The number and location of phenolic hydroxyl of the flavonoids will significantly influence the inhibition of tyrosinase activity. Phenolic hydroxyl is indispensable to the antioxidant activity of flavonoids. Isoeugenol, shikonin, baicalein, rosmarinic acid, and dihydromyricetin have respectively one, two, three, four, or five phenolic hydroxyls. The different molecular structures with the similar structure to This investigation tested the antityrosinase activity, the inhibition constant, and inhibition type of isoeugenol, shikonin, baicalein, rosmarinic acid, and dihydromyricetin. Molecular docking was examined by the Discovery Studio 2.5 . This experiment also examined the antioxidant effects of the five compounds on supercoiled pBR322 plasmid DNA, lipid peroxidation in rat liver mitochondria in vitro, and DPPH, ABTS, hydroxyl, or superoxide free radical scavenging activity in vitro.2+/vitamin C system in vitro, and supercoiled pBR322 plasmid DNA. The activity order is isoeugenol < shikonin < baicalein < rosmarinic acid < dihydromyricetin. The results showed the compounds with more phenolic hydroxyls have more antioxidant and antityrosinase activities.The compounds exhibited good antityrosinase activities. Molecular docking results implied that the compounds could interact with the amino acid residues in the active site center of antityrosinase. These compounds also exhibited antioxidant effects on DPPH, ABTS, hydroxyl, or superoxide free radical scavenging activity in vitro, lipid peroxidation in rat liver mitochondria induced by FeThis was the first study of molecular docking for modeling the antityrosinase activity of compounds. This was also the first study of the protective effects of compounds on supercoiled pBR322 plasmid DNA, the lipid peroxidation inhibition activity in liver mitochondria. These results suggest that the compounds exhibited antityrosinase and antioxidant activities may be useful in skin pigmentation and food additives.The online version of this article (10.1186/s13020-018-0206-9) contains supplementary material, which is available to authorized users. Flavonoids play a key role in the treatment of various diseases. Compounds with the ability to protect against DNA damage caused by reactive oxygen species (ROS) and inhibit tyrosinase may be useful for the treatment and prevention from ROS-related diseases. Flavonoids are a large type of compounds in natural products. Flavonoids already have been used widely as lead compounds or drugs.Some studies showed that the number and location of phenolic hydroxyl on the flavonoids will significantly influence the inhibition of tyrosinase activity –3. The n+, combined with Eoxy double oxygen, only come from the hydroxyl of tyrosine and dopamine. Phenol hydroxyl compounds, similar to tyrosine and dopamine, can inhibit the activity of tyrosinase [The tyrosinase inhibition mechanism of phenol hydroxyl compounds was analysed. Because the activity center of tyrosinase is hydrophobic, H rosinase .Phenolic hydroxyl is indispensable to the antioxidant activity of flavonoids. Many studies showed that the antioxidant activity increased with the phenol hydroxyl number in B ring of flavonoids. Seyoum  studied + combined with free radicals; (2) the phenolic hydroxyl has strongly denounce electronic effect, which result to the free radicals reaction; (3) the more phenolic hydroxyl number, the more hydrogen bonding, antioxidant activity is also enhanced obviously [The relationship between phenolic hydroxyl number and antioxidant activity of flavonoids is very significant. The reason may be: (1) the more phenolic hydroxyl number, the more Hbviously .l-3,4-dihydroxyphenylalanine (l-DOPA) were expected to the different antityrosinase and antioxidant activities.The number and location of phenolic hydroxyl of the flavonoids will significantly influence the inhibition of tyrosinase activity. Phenolic hydroxyl is indispensable to the antioxidant activity of flavonoids. Isoeugenol, shikonin, baicalein, rosmarinic acid, and dihydromyricetin have respectively one, two, three, four, or five phenolic hydroxyls. The different molecular structures with the similar structure to Tyrosinase (EC 1.14.18.1) plays a key role in the biosynthesis of melanin pigment . Under nEugenia caryophyllata Thunb., which has extensive pharmacological activities, such as antimicrobe, stomach-invigorating. The result of Jin [Isoeugenol is the major constituent of t of Jin  indicateArnebia euchroma(Royle)Johnst, which has extensive pharmacological activities. Shikonin has good antioxidant activities, which supports the use of shikonin as the new anti-aging candidate drug, cosmetic materials and food additives. The results of Chen [Shikonin is the major constituent of  of Chen  revealedBaicalein, a kind of oriental medicine, exhibits antioxidant and anti-inflammatory activities. The results of Li-Weber  revealedPerilla frutescens (L.) or Rosmarinus officinalis, exhibits many potent biological activities. The result of Zhu [Rosmarinic acid, isolated from t of Zhu  indicateDihydromyricetin can be used to scavenge the free radicals. It also has the effects of anti-oxidation and anti-tumour. Based on the results of Xin , dihydroThis investigation tested the antityrosinase activity, the inhibition constant, and inhibition type of compounds. Molecular docking can simulate the binding mode and binding affinity of the tyrosinase and compounds. This investigation also tested the antioxidant effects of isoeugenol, shikonin, baicalein, rosmarinic acid, and dihydromyricetin on supercoiled pBR322 plasmid DNA, lipid peroxidation, and DPPH, ABTS, hydroxyl, or superoxide free radical scavenging activity in vitro.l-3,4-dihydroxyphenylalanine (l-DOPA), tyrosinase (EC 1.14.18.1), phenanthroline, pyrogallol, 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), diphenyl-2-picrylhydrazyl (DPPH), thiobarbituric acid (TBA), and 2,2′-azobis(2-methylpropionamidine)dihydrochloride (AAPH) were purchased from the Sigma Chemical Company . C3606 reagent kit for organization mitochondria separation was purchased from Shanghai Biyuntian company. Disodium phosphate, sodium dihydrogen phosphate, K2S2O8, potassium sulfate, and ferrous sulfate were purchased from Sinopharm Chemical Reagent Co., Ltd . All other solvents and chemicals with analytical grade were commercially available. The Minimum Standards of Reporting Checklist contains details of the experimental design, and statistics, and resources used in this study  was used to dissolve the inhibitor samples. l-DOPA in PBS buffer (pH 6.8) was previously incubated at 30 °C. Then, 0.1 mL sample was mixed with 2.8 mL l-DOPA (0.5 mM). After 1 min, the mixture was added to 0.1 mL of tyrosinase solution (5.33 μg/mL) at 475 nm for 400 s, the absorbance was immediately monitored. The relative enzyme activity was regarded as the slope of the linear part. The inhibitory concentration 50 (IC50) was used to examined the antityrosinase activity. Each sample was examined in five times and averaged. The inhibitory rate was examined according to the formula:1 is the slope value with samples and S0 is the slope value without samples.According to the reference of Chen et al. , the tyrm/Vmapp or 1/Vmapp versus the concentration of the inhibitor.By Lineweaver–Burk plot, the inhibition type was assayed. The inhibition constant was assayed by the second plots of the apparent Khttp://www.rcsb.org/), the crystal structure of tyrosinase (PDB code: 2Y9X) was available [Molecular docking can predict the binding mode and binding affinity of the tyrosinase and compounds. From the Protein Data Bank  with the ABTS stock solution. The reaction lasted 12–16 h at room temperature in the dark. The absorbance of ABTS+ stock solution at 734 nm was 0.70 ± 0.02, diluted with methanol.According to the references of Wan et al. , ABTS fr+ (5 mL) for 6 min. The control group contains 0.5 mL of ethanol and 5 mL of ABTS+ solution. Each sample was examined in three times and averaged. The ABTS+ scavenging activity was examined according to the formula:AS is the absorbance value with samples and AC is the absorbance value without samples.Samples (0.5 mL) were added to ABTS4, 0.2 mL of 0.05% H2O2, 3.2 mL of ethanol were added in turn for 20 min in 37 °C. The wavelength used was 536 nm. Each sample was examined in three times and averaged. The hydroxyl free radical scavenging activity was examined according to the formula:AS is the absorbance value with samples and AC is the absorbance value without samples.According to the references of De Avellar IGJ et al. , hydroxyAccording to the references of Shen et al. , superoxSC is the slope value without samples and SS is the slope value with samples.Each sample was examined in three times and averaged. The inhibitory rate was examined according to the formula:Using the diagnostic kits from Biyuntian , liver mitochondria were obtained. The liver mitochondria from Sprague–Dawley (SD) rats were obtained, according to the references of Zuo et al. .2+ were added in turn. The positive control group contains 0.5 mL of 0.05 M PBS buffer, instead of the antioxidant solution. The blank group was added 1 mL of mitochondrial liquid and 1 mL of 0.05 M PBS buffer. The reaction lasted for 1 h at 37 °C. 2.5% hydrochloric acid solution and 2 mL of 20% CCl3COOH were added for 10 min, followed by 0.3% NaOH solution and 2 mL of 0.67% TBA were added. The test tubes were placed in the water for 30 min at 95 °C, then centrifuged for 10 min at 1372g. The wavelength used was 532 nm. Each sample was examined in three times and averaged. The lipid peroxidation inhibition activity was examined according to the formula:AS is the absorbance value with samples and AC is the absorbance value without samples.In the tubes, 1 mL of mitochondria liquid, 0.5 mL of antioxidant solution, 0.25 mL of 1 mM Vitamin C, and 0.25 mL of 0.1 mM FeAccording to the references of Lin et al., and Zuo et al. , 28, supOne-way ANOVA was used to analyze the differences among means, and statistically significant was considered by a P < 0.05 value .l-DOPA. The results showed that a group of lines with different slopes passing through the origin was the progress curve of enzyme reaction. The slope indicated the diphenolase activity. In the progress of oxidation of l-DOPA, the lag period did not exist. Isoeugenol, shikonin, baicalein, rosmarinic acid, and dihydromyricetin exhibited, with dose dependence, inhibitory effect on tyrosinase diphenolase activity. The IC50 values of the five compounds on the tyrosinase diphenolase activity were respectively 33.33 μmol/L, 26.67 μmol/L, 13.33 μmol/L, 6.67 μmol/L, and 3.33 μmol/L  versus the concentration of the inhibitor, KI was measured  versus the concentration of inhibitor, KIS was measured  were determined as 25.6 μM and 64.7 μM, respectively. Baicalein was the same inhibitor type as shikonin, and the inhibitor constants (KI and KIS) were determined as 16.5 μM and 38.4 μM, respectively. Rosmarinic acid was the same inhibitor type as shikonin, and the inhibitor constants (KI and KIS) were determined as 14.3 μM and 29.8 μM, respectively. Dihydromyricetin was the same inhibitor type as shikonin, and the inhibitor constants (KI and KIS) were determined as 10.26 μM and 23.6 μM, respectively.By Lineweaver–Burk double-reciprocal plots for the inhibition tyrosinase diphenolase, the inhibition type of the five compounds was examined. The enzyme kinetics in the presence of shikonin are shown in Fig. red Fig. b. From ared Fig. c. The vaFigure The docking score between tyrosinase and compound isoeugenol, shikonin, baicalein, rosmarinic acid, and dihydromyricetin was 33.14, 36.13, 37.93, 44.56, 50.98, respectively. The order of activity was: isoeugenol < shikonin < baicalein < rosmarinic acid < dihydromyricetin. The order of activity was very consistent with the experimental results , and alkane radical (ROO·) for dihydromyricetin was 83.9%, 90.0%, and 63.9% respectively.The result of Zhu  indicatet of Liu  showed tlt of Xu  showed t50 values of ABTS free radical scavenging capacity of isoeugenol, shikonin, baicalein, rosmarinic acid, and dihydromyricetin were respectively 36.36 μmol/L, 27.27 μmol/L, 9.09 μmol/L, 6.82 μmol/L, and 3.41 μmol/L  derived from AAPH.Isoeugenol is the major constituent of Hubungan  indicateHubungan  indicateArnebia euchroma(Royle)Johnst. The observed results revealed that shikonin demonstrated higher reducing ability (0.431%), and deoxy-shikonin showed maximum inhibition (0.440%) to DPPH-radical scavenging assay.Shikonin is the major constituent of Rheum officinale. The results of Nishioka [Baicalein is the major constituent of Nishioka  revealedNishioka  revealedNishioka  revealed50 of 4.0 μM). The result of Ding [Rosmarinic acid can inhibit the enzymatic browning of fruits and vegetables. The result of Ha  showed t of Ding  showed t of Ding  showed t of Ding  indicateSambucus williamsii Hance (Jiegumu) is traditionally used in Chinese medicine to treat bone and joint diseases. The major phytochemicals are phenolic acids, lignans, and terpenoids. This compounds may have the antioxidant, anti-inflammatory, bone fracture healing, and anti-osteoporotic effects [Zhao et al.  evaluate effects .Tyrosinase (EC 1.14.18.1) play a key role in melanin biosynthesis . Due to 2+/vitamin C(Vc) system in vitro, the protective effects on supercoiled pBR322 plasmid DNA. In a word, the results support the use of compounds as the new anti-aging candidate drugs, cosmetic materials and food additives.In conclusion, isoeugenol, shikonin, baicalein, rosmarinic acid, and dihydromyricetin exhibited good antityrosinase activities. These compounds also exhibited good antioxidant effects on lipid peroxidation, supercoiled pBR322 plasmid DNA, and DPPH, ABTS, hydroxyl, or superoxide free radical scavenging activity. The different molecular structures lead to the different antityrosinase and antioxidant activities. The activity order is isoeugenol < shikonin < baicalein < rosmarinic acid < dihydromyricetin. The results showed the compounds with more phenolic hydroxyls have more antioxidant and antityrosinase activities. This was the first study of molecular docking for modeling the antityrosinase activity of compounds. This was also the first study of the lipid peroxidation inhibition activity of compounds in liver mitochondria induced by FeAdditional file 1. Minimum Standards of Reporting Checklist.


Tilletia laevis Kühn [syn. T. foetida  Liro] and Tilletia tritici (Bjerk.) Wint. [syn. T. caries (DC) Tul.], is an important wheat disease worldwide. To quickly differentiate the closely related fungi T. laevis, T. tritici and Tilletia controversa (a pathogen that causes dwarf bunt of wheat and has been requested as a quarantined pathogen in many countries), a rapid diagnostic and detection method for an ISSR molecular marker was developed for the first time in this study. Based on the T. laevis-specific band (1300 bp) amplified by the primer ISSR860, a pair of SCAR primers (L60F/L60R) was designed to amplify a specific 660-bp DNA fragment from the isolates of T. laevis but not other related pathogens. The detection limit of the SCAR marker was 0.4 ng/μl of DNA from T. laevis; moreover, a SYBR Green I real-time PCR method was also successfully developed based on the SCAR marker with the detection limit of 10 fg/μl T. laevis DNA. This is the first report of a rapid, specific and highly sensitive SCAR marker and SYBR Green I real-time PCR method for detection of the teliospores of T. laevis based on ISSR technology. This method allows highly efficient, rapid and accurate differentiation of the pathogen from related pathogens, especially from the very similar pathogens T. tritici and T. controversa.Common bunt, caused by  Typically, yield loss is proportional to disease incidence because wheat kernels are replaced by bunt spores. If untreated seeds are used, the incidence of common bunt can reach 70% to 80%, with yield loss of 41% observed in Romania3, and yield losses reaching 10–20% and 25–30% in Turkey and Iran, respectively4.Contamination of wheat with common bunt spores has resulted in considerable loss of yield and seed quality accompanied by a fish-like odorTilletia laevis Kühn [syn. T. foetida  Liro] and Tilletia tritici (Bjerk.) Wint. [syn. T. caries (DC) Tul.], both could be variants of the same species together with the pathogen that causes dwarf bunt, Tilletia controversa Kühn, as proposed by several genetic, biochemical, and molecular studies5. In particular, T. laevis, and T. tritici, are very similar in terms of germination requirements, lifestyles, and disease symptoms2. Many traditional methods for pathogen detection have mostly been used for analysis of morphological and physiological features, such as microscopic examination of teliospores6, immunological detection methods7, and analyses of the polypeptide profiles8, triacylglycerol profiles9 and genetic properties of teliospores10. These characteristics are usually used as taxonomic criteria to diagnose and detect pathogens11, but these methods are time consuming and exhibit low diagnostic accuracy for the detection of T. laevis. For example, based on microscopic observation, the cell wall surface of the teliospore of T. laevis was smooth, whereas that of T. caries was reticular. The mean diameter of T. caries teliospores is 14–23.5 μm, occasionally 25 μm. Similarly, the diameter of T. laevis teliospores ranges from 13 to 22 μm12. T. controversa has a similar morphology and genetic structure to those of T. caries13. The development of an efficient method for rapid and accurate pathogen detection is urgently needed.Common bunt is caused by the pathogens T. tritici, T. laevis, T. controversa, and Tilletia bromi with species-specific primers in the ITS region, in the large subunit of the ribosomal RNA genes and RPB2 genes, but they failed because no variation was observed among the regions15. However, Kochanová et al.16 developed PCR primers for the ITS1 region of Tilletia species for simultaneous detection of T. controversa and T. tritici. Vesna et al.17 reported the differentiation of Tilletia species by repetitive sequence-based polymerase chain reaction (rep-PCR) fingerprinting. However, many molecular markers have been developed to differentiate T. controversa from similar species, including T. laevis and T. controversa, based on AFLP, RAPD, and ISSR analyses20. To date, there has been no report on molecular markers that could distinguish T. laevis from similar species of pathogens obtained by ISSR analysis, such as T. tritici and T. controversa.Detection technologies, such as molecular, enzymatic, chromatographic and spectroscopic detection, are more sensitive, precise and time saving than traditional detection methods. In recent studies, researchers tried to distinguish T. laevis from those of similar species  by ISSR technology, which will contribute to a diagnostic method based on a SCAR marker and SYBR Green I real-time PCR.Therefore, the main aim and objectives of this study were to develop a detection method that could be rapid, reliable and sensitive for distinguishing the teliospores of T. laevis was differentiated from T. controversa by a polymorphic profile (1300 bp), which was produced by the ISSR 860 primer   Fig. , and thevis Fig. .Figure 3T. laevis teliospore DNA was successfully developed in this study. The standard curve was generated with a linear range covering 6 log units . These results demonstrated that a sensitive real-time PCR detection method for T. laevis teliospores was successfully established with SYBR Green I.Based on the SCAR marker, the SYBR Green I real-time PCR method for identification of its Fig. . The melits Fig. . MoreoveT. laevis was successfully discovered using the ISSR 860 primer, and a specific 660-bp SCAR marker of T. laevis was developed that could successfully differentiate T. laevis from 9 related fungi (40 isolates) in a PCR assay, namely, U. maydis, U. horde, U. nuda, P. striiformis f. sp. tritici, P. triticina, B. graminis and F. graminearum, and, in particular, the very similar species T. tritici and T. controversa. The detection limit of the SCAR primer set was 0.4 ng/μl. This sensitivity is almost 10 times higher than that of the reported SCAR marker  of ay Table .22. A variety of strategies have been employed to identify pathogens and diagnose diseases by using DNA molecular marker technologies, which can distinguish sequence variations in specific genomic regions. This method provides a powerful tool for identification of pathogens with high efficiency23. Although the ITS region of the ribosomal subunit has been used as a target for species-specific primers24, in the case of wheat bunt, it was not possible to design species-specific primers in the sequenced ITS region, as there is no variation between the ITS2 regions of T. tritici, T. laevis, T. controversa15. Compared with several other types of DNA marker technologies, e.g., RFLP, AFLP, RAPD and SSR, ISSR analysis exhibits relatively high consistency and repeatability25, which has already been successfully used to differentiate the very similar species T. controversa19 from T. tritici and T. laevis. However, to date, there have been no reports on the SCAR marker of T. laevis using ISSR technology, and this study revealed the possibility of identifying T. laevis using this method. In this study, a rapid, simple and accurate detection method was developed to distinguish T. laevis from T. controversa and T. tritici with a SCAR marker.Methods for identification and monitoring of pathogens must be rapid, simple and sensitiveT. laevis from 9 related fungi (40 related isolates) by ISSR analysis, which will provide a highly efficient method for differentiation of the pathogen from very similar pathogens, including T. tritici and T. controversa. In the near future, we will focus on the development of a high-throughput kit for rapid, sensitive, and accurate detection of T. laevis from T. tritici and T. controversa, which will contribute significantly to for the great potential in the on-site detection.In summary, this is the first report of a rapid, specific and highly sensitive SCAR marker for detection of the teliospores of T. laevis; the very similar species T. controversa and T. tritici; the smut fungi Ustilago maydis, Ustilago horde, and Ustilago nuda; and fungal pathogens of wheat, including Puccinia striiformis f. sp. tritici, Puccinia triticina, and Blumeria graminis, in addition to Fusarium graminearum, were used in the study. Five isolates of T. controversa were collected in the United States by Blair Goates . All other fungi were isolated by Prof. Li Gao in IPP, CAAS . Genomic DNA was extracted according to Gao et al.12, with slight modifications ). The final genomic DNA solution was treated with RNase and analyzed on an agarose gel22.The pathogen The UBC primer set # 9  was tested for ISSR analysis. PCR amplification was conducted using a 25-μl reaction mixture containing 400 pmol of primer, 100 ng of template DNA, and 12.5 μl of 2 × EasyTaq PCR SuperMix (+dye)  on a Gradient Thermocycler  with the following program: an initial denaturing cycle (3 min at 94 °C), followed by 35 cycles of 30 s at 94 °C, 30 s at the annealing temperature (32–75 °C depending on the primer), and 2 min of elongation at 72 °C and a final extension (10 min at 72 °C). The amplification products were analyzed on 1.5% agarose gels at 180 V for 20 min in 0.5 × TBE buffer  containing 0.1 mg/ml ethidium bromide. Banding patterns were visualized under UV light and photographed using a gel imager .T. laevis DNA generated by the primer ISSR860 (5′-TGTGTGTGTGTGTGTGRA-3′) was excised from the gel and purified with the EasyPure Quick Gel Extraction Kit  according to the manufacturer’s instructions. The DNA fragment was cloned into the pEASY-Blunt Cloning vector . Ligated plasmids were transformed into Escherichia coli Trans5α competent cells according to the manufacturer’s protocol . The cloned fragment was sequenced by Sangon Biological Engineering Technology and Services, Ltd.  using the M13F and M13R vector-specific primers. Based on the sequence of the specific fragment, the SCAR marker (L60F/L60R) was designed by Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and synthesized by Sangon Biological Engineering Technology and Services, Ltd. . The amplification was carried out in a 25-μl reaction mixture, which included 1 μl of DNA template (100 ng), 1 μl of the SCAR primer L60F (10 μM), 1 μl of the SCAR primer L60R (10 μM), 12.5 μl of 2 × EasyTaq PCR SuperMix (+dye) , and 9.5 μl of ddH2O. The PCR amplification program was as follows: an initial denaturation (3 min at 94 °C), followed by 30 amplification cycles  and a final extension (10 min at 72 °C). We obtained a specific 660-bp band after the amplified products were separated by electrophoresis on a 1.0% (w/v) agarose gel (containing ethidium bromide) at 180 V for 20 min in 0.5 × TBE buffer, visualized on a UV transilluminator, photographed using a gel imager , and then sequenced by Sangon Biological Engineering Technology and Services, Ltd. .The specific band (1300 bp) generated of the T. laevis (5 isolates). First, we selected DNA from species that shared high similarity with T. laevis, including genomic DNA from T. controversa (5 isolates) and T. tritici (4 isolates). Second, we selected DNA from pathogens that cause disease in leaves and tassels of wheat, such as P. striiformis f. sp. tritici (3 isolates), P. triticina (3 isolates), B. graminis (5 isolates) and F. graminearum (5 isolates). Last, we selected DNA from species related to pathogens that cause smut disease in cereal crops, including U. maydis (5 isolates), U. horde (5 isolates), and U. nuda (5 isolates); ddH2O was used as a control. The DNA concentration of all tested isolates were adjusted to 1 ng/μl, and the amplification reaction mixture, the PCR amplification program and the amplified products were separated and sequenced as described above.To determine the specificity of the SCAR marker, we selected DNA from the pathogens based on three factors, except for the genomic DNA of T. laevis, the detection limit of the SCAR marker was determined. The amplification reaction mixture for PCR, amplification program and electrophoresis parameters were the same as those previously described.By using serial dilutions  of genomic DNA from 7–1.37 × 102 copies/μl, 0.01 ng–10 fg) were used as templates, which were established as follows: the volume of the TA cloning reaction system was 10 μL, consisting of 5 μL of 2 × Solution Buffer , 4 μL of recovered DNA bands, and 1 μL of T-vector ; ligation was performed at 22 °C for 4 hours. TA cloning of the recovered PCR products was carried out using DH5α competent cells . Colonies were picked and dissolved in 10 μL of sterile water, and 1 μL of the above solution was used as a template for colony PCR. The colony PCR products were sequenced by Sangon Biological Engineering Technology and Services Co., Ltd. . The EasyPure® Plasmid Mini Prep Kit  was used to extract plasmid DNA to be used as a standard for absolute quantification. For this purpose, the plasmid DNA standard was serially diluted 10-fold, and 2 μL of each dilution was used as a template to generate a standard curve. Then, RT-PCR was performed in a 20-μl reaction mixture containing 10 μl of 2 × PCR buffer (SYBR Premix Ex Taq II (RNaseH Plus), ROXplus), 0.5 μl of qL60F (10 μM), 0.5 μl of qL60R (10 μM), 7 μl of ddH2O and 2 μl of plasmid DNA sample. After the whole reaction system was mixed well and centrifuged, aliquots were loaded onto a 96-well PCR plate , and then three parallel RT-PCRs were designed for every sample. We also used 2 μL of nuclease-free water to replace the DNA template (2 μL) as a control on each plate. The ABI 7500 thermal cycler was used with the following reaction program: predenaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing at 60 °C for 40 s. In addition, to generate melt curves, the following program was used: 95 °C for 1 min, 65 °C for 1 min, and a temperature rise from 65 °C to 95 °C at 0.5 °C/5 s. The fluorescent signal was measured at the annealing step and the extension step (60 °C for 40 s) of each cycle.The fluorescence-based dye SYBR Green I was used for the real-time PCR (RT-PCR) experiment. The primer pair (qL60F: 5′-CTCTTCACTCGCATAGTCACTTG-3′/qL60R: 5′-GATGTTGGGTGGGTTACTGC-3′) was derived from the SCAR and synthesized by Sangon Biological Engineering Technology and Services, Ltd.  Fig. . To deveSubmit


Dictyostelium discoideum has been used as a model organism to study host-pathogen interaction in many intracellular bacteria. Francisella tularensis is a Gram-negative, highly infectious bacterium that causes the zoonotic disease tularemia. The bacterium is able to replicate in different phagocytic and nonphagocytic cells including mammalian, amoebae, and arthropod cells. The aim of this study was to determine the optimal temperature and infection dose in the interaction of Francisella novicida with D. discoideum in order to establish a model of Francisella infection in the social amoeba. The amoeba cells were infected with a different multiplicity of infection  and incubated at different temperatures . The number of intracellular bacteria within D. discoideum, as well as cytotoxicity, was determined at 2, 4, 24, 48, and 72 hours after infection. Our results showed that the optimal temperature for Francisella intracellular replication within amoeba is 30°C with the MOI of 10. We can conclude that this MOI and temperature induced the optimal growth of bacteria in Dictyostelium with low cytotoxicity.The amoeba  Francisella tularensis is a small Gram-negative bacterium that causes the zoonotic disease tularemia. Type A is designated as a category A select agent due to its low infectious dose [F. tularensis that cause human illness: tularensis (Type A), holartica (Type B), and mediasiatica [F. novicida, F. philomiragia, and F. hispaniensis can cause tularemia in immunocompromised people [F. noatunensis has been recognized as a fish pathogen, while recently new environmentally adapted species have been discovered including F. piscidida, F. guangzhouensis, F. opportunistica, F. Salina, F. uliginis, F. frigiditurris, and F. adeliensis [ous dose , 2. Therasiatica . F. novid people . F. noateliensis –7.Francisella is known as a fastidious organism with many growth requirements in vitro. The bacterium is difficult to culture and grows slowly at 37°C, requiring enriched growth medium [Francisella require different growth and medium conditions. Francisella requires medium supplemented with cysteine for cultivation. The bacterium grows on Buffered Charcoal-Yeast Extract Agar (BCYE), Chocolate Agar, Thayer–Martin Agar, and on the media containing hemoglobin, such as Cysteine Heart Agar Base (CHAB). Bacterial growth on blood agar is very slow with a narrow zone of alpha-hemolysis. Francisella can also be cultivated in a liquid medium; however it shows a lower replication rate. For example, the most used is Mueller–Hinton medium (MHM) in which bacteria efficiently replicates after incubation of 7 to 10 days. Francisella species can be incubated with 5% CO2, but it only enhances the growth of F. tularensis subsp. holarctica LVS (Live Vaccine Strain). F. novicida, F. noatunensis, and F. philomiragia require less nutritive supplements in the medium for in vitro growth, possibly due to their adaptation to environmental conditions [h medium . Each spnditions , 9.F. tularensis is 37°C. F. tularensis grows slowly at room temperature, in contrast to F. novicida and F. philomiragia, which survive at 25°C. Because of the complex growth requirement of Francisella, most of the human tularemia cases are misdiagnosed by serology or clinical features [Further, the optimal temperature for the growth of features , 11. Impfeatures .Francisella spp. in the environment has not been definitely determined. However, bacteria have been strongly associated with water environments [Francisella to survive and replicate within amoeba cells [in vitro studies showed the survival and replication of F. novicida in H. vermiformis and A. castellanii, showing the possible importance of protozoa in Francisella ecology [The exact reservoir of ronments . It is aba cells . Our and ecology –17.A. castellanii and H. vermiformis, D. discoideum has been used as a model organism to study phagocytosis, cell motility, and virulence factors for many bacterial pathogens, such as Pseudomonas, Legionella, Mycobacterium, Salmonella, and Klebsiella [Dictyostelium has been established as a model organism for studying the life cycle of the fish pathogen, F. noatunensis, but it has not been established for studying the strains of Francisella that cause the disease in humans [Salmonella requires O-antigen and the type VI secretion system (T6SS) for the survival within amoebae [Francisella uses the T6SS to avoid lysosomal fusion within the macrophages [F. novicida infection in Dictyostelium it would be interesting to investigate the role of T6SS in this social amoeba for prolonged survival of Francisella in nature.However, due to limited tools and antigens to study intracellular lifestyle in free-living amoeba, such as ebsiella –23. Dictn humans –26. It h amoebae . Francisrophages . After eFrancisella within macrophages and various cell types, little is known about the adaptation of Francisella to protozoa cells [F. novicida within D. discoideum. We examined the role of various incubation temperatures and the dose of infection on F. novicida capability to survive and replicate within D. discoideum.Although the previous studies demonstrated the survival and replication of oa cells , 29. OurF. novicida was kindly obtained from prof. Anders Sjöstedt . F. novicida was cultured on buffered charcoal-yeast extract (BCYE) agar  at 37°C with 5% CO2 atmosphere for 24 h. D. discoideum strain (AX2) was obtained from prof. Michael Steinert . D. discoideum cells were grown in HL5 medium at 25°C. HL5 medium contains peptone , yeast extract , glucose , monopotassium phosphate , and disodium phosphate .The wild type strain of F. novicida in vitro at different growing temperatures, the bacterial suspensions (103 CFU·mL−1) were inoculated in tubes with 50 mL of the buffered yeast extract (BYE) broth . The suspensions were incubated at 22, 25, 27, 30, 37°C, and 42°C for 5 days. Every 24 hours, the number of F. novicida was determined by plating the serial dilutions on BCYE agar at 37°C.To determine the survival of D. discoideum (105 amoebae·mL−1) was counted using a Neubauer chamber . The cells were seeded in 96-well plates, incubated overnight and infected with F. novicida at a multiplicity of infection (MOI) 5, 10, or 100. For infection, the HL5 medium was diluted with a phosphate buffer (1 : 1). In order to achieve synchronized infection, the cells were centrifuged immediately after infection at 240 g for 3 minutes at room temperature. The cells were then incubated at 27°C for 1 hour and washed 3 times with PBS to remove extracellular bacteria. This was considered as a time point zero. At each time point after infection , the cells were treated with 0.9% Triton X-100  for 10 minutes to lyse the cells. The number of intracellular bacteria was determined by plating the serial dilutions on BCYE agar.The number of To determine the influence of different incubation temperatures on the growth of bacteria within amoeba cells, the cells were infected with MOI 10 as described previously. The infected cells were incubated at 22, 25, 27, 30, or 37 °C and the number of intracellular bacteria was determined by plating the serial dilutions on BCYE agar.D. discoideum cells were infected with MOI 5, 10, or 100, washed 3 times and incubated for 1 hour (time point zero). At 2, 4, 24, 48, and 72 h after infection, 50 μL of culture supernatant was transferred to a 96-well plate for monitoring cytotoxicity. The CytoTox 96 Non-Radioactive Cytotoxicity Assay  was used to quantitatively measure lactate dehydrogenase (LDH) according to the manufacturer's instructions. The absorbance signal was measured at 490 nm using a microplate reader .t-test. Statistical analyses were performed using Statistica (Statsoft) software version 12 or with Graph Pad Prism version 6.0 software. p value < 0.05 were accepted as significantly different and were denoted by ∗. Exact p values are listed in the results.Statistical significances were determined using two-tailed Student's F. novicida in vitro was examined. The bacterial suspensions were incubated at temperatures of 22°C, 25°C, 27°C, 30°C, 37°C, or 42°C for 5 days. By every 24 hours, the number of F. novicida was determined by plating the serial dilutions on BCYE agar at 37°C.The effect of different incubation temperatures on the survival of F. novicida replicates with prolonged time of incubation at 22, 25, 27, 30, and 37°C. By day 5, the number of bacteria increased up to 8.0 × 105 CFU·mL−1 after incubation at 25°C. At 37°C the number of bacteria increased up to 1.5 × 105 CFU·mL−1, and at 22°C up to 8.0 × 106 CFU·mL−1, while the incubation temperatures of 27 and 30°C resulted in a higher replication of bacteria, 2.0 × 109 CFU·mL−1 and 5.0 × 1012 CFU·mL−1, respectively. In contrast, when the incubation temperature was 42°C, F. novicida was not able to survive after the second day of incubation , 25°C , 27°C , 37°C , and 42°C  was significantly different.We can conclude that the optimal temperature for bacterial replication D. discoideum was investigated. The amoeba cells were infected with F. novicida with MOI 5, 10, or 100. The infected amoeba cells were incubated at 27°C. At 2, 4, 24, 48, and 72 hours after infection, the number of intracellular bacteria was determined by plating the serial dilutions on BCYE agar.The influence of a different dose of infection on the survival and replication of bacteria within F. novicida did not replicate within Dictyostelium. In contrast, after infection of amoeba cells with MOI 100, bacteria started proliferation after 2 hours of infection and increased up to 106 CFU·mL−1 by 24 hours after infection  (7 CFU·mL−1)  . HoweverFU·mL−1) . At 72 htion 100 .p=0.002) but not with MOI 5 and MOI 10 (p=0.09). The number of intracellular bacteria after infection of Dictyostelium cells with MOI 5 and MOI 100 was statistically different at all observed time points (p < 0.001).Our results showed that the number of intracellular bacteria was statistically different after infection of amoeba cells with MOI 10 and MOI 100   with MOI 5 = 0.213, MOI 10 = 0.220, MOI 100 = 0.184. F. novicida induced the highest LDH release at 72 h after infection at MOI 100, in comparison to previous time points  (p=0.03), and MOI 10 with MOI 100 (p=0.01).At 2 and 4 hours after infection, the LDH levels in supernatants of  0.0625) . At 24 a= 0.464) . The LDHD. discoideum. Our results show that the MOI 10 induces the lowest level of cytotoxicity in Dictyostelium, so it will be used in our further experiments.We can conclude that the increase of the LDH activity in the culture supernatant of the infected amoeba cells is proportional to the intracellular growth of bacteria within F. novicida growth within D. discoideum was also examined. Based on our previous results, the optimal dose of infection for the linear growth of bacteria within amoeba with low cytotoxicity is MOI of 10. Therefore, the amoeba cells were infected with MOI 10 and incubated at different temperatures .The influence of different incubation temperatures on the F. novicida showed similar intracellular growth at 2 and 4 hours after infection and the bacterial number increased up to 104 CFU·mL−1. The bacterial cells continued to replicate within amoeba to 24 hours after infection. At 24 h after infection, the highest number of bacteria was determined at a temperature of 30°C (108 CFU·mL−1) (5 CFU·mL−1). At 72 hours after infection, the highest number of bacteria was determined after incubation on the temperature of 27 and 30°C  . In contand 30°C .Francisella intracellular growth within amoeba. In comparison with optimal temperature, the number of intracellular bacteria was statistically different at 24 hours after infection at 22°C (p=0.001), 25°C (p=0.001), 27°C (p=0.002), and 37°C (0.001). Also, the statistically different number of bacteria was found at 48 hours after infection at 22°C (p=0.001), 25°C (p=0.001), 27°C (p=0.002), and 37°C (0.002). At 72 hours after infection, only the number of intercellular bacteria observed after incubation of infected cells at 37°C was statistically different (p=0.001).Our results clearly show that the temperature of 30°C is an optimal temperature for F. tularensis, Legionella pneumophila, and Mycobacterium spp., has significant implication in their environmental persistence. It was also shown that after growing within the amoeba, nonpathogenic bacteria gained their pathogenic features and that bacteria become more resistant to antibiotics [Francisella tularensis is a unique pathogen that can survive different environmental conditions and animal reservoirs. To be able to understand this wide range of hosts and the diversity of virulence strategies, there is a need for establishing more models for studying mechanisms required for infecting diverse hosts. This was successfully accomplished using D. discoideum as a host model to study the pathogenesis of Klebsiella, Legionella, Pseudomonas, and Salmonella infections [Numerous studies have shown that the interaction between free-living amoeba and human pathogens, such as ibiotics –33. In aibiotics , 35. Manfections –23, 36.in vitro was previously discussed in many researches. Although previous studies showed that the optimal temperature for studying the host-pathogen interaction within Dictyostelium cells is 21–23°C [Francisella shows a little bit different behavior in comparison to other bacterial pathogens. The optimal growth of bacteria and D. discoideum was achieved at temperature 27–30°C. At lower temperatures, the growth of bacteria was significantly lower. Giving the concern that lower temperate may influence the expression of virulence traits, probably F. novicida somehow needs higher incubation temperature to be able to successfully replicate in this host. Our data show that F. novicida does not survive at 42°C, but it grows at 25 and 37°C. However, previous studies showed that subspecies holartica is more tolerant to a higher temperature (42°C) [Francisella requires different incubation temperatures. Various researches indicated that the findings of incubation temperature might improve the understanding of pathogenicity potential of the pathogens with the fastidious nature, such as L. pneumophila [The effect of incubation temperature on the replication of Gram-negative bacteria  21–23°C , Francise (42°C) . Each spF. novicida within D. discoideum. The growth of bacteria within amoeba was temperature-dependent. Higher survival of Francisella within Dictyostelium was noted at a higher temperature (30°C) at the dose of infection 10. Due to F. novicida adaptation to environmental conditions, bacteria also have the ability to survive within amoeba at lower temperatures (22°C). In contrast, studies showed that Legionella pneumophila is unable to replicate intracellularly at room temperature, while it grew significantly within amoeba at 30, 32, and 37°C [F. novicida growth within amoeba will result in a better understanding of F. novicida ecological niches.In addition, incubation temperature is crucial for the interaction between bacteria and amoeba. For the first time, in this study, we investigated the influence of incubation temperature on the replication of and 37°C , 39. SinF. novicida within amoeba with a high level of cytotoxicity. The optimal dose of infection is 10 at the incubation temperature of 30°C since it induces linear growth of F. novicida with low cytotoxicity in Dictyostelium.Our results demonstrate that the higher MOI (100) induce higher replication of F. novicida within amoeba depend on temperature and bacterial concentration. Since the free-living amoeba may be a reservoir for bacterial pathogens, growth factors in the interaction between bacteria and amoeba need to be clarified in future research for different Francisella subspecies.Altogether, the survival and replication of


Arctium lappa of the family Compositae. It is a commonly used veterinary herbal medicine, and arctigenin is the main active ingredient. The aim of this study was to characterize the absorption, distribution, metabolism, and excretion of arctigenin and Fructus arctii powder in piglets. These data were used to provide a theoretical reference for the development and clinical use of new veterinary drugs. Sixteen healthy piglets (mean weight 30.0 ± 5.0 kg) were divided into two groups. One group was administered 2.0 mg/kg body weight (bw) arctigenin intravenously, and the other was administered 1.0 g/kg.bw Fructus arctii powder by gavage. Blood samples were collected from the anterior vena cava at different time points, and the concentration of arctigenin in the plasma of the piglets was determined using high-performance liquid chromatography (HPLC). Arctigenin conformed to a two-compartment model with no absorption, and the main pharmacokinetic parameters were as follows: distribution half-life (t1/2α)−0.166 ± 0.022 h; elimination half-life (t1/2β)−3.161 ± 0.296 h; apparent volume of distribution (Vd)−0.231 ± 0.033 L/kg; clearance rate (CLb)−0.057 ± 0.003 L/(h.kg); and area under the curve (AUC)−1.189 ± 0.057 g.h/mL. The pharmacokinetic parameters of arctigenin following oral administration of the Fructus arctii powder were as follows: absorption half-life (t1/2ka)−0.274 ± 0.102 h, t1/2α−1.435 ± 0.725 h, t1/2β−63.467 ± 29.115 h, Vd−1.680 ± 0.402 L/kg, CLb−0.076 ± 0.028 L/(h kg), peak time (tmax)−0.853 ± 0.211 h, peak concentration (Cmax)−0.430 ± 0.035 g/mL, and AUC−14.672 ± 4.813 g/mL. These results indicated that intravenous arctigenin was sparingly distributed in tissues. In contrast, orally administered Fructus arctii powder was rapidly absorbed, more widely distributed, and more slowly eliminated than the intravenous arctigenin, which may indicate its sustained pharmacological effects.Fructus arctii, also known as great power seed, is the dried fruit of  Arctium lappa of the family Compositae. It is a commonly used veterinary herbal medicine and is cold, pungent, and bitter in taste. It is associated with the lung and stomach meridians and can eliminate wind-heat and promote healing lung rashes. Arctium lappa is used by veterinarians to treat external wind-heat, cough, asthma, sore throat, and other conditions  was purchased from Shanghai Ronghe Pharmaceutical Technology Co., Ltd. A standard solution containing 1.0 mg/mL arctigenin was prepared in a 5% methanol (aqueous) solution. An injection solution was prepared by dissolving arctigenin (10.0 mg/mL) in propylene glycol and water. Fructus arctii powder (0.33% arctigenin and 4.33% arctiin) was prepared by crushing Fructus arctii to 100 mesh granules. Arctiin and arctigenin content in Fructus arctii powder was determined per the methods in the veterinary pharmacopeia of the People's Republic of China. The dosage of arctigenin and Fructus arctii powder in piglets was determined according to the veterinary pharmacopeia of the People's Republic of China.The instruments used in this study were an Agilent 1200 high-performance liquid chromatography (HPLC) system equipped with an autosampler and ultraviolet detector (USA), a D3024R high-speed centrifuge , an electronic balance (0.0001 g) , and an N-EVAP™ 111 Nitrogen Blowing Apparatus .Sixteen healthy piglets  were purchased from Wuhan Bomu Biotechnology Co., Ltd. They were randomly divided into two groups (nos. 1–8 and nos. 9–16). Each group consisted of four males (nos. 5–8 and 13–16) and four females (nos. 1–4 and 9–12). The piglets were housed in the animal experiment center of Wuhan Academy of Agricultural Sciences. The animal study was conducted per lab animal ethical review number 20160521031. The temperature of the pigsty was controlled at (24.0 ± 1.0°C), and the piglets were allowed free access to water and food. The feed was a diet without any drugs to prevent confounding results due to other drugs. Prior to execution of experiments, the piglets were fed normally for 2 weeks. After completion of the experiments, normal feeding was carried out.18 , 280 nm, methanol/acetonitrile/water (31:20:49 V/V/V), 0.8 ml/min, 30 μL, and 30 ± 0.1°C, respectively  was injected onto the HPLC system for analysis (Plasma samples were stored in a −20°C freezer and were thawed at 20 ± 5°C. Samples (0.5 mL) were accurately transferred to 5.0-ml centrifuge tubes, 2.0 mL of dichloromethane was added, and the mixture was vortexed for 60 s. The mixture was centrifuged at 9,000 × analysis –18.x) and concentration (y) of arctigenin were plotted, and the standard curve in plasma was established. The regression equation was used to calculate the correlation coefficient (r).Blank plasma (500 μL) was added to 5.0-mL polypropylene centrifuge tubes, and this was followed by a series of diluted arctigenin working standard solutions. The resulting spiked plasma samples contained 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, and 5.0 μg/mL arctigenin. The samples were extracted using the procedure described in section Plasma Sample Treatment and analyzed using HPLC. The measured peak area (Blank plasma (500 μL) was transferred to 5.0-mL polypropylene centrifuge tubes. Diluted bovine aglycone working standard solutions at high, medium, or low concentrations were added, and the solutions were mixed well. The samples were prepared  according to the section Plasma Sample Treatment and analyzed using HPLC. Five concentrations were analyzed each day to determine the intraday coefficient of variation. Repeat analyses were evaluated on five different days to determine the interday coefficient of variation. Furthermore, the recovery, accuracy, precision, and sensitivity of the method were calculated according to the following formulae:Accuracy (extraction recovery) = peak area of drug injected after sample extraction/peak area after standard solution injection ×100%;Precision (intraday and interday coefficient of variation) = standard deviation/average ×100%;Limit of detection (LOD): The lowest concentration at which the signal-to-noise ratio (S/N) was ≥3;Limit of quantification (LOQ): The minimum concentration at which S/N was ≥10, and the accuracy and precision were both within the acceptance criteria..body weight (bw) arctigenin by intravenous injection, and the second group (nos. 9–16) was given 1.0 g/kg.bw Fructus arctii powder by gavage. In both groups, blood samples were collected from the anterior vena cava (5.0 mL) at 0.167, 0.333, 0.5, 0.75, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, and 8.0 h for the first group, and at additional time points of 12.0, 24.0, and 48.0 h for the second group (nos. 9–16 piglets). Blood samples were collected in heparinized polypropylene centrifuge tubes. The samples were centrifuged for 15.0 min at 3,000 × g, and the supernatants were collected and stored at −20°C until pharmacokinetic analysis  was administered 2.0 mg/kganalysis .The plasma samples collected at each time point after arctigenin administration were analyzed by HPLC (according to the section Plasma Sample Treatment), the peak area of arctigenin was calculated, and the standard curve regression equation was used to calculate the plasma concentration of arctigenin.Excel software was used to generate the plasma standard curve and the concentration–time curve. Pharmacokinetic model fitting and pharmacokinetic parameters were analyzed using the 3p97 pharmacokinetic software created by a mathematical professional committee of the Chinese Pharmacological Society.The retention time of arctigenin (ACT) was 14.0 min. The peak was symmetrical, the chromatogram showed a stable baseline, and the peak was well resolved from the impurity peak. The method also showed good sensitivity for arctigenin.r = 0.9998) across the range of 0.05–10.0 μg/mL. The regression equation was y = 0.0265 x – 0.0328 (where x represents the peak area of arctigenin and y represents the plasma concentration of arctigenin). The calibration curve of arctigenin in piglet plasma is shown in The plasma concentration curve for arctigenin was linear  is summarized in C = 1.479e−4.194t + 0.187e−0.221t. The distribution half-life (t1/2α) was 0.166 ± 0.022 h, the elimination half-life (t1/2β) was 3.161 ± 0.296 h, and the apparent volume of distribution (Vd) was 0.231 ± 0.033 L/kg. These results suggested that, in piglets, burdock glycosides were distributed rapidly, but distribution into tissues was low and elimination was rapid due to metabolism.After intravenous injection of arctigenin, the plasma concentration–time data of the arctigenin piglets conformed to the non-absorbed two-compartment model with the formula .bw) is shown in The time course of blood arctigenin concentration in piglets fed a single dose of Fructus arctii powder  was 0.430 ± 0.035 μg/mL, the absorption half-life (t1/2ka) was 0.274 ± 0.102 h, the elimination half-life (t1/2β) was 63.467 ± 29.115 h, and the apparent volume of distribution (Vd) was 1.680 ± 0.402 L/kg. The powder was rapidly absorbed, widely distributed, and was slowly eliminated.The blood arctigenin concentration–time data adhered to the absorption chamber model. The formula was Arctigenin has excellent therapeutic potential. This study evaluated the pharmacokinetics of arctigenin and Fructus arctii to characterize their absorption, distribution, metabolism, and excretion in piglets and provides a theoretical basis and reference for the development and clinical use of new veterinary drugs.t1/2α) (0.166 ± 0.022 h) was short and the apparent distribution volume (Vd) (0.307 ± 0.033 L/kg) was low, indicating that arctigenin was rapidly distributed, but distribution into tissues was relatively low. Arctigenin was mainly distributed in the blood and the extracellular fluid (t1/2β) (3.161 ± 0.296 h) was relatively short, indicating that elimination occurred rapidly and did not correlate with distribution.After intravenous injection of arctigenin, the distribution half-life (ar fluid . The elit1/2ka) of Fructus arctii powder was 0.274 ± 0.102 h and the peak time (tmax) was 0.853 ± 0.211 h, indicating that absorption was rapid and the time to reach the maximum blood concentration was short. The t1/2β (63.467 ± 29.115 h) was long, the Vd(1.680 ± 0.402 L/k) was large, and the peak concentration Cmax (0.430 ± 0.035 μg/mL) was relatively low. These results indicate that arctigenin was eliminated slowly and was distributed to tissues, resulting in a relatively low concentration of the drug in the blood, which may indicate a sustained pharmacological effect (t1/2β), apparent volume of distribution (Vd), and area under the concentration–time curve (AUC). These differences were significant (p < 0.01). These results indicated that the gastrointestinal system greatly influenced the absorption, distribution, metabolism, and excretion of arctigenin.The absorption half-life (l effect . A previl effect . This waTmax) of the first and second curves were 0.5 and 1.5 h, at which the peak concentrations (Cmax) were 0.606 ± 0.036 and 0.413 ± 0.036 μg/ml, respectively. This bimodal phenomenon was also observed in a pharmacodynamic study of Fructus arctii in mice by Yuan (O-glucuronide, and 4-O-demethylarctigenin were identified as three novel metabolites (After intravenous injection of arctigenin, the plasma concentration–time data in piglets conformed to the non-absorbed two-compartment model. These results were similar to those of a previous study, which evaluated intravenous injection of different doses of arctigenin and its valine ester derivative in Wistar rats . The blo by Yuan . Additio by Yuan . Ikeda e by Yuan . Gao et  by Yuan . Gao et abolites . These rCmax and AUC0−10h values of arctigenin administered orally to diabetic rats compared with normal rats, respectively. However, after intravenous injection, there were no significant differences in Cmax or AUC0−10h between normal and diabetic rats (Zeng et al. found that the function of p-glycoprotein was impaired in diabetic rats and that arctigenin is a substrate for p-glycoprotein, resulting in increases of 356.8 and 223.4% in the tic rats . The bloin vivo, and reabsorption resulted in a second absorption peak, which may have been indicative of enterohepatic circulation (Pharmacokinetic studies of traditional Chinese medicines often result in bimodal absorption time courses due to the complex nature of these medicines and conversion of components to the same parent molecule . In a stculation . The bimIn this study, HPLC was used to quantitate arctigenin in piglet plasma. The method was sensitive, specific, and rapid. This method can be used to support preclinical pharmacokinetic studies of arctigenin with new veterinary drugs. The pharmacokinetic characteristics of Fructus arctii and its main active ingredient arctigenin in piglets were characterized. The results showed that intravenous administration of arctigenin exhibited a two-compartment model without absorption in piglets and showed poor tissue distribution, short half-life, and rapid elimination. Oral administration of Fructus arctii exhibited a two-compartment model of arctigenin absorption characterized by fast absorption, wide distribution, and slow elimination, with potential hepatoenteric circulation, which suggested that arctigenin may exert effects for a prolonged period of time. Dosing amount and frequency should be controlled to prevent side effects caused by drug accumulation in the body due to slow elimination.All datasets generated for this study are included in the manuscript and/or the supplementary files.There is no violation of ethics or animal welfare in this study. The experiment is authorized by Wuhan academy of agricultural sciences and is willing to accept social supervision.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


To compare the axial length difference (ALD) and the estimated generational axial length shift (ALS) from parents to their children and its risk factors in urban and rural China.  Participants were enrolled from two longitudinal cohort studies, the Beijing Myopia Progression Study (BMPS) and the Handan Offspring Myopia Study (HOMS). Ocular biometry was performed in both parents and their children. ALD was defined as the difference between the children's axial length and the corresponding parental axial length. Generational ALS was estimated according to a binominal prediction model at 18 years of age. β = 0.26, p < 0.001; rural β = 0.11, p < 0.001) and males had larger ALD  in both areas. Furthermore, urban children with more educated parents  and more outdoor activity  had a less ALD.  237 and 380 urban and rural Chinese children (6–17 years) and their parents from the BMPS and HOMS, respectively, were enrolled. Children's axial length was estimated to be closest to the parental axial length at 11 and 9 years of age in the urban and rural areas, respectively; the estimated generational ALS would be 1.53 and 0.57 mm, respectively. Multivariable regression analysis revealed that older children (urban  The urban generational axial length shift was estimated to be approximately 1 mm longer than that of the rural area. These results suggest different environmental effects on the ocular development in these two populations of Chinese children. Myopia in school children is a major public health problem in both rural and urban populations in East Asia , 2. BothIt has been reported that axial length is longer, vitreous chamber is deeper, lens is thinner, anterior chamber is deeper, and the cornea is flatter in myopes , 8. SincThe purpose of the present study was to describe the quantitative, age-specific, axial length difference (ALD), and its generational shift from parents to their children, as well as the risk factors related to ALD, in urban (BMPS) and rural (HOMS) areas of northern China. Other ocular parameter differences (OPDs), including central anterior chamber depth difference (ACDD), length thickness difference (LTD), and vitreous chamber depth difference (VCDD), are also reported.The BMPS was a three-year, hospital-based, cohort study that primarily aimed to investigate the possible relationship between near work-induced transient myopia (NITM) and myopia progression in Chinese children . The HOMChildren and their parents who completed the ocular biometry examination were included in this study. The exclusion criteria were as follows: (1) children or parents with amblyopia or strabismus; (2) children or parents with a history of intraocular surgery or penetrating ocular trauma; and (3) children or parents with significant medical or ocular health problems. The participants (both children and their parents) received comprehensive vision examinations and the detailed related questionnaire.Subjects lay supinely on the testing bed. One drop of 0.5% proparacaine HCL  was instilled in each eye. They were then instructed to focus and fixate upon a red target on the ceiling directly above his/her head using the contralateral eye during the measurement. Ocular parameters including anterior chamber depth (ACD), lens thickness (LT), vitreous chamber depth (VCD), and axial length (AL) of both eyes were measured by a biometer A-scan ). Ten values of each parameter were obtained, and the average value was used for further analysis. One drop of tobramycin 0.3%  antibiotic ointment was instilled in each eye after the measurements were completed.The questionnaire used in the Sydney Myopia Study  was tranChildren's ocular parameters were defined and quantified as the average of the right eyes, while the parental ocular parameters were defined and quantified as the average of the fathers' right eyes and mothers' right eyes. The OPDs were defined as the difference between the ocular parameters of the children and their parents, i.e., the children's OPDs minus their parental average OPDs.R2 and smallest quasi-likelihood under the independence model criterion as compared to other comprehensive fitting functions, such as linear or logarithmic functions. Generational axial length shift was defined as the estimated ALD according to the binominal fitting function at the age of 18 years, since the age of myopia stabilization in the majority of children was reported to be less than 18 years [The data analysis methods in the current study were similar to our previous reports on generational myopic shift , 6. Sinct-test. Pearson correlation between the OPDs and the children's age was performed. Generalized linear models were performed to calculate the age- and gender-adjusted ALD and to determine the association between ALD and the putative risk factors assessed from the questionnaire. Statistical analysis was performed with Statistical Analysis System for Windows version 9.1.3 . A p value less than 0.05 was considered to be statistically significant.Data having a normal distribution were presented as the mean ± standard deviation and tested with the Student's A total of 234 urban families (234 pairs of parents and 237 children) with completed ocular parameter data were enrolled. No family was excluded in the urban area. A total of 246 rural families (246 pairs of parents and 400 children) had completed ocular parameter data. Twelve families (12 pairs of parents and 20 children) were excluded due to presence of amblyopia, strabismus, or significant ocular history of a family member. Thus, 234 rural families (234 pairs of parents and 380 children) were enrolled. There were 121 boys (51.1%) and 210 boys (55.3%) in the urban and rural areas, respectively. Characteristics of the parents are also presented in r = 0.41, p < 0.001; r = 0.61, p < 0.001; r = 0.62, p < 0.001) and rural  areas. p < 0.001). The ACDD increased from 0.21 ± 0.28 mm and 0.51 ± 0.29 mm in the urban and rural children at 6.0–7.9 years of age, respectively, to 0.54 ± 0.31 mm and 0.74 ± 0.31 mm at 16.0–17.9 years of age, respectively (both ptrend < 0.001). The urban children exhibited a shorter AL than their parents at age 6.0–9.9 years (both p < 0.001), no significant difference at 10.0–11.9 years (p=0.52), and had longer AL at 12.0–17.9 years old . Correspondingly, the rural children demonstrated a shorter AL than their parents at age 6.0–7.9 years (p=0.009), no significant difference at 8.0–9.9 years (p=0.43), and had longer AL at 10.0–17.9 years . The ALD increased from −0.96 ± 1.02 mm and −0.32 ± 0.74 mm in the urban and rural children at 6.0–7.9 years, respectively, to 1.17 ± 0.97 mm and 0.57 ± 0.73 mm at 16.0–17.9 years, respectively (both p < 0.001). Similar results were found for VCD in both areas. The LTD was negatively correlated with the children's age in both the urban  and rural areas . Children showed thinner LT than their parents at each age group . The LTD decreased from −0.56 ± 0.22 mm at 6.0–7.9 years to −0.76 ± 0.19 mm at 16.0–17.9 years in the urban children (ptrend < 0.001) and −0.70 ± 0.31 mm to −0.84 ± 0.24 mm, correspondingly in the rural children (ptrend=0.024).The ACDD, VCDD, and ALD were positively correlated with the children's age in both the urban  and 9 (ALD = −0.05 mm) years in the urban and rural areas, respectively. Furthermore, the ALD would be 1.53 mm and 0.57 mm at the age of 18 years in the urban and rural areas, respectively . The proβ = 0.26, p < 0.001; rural β = 0.11, p < 0.001) and were male  had larger ALD in both the urban and rural areas, after adjusting for children's gender and age, respectively. After adjusting for both age and gender, children with more educated parents  and more time in outdoor activity  had less ALD in the urban area. However, no significant association between ALD and near work time, books read, continuous reading time, and living environments in both urban and rural areas was found  share extremely close genetic backgrounds, including myopigenic ones. Hence, these children would be predicted to develop similar axial lengths, if there were no other factors involved. Before about the age of 11 years, the rural ALD fitting curve was above the urban one . This maβ = 0.10, p=0.16; β = −0.03, p=0.32). Hence, this trend of refraction and axial length between generations may not be attributed to a physiological decrease of ocular biometric parameters in adults younger than 45 years but predominantly due to increased exposure to myopigenic environmental factors between the two generations [The current study provides further evidence for the predominant cohort effect on myopia development. Epidemiological studies have found a remarkably increasing trend in the prevalence of myopia in the same area decades later in both children –29 and aThere were some potential limitations of this study. First, the representativeness of the urban area may be limited, since the BMPS was a hospital-based study. Second, there was a likely undersampling in some age groups. Third, only children aged 6 to 17 years were enrolled for this study. It would have been optimal to include young adults greater than 18 years of age as well, which will help to predict even better the generational axial length shift. Hence, further studies with a larger sample and wider age range are warranted.In summary, this study found different axial length developmental patterns between rural and urban children in China. The estimated generational axial length shift was approximately 1 mm more in the urban children compared to the urban cohort. Environment factors, such as parental education level and outdoor activity, may influence the axial length difference between the two generations.


The aim of this study was to assess the effects of residual anterior lens epithelial cell (LEC) removal by anterior capsule polishing on the effective lens position (ELP) and axial position stability of the intraocular lens (IOL) after cataract surgery via postoperative measurement of the anterior chamber depth.  We enrolled 30 patients (60 eyes) requiring bilateral cataract surgery for age-related cataracts. Meticulous anterior capsule polishing and removal of residual LECs under the capsule were performed using a bimanual irrigation/aspiration system for one randomly selected eye in each patient. The eye without polishing served as a control. ELP was measured at five different time points after surgery, and axial shifting of IOL was determined at each visit by comparison with the position at the previous visit. p=0.026) and 2 months  after surgery, the mean standard deviation for the five ELP values , and the root mean square of the change in ELP at each follow-up visit . The eyes in the control group exhibited a tendency for backward IOL movement with a concurrent hyperopic shift in refraction of approximately 0.2 diopter at 2 months after surgery.  The polishing and control groups showed significant differences with regard to the mean ELP at 1 (3.40 ± 0.29 versus 3.53 ± 0.32 mm, resp.;  Our findings suggest that residual anterior LEC polishing enhances the axial position stability of IOLs, without any complications, after cataract surgery. In recent years, phacoemulsification with concurrent foldable intraocular lens (IOL) implantation has taken the place of refractive surgery, with the development and increase in the popularity of various refraction-correcting IOLs, including multifocal or toric IOLs. In addition to an accurate IOL power calculation formula and ocular biometry, the precise determination of the effective lens position (ELP) is essential to optimize the postoperative refractive outcomes , 2. ELP,Residual lens epithelial cells (LECs) after cataract surgery play a significant role in the development and progression of capsule fibrosis and contraction –10. SeveWe enrolled 30 patients (60 eyes) from January 2016 to April 2017. The research was performed at the Department of Ophthalmology in Dongsan Medical Center, which is affiliated with Keimyung University in Daegu, Republic of Korea. All studies and measurements were in accordance with the tenets of the Declaration of Helsinki, and the study protocol was reviewed and approved by the Ethics Committee of Dongsan Medical Center . Informed consent for participation was obtained from all patients. The inclusion criteria were as follows: bilateral age-related cataract with a favorable clinical status and uneventful in-the-bag IOL implantation in both eyes. The exclusion criteria were as follows: a history of intraocular surgery or corneal laser surgery, a history of ocular trauma or uveitis, severe fundus pathology, an axial length (AL) of <22.0 mm or >24.0 mm, pseudoexfoliation syndrome (PEX), poor pupil dilation, zonular weakening or tension ring insertion, too large or too small continuous curvilinear capsulorhexis (CCC), eccentric CCC, radial tear in the anterior capsule, and inability to attend follow-up appointments on time.Before surgery, the opacity in each eye was evaluated using the Lens Opacities Classification System III (LOCS III)  after puAll study patients underwent the necessary laboratory tests and clinical examinations. The IOL power was set to achieve postoperative refraction between +0.25 and −0.25 diopter (D) using optical low-coherence reflectometry . The preoperative best-corrected visual acuity (BCVA) was confirmed and converted to logMAR units. The anterior chamber depth (ACD) was measured before surgery using A-scan . Approximately 30 min before surgery, 0.5% tropicamide/0.5% phenylephrine fixed combination eye drops  were instilled in the patients' eyes twice within 5 min for maximal pupil dilation. A single surgeon (JHJ) performed all surgeries with a 2.85 mm coaxial incision . After topical anesthesia with 0.5% proparacaine eye drops , a clear corneal incision was placed at the 9 o'clock (right eye) and 2 o'clock (left eye) positions. A centered CCC with a 5.5 mm diameter was prepared using capsulorhexis forceps. For the precise achievement of an equal CCC size, we used a 6 mm diameter capsulorhexis marker  for a 5.5 mm diameter CCC; too large, too small, or eccentric CCCs were excluded. Subsequently, thorough hydrodissection was performed to freely rotate the nucleus, following which phacochop nucleofractis was used for emulsification and removal of the nucleus.To eliminate any bias, the decision to perform anterior polishing or not was made only after the completion of cortex aspiration using an ordinary one-hand irrigation/aspiration system, without reference to the amount of visible residual LECs. Subsequently, the eyes were assigned to a polishing or control group using a table of random numbers generated by Microsoft Excel for Windows 2013, according to Monte Carlo calculations. For the control group eyes, no additional anterior capsular polishing was performed after routine cortex aspiration. For the polishing group eyes, residual LECs in the anterior capsule were aspirated using a bimanual irrigation/aspiration system . AccessiRMS) could determine the axial position stability of IOLs more precisely than the mean ELP could; we calculated ELPRMS for each group at 2 months after surgery using the following formula:ELP was defined as the distance from the anterior surface of the cornea to the anterior surface of IOL in the pupil center, along the optical axis. It was measured on an A-scan at 1 day, 3 days, 1 week, 1 month, and 2 months after surgery. Comparison of the mean ELP was considered inappropriate because forward and backward movements could be partly neutralized; therefore, we compared the mean standard deviation (SD) for the five ELP values, calculated at 2 months after surgery, between the two groups. Furthermore, on the basis of a report by Eom et al. , who repHere, 1D, 3D, 1W, 1M, and 2M represent 1 day, 3 days, 1 week, 1 month, and 2 months after surgery, respectively.The postoperative BCVA was recorded in logMAR units, and autorefraction  was performed at each visit to evaluate the surgical outcomes. To demonstrate the discrepancy between the preoperatively calculated refraction (Lenstar LS900®) and the postoperative refraction at each time point, we calculated the postoperative refraction error (PRE) as the postoperative spherical equivalent (SE) minus the preoperative SE (SE = sphere + cylinder/2).The preoperative central corneal endothelial cell density (ECD), coefficient of variation (CV), and percentage of hexagonal cells were measured with a specular microscope . To rule out the effect of anterior capsule polishing using a bimanual irrigation/aspiration system on corneal endothelial cell loss, the central corneal ECD, CV, and hexagonality were measured again at 1 and 2 months after surgery.t-test was used. A p value of <0.05 was considered statistically significant.The number of participants required to achieve a statistical power of 80% at a level of significance of 0.05 was 27. This sample size calculation was performed using the statistical freeware G∗Power (version 3.1.9.2) . Data arp > 0.05).Sixty eyes of 30 patients were included, and nine patients (33.3%) were men. All patients underwent uneventful surgeries with no intraoperative or postoperative complications and returned on time for measurements. The mean age of patients was 74.1 ± 8.9 years . p=0.026 and 0.004, resp.). The mean SD for the five ELP values was significantly smaller for the polishing group (0.087 ± 0.093 mm) than that for the control group . ELPRMS was also significantly smaller for the polishing group (0.124 ± 0.034 mm) than that for the control group .PRE was 0.29 ± 0.74, 0.23 ± 1.33, −0.03 ± 1.23, 0.02 ± 0.78, and 0.06 ± 0.60 D at 1 day, 3 days, 1 week, 1 month, and 2 months after surgery, respectively, in the polishing group. For the control group, these values were 0.27 ± 0.43, 0.25 ± 0.93, 0.13 ± 0.66, 0.23 ± 1.01, and 0.27 ± 0.93 D, respectively. There were no significant differences between the two groups .BCVA at 1 day, 3 days, 1 week, 1 month, and 2 months after surgery was 0.08 ± 0.10, 0.06 ± 0.09, 0.03 ± 0.06, 0.03 ± 0.06, and 0.02 ± 0.05, respectively, in the polishing group. These values were 0.07 ± 0.10, 0.05 ± 0.08, 0.03 ± 0.06, 0.02 ± 0.06, and 0.01 ± 0.04, respectively, in the control group. There were no significant differences between groups (1 day: p > 0.05).There are many studies concerning the influence of anterior capsule polishing during surgeries involving the implantation of different IOLs on anterior capsule opacification (ACO), posterior capsule opacification (PCO), and the size of the capsulorhexis opening , 14, 17.α-smooth muscle actin elements that lead to anterior capsule contraction, constriction of the capsulorhexis and ACO [The mechanism underlying backward IOL movement in the absence of anterior capsule polishing remains uncertain. After cataract surgery, residual anterior LECs undergo fibrous metaplasia after contact with the anterior IOL surface . These m and ACO . This en and ACO . In fact and ACO . TherefoThe axial position stability is also related to the mechanical characteristics of IOL, such as the design and material of the optic/haptic, optic-haptic angulation, and diameter . In the t-test that we used. A larger population size, as well as follow-up periods that extend beyond 1 year , are necessary in future studies. Furthermore, we measured postoperative ELP using A-scan ultrasound images, not AS-OCT. Nemeth et al. [The main limitation of this study is the short follow-up period of 2 months after surgery, during which few instances of ACO, as well as no PCO, were detected. Moreover, our study population size is somewhat small despite the high statistical power of the paired h et al.  reportedIn the present study, we polished the anterior capsule to eliminate residual LECs using a bimanual irrigation/aspiration system to assess the effects of this procedure on the axial position stability of hydrophobic, spherical, one-piece IOLs. We found that anterior capsule polishing enhanced the axial position stability of IOL without any complications. Without anterior capsule polishing, IOL tended to move backwards by approximately 0.2 mm, with a concurrent hyperopic shift in refraction of approximately 0.2 D, at 2 months after surgery. With the advancement of various refraction-correcting IOLs, the axial position stability becomes an important aspect. Therefore, anterior capsule polishing using a bimanual irrigation/aspiration system during cataract surgery may be considered a useful procedure, particularly for eyes receiving refraction-correcting IOLs.


Translocation of N and DM in the post-anthesis periods were enhanced by 38.4% and 50.9%, respectively. NC could also stimulate enzyme activities, and increased 39.8% and 57.1% in flag leaves, and by 36.0% and 58.8% in spikes, respectively, at anthesis. SPS and PEPC increased by 28.2% and 45.1% in flag leaves, and by 42.2% and 56.5% in spikes, respectively, at 15 days after anthesis. The results indicated that the NC promoted N metabolism more than C metabolism, and resulted in the enhancement of grain yield by 27.56% and of crude protein concentration in grain by 13.26%, respectively.Nanochitin whisker (NC) with a cationic nature could enhance plant photosynthesis, grain yield, and quality of wheat, but have not been systematically studied. This study was designed to investigate the stimulatory effects of NC on dry matter (DM) and nitrogen (N) accumulation and translocation, and on the metabolism of carbon (C) and N in later growth stages of winter wheat to reveal the enhancement mechanism of grain yield and crude protein concentration. Different parts of NC-treated plants from pot grown experiments were collected at the pre- and post-anthesis stages. The accumulation, translocation, and contributions of DM and N from pre-anthesis vegetation organs to grains, as well as key metabolic enzyme activities, including sucrose phosphate synthase (SPS) and phosphoenolpyruvate carboxylase (PEPC), were examined. The results showed that, at an application rate of 6 mg·kg Metabolism of carbon (C) and nitrogen (N) is the most basic metabolic process in plants . The accPlant growth and development are not only dependent on the absorption of carbon, nitrogen, and other essential elements for photosynthesis, but are also related to interdependence between C- and N-metabolism. Sucrose phosphate synthase (SPS), phosphoenolpyruvate carboxylase (PEPC), glutamine synthetase (GS), and nitrate reductase (NR) are important metabolic enzymes in N assimilation, anaplerotic C flow, and sucrose synthesis in the leaves of many higher plant species ,13. The Arabidopsis root, the plant hormones regulating plant growth and development [Chitin, the second most abundant polysaccharide on earth, is composed of N-acetylglucosamine oligosaccharide monomer. Chitosan and chitooligosaccharide, derivatives of chitin, have been studied for their effects on crop growth promotion, C or N metabolism, stress resistance, and plant disease defense elicitation ,18,19. Aelopment . The bioelopment ,26.2 concentrations, and transpiration rate in flag leaf at the grain-filling stage [In our previous studies, we found that cationic nanochitin whisker when applied at an optimal rate could inhibit root rot diseases of tobacco and wheat ,27, promng stage . Grain pThe characteristics, including size and size distribution, zeta potential, and morphology of the nanochitin particles, were characterized by DLS and transmission electron microscopy  respectively. The results showed that the final product of the nanoparticle was positively charged, with an average hydrodynamic diameter of 138.9 nm , and a z−1, the treatments with 2 mg·kg−1 and 6 mg·kg−1 of nanochitin in the soil significantly increased grain yield and protein concentration in wheat, in which the grain yield was increased by 19.14% and 27.56%, respectively, and the crude protein concentration was increased by 9.92% and 13.26%, respectively, which are in agreement with our previous report [−1, the nanochitin had less effects on the yield components of wheat, even though the kernel weight still increased slightly at this application rate. When the 20 mg·kg−1 application rate was compared to the 0 mg·kg−1 rate, there were no significant changes in the number of spikes, the number of grains per ear, yield, and the crude protein concentration in grain.The yield and protein concentration are the most important factors for wheat production, which are governed by the accumulation and translocation of dry matter and nitrogen. In this study, the grain yield and crude protein concentration of the winter wheat treated with nanochitin first rose and then dropped with the increase in nanochitin application rate, because the number of spikes, grains per spike, and kernel weight (1000 grain weight) were all enhanced with the same dynamic trend according to the application rate of nanochitin in soil . Compares report . However−1 of nanochitin  was the same as those of DMA-A and DMA-M. From −1 and 6 mg·kg−1 nanochitin application rate, the DMA-PA was increased by 7.68% and 15.35%, respectively, while applying 20 mg·kg−1 nanochitin had less effect on DMA-PA.The grain yield and quality could be affected by dry matter and nitrogen accumulation in winter wheat in pre- and post-anthesis. Post-anthesis describes the accumulation of nutrients during the period from flowering to maturity. The total dry matter accumulation at anthesis (DMA-A) and at maturity (DMA-M) in this study were enhanced first and then dropped with the increase in the nanochitin application rate, and reached a peak rate of 6 mg·kgnochitin . Compare−1 and 6 mg·kg−1 nanochitin significantly increased NA-A by 26.21% and 38.83%, and NA-M by 25.20% and 37.40%, respectively. When the nanochitin application rate reached 20 mg·kg−1, N accumulation had no significant enhancement when compared to the control. The effect of the nanochitin application rate on N accumulation in the post-anthesis period (NA-PA) was almost the same as N accumulation at anthesis (NA-A) and mature (NA-M) periods. However, the NA-PA under 2 mg·kg−1 and 6 mg·kg−1 nanochitin application rates was increased by 20.00% and 30.00% respectively, but the nanochitin application rate at 20 mg·kg−1 only enhanced the N accumulation by 15.00%.The total N accumulations in winter wheat treated with nanochitin at anthesis (NA-A) and at maturity (NA-M) were increased first and then decreased with the increase in the nanochitin application rate as well. Compared with the control, the treatment rates of 2 mg·kg−1 of nanochitin application rate , the dry matter translocation (DMT), and percentage of translocation from pre-anthesis vegetative organs to grains (DMTP), as well as the N accumulation in vegetative organs at maturity (NA-VO-M), translocation of N (NT), and percentage of N translocated from pre-anthesis vegetative organs to grains (NTP), and reached a peak at the 6 mg·kgion rate .−1 and 6 mg·kg−1 of nanochitin significantly enhanced DMT by 31.12% and 38.35%, respectively, and DMTP by 3.90 percentage points and 4.17 percentage points, respectively. However, the nanochitin rate of 20 mg·kg−1 had less effects on DMT and DMTP. A similar trend was found for NT and NTP when treated at different rates of nanochitin. As −1 and 6 mg·kg−1 of nanochitin application rates significantly increased NT by 36.36% and 50.91%, respectively, and NTP by 4.07 percentage point and 4.17 percentage point, respectively. When the nanochitin application rate reached 20 mg·kg−1, the NT and the NTP were all below those of 0 mg·kg−1.Compared with the control, both treatments of 2 mg·kgBoth the accumulations of grain dry matter and N are derived from two processes: translocation of vegetative organs in pre-anthesis and the new accumulation in the post-anthesis period. The contributions of dry matter (CR-DMT) and N translocation (CR-NT) from the pre-anthesis vegetative organs to grain, as well as the accumulation of dry matter (CR-DMA) and N (CR-NA) in the post-anthesis period to grain, reflect the relative importance of the sources of dry matter or N in grain, respectively. However, with nanochitin application rate increase, the dynamic trends of the CR-DMA and CR-NA response to nanochitin rate were opposite to that of CR-DMT and CR-NT.−1 and 6 mg·kg−1 of nanochitin application rates significantly decreased by 4.85 percentage points and 4.49 percentage points, respectively, and the CR-NA decreased by 2.16 percentage points and 2.72 percentage points, respectively  and the ratio of N to dry matter translocation from pre-anthesis vegetative organs (NT/DMT) in wheat both increased when treated with nanochitin. −1 of nanochitin significantly enhanced the activities of SPS and PEPC at the anthesis, which increased SPS and PEPC in the flag leaves by 39.75% and 57.09%, and 35.97% and 58.79% in spikes, respectively. At 15 days after the anthesis of the wheat, nanochitin applied at a rate of 6 mg·kg−1 increased the SPS and PEPC in the flag leaves by 28.20% and 45.08%, respectively, and 42.23% and 56.52% in spikes, respectively. The results from enzymatic studies showed that nanochitin could simultaneously improve C and N metabolism in flag leaves and spikes during the anthesis and grain filling stages of wheat. To understand the mechanism of the enhancement of yield and quality, carbon and N metabolism were key factors to explore. In this study, we investigated two important metabolic enzymes, SPS and PEPC, and how their activities were stimulated by nanochitin. From −1 nanochitin decreased the ratio of SPS to PEPC in flag leaves and spikes at the anthesis and the 15-days after anthesis stages of winter wheat. Among them, the decline in flag leaves and spikes were 11.11% and 14.58% at anthesis, and 11.69% and 8.45% at 15 days after anthesis, respectively. The results showed that nanochitin promoted nitrogen metabolism more than carbon metabolism.The ratio of SPS/PEPC indicated the degree of enhancement in C metabolism when N was strengthened. Compared with the control, applying 6 mg·kgC allocation might be an important prerequisite for plant growth in an N-rich environment . Deficie−1 in soil. This phenomenon was attributed to nanochitin whisker’s unique properties, such as its spindle-like structure, nano-scale size, cationic property, and high surface area [−1, nanochitin whisker had less of an effect on the dry matter and/or N accumulation in pre- and post-anthesis, and the yield components of wheat [The results showed that applying nanochitin whisker at an appropriate dosage in the soil could promote the accumulation of dry matter and N in wheat, both at pre- and post-anthesis stages , which wace area ,28. Chitace area ,36. Nanoace area , which iace area ,27,28. Aof wheat .−1, 2 mg·kg−1 and 6 mg·kg−1 of nanochitin whisker in the soil increased the dry matter accumulation at anthesis by 11.2% and 16.12%, and the N accumulation by 26.2% and 38.8%, respectively , and hydrogen peroxide  were purchased from Zhengzhou Shenghe Instruments Co., Ltd. . The water used in the experiment was deionized (DI) water from the Millipore Direct-Q 5 ultrapure water system. All other chemicals used in the nanochitin preparation were analytical grade.Chitin  was purchased from Sigma Aldrich . Hydrochloric acid (HCl) (36–38%), sulfuric acid (Hw/v) was diluted with DI water from stock solution of the nanochitin suspension without adjustment of the pH or ionic strength. For morphology of the particles, a drop of the nanochitin dilution was stained with a 2% uranyl acetate negative stain on a carbon-coated copper grid (200 meshes). The sample was visualized by a JEOL transmission electron microscope (JEM 1400)  with an accelerating voltage of 80 kV.A nanochitin suspension was obtained by acidic hydrolysis following the method described by Xue et al. . Briefly−1 of organic matter in soil, 0.98 g·kg−1 of total N, 19.34 mg·kg−1 of available P, and 108.52 mg·kg−1 of available K. The pH of the soil was 7.32.The pot experiment was conducted in the winter wheat growing season at the experimental station at Henan Agricultural University, Zhengzhou, China. A multi-spike variety of winter wheat of Zhou Mai 32 was used in this study. The soil was fluvo-aquic soil with medium texture, collected from the top layer of the planting area at the North China Scientific Observation and Experimental Station for Nutrition and Fertilization of Wheat/Maize Rotation, Ministry of Agriculture, Xuchang County, Henan province, China. The basic physical and chemical properties of the soil included a concentration of 13.07 g·kg−1 of nanochitin in the soil were set in this experiment as a comparison. Each pot received the same amount of fertilizer with pure 0.15 g·kg−1 N, 0.2 g·kg−1 P2O5, and 0.1 g·kg−1 K2O. All P2O5 and K2O were applied as a starter. N was applied as starter by 2/3 and as a topdressing by 1/3 at the jointing stage. Twelve wheat seeds were planted in each pot at approximately a 2 cm sowing depth. After seedling setting, 2 L of nanochitin suspension, based on the designed application rate, was applied to each corresponding pot. DI water served as a control. During the growth period, general management for pest control and irrigation were conducted as necessary. All experiments were carried out in triplicate for each sampling.Plastic pots, 30 cm × 30 cm in size, were employed and accommodated 10 kg of air dried soil for the test. During the test period, the pots were placed outdoors and arranged randomly with frequent position changes. Based on a previous study for dose response , four ra2SO4-H2O2, and the nitrogen content was determined using a Type AA3 continuous flow analyzer.At anthesis and maturity, the above ground parts of the plants were sampled and divided into different parts by organs. The fresh samples were dried at 105 °C for 30 min, then continuously dried at 65 °C and weighed at 2h intervals until the weight remained unchanged in a period of 24 h. After crushing and sieving through a 60 mesh, the samples were digested by H−1 Tris-HCl buffer (pH 7.2), 10 mmol·L−1 MgCl2, 1 mmol·L−1 EDTA, 10 mmol·L−1 β-Mercaptoethanol, 2% ethylene glycol, and 1% pyrrolidone in a pre-cooled mortar and rapidly ground. The homogenate was centrifuged at 15,000× g, at 4 °C for 10 min and then the supernatant was collected for the measurement. About 0.4 mL of reaction buffer, containing 100 mmol·L−1 Tris-HCl (pH 7.2), 10 mmol·L−1 MgCl2, 5 mmol·L−1 UDPG, and 5 mmol·L−1 fru-6-P, was mixed with the supernatant and incubated first at 30 °C for 30 min, and then in boiling water for 5 min. After adding 0.1 mL of 2 mmol·L−1 NaOH, the reaction liquid was further boiled for 10 min. About 1 mL of 0.1% resorcinol and 3.5 mL of 30% HCl were then added into the reaction and incubated at 80 °C for 10 min. When the reaction finished, the mixture was cooled down and absorbance was measured using a spectrophotometer  at 480 nm wavelength.The metabolic key enzymes, including sucrose phosphate synthase (SPS) and phosphoenolpyruvate carboxylase (PEPC), were extracted and measured for flag leaves and spikes at the flowering stage and 15 days after grain filling. The flag leaves and ears of wheat at flowering and filling stage were selected randomly. The activities of SPS and PEPC were determined following the method described by Wang and Huang . In deta−1 Tris-HCl buffer, containing 5% glycerol, 1% PVP, 1 mmol·L−1 EDTA, and 7 mmol·L−1 mercaptoethanol, at pH 8.2 in a pre-cooled mortar. After rapid grinding, the homogenate was centrifuged at 15,000× g, at 4 °C for 20 min and the supernatant was collected for measurement. About 1 mL of reaction buffer, containing 50 mmol·L−1 Tris-HCl (pH 7.8), 10 mmol·L−1 MgCl2, 0.25 mmol·L−1 EDTA, 5.0 mmol·L−1 NaHCO3, 2.0 mmol·L−1 DTT, 4 U MDH, 0.1 mmol·L−1 NADH, and 2.0 mmol·L−1 PEP, was added and incubated at 28 °C for 10 min. Then, about 200 μL of PEPC extraction solution was added into the reaction. The absorbance at 340 nm wavelength was measured spectrophotometrically.For PEPC measurement, about 0.5 g of wheat sample was mixed with 3 mL of pre-cooled 100 mmol·L−1).−1).−1).−1).−1).The following formulas were adopted from Chen et al.  for the p < 0.05 were considered to be statistically significant.Variance analysis and multiple comparison for the data were conducted using SPSS 22.0 software . Differences at −1 in soil could significantly promote dry matter and N accumulation, and translocation from vegetative organs to grains in post-anthesis, which could result in the considerable enhancement of grain yield and crude protein concentration in grain. This is also attributed to the stimulation of key metabolic enzymes, SPS and PEPC activities, in flag leaves and spikes. As a green nanobiomaterial, there is a great potential for the enrichment of crop production and the reduction of chemical fertilizer use in future agriculture sustainability.Nanochitin whisker at an application rate of 6 mg·kg