performed for each sample and the concentration that inhibited viability by 50% (IC50) was determined through dose-response regression analysis, plotted by GraphPad Prism 6 .The antiparasitic effect of EE and (ethod of . Log phaR)-ASME and morphological alterations were investigated by optical microscopy using direct examination of live microorganisms (hanging drop) and after Giemsa stain. Briefly, exponentially grown of L. infantum (2 × 106 cells mL−1) were treated with the compound at IC50 concentrations for 2 h, 4 h, 6 h and 24 h at 26 °C. After incubation, cells were pelleted by centrifugation at 3000 rpm for 5 min and the supernatant was discarded by aspiration. The cell pellet was suspended in fresh medium and approximately 10 μL were placed on a Koch slide and directly observed under the optical microscope phase contrast . In addition, a total cell counting was performed using a haemocytometer and a smear was made which was submitted to Giemsa stain. The smear was fixed with methanol for 5 min, stained with aqueous solution of Giemsa  for 10 min at room temperature and finally washed with water and air dried. The stained smear was observed under the microscope with a 100× lens .Parasites cells were exposed to (L. infantum (2 × 106 cells mL−1) were treated with (R)-ASME at IC50 concentration for 2 h, 4 h, 6 h and 24 h at 26 °C. At each time point, cells were fixed in 200 μL of 70% ethanol for 30 min. at 4 °C. After washing cells with 2 mL of PBS, enriched with 2% of bovine serum albumin (BSA), the pellets were suspended in 0.5 mL of propidium iodide (PI) solution  and incubated for 15 min at 37 °C [For the analysis of DNA content, exponentially grown L. infantum promastigotes (2 × 106 cells mL−1) were exposed to (R)-ASME at IC50 concentrations for 2 h, 4 h, 6 h, and 24 h at 26 °C. Cells were then washed with PBS and re-suspended in binding buffer . To 100 μL of this suspension were added 5 μL of annexin V-FITC and 5 μL of PI . After 15 min incubation in the dark at room temperature, 400 μL of binding buffer were further added and cells were analyzed by flow cytometry . Data analysis was carried out using the program Paint-a-gate, and values are expressed as a percentage of positive cells for a given marker, relatively to the number of cells analyzed.Double staining for annexin V-FITC and PI was performed as described previously . Brieflym), a cell-permeable cationic and lipophilic dye, JC-1 , was used as previously described [m. However, at lower ∆ψm, JC-1 cannot accumulate within the mitochondria and instead remains in the cytosol as monomers, which fluoresces green (490 nm). Therefore, the ratio of red to green fluorescence gives a measure of the transmembrane electrochemical gradient. L. infantum promastigotes (106 cells) were exposed to (R)-ASME at the IC50 concentrations for 24 h at 26 °C. Promastigotes were then incubated with JC-1 (5 µg·mL−1)  in the dark for 15 min at RT. Then, cells were washed in PBS, suspended in 400 µL of PBS and analyzed by flow cytometry. Data analysis was carried out using the program Paint-a-gate.To assess mitochondrial membrane potential (∆ψescribed . This prL. infantum (2 × 106 cells mL−1) were exposed to (R)-ASME at IC50 concentration or to dissolution vehicle (DMSO), and incubated at 26 °C for 24 h. The Leishmania DNA extraction was carried out according to the procedure in DNeasy Blood & Tissue . DNA integrity analysis was done by electrophoresis, running DNA through an EtBr-treated agarose gel and visualizing it with UV light.Promastigotes of R)-ASME at IC50 concentrations. After incubation, control and treated cells were washed with PBS, pH 7.2, and 450 μL of PBS and 50 μL MTT solution (5 mg·mL−1) were added to each well and incubated at 37 °C for 1 h. The cells were then washed with PBS, 500 μL of DMSO were added to the wells and absorbance was measured at 530 nm on an ELISA plate reader . The percentage of viable cells was determined as described in the viability assay.For cytotoxicity assays on mammalian cells, log phase of macrophages (RAW 264.7) were trypsinized and incubated at 37 °C in 24-well tissue culture plates in Dulbecco’s Modified Eagle Medium (DMEM), enriched with Glutamax and supplemented with 10% FBS, under microaerophilic conditions. As soon as the monolayers reached confluence, the medium was removed and cells were incubated at 37 °C for 24 h with fresh medium and , followed by Dunnett´s test, using GraphPad Prism, version 6.0d . The level of significance was * Eremurus persicus roots combined with biological evaluation led to the identification of an active fraction with antileishmanial activity corresponding to the (R) enantiomer of Aloesaponol III 8-methyl ether ((R)-ASME). Interestingly, this is the first time that the (R)-ASME has been isolated from natural sources, while the (S) enantiomer had already been reported as found in Kniphofia foliosa [Eremurus chinensis [Asphodelus microcarpus [In this work, we used a bioassay-guided fractionation as a strategic approach to natural source product discovery and isolation. Liquid–liquid extraction of the ethanolic extract (EE) of  foliosa , Eremuruhinensis  and Asphrocarpus .R)-ASME possesses a remarkable antiprotozoal effect against L. infantum. We demonstrated that it is active with an IC50 of 73 µg/mL, it is able to induce alterations on both morphology and mitochondrial potential of L. infantum promastigotes, and does not produce significant toxicity in a macrophage cell line. Results showed that (R)-ASME seems to be safe for mammalian cells at tested concentrations, since only a slight decrease in macrophages viability was observed. These results strongly suggest that (R)-ASME may represent a valuable lead against Leishmania infections. Although our preliminary results are encouraging, additional studies should be planned both in vitro and in vivo to get further insight into the mechanisms of action behind the antileishmanial activity of (R)-ASME.(


In these assays, acacetin had consistently higher IC50 values than isoginkgetin. Subsequently, the acacetin was incubated with 4-methoxy-2,2,6,6-tetramethylpiperidine-1-oxy radicals (4-methoxy-TEMPO) and then analyzed by ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC−ESI−Q−TOF−MS) technology. The results of the UHPLC−ESI−Q−TOF−MS analysis suggested the presence of a dimer with m/z 565, 550, 413, 389, 374, 345, 330, and 283 peaks. By comparison, standard isoginkgetin yielded peaks at m/z 565, 533, 518, 489, 401, 389, 374, and 151 in the mass spectra. Based on these experimental data, MS interpretation, and the relevant literature, we concluded that isoginkgetin had higher electron transfer potential than its monomer because of the 3′,8″-dimerization. Additionally, acacetin can produce a dimer during its antioxidant process; however, the dimer is not isoginkgetin.To probe the effect of 3′,8″-dimerization on antioxidant flavonoids, acacetin and its 3′,8″-dimer isoginkgetin were comparatively analyzed using three antioxidant assays, namely, the  Flavonoids are an important source of natural phenolic antioxidants . ThousanO,6″-linkage biflavonoids, and 4′-O,3‴-linkage biflavonoids  scavenging effects. ·O2– is an important form of reactive oxygen species (ROS)  was obtained from the Amresco Chemical Co. . Tris-hydroxymethyl amino methane (Tris) was obtained from Dingguo Biotechnology, Ltd . Water, methanol, and acetonitrile were of HPLC grade and the other reagents were purchased as analytical grade from Guangdong Guanghua Chemical Plants Co. Ltd .Acacetin (C·O2–) scavenging activity was determined using the improved pyrogallol autooxidation method [x = 5–25 μL) was mixed with Tris-HCl buffer  containing Na2EDTA (1 mM). Pyrogallol  was added, and the mixture was thoroughly shaken at room temperature for 3 s. The absorbance of the mixture was measured  at 325 nm every 30 s for 5 min. Tris-HCl buffer was applied as a blank. The ·O2– inhibiting ability was calculated as follows: A325nm, control is the increment in the absorbance at 325 nm of the mixture without the sample and ΔA325nm, sample is the increment of the mixture with the sample; T = 5 min.The superoxide anion radical (n method . In brie4 solution (0.01 M) and 12 μL of ethanolic neocuproine solution (7.5 mM) were added to a 96-well plate and mixed with different concentrations of samples (4–20 μg/mL). The total volume was then adjusted to 100 μL with a CH3COONH4 buffer solution (0.1 M), and mixed again to homogenize the solution. The mixture was maintained at room temperature for 30 min, and the absorbance was measured at 450 nm on a microplate reader . The relative reducing power of the sample was calculated as follows:maxA is the maximum absorbance, minA is the minimum absorbance, and A is the absorbance of the sample.The cupric ion reducing antioxidant capacity (CUPRAC) assay was determined based on the method proposed by Apak , with sm+· cation radical was generated from the reaction of (NH4)2ABTS aqueous solution (7.4 mM) with K2S2O8 aqueous solution (2.6 mM). In brief, the (NH4)2ABTS aqueous solution was mixed with the K2S2O8 aqueous solution at room temperature. After an incubation of 12 h, the resulting mixture became dark green. Then, it was diluted about 50 times with methanol. The diluted solution was scanned by using a UV–Vis spectrophotometer  from 200 to 900 nm and used as the working solution for this assay [The ABTSis assay .+· working solution (80 μL), the test sample  and methanol (20 − x μL) were added to adjust the total reaction system to 100 μL. After incubating for 6 min, the absorbance of the 100 μL mixture was measured at 734 nm by using a microplate reader . The percentage inhibition was calculated based on the formula as follows:A0 is the absorbance of the control without the sample and A is the absorbance of the reaction mixture with the sample.To the above ABTSThe reaction proceeded under the conditions described in previous reports ,32. In b18 column . The mobile phase in the chromatography was made up of a mixture of acetonitrile (phase A) and 0.1% formic acid in water (phase B). The column was eluted at a flow rate of 0.2 mL/min with the following gradient elution program: 0–2 min, 30% B; 2–10 min, gradient decreasing from 30% to 0% B; 10–12 min, gradient increasing from 0% to 30% B. The sample injection volume was 5 μL. The Q–TOF–MS analysis was performed under the negative ionization mode in a Triple TOF 5600plus mass spectrometer  equipped with an ESI source. The scan range was set at 100–2000 Da. The system was run with the following parameters: Ion spray voltage, −4500 V; ion source heater, 550 °C; curtain gas , 30 psi; nebulizing gas , 50 psi; and Tis gas , 50 psi. The declustering potential (DP) was set at −100 V, whilst the collision energy (CE) was −45 V with a collision energy spread (CES) of 10 V. For comparison, the standard isoginkgetin solution  was also analyzed using the above UHPLC–ESI–Q–TOF–MS conditions.The UHPLC−ESI−Q−TOF−MS analysis apparatus was equipped with a Phenomenex Luna C50 value was defined as the final concentration required for 50% radical inhibition (or relative reducing power) [n = 3). The linear regression was analyzed using Origin 6.0. The determination of significant differences between the mean IC50 values was performed by using one-way ANOVA analysis and the t-test. The analysis was performed using SPSS software 13.0  for Windows. p < 0.05 was considered statistically significant.The concentration response curves were analyzed using Origin 6.0 professional software . The ICg power) . It was Acacetin and its 3′,8″-dimer isoginkgetin may mediate ET to exert antioxidant action. However, when compared with acacetin, isoginkgetin has an enhanced ET antioxidant capacity; this enhancement may be attributed to the 3′,8″-dimerization, which allows partial π–π conjugation. Acacetin can produce a dimer during the antioxidant process; however, this dimer is not isoginkgetin.


Fulminant type 1 diabetes is characterized by an intrinsic insulin deficiency resulting from the severe destruction of pancreatic β cells and it rapidly leads to ketoacidosis. However, the association between fulminant type 1 diabetes in pregnancy and specific viral infections has not been reported.The patient in this study was a 31-year-old Japanese woman, and at 30 weeks of pregnancy she was admitted with marked fatigue. Fetal bradycardia was noted, and the child was delivered by emergency cesarean section but was stillborn. The maternal blood sugar level was high (427 mg/dL), but the glycated hemoglobin value was 6.2%; therefore, fulminant type 1 diabetes was suspected. Serum antibody testing confirmed a Coxsackievirus B1 infection. The patient in this case had fulminant type 1 diabetes in pregnancy associated with Coxsackievirus B1.This case highlights that fulminant type 1 diabetes in pregnancy may be associated with Coxsackievirus B1 infection. Fulminant type 1 diabetes (FT1D) is a subtype of type 1 diabetes (T1D); it is characterized by the abrupt onset of insulin-deficient hyperglycemia and ketoacidosis within a few days [The specific etiology and pathogenesis of FT1D are unclear and may be related to genetic predisposition, autoimmunity, viral infection, and pregnancy.Viral infection is strongly associated with fulminant diabetes, as reported previously, and can be associated with frequent flu-like symptoms along with a seasonal variation in time of onset. A viral infection is suggested to play a role in rapid β cell destruction.Epidemiological studies have indicated an association between enteroviruses (EVs) and T1D . CertainWe report the case of a patient who developed FT1D in pregnancy accompanied by Coxsackievirus B1 infection.Our patient was a 31-year-old Japanese woman, gravida 1, para 1. She had no remarkable medical history, and there were no abnormalities during her medical examination. Her father and uncle had type 2 diabetes requiring insulin therapy. However, her sister did not have diabetes. The weight gain during her first 3 months of pregnancy was 1.1 kg. A regular examination at 29 weeks and 5 days was normal; her blood glucose level was 73 mg/dL, with negative levels of urinary glucose, and 3+ urinary ketone bodies.At 30 weeks and 6 days, she was admitted with marked fatigue and dyspnea. However, she did not present with fever, headache, costochondritis, pharyngitis, diarrhea, maculopapular non-pruritic rash, nausea, or flu-like symptoms such as a sore throat, cough, or rhinorrhea.DR8,12/DQ7,4. These results were indicative of a FT1D diagnosis , 36.7 °C; pulse rate (P), 94 beats per minute (bpm); respiratory rate (R), 18/minute; and blood pressure (BP), 121/65 mmHg; fetal bradycardia was also observed, for which emergency cesarean section was performed, but the child was stillborn. The laboratory findings revealed diabetic ketoacidosis (DKA), with random sample glucose of 427 mg/dL, C-peptide reactivity (CPR) of 0.04 ng/mL, arterial pH of 6.91, bicarbonate of 6.7 mEq/L, and 3+ urinary ketone bodies. Despite the presence of DKA, the glycated hemoglobin (HbA1c) value was within the normal range (6.2%), and urinary CPR was extremely low at 0.5 μg/day. In addition, the serum elastase-1 and lipase levels were increased to 1939 (< 300) ng/dL and 119 (11–53) U/L, respectively. Antibody level against glutamic acid decarboxylase was 1.2 (< 1.5) U/mL. Neither anti-insulin nor anti-insulinoma-associated antigen-2 antibodies were detected. HLA II haplotypes showed She was diagnosed as having DKA due to FT1D. She was simultaneously treated with fluid replacement and continuous insulin infusion to maintain vital signs, plasma glucose, and electrolyte levels. On day 2, ketonuria, electrolytes, and vital signs had normalized. On day 3, the continuous infusion of insulin was withdrawn, and daily multiple insulin injections were administered with blood glucose monitoring. On day 24, she continued to undergo treatment with multiple daily insulin injection therapy with insulin aspart (25 U/day) and insulin degludec (12 U/day).To investigate the association between viral infection and FT1D, we performed serological testing for several viruses such as parainfluenza virus 1–3, Coxsackievirus A2–7, 9, 10, 16, B1–6, cytomegalovirus (CMV), Epstein–Barr virus (EBV), and human herpes virus (HHV) 6 at day 3, day 17, and day 38 (Table FT1D is a special type of T1D that was first proposed by the Japanese scholar, Imagawa, in 2000 . A natioIt is well known that the immunological milieu is significantly changed during pregnancy. However, the issue of whether the clinical and immunogenetic characteristics are different between the cases associated with and without pregnancy remains to be determined.DRB1*04:05- DQB1*04:01 strongly confer susceptibility to the development of FT1D [DRB1*09:01-DQB1*03:03 tended to be higher in pregnant women with FT1D than in non-pregnant patients with FT1D [DR8,12/DQ7,4. To the best of our knowledge, these haplotypes have not been reported as conferring susceptibility to FT1D.Although multiple genes have been implicated, HLA class II genes, especially the HLA-DR and DQ genes, are the most important. HLA class II genotypes  of FT1D . In contith FT1D . The invPicornaviridae family. Coxsackie B viruses have a tropism for muscle cells and are most common in children but may occur in adults. Clinical manifestations include fever, aseptic meningitis, costochondritis, pharyngitis, myocarditis, diarrhea, and maculopapular non-pruritic rash [Viral infection is strongly associated with fulminant diabetes, as reported previously, and is evidenced by frequent flu-like symptoms, and it possibly has a seasonal variation in time of onset. Coxsackie B is a group of six types of EVs belonging to the tic rash .Epidemiological studies have indicated an association between EVs and T1D. These viruses have a strong tropism for insulin-producing β cells; the destruction of these cells leads to T1D . The exaRecent studies have reported a variety of viral infections, including Coxsackievirus B1, B3, B4, A4, A5, A6, CMV, EBV, HHV6, 7, mumps, and parvovirus B19 to be involved in the development of FT1D –12. HoweTherefore, this case suggests that Coxsackievirus B infection is associated with FT1D with pregnancy and without pregnancy. DKA is a medical emergency during pregnancy and has high rates of fetal mortality. A prophylactic measure should be available to avoid the development of FT1D in pregnancy. Recent studies have narrowed down the epidemiological association to a subset of EVs: group B Coxsackieviruses; clinical development of a vaccine against T1D-associated EV types has been started . A CoxsaWe should assess pregnant women with symptoms of both fatigue and acute hyperglycemia for the presence of DKA, even if they are alert when presenting to the hospital. This case highlights that FT1D in pregnancy may be associated with Coxsackievirus B1 infection.