Of the 191 metabolites tentatively identified, 154 were new metabolites, 69 were new compounds and 32 were dimers. This is the first report of the in vivo biotransformation of a single compound into more than 100 metabolites. Furthermore, acetylamination and pyroglutamic acid conjugation were identified as new metabolic reactions. Seventeen metabolites were found to have various taxifolin-related bioactivities. The potential targets of taxifolin and 63 metabolites were predicted using PharmMapper, with results showing that more than 60 metabolites have the same five targets. Metabolites with the same fragment pattern may have the same pharmacophore. Thus these metabolites may exert the same pharmacological effects as taxifolin through an additive effect on the same drug targets. This observation indicates that taxifolin is bioactive not only in the parent form, but also through its metabolites. These findings enhance understanding of the metabolism and effective forms of taxifolin and may provide further insight of the beneficial effects of taxifolin and its derivatives.Taxifolin is a ubiquitous bioactive constituent of foods and herbs. To thoroughly explore its metabolism in vivo, an HPLC-ESI-IT-TOF-MS Quercetin (C15H10O7) showed characteristic fragment ions at m/z 229.0526, m/z 211.0386, m/z 179.0015, m/z 151.0061 and m/z 107.0230 in its MS2 spectrum. Dihydrokaempferol (C15H12O6) showed characteristic fragment ions at m/z 269.0431, m/z 259.0613, m/z 243.0663, m/z 201.0564, m/z 173.0622 and m/z 125.0275 in its MS2 spectrum.Taxifolin , −2.01 Da (H2), +2.01 Da (H2), −18.01 Da (H2O), +18.01 Da (H2O), +79.95 Da (SO3) and +176.03 Da (C6H8O6) indicated hydroxylation, dehydroxylation, methylation, dehydrogenation, hydrogenation, dehydration, hydration, sulphation, and glucuronidation, respectively. The molecular formulae were predicted based on HRMS data, and the specific type and structure of metabolites were identified by the fragmentation characteristics in their NI MSn spectra. In total, 191 metabolites  of taxifolin were tentatively identified  of drug and blank groups. The detailed LC-MS data are summarized in Metabolites of taxifolin in rats were identified on the basis of knowledge of taxifolin metabolism and the strategy proposed in our previous study . The metentified  by carefM1 and M2 had the same molecular formula and similar fragmentation behaviour as taxifolin. Because C-2 and C-3 are chiral centres, taxifolin has four stereoisomers − (m/z 303.05) was observed, with identical molecular formula and fragmentation behaviour to taxifolin. We therefore deduced that the metabolites were conjugates of taxifolin. According to their characteristic neutral losses, M3–M11 were identified as sulphates of taxifolin or its isomers. Conjugates M12–M15 were identified as taxifolin disulphates. The molecular formula of M16 was determined to be C20H19NO13S from its HRMS data. The base peak ion at m/z 383.0083 was formed by neutral loss of 129.05 Da (C5H7NO3). According to our previous research − at m/z 349.06. Their molecular formulae were calculated to be C16H14O9, 31.98 Da (2O) more than that of methyl taxifolin and resulting in their temporary identification as methylated and dihydroxyled taxifolin. M66–M69 showed [M − H]− at m/z 525.09 and then yielded [aglycone − H]− at m/z 349.06 by neutral loss of 176.03 Da; the aglycones were identical to M64–M65. Hence, these metabolites were tentatively identified as glucuronides of methylated and dihydroxylated taxifolin.In total, 34 metabolites having the aglycone of quercetin were found from the drug group, including quercetin, isorhamnetin, nine quercetin conjugates, 11 isorhamnetin conjugates and 12 conjugates of hydroxylated quercetin.M70 was formed through dehydrogenation. The [M − H]− of M70 was at m/z 301.0349 (C15H9O7), which is 2.01 Da (H2) less than taxifolin, and the retention time and characteristic fragment ions were the same as those for the reference compound quercetin. M70 was thus determined to be quercetin.Metabolite 2 spectra of M71–M79, the same [aglycone − H]− (m/z 301.04) was observed with identical molecular formula and fragmentation behaviour as quercetin. We therefore deduced that they were conjugates of quercetin. Based on characteristic neutral losses, M71–M75 were identified as quercetin sulphates. According to the ClogP rule, M71 (tR = 51.583) was quercetin-5-O-sulphate (ClogP = −0.897894), M72 (tR = 52.647) was quercetin-7-O-sulphate (ClogP = 0.00210607), M73  and M74  were quercetin-3′/4′-O-sulphate (ClogP = 0.0554161) and M75 (tR = 58.173) was quercetin-3-O-sulphate (ClogP = 0.160939). According to the literature − (m/z 315.05) was observed with identical molecular formula and fragmentation behaviour to isorhamnetin. Hence, these metabolites were considered as conjugates of isorhamnetin. M81–M84 were isorhamnetin sulphates. Based on the ClogP rule, M81 (tR= 48.633) was isorhamnetin-5-O-sulphate (ClogP = −0.452683), M82 (tR = 56.917) was isorhamnetin-7-O-sulphate (ClogP = 0.447317), M83 (tR = 58.042) was isorhamnetin-3-O-sulphate (ClogP = 0.605693) and M84 (tR = 58.922) was the isorhamnetin-4′-O-sulphate (ClogP = 0.631748). M85 was identified as isorhamnetin disulphate and M86–M87 were identified as glucuronides of isorhamnetin. According to the literature −), m/z 259.0621 ([M − H − CO]−), m/z 243.0647 ([M − H − CO2]−), m/z 201.0554 ([M − H − CO2 − C2H2O]−), m/z 173.0683([M − H − CO − CO2 − C2H2O]−) and m/z 125.0290  were consistent with the reference compound dihydrokaempferol. Hence, M105 was identified as dihydrokaempferol.The molecular formulae of 2 spectra of M106–M112, the same [aglycone − H]− (m/z 287.05) with identical molecular formula and fragmentation behaviour to dehydroxylated taxifolin was observed, we therefore deduced that these were conjugates of dehydroxylated taxifolin. The characteristic fragment ions of the [aglycone − H]− of M106 and M108 were the same as those of eriodictyol. Because the main sulphation sites were located at C-3′ and C-7, and based on the ClogP rule, M106 (tR = 37.325) was tentatively identified as eriodictyol-7-O-sulphate (ClogP = 0.224621) and M108 (tR= 37.708) as eriodictyol-3′-O-sulphate (ClogP = 0.398051). The characteristic fragment ions of the [aglycone − H]− of M107 and M109 were identical to those of dihydrokaempferol. Hence, M107 (tR = 38.200) was dihydrokaempferol 7-O-sulphate (ClogP = −0.255279) and M109 (tR = 40.383) was dihydrokaempferol 4′-O-sulphate (ClogP = −0.192048). M110–M112 yielded [aglycone − H]− by neutral loss of 176.03 Da (C6H8O6), which suggested that M110–M112 were glucuronides of dehydroxylated taxifolin. The characteristic fragment ions of M112 were consistent with dihydrokaempferol, so M112 was considered to be dihydrokaempferol glucuronide.In the NI MSM113–M115 showed [M + NH3 − H]− at m/z 478.10 (predicted to be C21H20O12N) in their HRMS data. The [aglycone + NH3 − H]− was formed by the neutral loss of 176.03 Da in the NI MS2 spectra and the aglycone had the molecular formula of C15H10O6, which is 18.01 Da (H2O) less than taxifolin (C15H12O7). The characteristic fragment ions of the aglycone were m/z 217.06 ([M − H − C3O2]−), m/z 175.03 ([M − H − C3O2 − C2H2O]−) and m/z 177.03 , indicating that there were two hydroxyl groups linked to the A-ring and B-ring, respectively. Accordingly, the aglycone was considered as the dehydration metabolite of taxifolin and tentatively identified as luteolin. As a result, M113–M115 were glucuronides of luteolin. Because C-5 was not easily conjugated, the sites of glucuronidation were considered to be the hydroxyl groups of the C-7, 3′ and 4′ positions of luteolin. Based on the ClogP rule, M113 (tR = 16.017) was luteolin-7-O-glucuronide (ClogP = 0.335925), and M114 (tR = 16.583) and M115 (tR = 17.483) were luteolin-3′/4′-O-glucuronide (ClogP = 0.188342). M116 showed [M + NH3 − H]− at m/z 492.1165 (predicted to be C22H23O12N) in the HRMS data. In the MS2 spectrum, the neutral loss of 176.03 Da (C6H8O6) was observed and the aglycone was 14.01 Da (CH2) more than luteolin. Hence, the aglycone was methyl luteolin, and M116 was identified as the glucuronide of methyl luteolin.Four metabolites were identified, including three luteolin glucuronides and one methyl luteolin glucuronide. M117 showed [M − H]− at m/z 305.0652, which was 2.01 Da (H2) more than taxifolin, and the characteristic fragment ions were at m/z 287.0565 (C15H11O6), m/z 183.0309 (C8H7O5), m/z 165.0249 (C8H5O4), m/z 161.0287 (C9H5O3) and m/z 137.0301 (C7H5O3). Therefore, M117 was tentatively identified as a hydrogenated product. The molecular formula of M118 was calculated to be C16H16O7, which is 2.01 Da (H2) more than that of methyltaxifolin; hence, M118 was identified as hydrogenated methyl taxifolin. M119–M121 yielded [aglycone − H]− at m/z 305.06 by neutral loss of 79.95 Da (SO3), indicating that they were hydrogenated taxifolin sulphates.In total, five metabolites including hydrogenated taxifolin, hydrogenated methyltaxifolin and three hydrogenated taxifolin sulphates were detected. In total, 38 metabolites (four new compounds) having the aglycone of phenolic acid derivatives were found in the drug group, including phenolic acids and their conjugations.M122–M130. The [M − H]− of M122–M123 were at m/z 165.06, and characteristic fragment ions at m/z 121.07 and m/z 119.04 were observed in their MS2 spectra. According to a previous report [M122–M123 as 3/4-hydroxyphenylpropanoic acid. M124–M127 yielded [aglycone − H]− at m/z 165.06 by neutral loss of 79.95 Da or 176.03 Da. Hence, M124–M125 were identified as sulphates of hydroxyphenylpropanoic acid. M126–M127 were glucuronides of hydroxyphenylpropanoic acid. M128–M130 yielded [aglycone − H]− at m/z 163.04 (C9H8O3) by the loss of SO3 (79.96Da) and produced characteristic fragment ions at m/z 163.04 (100.0) and m/z 119.06 (17.51). According to a previous report − at m/z 167.0349 (predicted to be C8H7O4), and characteristic fragment ions at m/z 123.0458 were observed in NI MS2 spectrum. According to a previous report − at m/z 167.04 by neutral loss of 79.95 Da, indicating that these were sulphates of dihydroxyphenylacetic acid. M135 showed [M − H]− at m/z 261.0073 and yielded [aglycone − H]− at m/z 181.0569 by neutral loss of 79.95Da (SO3) with characteristic fragment ions at m/z 217.0189 ([M − H − CO2]−), 181.0569 ([M − H − SO3]−), 137.0659 ([M − H − SO3 − CO2]−) and 123.0520 ([M − H − SO3 − CO2 − CH2]−). According to the previous report − at m/z 181.0504 (predicted to be C9H9O4) and characteristic fragment ion at m/z 137.0642 ([M − H − CO2]−) was observed in the NI MS2 spectra. According to a previous report [M136 as dihydrocaffeic acid. M137–M138 yielded [aglycone − H]− at m/z 181.05 by neutral loss of 79.95 Da and were tentatively identified as dihydrocaffeic acid sulphate.Metabolites having the aglycone of dihydrocaffeic acid: s report , we idenM139–M145. M139–M141 showed [M − H]− at m/z 238.07 (predicted to be C11H12NO5) and yielded [aglycone − H]− at m/z 179.04 in the MS2 spectra by neutral loss of 59.03 Da (C2H5NO). The aglycone had the same molecular formula and characteristic fragment ions as caffeic acid. Therefore, M139–M141 were designated caffeic acid acetyl amination metabolites. M142–M143 showed [M − H]− at m/z 197.05, which is 18.01 Da (H2O) more than caffeic acid; thus, they were tentatively identified as hydration metabolites of caffeic acid. Based on the ClogP rule, M142 (tR = 11.692) was 3--3-hydroxypropanoic acid (ClogP = −0.6414) and M143 (tR = 12.658) was 3--2-hydroxypropanoic acid (ClogP = −0.5798). M144–M145 showed [M − H]− at m/z 277.00, and the [aglycone − H]− at m/z 197.05 was formed by the loss of 79.95 Da. Therefore, M144–M145 were tentatively identified as the sulphates of caffeic acid hydrate.Metabolites having the aglycone of caffeic acid: M146–M148. The molecular formula of M146 was calculated to be C10H12O7S. The [aglycone − H]− at m/z 195.0681 (C10H11O4) was formed by the loss of SO3 (79.95Da). Characteristic fragment ions at m/z 195.0681, 151.0845, 149.0632, 136.0607 and 119.0578 were observed in NI MS2 spectra. According to a previous report [M146 as dihydrogen ferulic acid sulphate. M147–M148 showed [M − H]− at m/z 291.02 and the [aglycone − H]− at m/z 211.06 (C10H11O5) was formed by loss of SO3 (79.95 Da), which was 18.01 Da (H2O) more than ferulic acid (C10H9O4). Therefore, these metabolites were tentatively identified as the sulphates of ferulic acid hydrate.Metabolites having the aglycone of ferulic acid: 2 spectra of M149–M155, the same [aglycone − H]− (m/z 123.05) was observed with a molecular formula identical to hydroxybenzyl alcohol. We therefore deduced that they were conjugates of hydroxybenzyl alcohol − at m/z 217.02 (predicted to be C8H9O5S), and yielded [aglycone − H]− at m/z 137.07 by neutral loss of 79.95 Da. The aglycone was 14.01 Da (CH2) more than hydroxybenzyl alcohol, so the compounds were tentatively identified as sulphates of methyl hydroxybenzyl alcohol.M156–M159. M156–M159 yielded [aglycone − H]− by loss of 79.95 Da and so were sulphate conjugates. From the [aglycone − H]− of M156–M157 at m/z 137.03 (C7H6O3), these were identified as 3/4-hydroxybenzoic acid sulphates according to a previous report [− of M158–M159 at m/z 167.04 (C8H8O4), they could identify as vanillic acid sulphate and isovanillic acid sulphate according to the previous report [Metabolites having the aglycone of hydroxybenzoic acid: s report . From ths report .In total, 32 metabolites of dimerization (31 new compounds), including 10 taxifolin dimer derivatives and sulphates and 22 methyl taxifolin dimer derivatives and sulphates, were identified.M160–M169. The characteristic fragment ions of taxifolin at m/z 303.05, m/z 285.04 and m/z 241.05 were observed in the NI MS2 spectra of M160–M169. We therefore deduced that their structures contained taxifolin, and that they were taxifolin dimer derivatives. The molecular formula of M160 was calculated to be C31H24O13 and, when compared with the molecular formula (C15H12O7) of taxifolin, we predicted that M160 might be a dimer of taxiflolin and dehydroxylated methyl taxifolin. However, the site of dimerization was ambiguous. Only two forms of coupling bond are found between monomers of biflavonoids, namely C-C coupling and C-O coupling. In the NI MS2 spectra of M160, the relative abundance of m/z 303.0557 was less than 5% (4.08%), thus implying that the coupling bond between two monomers was extremely difficult to cleave [M160 and its fragmentation pathways are shown in M160, we predicted that M161–M162 might be the dimers of taxiflolin and methyltaxifolin formed through C-C coupling. M163 might be a dimer of taxiflolin and dimethyltaxifolin formed through C-C coupling. M164–M166 were tentatively identified as sulphates of dimers of taxiflolin and methyltaxifolin. M167–M169 were tentatively identified as sulphate of dimers of taxiflolin and dimethyltaxifolin.Dimers having the aglycone of taxifolin: o cleave . TherefoM170–M191. The characteristic fragment ions of methyl taxifolin at m/z 317.06, m/z 299.05 and m/z 289.07 were observed in the NI MS2 spectra of M170–M191 . Similar to M160, we predicted that M170–M172 might be dimers of methyltaxiflolin and dehydroxylated methyltaxifolin. M171 was identified as a dimer formed through C-O coupling. M173–M176 might be dimers of methylquercetin and methyl-taxifolin. Among these, M174 was identified as a dimer formed through C-C coupling while M173, M175 and M176 were identified as dimers formed through C-O coupling. M177–M179 might be dimers of methyl taxiflolin and dehydroxylated dimethyltaxifolin formed through C-O coupling. M180 and M181 might be dimers of methyltaxiflolin and methyltaxifolin formed through C-O and C-C coupling, respectively. M182–M183 might be dimers of methyltaxiflolin and dimethylquercetin formed through C-O coupling. M184–M190 might be dimers of methyltaxiflolin and dimethyl- taxifolin; M190 was formed through C-C coupling while the other metabolites were formed through C-O coupling. M191 was tentatively identified as a sulphate of dimers of methyltaxiflolin and dehydroxylated methyltaxifolin.Dimers having the aglycone of methyltaxifolin: g supernatant incubation system [In total, 32 dimers were newly identified as metabolites of taxifolin, and this is the first report of dimers formed as metabolites of flavanonol in vivo. To the best of our knowledge, the number of dimers found is the largest in metabolism studies to date, although six honokiol dimers were previously identified from the faeces of rats  and seven system . Dimers n system . TherefoUnequivocal structure identification of the metabolites (known as the level 1 metabolite identification) is a fundamental issue in the field of drug metabolism research. Generally speaking, to solve this issue, the metabolites have to be prepared and purified from complex biological or chemical matrix, and then be analyzed by modern spectroscopic techniques such as NMR, circular dichroism (CD) and even X-ray diffraction. Unfortunately, the process is usually very difficult, because the contents of these metabolites in the biological matrix  are very low.n becomes the most common and effective method for quickly profiling and tentative identification of the metabolites to get a preliminary global view of the metabolic pathways of the original compound.Since the substrate  is known in drug metabolism research, i.e., the exact chemical structure of the substrate is definite, the LC-HRMSn data. However, it′s usually difficult or even impossible to determine regioisomers, stereoisomers and the exact metabolic site only by current MS techniques. Moreover, it is still a difficult problem to determine the exact sulphation site in flavonoids bearing a catechol moiety even by NMR technique. Fortunately, Purchartova et al. recently proposed a novel approach to solve this problem. They found that the methylation of flavonoid sulphates could be used for the direct and unequivocal determination of the position of sulphates in quercetin derivates by NMR [O-sulphate and 3′-O-sulphate in a ratio of 80:20 by bacterial aryl sulfotransferase from Desulfitobacterium hafniense. Besides, rat aryl sulfotransferase AstIV (EC 2.8.2.1) expressed recombinantly in Escherichia coli can biotransform taxifolin into taxifolin 3′-O-sulphate and quercetin 3′-O-sulphate [M3–M11) and quercetin sulphates , which is consistent with the results of rat AstIV, indicating the similarity between rat and recombinant rat AstIV.In this study, 191 metabolites of taxifolin were tentatively identified by their high resolution LC-MSs by NMR . This mesulphate . These rM1, M2) of taxifolin in this study. Since taxifolin has five hydroxyl groups, five sulphates could be formed at most. However, we have found nine taxifolin sulphates (M3–M11) based on LC-HRMSn data, which indicates that the metabolites should include optical isomers. Because the amount of metabolites are small, we were not able to isolate sufficient metabolites and determine their exact structures. It needs more work and time to determine their exact structures by moder spectroscopic techniques in future.There are four optical isomers of taxifolin because C-2 and C-3 are chiral centers, and we found two isomers metabolites  were reported for the first time shown in .M33 was observed in all eight organs. M11, M18, M19, M34 and M49 were detected in seven organs. In total, 19 metabolites  can be found in more than three organs. Therefore, these 19 metabolites were distributed more widely than the other metabolites, and they might contribute to the pharmacological activities of taxifolin in vivo.In total 46 metabolites were detected in eight organs, and there were 35 metabolites in the small intestine, 31 in the kidneys, 29 in the stomach, 22 in the liver, 12 in the lungs, 10 in the spleen, seven in the heart, and three in the brain. Therefore, the small intestine, kidney, stomach and liver were the main organs for the distribution of the 46 metabolites of taxifolin. The methylated metabolite M1 and M2), quercetin (M70), eriodictyol (M104), dihydrokaempferol (M105), 3/4-hydroxyphenylpropionic acid , dihydroxyphenylacetic acid (M131) and dihydrocaffeic acid (M136), and the eight phase II metabolites, quercetin-4′-O-sulphate (M73), quercetin-3′-O-sulphate (M74), quercetin-3-O-sulphate (M75), quercetin glucuronide (M76), isorhamnetin (M80), isorhamnetin-3-O-sulphate (M83), isorhamnetin disulphate (M85) and luteolin-7-O-glucuronide (M113), have similar bioactivities to taxifolin according to the literature  to suppress cancer [Among the 191 metabolites, the specific structures of 63 were identified tentatively by their HRMS data, reference compounds, and previous studies  are reported in the literature: angiotensin-converting enzyme [Five of the top 300 PharmMapper-predicted target proteins of quercetin ; six metabolites, M70, M73, M74, M80, M84 and M86, include fragment 2 (in red); eighteen metabolites, M33, M34, M36, M70, M73, M74, M75, M80, M83, M84, M86, M104, M105, M107, M109, M112, M114 and M115, contain fragment 3 (in red); and fourteen metabolites, M35, M36, M71, M72, M75, M80, M104, M106, M113, M131, M134, M136, M142 and M143, include fragment 4 (in red). Metabolites with the same fragment may contain the same pharmacological groups in their structures and act at the same targets with the same effects. For example, according to the literature, among the eighteen metabolites with fragment 3, eight metabolites  exhibitM70 [M75 , M80 [6380 [M104  and M10504 [M105 ) exhibit-(+)-Taxifolin (purity > 98%) was purchased from Chengdu Must Bio-technology Co., Ltd  and used as the experiment source of taxifolin in the study. Quercetin and dihydrokaempferol were isolated in our laboratory, and the purities of these two standards were >98% as determined by high-performance liquid chromatography coupled with diode array detector analysis . Formic acid , acetonitrile , and methanol  were of HPLC grade. Ultrapure water was prepared using a Milli-Q water purification system . Analytical-grade sodium carboxymethyl cellulose (CMC-Na) was purchased from Tianjin Guangfu Fine Chemical Research Institute . All other reagents and chemicals were of analytical grade.ad libitum. The rats were randomly divided into two groups (six rats per group), a drug group and a blank group. Taxifolin was suspended in 0.5% CMC-Na solution and orally administered to the drug group at a dose of 200 mg/kg body weight, while blank group rats were orally administered 0.5% CMC-Na solution at the same volume. All rats were dosed once a day (at 9:00 a.m.) for 3 days. All animal treatments were conducted according to the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health. The animal research protocols were approved by the Biomedical Ethical Committee of Peking University .Twelve male Sprague-Dawley rats (weighing 180–220 g) were obtained from the Experimental Animal Center of Peking University Health Science Center . The rats were maintained in metabolic cages  and acclimatized to the facilities for 5 days prior to experiments. All rats were housed in an environmentally controlled animal room, with food and water provided During the administration period, urine and faeces samples from animals in the drug and blank groups were collected at 0–24 h after the first and second dosing, respectively. The urine samples were collected every 6 h from the urine collection tube , a 1-fold volume of methanol was added, and samples were temporarily stored at 4 °C. Finally, all urine samples from the same group were merged into one sample and immediately evaporated to dryness at 40 °C under reduced pressure by a rotator evaporator. The dried sample was then extracted ultrasonically with a 4-fold volume of methanol for 30 min using an ultrasonic cleaner (at about 25 °C) and the extract was centrifuged at 5000 rpm for 15 min. Subsequently, the supernatant was dried in a vacuum at 40 °C. Each 1 g residue was reconstituted in 2.0 mL methanol and filtered through a 0.45-μm Millipore filter before undergoing LC-MS analysis.Faecal samples were collected every 6 h and dried immediately using an electro-thermostatic blast oven at 40 °C. Finally, all faecal samples from the same group were merged into one sample. The dry sample was ground to powder, and 3.0 g powder from each group was mixed with 15 mL of methanol and extracted ultrasonically for 30 min three times. Next, the extracts were centrifuged at 5000 rpm for 15 min and the three supernatants were combined and evaporated to dryness under reduced pressure at 40 °C. The resulting residue was dissolved in 3.0 mL methanol and filtered through a 0.45-μm Millipore filter, and the filtrate was then subjected to LC-MS analysis.Blood samples were collected into heparinized tubes using a heart puncture technique under anaesthesia at 0.5, 1, and 1.5 h (two rats were sacrificed at each time point) after the last administration and were centrifuged at 5000 rpm, 4 °C for 10 min to obtain plasma. Plasma samples from the same time point within each group were combined into one sample and stored at −80 °C until processing. Upon thawing, 24 mL methanol was added to 6 mL of plasma (2 mL plasma from each of the three time points combined) in an ultrasonic bath for 30 min at about 25 °C and samples were then centrifuged at 5000 rpm for 15 min to remove precipitated protein. Next, the supernatant was concentrated to a small volume under reduced pressure at 40 °C, transferred to a clean conical tube and dried under a gentle stream of nitrogen gas at ambient temperature. The residue was then reconstituted in 300 μL of methanol and filtered through a 0.45-μm Millipore filter before undergoing LC-MS analysis.After collection of blood samples and rapid removal of blood from organs via heart perfusion (until the liver became yellow in colour), the heart, liver, spleen, lungs, kidneys, brain, stomach and small intestine were rapidly removed and flushed with cold normal saline , dried with filter paper, and weighed. All organ samples were stored at −80 °C until further processing. The same organ samples from each group were combined into one sample and processed using a homogenizer following suspension in a four-fold (volume/mass organ wet weight) volume of cold normal saline. Next, a 6 mL aliquot of homogenate from each organ sample was added to 48 mL of methanol, extracted ultrasonically for 30 min at about 25 °C, and centrifuged at 5000 rpm for 15 min to remove the protein. The supernatant was evaporated to a small volume under reduced pressure at 40 °C and transferred into a clean tube. The supernatant was then dried under a gentle flow of nitrogen at ambient temperature, the residue was reconstituted in 1 mL methanol, and filtered through a 0.45-μm Millipore filter, and the filtrate was subjected to LC-MS analysis.n analysis was performed on a Shimadzu HPLC instrument  coupled to an IT-TOF mass spectrometer  through an ESI interface. The chromatographic separations were carried out on an Agilent Zorbax SB-C18 column  maintained at 30 °C and protected using an Agilent Security Guard column . The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B). The gradient was as follows: 0 min, 2% B; 15 min, 10% B; 30 min, 15% B; 45 min, 22% B; 60 min, 35% B; 70 min, 55% B; 85 min, 82% B; 86 min, 100% B; 95 min, 100% B; 96 min, 2% B; and 105 min, 2% B (v/v). The volume injected was 20 μL. High-resolution mass spectra were recorded using an IT-TOF mass spectrometer programmed to carry out a full scan over m/z 100–1500 Da (MS) and m/z 50−1000 Da (MS2 and MS3) in both positive ion (PI) and negative ion (NI) detection mode. The flow velocity was maintained at 1.0 mL/min and was spilt at 0.2 mL/min through a flow divider to flow into the mass spectrometer. A trifluoroacetic acid sodium solution (2.5 mM) was used to calibrate the mass range of 50−1500 Da. The other operating parameters were set as follows: interface voltage was (+), 4.5 kV; (−), −3.5 kV; nebulizing nitrogen gas flow was 1.5 L/min; detector voltage was 1.70 kV; relative collision-induced dissociation energy was 50%; and heat block and curved desolvation line temperature was 200 °C. All data were recorded and processed using LCMS solution version 3.60, Formula Predictor version 1.2 and Accurate Mass Calculator software .HPLC-ESI-IT-TOF-MShttp://59.78.96.61/pharmmapper.The potential targets of the metabolites of taxifolin were predicted using PharmMapper server . PharmMapper is available at Level 1: The metabolites are identified by comparison with reference compounds.Level 2: The metabolites are identified by comparison with reference literature or can be found in the Scifinder database.Level 3: New metabolites/compounds that could not be found in the SciFinder database.The definition of metabolite identification level reported in the literature was generally adopted . HoweverA total of 191 metabolites (including 153 flavonoids and 38 phenols) of taxifolin were tentatively identified, 154 of whom were new metabolites of taxifolin. Furthermore, 69 metabolites were new compounds that were not found in the SciFinder database, including 12 taxifolin conjugates, 22 methyl taxifolin derivatives, four phenolic acid derivatives and 31 dimers. To our knowledge, this is the first report of a single compound biotransformed into more than 100 metabolites in vivo.The major metabolic reactions of taxifolin in rats included ring-cleavage, sulphation, glucuronidation, methylation and dimerization. Furthermore, acetylamination and pyroglutamic acid conjugation were new metabolic reactions not described in any previous metabolism studies.A total of 17 metabolites had similar bioactivites to taxifolin. The PharmMapper prediction showed that more than 60 metabolites had the same five targets. This suggested that the effective forms  of taxif


Chaenomeles speciosa (Sweet) Nakai is a medicinal plant. Until date, there are no studies focusing on comparing the chemical profiles, antioxidant activity and α-glucosidase inhibitory activity of the dried fruits of C. speciosa from different production regions. In the study, we investigated the chemical components of dried fruits of C. speciosa from Yunnan, Chongqing, Zhejiang and Anhui provinces in China in relation to the antioxidant activity and α-glucosidase inhibitory activity. C. speciosa from Yunnan had higher total flavonoid (47.92 ± 3.79 mg/g), total polyphenol (29.15 ± 0.29 mg/g) and polysaccharide (27.60 ± 1.56 mg/g) contents than plants from other production areas. Samples from Yunnan, Zhejiang and Anhui  had higher free amino acid contents than those from Chongqing (2286.66 mg/kg). Oleanolic acid and ursolic acid levels were highest in samples from Zhejiang (555.98 ± 20.88 μg/g) and Anhui (321.06 ± 14.64 μg/g), respectively. C. speciosa from Chongqing had low total flavonoid, total polyphenol, polysaccharide, free amino acid, oleanolic acid and ursolic acid contents but high levels of palmitic acid (12.04 ± 0.02 mg/g) and stearic acid (2.23 ± 0.08 mg/g). Among four production areas, Yunnan represented the highest antioxidant activity and α-glucosidase inhibitory activity. In addition, correlation analysis revealed that total flavonoid, total polyphenol, polysaccharide and ursolic acid were the major components responsible for the antioxidant activity of C. speciosa, while total flavonoid and polysaccharide were the main contributors for α-glucosidase inhibitory activity of the plant. These results would be helpful for evaluating the quality of C. speciosa in the different production areas. Chaenomeles, belonging to the Rosaceae family, is mainly distributed in Korea, Japan and China. There are five wild Chaenomeles species in China including C. speciosa (Sweet) Nakai, C. thibetica Yü, C. cathayensis (Hemsl.) Schneider, C. sinensis (Thouin) Koehne and C. japonica (Thunb.) Lindl. ex Spach. C. speciosa has traditionally been cultivated in Yunnan, Chongqing, Zhejiang and Anhui provinces and its dried fruits are commonly used in traditional medicine for the treatment of the common cold, asthma, hepatitis and rheumatoid arthritis [C. speciosa have reported anti-inflammatory, antinociceptive, antimicrobial, antioxidant, immunoregulatory, antitumor, hepatoprotective and antiparkinsonian activities [The genus rthritis . The frutivities ,3,4,5. C. speciosa. The pharmacological properties of C. speciosa can be understood by identifying the chemical constituents of the plant. For example, phenolic compounds such as anthocyanins and flavonoids have shown anti-inflammatory, antitumoral, antimicrobial and antioxidant effects [α-glucosidase inhibitory effects [α-glucosidase inhibitory activity of C. speciosa have attracted more and more attention from researchers. Flavonoids, polyphenols, polysaccharides, free amino acids, organic acids and triterpenic acids have been isolated from the fruits of  effects . The phe effects , which a effects ,9. Olean effects , antitum effects ,12 and α effects ,14. AmonC. speciosa. 4’-Nitrophenyl-beta-d-glucopyranoside (PNPG) is usually adopted for exploring the α-glucosidase inhibitory activity of the plant because it could be decomposed by α-glucosidase into 4-nitrophenol and the product could be further detected by microplate reader at 405 nm [α-glucosidase inhibitory activity of C. speciosa. For examples, Xie et al. [C. speciosa. The purified polysaccharides exhibited remarkable antioxidant activity to scavenge the DPPH, ABTS, SOA and hydroxyl radicals. Gao et al. [α-glucosidase inhibitory activity of C. speciosa. The results revealed that 60% ethanol was the best choice for industrial production of C. speciosa extract with good antioxidant activity and α-glucosidase inhibitory activity. In addition, the same group [C. speciosa performed better FRAP, DPPH and α-glucosidase inhibitory activities than the fleshes. Ferric reducing/antioxidant power (FRAP), 2,2-diphenyl-1-picrylhy-drazyl (DPPH), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), super oxygen anion (SOA) and hydroxyl free radical assays were the commonly used approaches for evaluating the antioxidant activity of t 405 nm . Recentle et al.  isolatedo et al.  presenteme group  reportedα-glucosidase inhibitory activity of the dried fruits of C. speciosa from different production regions. To address this issue, in the present study, we compared the contents of total flavonoid (TF), total polyphenol (TP), polysaccharide, free amino acid, organic acid, OA and UA as well as antioxidant activity and α-glucosidase inhibitory activity of C. speciosa from Yunnan, Chongqing, Zhejiang and Anhui provinces and examined the correlation of these chemical components with the antioxidant activity and α-glucosidase inhibitory activity of the plant. Until date, there are no studies focusing on comparing chemical profiles, antioxidant activity and C. speciosa from Yunnan had the highest TF content (47.92 ± 3.79 mg/g), which was about 2-fold higher than the level in plants from Chongqing (22.36 ± 0.53 mg/g) (C. speciosa from Yunnan had a higher TP content (29.15 ± 0.29 mg/g) than samples from Chongqing (18.19 ± 0.37 mg/g), Zhejiang (18.85 ± 0.24 mg/g) and Anhui (22.27 ± 0.49 mg/g). The TP contents from four production areas were significantly higher than the results (13.58 ± 0.13 mg/g) reported by Li et al. [C. speciosa. It is found that all the fruits of C. speciosa processed by both the drying methods have lower TP content (ranging from 2.14 ± 0.10 to 3.28 ± 0.47 mg/g) than the samples from Chongqing in the work. The dried fruits of C. speciosa from Yunnan (27.60 ± 1.56 mg/g), Zhejiang (24.45 ± 3.70 mg/g) and Anhui (25.26 ± 1.31 mg/g) had similar polysaccharide contents, which were higher than that of plants from Chongqing (19.95 ± 1.88 mg/g). Thus, dried fruits of C. speciosa in Yunnan had the highest TF, TP and polysaccharide levels in four production areas.The dried fruits of 53 mg/g) . TF conti et al. . Gao et C. speciosa from Yunnan than in plants from the other three provinces, whereas the opposite trend was observed for methionine (30.29 ± 4.54 mg/kg), isoleucine (13.78 ± 0.75 mg/kg) and leucine (9.41 ± 1.49 mg/kg). The highest levels of isoleucine (30.43 ± 3.43 mg/kg) were found in samples from Chongqing, whereas leucine (34.23 ± 1.40 mg/kg) and lysine (44.07 ± 2.65 mg/kg) levels were highest in samples from Zhejiang. Methionine content (35.79 ± 0.62 mg/kg) was highest in plants from Anhui. Samples from Chongqing had the lowest threonine (5.94 ± 0.56 mg/kg) and phenylalanine (43.60 ± 2.46 mg/kg) contents, whereas valine (30.40 ± 0.62 mg/kg) and lysine (21.03 ± 1.74 mg/kg) levels were lowest in Zhejiang and Anhui samples, respectively.The total amino acid content in samples from Chongqing (2286.66 mg/kg) was lower than that of samples from Yunnan (3411.60 mg/kg), Anhui (3349.83 mg/kg) and Zhejiang (3226.30 mg/kg) . On the C. speciosa samples from four production areas, which is consistent with results reported in the literature [We identified 19 organic acids in terature  (Table 2C. speciosa samples are shown in C. speciosa from Yunnan (3.20 ± 0.05 mg/g) and Anhui (1.33 ± 0.08 mg/g) had the highest and lowest levels of oxalic acid, respectively. Malic acid levels in samples from Yunnan (124.96 ± 7.43 mg/g) and Anhui (139.91 ± 4.24 mg/g) were three times higher than those in plants from Chongqing (34.81 ± 0.35 mg/g) and Zhejiang (32.86 ± 0.05 mg/g). Citric acid content in plants from four production areas varied from 10.26–30.91 mg/g. C. speciosa from Chongqing had a higher palmitic acid content (12.04 ± 0.02 mg/g) than samples from Zhejiang (7.41 ± 0.07 mg/g), Anhui (7.45 ± 0.13 mg/g) and Yunnan (5.78 ± 0.28 mg/g). Stearic acid concentration was about two times higher in plants from Chongqing (2.23 ± 0.08 mg/g) than in those from Yunnan (1.05 ± 0.04 mg/g), Zhejiang (1.16 ± 0.10 mg/g) and Anhui (1.09 ± 0.01 mg/g). Representative chromatograms of organic acids in C. speciosa. The dried fruits of C. speciosa from Zhejiang had the highest concentration of total triterpenic acids (720.89 μg/g), which was over twice that in samples from Chongqing (331.51 μg/g)  . Plants C. speciosa (C. speciosa from Yunnan had the highest DPPH (273.30 ± 15.59 mg/g), ABTS (114.37 ± 0.31 mg/g) and SOA (52.36 ± 1.51 mg/g) radical-scavenging activities. The DPPH activity for Yunnan samples was 8- to 11-fold higher than that of plants from other production areas. In addition, Zhejiang had the lowest level of DPPH (24.32 ± 2.55 mg/g) radical-scavenging activity, while Chongqing samples represented the lowest levels of ABTS (41.68 ± 4.73 mg/g) and SOA (8.11 ± 0.06 mg/g) radical-scavenging capacities. Samples from Zhejiang (43.57 ± 0.83 mg/g) and Anhui (44.79 ± 1.05 mg/g) had similar SOA activity. All these results demonstrate that the dried fruits of C. speciosa from Yunnan had higher DPPH, ABTS and SOA radical-scavenging capacities than plants from other production areas. In addition, α-glucosidase inhibitory activities of C. speciosa from four production areas were also investigated and their results varied vigorously from each other. As shown in α-glucosidase inhibitory activity was in the order of Chongqing (56.59 ± 6.50 mg/g) < Zhejiang (86.41 ± 4.57 mg/g) < Anhui (172.62 ± 3.69 mg/g) < Yunnan (207.23 ± 0.26 mg/g). It could be deduced that some environment factors [C. speciosa, which could further affect the antioxidant and α-glucosidase inhibitory activities of C. speciosa.DPPH, ABTS and SOA radical-scavenging assays were adopted for evaluating the antioxidant capacities of the dried fruits of speciosa A from fo factors , such asα-glucosidase inhibitory activity of C. speciosa were examined (p < 0.01) was positively correlated with DPPH (r = 0.88), ABTS (r = 0.94), SOA (r = 0.74) and α-glucosidase inhibitory (r = 0.97) activities. The same was true for TP with DPPH , ABTS , SOA  radical-scavenging activities but not for α-glucosidase inhibitory activity. These results demonstrated that TF and TP possess strong DPPH and ABTS radical-scavenging capacities. The correlation coefficient of TF (or TP) with SOA was lower than that with DPPH or ABTS. The phenolic group in TF and TP would react more easily with DPPH or ABTS radicals than with SOA radicals. Polysaccharide content was correlated with ABTS , SOA  radical-scavenging activities and α-glucosidase inhibitory activity . The lower correlation coefficients of polysaccharide with ABTS than TF or TP might account for the different reaction rates of alcohol and phenolic groups with ABTS radical. A comparison of the correlation coefficients of polysaccharide and TP (or TF) with SOA suggested that polysaccharide could better inhibit the formation of superoxide radical and exhibit stronger SOA radical-scavenging activity than TP (or TF). Furthermore, in the present study, there was no correlation of OA level with DPPH, ABTS, SOA and α-glucosidase inhibitory activities, whereas UA content was correlated with DPPH (p < 0.05) and ABTS (p < 0.05) activities but not with SOA radical-scavenging and α-glucosidase inhibitory activities [C. speciosa, while TF and polysaccharide were the main components for α-glucosidase inhibitory activity of the plant.The correlations of TF, TP, polysaccharide, OA and UA contents with antioxidant activity and examined . TF conttivities . All aboC. speciosa were collected during the mature period from Yunnan, Chongqing, Zhejiang and Anhui provinces. The samples were cut in half vertically and dried under sunlight for 10 days. They were then powdered and stored at 4 °C until use.The fruits of d-glucopyranoside (PNPG), α-glucosidase  and standard free amino acids  were purchased from Sigma-Aldrich . Methanol, ethanol, acetone, acetic acid, sodium nitrite, sodium hydroxide, hydrochloric acid, phenyl isothiocyanate, triethylamine, sodium acetate, d-(+)-glucose, phenol, 1,4-dioxane, phenylisothiocyanate, sulfuric acid and high-performance liquid chromatography-grade acetonitrile (ACN) were from Sinopharm Chemical Reagent Co. . Aluminum chloride, potassium persulfate and UA were from Aladdin . Gallic acid was from J&K Co. . DPPH, OA, oxalic acid, oleic acid, malic acid, citric acid, linoleic acid, palmitic acid and stearic acid were from Macklin Biochemical Co. . Chloroform, sulfosalicylic acid, hexane and quercetin were obtained from Solarbio . ABTS was from Tokyo Chemical Industry . Deionized water was prepared using a Unique-R20 purification system .Folin-Ciocalteu reagent, phenazine methosulfate (PMS), nicotinamide adenine dinucleotide (NADH), Tris buffer, nitrotetrazolium blue chloride (NBT), 4’-nitrophenyl-beta-C. speciosa samples (0.1 g) were extracted twice with 80% ethanol solution  in an ultrasonic bath at 60 °C for 40 min. The extracts were combined and stored at 4 °C until use.3 solution  was added and the mixture was incubated at room temperature for 6 min. A 0.5 mol/L NaOH solution (100 μL) was added to terminate the reaction and the absorbance of the mixture at 510 nm was measured with a microplate reader. TF content is expressed as milligrams of quercetin per gram of dry weight of the plant material.TF content was measured as previously described , with so2O  solution were mixed in a 96-well plate. After 5 min, H2O (100 μL) and 20% Na2CO3 solution  were added. The plate was maintained in the dark for 30 min and the absorbance was measured at 760 nm with a microplate reader. The results are expressed as milligrams of gallic acid equivalent (mg GAE) per gram (g) of the dry weight of the plant material.TP content was determined according to the Folin-Ciocalteu method , with soC. speciosa sample (0.2 g) was extracted twice with 80% ethanol/H2O solution  in an ultrasonic bath at 80 °C for 30 min. The extracts were combined and the mixture was centrifuged at 4000 rpm for 10 min; the supernatant was discarded. The insoluble material was extracted three times with H2O (10 mL) by bath-sonication at 120 W and 80 °C for 30 min. The extracts were combined and the mixture was diluted to 100 mL with H2O.The polysaccharide content was determined according to the Entry-Exit Inspection and Quarantine industry standards of China (SN/T 4260-2015) . Powdere2O  and sulfuric acid (5 mL) were mixed in centrifuge tubes. After 10 min, the centrifuge tubes were incubated in a water bath at 30 °C for 20 min, the reaction solution was removed to 96-well plates and the absorbance was measured at 490 nm with a microplate reader. d-(+)-glucose was used to generate the calibration curve.Polysaccharide content was analyzed according to the phenol-sulfuric acid method. Sample solution (1 mL), 5% phenol/HC. speciosa (3 g) were extracted three times with 0.02 mol/L HCl solution (1.5 mL) in an ultrasonic bath at 30 °C for 5 min. The pooled extracts were allowed to stand in the dark for 2 h before centrifugation at 4000 rpm for 2 min. The supernatant (1 mL) was mixed with 7% sulfosalicylic acid  solution with shaking for 1 min. The mixture was maintained in the dark for 1 h and centrifuged at 15,000 rpm for 15 min. The supernatant (500 μL) was mixed with 1 mol/L triethylamine/ACN solution (250 μL) and 0.1 mol/L phenylisothiocyanate/ACN solution (250 μL). After 1 h, hexane (2 mL) was added and the mixture was shaken vigorously. The bottom layer was passed through a 0.22-μm filter and stored at 4 °C until use.Powdered samples of 2O) and B  were used for gradient elution as follows: 0–14 min, 0–15% B; 14–29 min, 15–34% B; 29–30 min, 34–100% B; 30–37.1 min, 100%–0 B; 37.1–45 min, 0% B. The flow rate was 1.0 mL/min and the temperature was maintained at 40 °C. The absorbance was measured at 254 nm.Free amino acid contents were determined according to the previous method with some modifications . SamplesC. speciosa (0.5 g) sample was extracted twice with 7% sulfuric/methanol  solution at 0 °C for 30 min. The extracts were combined and the mixture was immersed in a water bath at 60 °C for 24 h and then centrifuged at 3000 rpm. The supernatant was sequentially combined with H2O (20 mL) and chloroform (20 mL). After 2 h, sodium sulfate (3 g) was added to the sample solution and the bottom layer was used for analysis.Organic acid content was estimated based on Liu’s method  with somGC-MS analysis of organic acids was performed with a 5977A mass spectrometer system  and an HP-5ms ultra-inert column (30 m × 250 μm × 0.25 μm). Helium was used as the carrier gas at a flow rate of 1 mL/min. The temperature program was as follows: the column was held at 220 °C for 1 min before increasing the temperature to 228.5 °C at a rate of 5 °C/min and holding for 5 min. The column temperature was increased to 240 °C at a rate of 5 °C/min and held for 1 min, then increased to 290 °C at a rate of 10 °C/min and held for 3 min. The injection volume was 1 μL.2O, acetic acid and triethylamine at a volume ratio of 265:35:0.1:0.05. The flow rate and injection volume were 0.3 mL/min and 5 μL, respectively. Absorbance was measured at 210 nm with a microplate reader.OA and UA were extracted using the same method as TP and TF. OA and UA contents were determined with a UPLC system  equipped with an ultraviolet detector. The analysis was performed according to the Pharmacopoeia of the People’s Republic of China , with soC. speciosa was the same as that used for TF and TP extractions. The DPPH radical-scavenging activity was evaluated based on a previous report [A = (0A − iA)/0A × 100, where A, 0A and iA represent the scavenging rate and absorbance of blank and sample, respectively.The extraction method for assessing the antioxidant activity of s report . The extABTS free radical-scavenging capacity was determined as previously described . Equal vThe SOA radical-scavenging effect was determined according to a published method . BrieflyDPPH, ABTS and SOA radical-scavenging activities are all evaluated with a microplate reader and expressed as milligrams of quercetin equivalents per gram dry weight of the plant material.α-glucosidase inhibitory activity of C. speciosa was the same as that used for TF and TP extractions. The α-glucosidase inhibitory activity was determined according to Hattori’s method [2CO3 (150 μL) was added to end the reaction. The absorbance was determined at 405 nm with a microplate reader. α-Glucosidase inhibitory activity is expressed as milligrams of quercetin equivalents per gram dry weight of the plant material.The extraction method for evaluating s method  with somp < 0.05 by analysis of variance analysis (Duncan’s multiple range test) and the least significant difference method.Data are expressed as mean ± standard error of mean of triplicate measurements. Statistical analysis was performed using SAS v.9.2 software . Significant differences were determined at α-glucosidase inhibitory activity of the dried fruits of C. speciosa from four production areas  and examined the relationship of the chemical composition of the plant with antioxidant activity and α-glucosidase inhibitory activity. The results show that C. speciosa has rich chemical composition, good antioxidant activity and α-glucosidase inhibitory activity and C. speciosa from different production areas exhibit variations in chemical composition, possibly due to factors such as sunlight, soil, rainfall and agricultural operations. The abundant light and heat resources in Yunnan might contribute to the high contents of TF, TP and polysaccharides. A correlation analysis indicated that TF, TP, polysaccharides and UA were the major components responsible for the antioxidant activity of C. speciosa, while TF and polysaccharide were the main contributors for α-glucosidase inhibitory activity of the plant. The results reveal that C. speciosa could be a potential source of natural antioxidants and hypoglycemics in the food industry. The study would be helpful for evaluating the quality of C. speciosa in the different production areas. In this study, we compared the chemical composition, antioxidant activity and


Dasypyrum villosum is an important wild species of wheat (Triticum aestivum L.) and harbors many desirable genes that can be used to improve various traits of wheat. Compared with other D. villosum accessions, D. villosum#4 still remains less studied. In particular, chromosomes of D. villosum#4 except 6V#4 have not been introduced into wheat by addition or substitution and translocation, which is an essential step to identify and apply the alien desired genes. RNA-seq technology can generate large amounts of transcriptome sequences and accelerate the development of chromosome-specific molecular markers and assisted selection of alien chromosome line.D. villosum#4 via a high-throughput sequencing technique, and then developed 76 markers specific to each chromosome arm of D. villosum#4 based on the bioinformatic analysis of the transcriptome data. The D. villosum#4 sequences containing the specific DNA markers were expected to be involved in different genes, among which most had functions in metabolic processes. Consequently, we mapped these newly developed molecular markers to the homologous chromosome of barley and obtained the chromosome localization of these markers on barley genome. Then we analyzed the collinearity of these markers among D. villosum, wheat, and barley. In succession, we identified six types of T. aestivum-D. villosum#4 alien chromosome lines which had one or more than one D. villosum#4 chromosome in the cross and backcross BC3F5 populations between T. durum–D. villosum#4 amphidiploid TH3 and wheat cv. Wan7107 by employing the selected specific markers, some of which were further confirmed to be translocation or addition lines by genomic in situ hybridization (GISH).We obtained the transcriptome of D. villosum#4 based on transcriptome data were developed in the current study and their collinearity among D. villosum, wheat, and barley were carried out. Six types of Triticum aestivum-D. villosum#4 alien chromosome lines were identified by using 12 developed markers and some of which were further confirmed by GISH. These novel T. aestivum-D. villosum#4 chromosome lines have great potential to be used for the introduction of desirable genes from D. villosum#4 into wheat by chromosomal translocation to breed new wheat varieties.Seventy-six PCR markers specific to chromosomes of The online version of this article (10.1186/s12864-019-5630-4) contains supplementary material, which is available to authorized users. Dasypyrum villosum  is a cross-pollinating annual grass in the tribe Triticeae, which is native to the north-eastern part of the Mediterranean region (from southern France to the Caspian Sea), south-western Asia, Russia and the Caucasus areas. It is a vigorous ruderal plant that can grow in harsh and moisture-stressed environments [D. villosum is resistant to many major wheat diseases including stripe rust (Puccinia striiformis), leaf rust (P. recondita), stem rust (P. graminis), powdery mildew (PM) caused by Blumeria graminis (Bgt), eyespot (Pseudocercosporella herpotrichoides) and wheat spindle streak mosaic virus transmitted by Polymyxa graminis [D. villosum is tolerant to cold, salt, and drought stresses, has a high tillering rate, produces large amounts of seed, and has high protein content [D. villosum has great potential as a genetic source for improving cultivated wheat with more desirable traits. As of now, more than 300 D. villosum accessions have been collected [D. villosum#1, D. villosum#2, D. villosum#3, D. villosum#4, and D. villosum#5 have been introduced into wheat backgrounds [sum 2n = 4, VV is sum 2n = 4, VV is graminis . In addisum 2n = 4, VV is ollected , but onlkgrounds –11.D. villosum into wheat background is an important step towards exploiting the desirable genes in D. villosum. To breed wheat with the aforementioned desirable traits of D. villosum and of the T. durum-D. villosum amphidiploid, some T. aestivum-D. villosum addition, substitution, and translocation lines have been developed using different D. villosum accession-derived materials as donors [D. villosum#1 [D. villosum#2, a complete set of disomic addition lines (DA1V#2 to DA7V#2), substitution lines 2V#2(?), 3V#2(3D), 4V#2(4D), 5V#2(5D), and 6V#2(6A), and several translocation lines including T1V#2S·1BL, T1V#2L·1DL, T1V#2S·1DS, T1V#2S-6BS·6BL, T2V#2S·2DL, T4V#2S·4DL, T5V#2S·5AL, T5V#2S·5DL, T6V#2S·6AL, and T6V#2L·6AS have been obtained [D. villosum#3, a complete set of disomic addition lines (DA1V#3 to DA7V#3), and some translocation lines including T1V#3S·1DL, T1V#3L·1DS, T2V#3L·2BL, T2V#3S·4BS, T2V#3S·4AL, T3V#3S·3DL, T3V#3L·3DS, T4V#3S·4DL, T4V#3L·4DS, T5V#3S·5DL, T6V#3S·6AL, T6V#3L·6AS, T7V#3S·7DL, and T7V#3L·7DS were developed [D. villosum#4, one substitution line 6V#4(6D) and three T6V#4S·6DL translocation lines  were developed [D. villosum#5, only one disomic addition line, DA2V#5, one disomic substitution line, 2V#5L(2D), and one translocation line, T2V#5L·2BS have been obtained [D. villosum#4 have not yet been created. By using a complete set of addition, substitution or translocation lines, the effect of an individual D. villosum#4 chromosome on wheat growth and development can be identified and some useful genes in D. villosum#4 can be located for wheat improvement [The successful transfer of an arm or fragment of a chromosome from s donors –11. For llosum#1 , 13. Forobtained , 14–19. eveloped , 21. As eveloped . For D. rovement .D. villosum has many excellent genes, and the exact locations of some agronomic important genes on chromosomes are becoming clear . The results obtained in this study will be useful to breed addition, substitution and translocation lines of T. aestivum–D. villosum#4 and subsequent wheat varieties by marker assisted selection (MAS).In this study, we obtained the transcriptome data of D. villosum#4 accession No. 1026, and obtained 7.14 G of clean bases and 47,619,528 clean reads in total. The rate of base mismatch was 0.02% , No. 1026 and CS were amplified with the selected candidate primers, in which CS was used as the negative control, and No. 1026 was used as the positive control. Results showed that some primers amplified a unique band in D. villosum#4 and their corresponding addition line (one of the seven addition lines from DA1V#3 to DA7V#3) containing the targeted D. villosum#3 chromosome, and did not amplify the same band in CS , which is a gene that regulates brassinosteroid levels and light responsiveness in Arabidopsis [D. villosum#4. Additionally, marker 5V-9 was annotated to E3 ubiquitin ligase which can catalyze the protein.The 76 specific molecular markers were developed based on es Table . The annbidopsis . HoweverT. aestivum–D. villosum#4 alien chromosome lines which contain D. villosum#4 chromosomes 1V to 7V, a BC3F5 population consisting of 409 plants from the cross and backcross between the T. durum–D. villosum#4 amphidiploid TH3 and the wheat line Wan7107 was screened using 12 developed PCR markers specific to D. villosum#4 chromosomes 1V to 7V. Six types of T. aestivum-D. villosum#4 alien chromosome lines were obtained except 4V , and the T. aestivum-D. villosum#4 candidate alien chromosome lines; however, they did not amplify any band in wheat line Wan7107 , exploring RNA alternative splicing and gene structural variation due to its high sequencing depth [Bgt). For non-model plants with limited genomic sequence information, application of RNA-seq can be used to discover gene coding regions despite the small number of repetitive elements and high GC contents compared to whole genomes, which makes the assembly of transcriptome data relatively easy [This study was conducted based on transcriptome data from ng depth –50. Partely easy . RNA-seqCompared with general molecular markers, the PCR markers developed from transcription data have particular advantages in plant genetics and breeding . FirstlyD. villosum focuses primarily on germplasm innovation. Wheat distant breeding using D. villosum will allow introduction of some desired genes into the general wheat background. This process is likely to be more successful when wheat-D. villosum alien chromosome lines are used as a bridge. Currently, wheat-D. villosum amphidiploid, addition, substitution, and translocation lines have been bred already. In order to efficiently identify D. villosum chromatin in the wheat background, including its targeted chromosomes and arms or fragments, it is necessary to develop a large number of markers specific to each chromosome arm or region of D. villosum. Current research on D. villosum, including marker development, is mostly focused on the accession D. villosum#2. Although some preliminary investigations on D. villosum#4 have been performed [D. villosum#4 genome will accelerate the application of the potential genes in D. villosum#4 and further expand the genetic background of wheat.The transfer of the foreign chromosomes into wheat through alien chromosome lines to confer desirable new traits also brings undesirable foreign genes. Nevertheless, wheat alien chromosome lines are very important foundational materials in chromosome engineering and wheat breeding programs. At present, research regarding the utilization of erformed , 39, 56,D. villosum#4 using RNA-seq without knowing the chromosome locations of all the unigene sequences obtained by analyzing the transcriptome data, we initially submitted the unigene sequences to the URGI BLAST database https://urgi.versailles.inra.fr/blast/ to screen for unigene sequences which have high similarity to the sequences on the chromosomes of wheat groups 1 to 7. It was thus assumed that these unigene sequences were located on the chromosomes 1V to 7V of D. villosum#4. This might be a convenient approach to develop markers specific to the chromosomes of D. villosum#4 in the wheat background. Following this strategy, we located the D. villosum#4 unigenes to wheat genome, and then designed the specific molecular markers based on the differences between these unigenes in wheat and D. villosum#4 genomes.To develop molecular markers specific to D. villosum#4. Among them, enough markers specific to the chromosomes 2V, 3V, 4V, 5V, and 6V were obtained. The results revealed by transcriptome analysis indicated that these unigene sequences had the most homology with the corresponding sequences from Hordeum vulgare  have been used to develop wheat-D. villosum chromosome addition, substitution, and translocation lines [D. villosum accessions [D. villosum#4 and wheat, and the polymorphism between different sources of D. villosum. While developing the markers specific to 1V and 7V of D. villosum#4, we found that most primers (86.67% in 1V and 70.59% in 7V) could not amplify any unique band in DA1V#3-DA7V#3, but that they could amplify bands in D. villosum#4. This indicated that there might be polymorphisms in the 1V and 7V between the accessions of D. villosum#4 and D. villosum#3. The specific reasons for this phenomenon need to be further studied. We will also try to map the correspondence D. villosum#4 unigenes to H genome for the development 1V- and 7V-specific markers in the near future.Update, more than 300 on lines –21. Thercessions , 31, 39.Dasypyrum villosum is an important wild species of wheat and contain a lot of novel genes that can be used to improve agronomic traits of wheat. Most studies on D. villosum have focused on D. villosum#1, D. villosum#2, and D. villosum#3, whereas the number of investigations on D. villosum#4 is relatively less. We obtained the transcriptome data of D. villosum#4 by using RNA-seq. Then, 76 molecular markers specific to D. villosum#4 chromosomes 1V to 7V were developed based on transcriptome data. Furthermore, six types of T. aestivum-D. villosum#4 alien chromosome lines were identified by using 12 developed markers and some of which were further confirmed by GISH. These novel T. aestivum-D. villosum#4 chromosome lines have great potential to be used for the introduction of desirable genes from D. villosum#4 into wheat by chromosomal translocation to breed new wheat varieties.D. villosum#4 accession No. 1026 and wheat lines Chinese Spring (CS) and Wan7107, were maintained in our laboratory. T. aestivum-D. villosum#3 addition lines DA1V#3, DA2V#3, DA3V#3, DA4V#3, DA5V#3, DA6V#3, and DA7V#3 in CS background were kindly provided by Prof. Wenxuan Liu of Henan Agricultural University. A backcross BC3F5 population derived from the cross of T. durum–D. villosum#4 amphidiploid TH3 and wheat line Wan7107 was made by our laboratory. T. durum–D. villosum#4 amphidiploid TH3  is derived from the cross between T. durum cv. Mexicali75  and the D.villosum#4 accession No.1026 that originated from the former USSR. Wheat lines CS and Wan7107 and D. villosum#4 No. 1026, DA1V#3, DA2V#3, DA3V#3, DA4V#3, DA5V#3, DA6V#3, and DA7V#3 were grown in a greenhouse. The backcross population of plants from the cross of TH3 and Wan7107 were grown in the field.D. villosum#4 accession No. 1026 were collected at seedling stage and RNA was extracted using TRIZOL . RNA quality, purity, integrity, and concentration were tested by the methods described in our previous publications [http://www.novogene.com/). Libraries for sequencing were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® . Then, the libraries were sequenced and paired-end reads were generated on the Illumina Hiseq platform. Clean data were obtained by removing the low quality reads after the raw data was refined through in-house Perl scripts. The high quality clean data were used for further analyses. Transcriptome assembly was conducted using the Trinity software with the ‘--min_kmer_cov’ parameter for a mismatch rate set to 2 by default, and all other parameters were also set to the default [Fresh leaves of ications , 43. The default .Gene function annotation was performed using Nr, Nt, Pfam, KOG/COG, Swiss-Prot, KO, and GO databases.D. villosum#4 was previously described by Li et al. [http://www.premierbiosoft.com/primerdesign/) based on those candidate unigene sequences which have the highest similarity to the sequences on the chromosomes of wheat groups 1 to 7.The strategy for screening candidate markers specific to the genome of i et al. . PrimersD. villosum accession#4 No. 1026, wheat-D. villosum#3 addition lines DA1V#3, DA2V#3, DA3V#3, DA4V#3, DA5V#3, DA6V#3, and DA7V#3, and the backcross population of TH3 and Wan7107, and stored at − 80 °C. Then, the genomic DNA for developing specific makers and alien chromosome lines was isolated from all samples using a NuClean Plant Genomic DNA Kit . And the genomic DNA used for probe in GISH was extracted from D. villosum#4 using the CTAB method. Finally, the DNA was dissolved in 50 μL ddH2O.Leaves were sampled from two-month-old seedlings of wheat lines CS and Wan7107, − 1), and 7.5 μL 2 × Taq MasterMix . Amplification reaction was performed in a MiniAmp Plus thermal cycler (USA) with a PCR program including 95 °C for 5 min, 35 cycles of 20 s at 95 °C, 30 s at 60 °C, 1 min at 72 °C, and 8 min for 72 °C. Amplification products were run on a 2% agarose gel, and observed under UV light after Genecolour II  staining.The PCR reactions of 15 μL volume included 100 ng of template DNA, 0.3 μL primer . Then, the root tips were treated with nitrous oxide for 2 h in a special container and fixed in 90% acetic acid for 5 min. They were rinsed twice with sterile water. The apical milky parts of the root tips were excised and digested with cellulase and pectinase for 55 min. Cell spreading preparations at mitosis were made as previously described [D. villosum#4 genomic DNA was labeled with fuorescein-12-dUTP as a probe using the nick translation method according to the manufacturer’s instructions. GISH was performed as previously description [Wheat seeds were soaked in water containing 0.7% Hescribed , 59, 60.cription , 59–61. Additional file 1:T. aestivum-D. villosum#3 addition lines, and D. villosum#4 accession No. 1026. M: marker 2000+; CS: Chinese Spring; 1: DA1V#3; 2: DA2V#3; 3: DA3V#3; 4: DA4V#3; 5: DA5V#3; 6: DA6V#3; 7: DA7V#3; 8: D. villosum#4 accession No.1026. (DOCX 95 kb)Amplification patterns of six 1V and 7V candidate chromosome-specific primers in Chinese (CS), seven Additional file 2:D. villosum#4, and their primer sequences and amplified sizes. 1: Chromosomal localization of the corresponding homologous sequences of the developed markers in wheat; 2: Chromosomal localization of the corresponding homologous sequences of the developed markers in barley; 3: Chromosomal localization of the corresponding sequences of the developed markers in D. villosum#4. (DOCX 24 kb)Localization of the 76 developed PCR markers in wheat, barley, and


The CxS1–xBT FeFETs with x = 0, 0.1, 0.2, 0.5, and 1 were characterized by the EBSD inverse pole figure map. The maps of x = 0, 0.1, and 0.2 showed more uniform and smaller grains with more inclusion of the a-axis component along the film normal than the maps of x = 0.5 and 1. Since spontaneous polarization of CxS1–xBT is expected to exist along the a-axis, inclusion of the film normal a-axis component is necessary to obtain polarization versus electric field (P–E) hysteresis curves of the CxS1–xBT when the E is applied across the film. Since memory windows of FeFETs originate from P–E hysteresis curves, the EBSD results were consistent with the electrical performance of the FeFETs, where the FeFETs with x = 0, 0.1, and 0.2 had wider memory windows than those with x = 0.5 and 1. The influence of annealing temperature for C0.1S0.9BT poly-crystallization was also investigated using the EBSD method.Electron backscatter diffraction (EBSD) was applied to investigate the grain size and orientation of polycrystalline Ca F2 cell size (F: feature size) [2Ta2O9(SBT)/Hf–Al–O(HAO)/Si gate stacks, where the HAO is (HfO2)y(Al2O3)1–y, often with y = 0.75 [xSr1–xBi2Ta2O9 or CxS1–xBT, was introduced as a ferroelectric material in MFIS FeFETs that attained larger memory windows of the FeFETs than the conventional SBT [xS1–xBT were developed, 3.3 V-write-voltage FeFETs [As a memory device, the ferroelectric-gate field-effect transistor (FeFET) has attracted much interest ,4,5,6,7.re size) ,3. FeFETre size) ,9,10,11,re size) ,13,14. Ty = 0.75 ,16,17. Oy = 0.75 . In 2013onal SBT . After te FeFETs . Memory-e FeFETs .xS1–xBT hidden inside the MFIS gate stacks. The EBSD method exhibits a two-dimensional map of the CxS1–xBT grains, whose size and crystal orientation can be understood visually. The minimum size detectable by the EBSD is as small as 10 nm, which can be observed in situ by the field-emission-type scanning electron microscope (FESEM) [xS1–xBT layers in the FeFETs were performed after the manufacturing processes were completed and the electrical properties were identified. In the processes, the whole MFIS stacks were annealed for the CxS1–xBT crystallization with the Pt electrodes formed on the CxS1–xBT by photo-lithography and ion-beam etching [xS1–xBT underneath the gate-electrode Pt could not be directly observed. How to remove the Pt layer was an important technique in this work.Ferroelectricity of materials strongly depends on the crystallinity, which has been conventionally characterized by X-ray diffraction (XRD) ,24. Rece (FESEM) . The spa (FESEM) ,28, micr etching . TherefoP-type Si substrates, with n+ source and drain regions formed in advance, were used to fabricate n-channel MFIS FeFETs with Pt/CxS1–xBT/(HAO)/Si gate stacks [xS1–xBT, and Pt were 7 nm, 200 nm, and 200 nm, respectively. HAO and CxS1–xBT were deposited by pulse laser deposition. Pt was deposited by electron-beam evaporation. Further details of the fabrication process were reported elsewhere [L) was 10 μm, and the gate width (W) was 200 μm [x was varied. Namely, CxS1–xBT FeFETs with x = 0 (SBT), x = 0.1, x = 0.2, x = 0.5, and x = 1 , were characterized. The SBT FeFET was annealed at 813 °C. The other CxS1–xBT FeFETs were annealed at 800 °C. In the second group, x was fixed at x = 0.1 and the C0.1S0.9BT FeFETs were annealed at nine different temperatures in O2 ambient, which were 748, 755, 763, 775, 788, 800, 813, 823, and 833 °C. All the temperatures were set-temperatures, which were about 10 °C higher than the real sample temperatures according to a temperature sensor. The sensor was an R-type thermocouple buried in a 20 mm2 diced Si wafer. Drain current versus gate voltage (Id–Vg) curves were measured using an Agilent 4156C transistor analyzer. Memory window (MW) was defined as the difference of the threshold voltages when Id/W = 10−8 A/μm on the Id–Vg hysteresis loops [P) versus gate voltage (P–Vg) hysteresis curves were obtained using the virtual ground mode of the Radiant RT6000S ferroelectric test system.e stacks . The thilsewhere . The mets 200 μm . Here, tis loops . PolarizxS1–xBT/HAO/Si FeFETs were manufactured successfully with good reproducibility [xS1–xBT, the whole MFIS stacks were annealed. Due to the coverage by the Pt gate metal, the ferroelectric CxS1–xBT film was hidden in the MFIS stacks. To observe the CxS1–xBT by EBSD, therefore, the Pt layer was removed using polishing cloths with 0.25 μm-size fine diamond pastes of SCAN-DIA. After the polishing, the samples were ultrasonically cleaned in acetone and in deionized water. Although the polishing made linear scratches to some extent on the surface of the CxS1–xBT layer, such scratches had no influence on crystal-orientation characterization by EBSD.High quality Pt/Ccibility . For cryXS1–xBT is orthorhombic (A21am) [a and b, we assumed it to be a pseudo-tetragonal structure (I4/mmm), with a = b = 0.5523 nm and c = 2.5026 nm, to index the Kikuchi patterns [a, b, and c were the lattice parameters of the crystal unit cell. During the scanning in this work, the FESEM accelerating voltage, work distance, and magnification were set to 15 kV, 25 mm, and 20,000, respectively. The scanning area and step were 4.4 × 6.6 μm2 and 15 nm, respectively. The EBSD is a good method to know the relationship between electrical properties and crystal orientations of ferroelectric materials [An EBSD system  was installed in the FESEM . During EBSD scanning, the samples were kept 70° tilted from the horizontal position in order to optimize the fraction of electrons scattered and the contrast in the diffraction pattern. When the electron beam hit the sample surface region, some of the scattered electrons agreed with the Bragg’s law and were diffracted from lattice planes. The diffraction patterns in the space, which are well known as Kikuchi bands, were captured by a phosphor screen equipped inside the FESEM. The Kikuchi band of every sample point was collected by the EBSD software and outputted into EBSD maps after Miller indexing. The crystal structure of C (A21am) . Howeverpatterns , where aaterials .xS1–xBT/HAO/Si FeFETs with x = 0, 0.1, 0.2, 0.5, and 1 were reported in our former work [Id–Vg curves are shown in Vg scanning from −4 V to 6 V, and then back to −4 V. During the scanning, the drain voltage (Vd) was set to 0.1 V. The source voltage (Vs) and substrate voltage (Vsub) were kept to zero. Drawing directions of the Id–Vg hysteresis loops were counter-clockwise in the cases of x = 0, 0.1, 0.2, and 0.5 V, indicating that the origins of the loops were ferroelectric. MWs of the CxS1–xBT FeFETs were 0.75 V , 0.89 V (x = 0.1), 0.84 V (x = 0.2), and 0.43 V (x = 0.5). In the case of CBT (x = 1), the MW was almost zero, but, more exactly, the loop direction was clockwise, indicating that the CBT was not ferroelectric and that the CBT FeFET showed weak charge-injection behavior in the Id–Vg measurement.The electrical properties of Pt/Cmer work . Id–Vg cP, of the CxS1–xBT FeFETs was also measured as a function of Vg, as shown in P–Vg measurements, Vd, Vs, and Vsub were set to 0 V. Note that the Vg was applied across all the stacked layers, which included CxS1–xBT, HAO, and the interfacial layer between HAO and Si, and the surface potential of the silicon. While Vg changed between −4 V and 6 V, half the peak-to-peak amplitudes of polarization (Phppa) [2 (x = 0), 2.22 μC/cm2 (x = 0.1), 2.12 μC/cm2 (x = 0.2), 1.46 μC/cm2 (x = 0.5), and 0.93 μC/cm2 (x = 1). The MWs on P–Vg curves, i.e., the voltage differences when P = 0, were 0.72 V (x = 0), 1.18 V (x = 0.1), 0.86 V (x = 0.2), 0.56 V (x = 0.5), and 0.1 V (x = 1). Drawing directions of the P–Vg hysteresis loops for all cases were counter-clockwise. As a non-volatile memory FeFET cell, a large coercive field of the CxS1–xBT is preferred rather than a spontaneous or remnant polarization if an appropriate Phppa  is attained [The polarization,  (Phppa) ,30, werexS1–xBT was shaded with a color guided by the color code unit triangle according to its orientation  means that the a-axis of the grain is parallel to the film normal. When the points with the same crystal orientation are accumulated together and make an area of one color, this is a grain of CxS1–xBT observed by EBSD. A neutron diffraction showed that SBT and CBT have spontaneous polarization along the a-axis [P–E) curves of epitaxial SBT thin films supported the a-axis directed spontaneous polarization [xS1–xBT can be regarded as the a-axis, and the more green color in the EBSD map , C0.1S0.9BT (x = 0.1), and C0.2S0.2BT (x = 0.2) are broadly distributed in the range of 0° < ψ ≤ 90°. On the other hand, the distribution of C0.5S0.5BT (x = 0.5) shows a large distribution below 20°, representing the near-c-axis oriented crystallization. Average values of the grain diameters  characterization for the CBT as shown in j and k shown in j and k in j and k  and (200). They do not have the c-axis oriented (008) peak. On the other hand, C0.5S0.5BT shows the (008) c-axis peak as well as the (115) peak but does not have the (200) peak. The XRD measurements of the c-axis orientation more in the C0.5S0.5BT than in the CxS1–xBT with x = 0, 0.1, and 0.2 are in good agreement with the EBSD characterization, which showed the C0.5S0.5BT orientated near the c-axis direction  and (200) peaks did not appear. The insufficient crystallization of the CBT was supported by EBSD observation of a major area occupied by multi-color dots  hysteresis curves of the CxS1–xBT, where the E is applied across the CxS1–xBT film. Regarding the CxS1–xBT with x = 0, 0.1, and 0.2, the wide range ψ distributions observed by EBSD  peak. 0.5S0.5BT had the (115) peak but had no (200) peak corresponding to the a-axis orientation. The c-axis-preferred crystal orientations are expected to show very small ferroelectric-polarization switching by imposing bipolar Vg pulses. Actually, the C0.5S0.5BT FeFET had smaller MW than the CxS1–xBT with x = 0, 0.1, and 0.2  on the C0.1S0.9BT crystallization. 0.1S0.9BT in the FeFET annealed at Ta = 748, 755, 763, 775, 788, 800, 813, 823, and 833 °C. For Ta = 748 °C, several grains were found, but noisy multi-color-dot areas covered most parts of the scanning area. For Ta = 755 °C, although the multi-color-dot areas still occupied more than half the map, grain size and number were increased in comparison with those for Ta = 748 °C. In the case of Ta ≥ 763 °C, grains with certain crystal orientations covered the most scanning area. In the temperature range from Ta = 775 °C to 833 °C  grown between the I layer and the S surface in the MFIS of the FeFETs. As shown in [xS1–xBT crystallization, the IL is grown to normally a few nano-meters-thick. The IL may get thick as the Ta increases from 800 °C to 833 °C. In the MFIS, three capacitances of F, I, and IL and a capacitance of S (appearing when the depletion layer is formed) are connected in series and share the Vg. The voltage across the CxS1–xBT is reduced when the IL becomes thick because the voltage across the IL is increased. With the voltage decrease across the CxS1–xBT, the MW may become narrow as Ta is increased from 800 °C to 833 °C. The increasing MW with increasing shown in ,16,17,19xSr1–xBi2T2O9 layer inside the FeFET stacks. The distributions of grain size and orientation of CxS1–xBT with x = 0 (SBT), 0.1, 0.2, 0.5, and 1 (CBT) were presented in the inverse polar figure maps and the unit triangles of EBSD. The grain size and crystal orientation distributions depended on the content x of CxS1–xBT. There were more uniform and smaller grains with random orientation in SBT, C0.1S0.9BT, and C0.2S0.8BT. In C0.5S0.5BT, grains got larger and the major grains were orientated near the c-axis direction. Since spontaneous polarization exists along the a-axis, these EBSD results explain well the reason that the memory windows of SBT, C0.1S0.9BT, and C0.2S0.8BT FeFETs were much wider than that of the C0.5S0.5BT FeFET. EBSD investigation also revealed the existence of noisy multi-color-dot areas on which no Kikuchi-bands appeared, indicating that the areas were non-ferroelectric. EBSD characterizations of the C0.1S0.9BT FeFETs with various annealing temperatures from Ta = 748 to 833 °C were performed. As the Ta was raised from 748 °C toward 763 °C, grain size and number increased, and the noisy multi-color-dot areas decreased. When Ta was increased from 763 °C to 833 °C, the EBSD maps showed the C0.1S0.9BT mostly composed of grains with rather uniform sizes and random crystal orientations. Good consistency among the EBSD investigations of the CxS1–xBT and memory window widths of the CxS1–xBT FeFETs could be discussed.The EBSD method was used to characterize the Ca


Dear Editor,Intensity-modulated radiotherapy (IMRT) has a distinct advantage of high conformity and is an appropriate technique for treating nasopharyngeal carcinoma (NPC). Previous studies have demonstrated that anatomical changes in the external contour, shape, and location of the target and critical structures are significant and result in dosimetric changes , 2. PatiWe analyzed the data of 54 patients with newly diagnosed NPC between October 2013 and June 2016. The mean age was 45.5 years (range 18–67 years). All patients had undifferentiated non-keratinized carcinoma. According to the 7th edition of the Union for International Cancer Control (UICC)/American Joint Committee on Cancer (AJCC) staging system, 6 (11.1%), 17 (31.5%), 29 (53.7%), and 2 (3.7%) patients had stage IVb, IVa, III, and II diseases, respectively. Twenty-four (44.4%) patients received induction chemotherapy and concurrent chemoradiotherapy, 29 (53.7%) received concurrent chemoradiotherapy, and 1 (1.9%) received radiotherapy alone. The median duration of radiotherapy was 47 days (range 41–71 days). The median duration of interruption between two plans was 2 days (range 1–24 days). The baseline clinical characteristics are shown in Table In plan-I radiotherapy, computed tomography simulation (CT-I) was performed for target delineation. The gross target volume of the nasopharynx (GTVnx)-I was defined as all gross lesions determined with clinical and imaging examinations. The high-risk clinical target volume (CTV1-I) was delineated with a 1-cm margin surrounding the GTVnx-I area. The low-risk clinical target volume (CTV2-I) was delineated with a 0.5-cm margin surrounding the CTV1-I. Any metastatic retropharyngeal lymph nodes and cervical lymph nodes were delineated as GTVrpn-I and GTVnd-I . CTVrpn1On the basis of results of previous studies , 2, we cA 3- to 5-mm margin surrounding the above targets was required for the delineation of the planning target volumes .Before June 2014, the doses prescribed were as follows: GTVnx/rpn/nd-I, 57 Gy in 26 fractions at 2.19 Gy/fraction; PCTV1/rpn1/nd1-I, 50 Gy in 26 fractions at 1.92 Gy/fraction; PCTV2/rpn2/nd2-I, 46–47 Gy in 26 fractions at 1.77–1.81 Gy/fraction; PGTVnx/rpn/nd-II, 11 Gy in 5 fractions at 2.2 Gy/fraction; PCTV1/rpn1/nd1-II, 10 Gy in 5 fractions at 2.0 Gy/fraction.After June 2014, the doses prescribed were as follows: PGTVnx/rpn/nd-I, 53–54 Gy in 25 fractions at 2.12–2.16 Gy/fraction; PCTV1/rpn1/nd1-I, 47.5 Gy in 25 fractions at 1.90 Gy/fraction; PCTV2/rpn2/nd2-I, 45 Gy in 25 fractions at 1.8 Gy/fraction; PGTVnx/rpn/nd-II, 15–15.5 Gy in 7 fractions at 2.14–2.21 Gy/fraction; PCTV1/rpn1/nd1-II, 13.5 Gy in 7 fractions at 1.93 Gy/fraction.tolerance limit) by the percentage of dose of each plan in total dose.The target delineation and dose prescription of organs at risk (OARs), including the brain stem, spinal cord, and optic chiasm, were performed according to Radiation Therapy Oncology Group (RTOG) 0225 protocol . Under tP > 0.05). GTVnx, GTVnd-R, volumes of bilateral parotids, and volumes of bilateral submandibular glands showed significant reductions after 22 fractions of irradiation , whereas other volume changes were not significant   in the total dose (Dmean%) of PGTVnx, bilateral PGTVrpn, or bilateral PGTVnd were observed between the two plans. Among the evaluated OARs, Dmean% values of the brain stem, spinal cord, optic chiasm, pituitary, oral cavity, oropharynx, hypopharynx, and thyroid gland were significantly different between plan-I and plan-II , 13 (24.07%), and 5 (9.25%) patients, respectively; 1 (1.85%) developed grade 4 neutropenia.The median follow-up period was 30 months (range 3–44 months). Three patients developed distant metastasis, and 4 developed locoregional failure, but none occurred in the regression area. The 3-year overall survival, local recurrence-free survival, and distant metastasis-free survival rates were 93.3%, 90.5%, and 91.4%, respectively.Few studies have described the target redelineation in detail for replanning or modified dose prescription for tumor regression areas. Hansen et al.  used theAccording to the principle of radiobiology, the tumor-killing effect of radiation is related to not only the fractionated dose but also the total dose. Wang et al.  and YangIn conclusion, our adaptive replanning IMRT for patients with NPC provides a new perspective on target redelineation and dose prescription, as it would demonstrate a significant dosimetric and clinical benefits without recurrence and reduction in survival.Additional file 1: Table S1. Changes in target volumes and volumes of OARs between plan-I and plan-II. Table S2. Relative doses for targets and OARs between plan-I and plan-II.


Doxorubicin is a typical anticancer drug that causes cardiomyopathy and heart failure (HF). The aim of our study was to investigate incidence, risk factors for doxorubicin‐induced HF in Korean cancer patients and their survival rate, utilizing a nationwide population‐based cohort.We analyzed 58 541 cancer patients who received doxorubicin between 2003 and 2010. Descriptive analysis was performed in patients with breast cancer, hematologic malignancy, gynecological malignancy, and sarcoma. Risk factors associated with doxorubicin‐induced HF were investigated using a Cox proportional hazards model. The survival rate of doxorubicin‐induced HF patients was compared with that of patients without doxorubicin‐induced HF.P < 0.001).A total of 2324 (4%) were diagnosed with doxorubicin‐induced HF. In patients with breast cancer, predictive risk factors for doxorubicin‐induced HF included age over 65 years , hypertension [HR 2.45 (2.12‐ 2.84)], diabetes mellitus [HR 1.26 (1.05‐1.51)], coronary artery disease [HR 2.08 (1.63‐2.66)], advanced stage [HR 1.31 (1.13‐1.50)], and trastuzumab administration [HR 2.94 (2.54‐3.40)]. In patients with hematologic malignancy, predictive risk factors included age over 65 years [HR 1.75 (1.49‐2.07)], hypertension [HR 1.62 (1.37‐1.92)], and coronary artery disease [HR 2.28 (1.80‐2.89)]. Five‐year survival rates of patients with doxorubicin‐induced HF were significantly lower relative to those of patients without HF in breast cancer and hematologic malignancy: 80% vs 84% and 69% vs 75%, respectively (In cancer patients treated with doxorubicin, management of risk factors, early detection, and treatment for doxorubicin‐induced HF might be critical for patient survival. It is also the prototype for type 1 chemotherapy‐induced cardiotoxicity characterized by myocardial cell death, rendering dose‐dependent, and irreversible injury.22.1The study protocol was approved by the Institutional Review Board of the National Cancer Center in Korea (NCC2016‐0142). Our study included cancer incidence data from the Korean Central Cancer Registry (KCCR). Briefly, patients diagnosed with cancer at a hospital are entered into the KCCR, and their data are used to calculate cancer registration statistics such as cancer incidence, survival rate, and prevalence. These data cover approximately 95% of all newly diagnosed malignant tumor cases in Korea.2.2The present study used cancer incidence data registered with KCCR (2003‐2010), health insurance claims and health care utilization data from NHIS (2003‐2010), and cause of death data from Statistics Korea (2003‐2011).Among the 1 225 218 patients diagnosed with cancer and registered with the KCCR between 2003 and 2010, 146 751 patients were selected who were ≥18 years and diagnosed with any of the following: breast cancer, hematologic malignancy , gynecologic malignancy , and sarcoma (soft tissue sarcoma and osteosarcoma). Patients with multiple primary cancers were excluded. Among the 146 751 patients with a single primary cancer, 58 541 patients were selected who underwent chemotherapy after cancer diagnosis but did not show HF prior to or within one day of chemotherapy administration . Patients were assigned to the HR group if they experienced cardiomyopathy due to drugs and other external agents (I42.7), cardiomyopathy (I42), other cardiomyopathies (I42.8), unspecified cardiomyopathy (I42.9), HF (I50), congestive HF (I50.0), congestive HF with systolic dysfunction (I50.04), other and unspecified congestive HF (I50.08), LV failure (I50.1), and unspecified HF (I50.9). All others were assigned to non‐HF group. Other diseases were classified and defined according to ICD‐10: I10, I11, I12, and I13 for hypertension; E10, E11, E13, and E14 for diabetes mellitus; E78 for dyslipidemia; and I20, I21, I22, I23, I24, and I25 for coronary artery disease.The subjects were divided by cancer disease codes: C50 for breast cancer; C81, C82, C83, C85, C86, C91, and C92 for hematologic malignancy; C54, C55, and C56 for gynecologic malignancy; and C40, C41, and C49 for sarcoma. For each cancer type, subjects were divided into HF and non‐HF groups. The general health screening data collected closest to the date of cancer onset were used to obtain their body mass index (BMI), frequency of smoking, smoking cessation, and nonsmoking status.2.4t tests were performed to evaluate statistically significant differences between HF and non‐HF groups.Descriptive analysis was performed on age, gender, BMI, smoking, hypertension, diabetes mellitus, dyslipidemia, coronary artery disease, Surveillance, Epidemiology, and End Results (SEER) summary stage, radiation therapy, and number of cycles of doxorubicin in patients with breast cancer, hematologic malignancy, gynecologic malignancy, and sarcoma. In breast cancer patients only, frequency analysis was performed on trastuzumab after doxorubicin administration. Chi‐square tests and Student's P < 0.05 considered significant.To investigate the risk factors that affect HF patients and survivors, a Cox proportional hazards model was used to estimate hazard ratios (HRs). Age, gender, hypertension, diabetes mellitus, dyslipidemia, coronary artery disease, SEER summary stage, radiation therapy, and number of cycles of doxorubicin were adjusted in the Cox proportional hazards model, while smoking and BMI were also added to independent variables for analysis. Analyses were performed with gender removed and trastuzumab after doxorubicin added for breast cancer, SEER summary stage removed for hematologic malignancy, and gender removed for gynecologic malignancy. We analyzed survival rates, 95% confidence intervals (CIs), and adjusted HRs (aHRs) of HF in breast cancer, hematologic malignancy, gynecologic malignancy, and sarcoma. All data were analyzed using SAS Windows Ver. 9.4 , with 3Table 3.1Of the 43 586 patients with breast cancer, 1482 (3.4%) were diagnosed with doxorubicin‐induced HF. Compared with breast cancer patients who did not develop HF, patients with HF were older and had more advanced stage and many comorbidities. Among the 11 768 patients with hematologic malignancy, 725 (6.2%) were diagnosed with doxorubicin‐induced HF. Compared with patients who did not develop HF, they were older and had more comorbidities Table .3.2When adjustments were made except for BMI and smoking due to missing data, the following were predictive risk factors for doxorubicin‐induced HF in patients with breast cancer: age >65 years [aHR 1.34 (1.05‐1.72)], hypertension [aHR 2.45 (2.12‐2.84)], diabetes mellitus [aHR 1.26 (1.05‐1.51)], coronary artery disease [aHR 2.08 (1.63‐2.66)], advanced stage [aHR 1.31 (1.13‐1.50)], and additional trastuzumab administration [aHR 2.94 (2.54‐3.40)]. In patients with hematologic malignancy, the predictive risk factors were age >65 years [aHR 1.75 (1.49‐2.07)], hypertension [aHR 1.62 (1.37‐1.92)], and coronary artery disease [aHR 2.28 (1.80‐2.89)] Table .2 [aHR 1.28 (1.03‐1.59)], hypertension [aHR 1.49 (1.18‐1.89)], and coronary artery disease [aHR 2.34 (1.69‐3.23)] ], coronary artery disease [aHR 2.39 (1.74‐3.28)], and additional trastuzumab administration [aHR 3.15 (2.59‐3.83)]. For patients with hematologic malignancy, the predictive risk factors were age >65 years [aHR 1.84 (1.47‐2.31)], BMI ≥25 kg/m)] Table . Hyperte)] Table .3.3P < 0.001).As shown in Figure P < 0.001)  , B (asymptomatic), C (symptomatic), and D (refractory).2) or obese (BMI ≥30 kg/m2) is previously reported risk factors for anthracycline‐induced cardiotoxicity,Previous studies indicate that risk factors for doxorubicin‐induced HF include old age (>65 years), female, preexisting cardiac disease, hypertension, accumulated dose, mediastinal radiation therapy, and concomitant chemotherapy .The 5‐year survival rate stratified by HF stage is 97% for stage A, 96% for stage B, 75% for stage C, and 20% for stage D.2 for breast cancer, 6‐8 cycles of 40‐50 mg/m2 for lymphoma, and 6‐8 cycles of 75‐90 mg/m2 for sarcoma.y = x2/16 where x is the number of cycles of 50 mg/m2 each and y is the percent of prevalence of doxorubicin‐induced HF, the incidence of HF is 4% at eight cycles.Our study has several limitations. First, there is a lack of data on echocardiographic findings  or biomarkers. Because our study was based on NHIS, it was difficult to know the detailed test results and of individual patients. Second, because we primarily included patients with stage C (symptomatic) HF, we presume that stage B (asymptomatic) HF was excluded from this study. According to the Multi‐Ethnic Study of Atherosclerosis, 18.6% of participants with asymptomatic HF developed overt HF within 9 years.In conclusion, management of risk factors, early detection, and treatment for doxorubicin‐induced HF in cancer patients treated with chemotherapy including doxorubicin might be critical for patient survival.The authors declare no conflict of interests. Click here for additional data file. Click here for additional data file.