Dalbergia odorifera T. Chen, known as Jiang Xiang in traditional Chinese medicine, is formed very slowly, and there is a need to better understand the process and promote heartwood formation. Chemical induction is considered to be one of the promising methods to induce heartwood formation. However, to date no method has been proved effective for D. odorifera as little is known about biochemical and physiological changes during heartwood development. Three potential heartwood induction substances viz. acetic acid, sodium chloride, and hydrogen peroxide solutions were injected into the trunk of D. odorifera to determine the effect on heartwood formation and physiological activity. Non-structural carbohydrates, lipids, wood properties, and essential oil were assessed in the post-treatment period. As also observed in the formation of natural heartwood, chemical-induced Jiang Xiang production was accompanied by sapwood dehydration, non-structural carbohydrates consumption, and synthesis of heartwood substances. As the heartwood substances accumulated, basic density and essential oil content increased gradually, thereby Jiang Xiang was finally produced. In this process, physiological parameters of discolored sapwood gradually evolved to resemble those of natural heartwood. Hydrogen peroxide-induced Jiang Xiang was closest to natural heartwood, and the essential oil components met the standards for high-quality Jiang Xiang, while the induction effects of acetic acid and sodium chloride were unsatisfactory. Thus, this study indicates that hydrogen peroxide has the potential to induce Jiang Xiang production in Dalbergia odorifera.The highly valuable heartwood of  Dalbergia odorifera T. Chen (Leguminosae) is a medium-sized tree native to Hainan Island, southern China -beta-Farnesene 0–3%, and alpha-Bisabolol 0.1–6.0% in the essential oil  Gilg. (Acacia auriculiformis A. Cunn. ex Benth. (Samanea saman (Jacq.) Merr. , which have been applied to stimulate the production of agarwood, the heartwood of .) Gilg. . In addix Benth. , Samanea.) Merr. , Quercusa Thunb. , Santalualbum L. , D. odordorifera , and somdorifera . HoweverJiang Xiang in D. odorifera (2O2) could be an important wound signal in D. odorifera that may help induce vessel occlusions and production of Jiang Xiang (2O2), acetic acid (CH3COOH), and sodium chloride (NaCl) solutions.Mechanical wounding has been reported to induce the production of dorifera . Moreoveng Xiang . Experime.g. sucrose, fructose, glucose, arabinose, galactose, stachyose). The arabinose and galactose contents were reported to be related to the synthesis and hydrolysis of hemicellulose in the cell wall during the formation of heartwood  the physiological changes in xylem during chemical-induced Jiang Xiang production in D. odorifera.In order to better understand heartwood formation induced by chemicals, this study was conducted to investigate: 1) whether HD. odorifera plantation located in Xiashi Arboretum , Pingxiang City, Guangxi Zhuang Autonomous Region (GZAR) was selected for this study. The site is characterized by the south subtropical monsoon climate with a mean annual temperature of 20.5–21.7°C, mean annual rainfall 1,200–1,500 mm yr−1, mean annual evaporation 1,261–1,388 mm yr−1, and mean annual relative humidity 80–84%. The soil is lateritic with an average depth of more than 1 m. The D. odorifera plantation was established in 2012 with 2 m × 2 m spacing. The status of stand growth in 2017 was as follows (mean ± standard deviation): tree height 5.03 ± 0.81 m, diameter at breast height (DBH) 6.54 ± 1.35 cm, and north–south crown diameter 3.21 ± 0.68 m.A 5-year-old −1 hydrogen peroxide (H2O2), 1.0 mol L−1 (pH≈2.4) acetic acid (CH3COOH) and 1.0 mol L−1 sodium chloride (NaCl). Distilled water was injected as a control. Sixty trees of similar size, all without heartwood were selected for a single-tree plot experiment with 15 replicates for each treatment. A tiny drill was used to detect which trees have or don’t have heartwood. In May 2017, an injection hole of 1 cm diameter and 5 cm deep was drilled at a downward angle of 45° at 1.3 m from the ground on each tree. An amount of 50 ml solution was injected into each tree over 8 h period with a 5 ml syringe, and the hole was sealed with a cork after injection.Three chemicals used were: 0.1 mol LThe DBH, tree height, and north–south crown diameter of the sampled trees were measured before and at 6 months after commencement of treatment, and the growth increment was calculated.Five trees per treatment were harvested at one, three and six months after injection treatment. At the same time, three trees with heartwood were selected to collect natural heartwood. At each harvest, the trees were felled, and the boles were immediately dissected Figure 1The length of discoloration in the transverse .−1 pH 7.0 phosphate buffer solution, acetonitrile:water = 82:18 (v:v); flow rate 1.0 ml min−1; column temperature 25°C; injection volume 10 μl; wavelength 245 nm (after degraded by 4 mol L−1 trifluoroacetic acid and derivatives with 1-phenyl-3-methyl-5-pyrazolone). Glucose, fructose, sucrose, and stachyose were determined by high performance liquid chromatography (HPLC) with the following parameters: Shodex NH2P column ; mobile phase: acetonitrile:water = 75:25 (v:v); flow rate 1.0 ml min−1; injection volume 10 μl; column temperature 35°C; detector: differential refractive index (DRI), temperature: 35°C. The starch content was determined by an anthrone colorimetric method ; mobile phase: 0.1 mol Lc method . The lipFresh wood samples were weighed immediately three times, and basic density (bone-dry weight per unit of fresh volume) was determined using the water displacement method. Dry weight was obtained after drying at 105°C to constant weight, and relative moisture content was calculated .For each treatment, 5 g powdered wood samples were immersed in 50 ml petroleum ether and shaken for 24 h. After filtration and concentration , essential oils were obtained, and the oil content was calculated . The oil−1, and this temperature held for 15 min. Detailed operating parameters are described by GC–MS analysis was performed by Agilent coupled with a 6890N-5975I system equipped with flame ionization detector (FID) and a DB-5MS . The temperature program included a starting temperature of 70°C which was then increased to 250°C at the rate of 8°C min2O2, CH3COOH, NaCl, and control treatments at one, three, and six months after treatment were evaluated using Duncan’s multiple range tests using the data processing software SPSS 17.0 . Significant differences in non-structural carbohydrates and lipid contents in each part of the xylem among H2O2, CH3COOH, NaCl, and control treatments were evaluated by one-way ANOVA with asterisks, *p < 0.05, **p < 0.01. The plots for the graphs were generated in SigmaPlot 10.0 .Data were subjected to one-way analysis of variance (ANOVA), and any significant differences among Hp < 0.01). Compared to control, the increment in height, DBH, and crown diameter of the CH3COOH treated trees was significantly decreased by 25.53, 34.92, and 32.78%, respectively . For position II, except for starch, significant differences in NSCs were observed between different treatments, with the H2O2 treatment being the lowest. The lipid content showed the opposite trend. Contents of starch, soluble sugars, and lipids in position III were all significantly different among the treatments, and their patterns were similar to those of position II. In addition, there were lower concentrations of stachyose, galactose, arabinose, and sucrose than other NSCs in position III, especially for the H2O2 treatment. Correspondingly, the lipid content in position III was higher in the H2O2 than in other treatments.One month after injection, significant changes occurred in the starch, soluble sugars, and lipids in various positions of the xylem. For all treatments, NSCs gradually decreased while lipids increased from position I to position III Figure 32O2 treatment being the lowest, while arabinose, galactose, and fat were not significantly different. Except for sucrose and stachyose, the other carbohydrates in position II almost continued the patterns of the position I, and the lipid content was also significantly different between treatments. Furthermore, compared to one month after treatment, fructose, glucose, sucrose, stachyose, and starch were depleted in position III in all treatments, whereas the arabinose and galactose contents significantly increased . The lipid content in positions II and III increased significantly in all treatments over time. In particular, the H2O2 treatment induced the xylem to consume more NSCs and synthesize more lipids than the other treatments.After six months of treatment, significant changes were observed in each treatment Figure 43COOH treatment, followed by the H2O2 treatment. No significant changes in the transverse discoloration length and area were found with chemical treatments over time. In contrast, the transverse discoloration of the control treatment nearly doubled after three months, and the subsequent increase was small .In this study, all treatments caused sapwood discoloration Figure 53COOH treatment, the discoloration length and area after three months were more than double those at one month. The discoloration length and area of the NaCl, H2O2, and CK plants increased significantly after three and six months. Hence, NaCl, and H2O2 had a lengthier period of induction effect compared with CH3COOH.The length and area of axial discoloration of all treatments increased significantly with the treatment time (p < 0.05). The relative moisture content of each treatment was significantly reduced after three months compared to one month of treatment (p < 0.05), which were not significantly different from that of NH   in H2O2 and control treatment but not in CH3COOH and NaCl treatments.As shown in 2O2 and control treatments was significantly higher than that of NH after six months, while the density of the discolored sapwood in CH3COOH and NaCl treated wood remained slightly lower than that of NH  than that of NH (0.82%). However, the oil content of the discolored sapwood induced by the CH3COOH and NaCl treatments was still significantly lower than that of NH.Compared with natural heartwood, the oil content of induced heartwood was significantly lower after one and three months of treatment , had the total relative amount of the essential oil components at 46.47%.Twenty-one essential oil components in the natural heartwood were used as a reference set for comparison with the essential oil components of the natural heartwood. The composition and relative content of essential oil components induced by chemicals increased with the treatment time Table 1.2O2 treatment with the relative amount reaching 48.59%. Moreover, the relative amount of trans-Nerolidol (39.14%), (E)-beta-Farnesene (0.04%), and alpha-Bisabolol (0.12%) in the H2O2-induced essential oil met the standards for high-quality Jiang Xiang, i.e. 25–60%, 0–3%, and 0.1–6.0%, respectively. The relative amount of alpha-Bisabolol in the other treatments was considerably less at 0.03 and 0.04%. Therefore, a greater diversity of heartwood oil components was induced by the H2O2 treatment than by the CH3COOH and NaCl treatments. H2O2 was the only inducer of essential oil components that reached the level of NH and met the standards of high-quality Jiang Xiang.All 21 essential oil components in the natural heartwood were detected in the HQuercus serrata , (E)-beta-Farnesene (0.04%), and alpha-Bisabolol (0.12%) in the H2O2-induced essential oil met the standards for high-quality Jiang Xiang which are 25–60%, 0–3%, and 0.1–6.0%, respectively. Thus, H2O2 is a promising inducer for Jiang Xiang production in D. odorifera. The superior effect of H2O2 treatment may be related to its involvement in the wound signal of D. odorifera -induced Jiang Xiang was closest to natural heartwood and met the standards for high-quality Jiang Xiang, while the induction effects of CH3COOH and NaCl were unsatisfactory. Thus, this study supports the hypothesis that H2O2 has the potential to induce formation of Jiang Xiang in D. odorifera.All chemical inducers in this study are effective in inducing discoloration of sapwood. In this process, physiological parameters of discolored sapwood gradually evolved to resemble those of natural heartwood. Comparative analysis indicated hydrogen peroxide  and the National Key Research and Development Program of China (2016YFD0600601).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bacillus coagulans in a continuous fermentation, 2781.01 g of lactic acid was produced from 3916.91 g of sugars from hydrolyzed sugar beet pulp, with a maximum productivity of 18.06 g L−1h−1. Without interfering in the sugar production, ethanol, or lactic acid, it is also possible to produce pectin and phenolic compounds in the biorefinery. The lactic acid produced was purified by a bipolar membrane electrodialysis and the recovery reached 788.80 g/L with 98% w/w purity.Lactic acid is a high-value molecule with a vast number of applications. Its production in the biorefineries model is a possibility for this sector to aggregate value to its production chain. Thus, this investigation presents a biorefinery model based on the traditional sugar beet industry proposing an approach to produce lactic acid from a waste stream. Sugar beet is used to produce sugar and ethanol, and the remaining pulp is sent to animal feed. Using  Sugar beet provides 16% of the sugar produced worldwide . In 2017In the traditional process, sucrose is extracted from sugar beet using hot water. The juice is then purified, concentrated, and crystallized. The remaining pulp is sent to animal feed or biogas production. The molasses is generally used for ethanol production, in animal feed, or as a medium for yeast biomass production . NowadayA biorefinery is an installation similar to an oil refinery that uses a conversion of biomass to produce biofuels, energy, and chemicals . ConsideThe production of sucrose and ethanol is a long-time known process for the sugar beet industry. However, the concept of biorefinery to aggregate value to its residues has just recently come into highlight ,6. The mThe current study presents a new possibility to produce second-generation (2G) lactic acid from SBP. Lactic is an organic acid with widespread applications mainly in pharmaceutical, cosmetic, chemical, as well as food industries ,19. In 2B. coagulans is a homofermentative microorganism that efficiently converts the hydrolyzed SBP into lactic acid. The main results from the carried-out fermentations are presented below.g/g, an average productivity of 1.25 g L−1h−1, and a maximum productivity of 12.48 g L−1h−1. Yeast extract is used as an effective source of nutrients for high lactic acid production, especially due to the presence of vitamin B and amino acids used for cell growth [−1h−1, and maximum productivity of 14.67 g L−1h−1. Comparing batches B1 and B2 (Two batch fermentations (named B1 and B2) were performed simultaneously in identical bioreactors, to evaluate the need for yeast extract addition. In batch B1, the use of yeast extract resulted in a total lactic acid production of 29.90 g, with a yield of 0.83 l growth . Howeverl growth . Bearingl growth . Using t1 and B2 , it is n−1. −1h−1 and 0.38 g/g , and the feed started after 10 h when the glucose was almost depleted (around 2 g/L). The maximum lactic acid concentration reached 34.79 g/L after 33 h of fermentation  was performed using an annexed cell retention system. The dilution rate was kept the same as experiment C1 , and also the remaining sugars as follows  with a dilution rate of 0.10 hre 104 h . The prohe tests .B. coagulans without any extra supplementation of nutrients, even for a long time with a running process. Additionally, the process temperature settled at 52 °C helped to avoid further contamination for everyday strains, making it possible to run the process for longer times without by-product production.This latest fermentation showed that SBP may be an excellent substrate to be employed as a substrate for lactic acid production, since its composition suited the needs of l-lactic acid from hydrolyzed municipal solid waste on a batch mode, noticing a high positive impact of yeast extract addition on the media. Balakrishnan et al. [d-lactic acid from hydrolyzed millet bran residue and hydrolyzed casein, with a productivity of 0.45 g L−1h−1. Tian et al. [−1h−1 on a simple batch mode. Using simultaneous saccharification and fermentation of lignocellulosic corncob residue, Jiang et al. [l-lactic acid with a yield of 0.76 g/g cellulose in fed-batch mode. Solid carob waste from the Lebanese industry was also suggested by Bahry et al. [Lactobacillus rhamnosus immobilization in alginates beads for lactic acid production, reaching a productivity of 1.22 g L−1h−1. d-lactic acid production from orange peel waste hydrolysates was shown by de la Torre et al. [−1h−1 of lactic acid productivity. Cell adaptation was also used as a strategy by Aulitto et al. [Considering the recent literature, López-Gómez et al.  producedn et al.  producedn et al.  producedg et al.  producedy et al. , who stue et al. , using co et al.  to produSugar beet pulp has gained attention due to its high amount of hemicellulose and pectin, moderate cellulose, and low lignin amounts . Some prThe biorefinery concept can be applied to lactic acid production from SBP, as it has been done for sugar and ethanol production. One of the advantages of using a byproduct of sugar production for lactic acid production is that its industrial plant can be easily integrated into a mill following the biorefinery concept. This may be also interesting for the economical point of view, since it provides a larger range of business opportunities. A simplified mass balance of an integrated first (1G) and 2G sugar beet proposed biorefinery was performed in Microsoft Excel based on literature data ,33,34,35All 1G products and inputs were gathered from Dorst, Krajnc et al., Lorenz, Marzo et al., and Zeist et al. ,35,36,37For this study, a medium-sized plant with a milling capacity of 340 days of operation was adopted. This plant has the potential to produce around 315,000 t of sugar and 13 million L of anhydride ethanol per year, considering 8 t molasses produced per hour. From this operation, 405,000 t of SBP is produced, to be designated to lactic acid production and other high-value products.Finally, considering a goal of a minimum of 20,000 t of lactic acid production per year with 85% purity, an integrated process for a biorefinery was designed considering the experimental data from C2, C3, and C4. In this case, the industrial plant has the capacity of processing an average of 110 t of SBP per year, resulting in average production of 8600 t/year of phenolic compounds, 33,300 t/year of pectin, and 23,000 t/year of animal feed with 20% of protein content, as well as 20,000 t of lactic acid. The produced lactic acid is then recovered employing a liquid−liquid extraction process and a stripping separation, as presented by Udachan and Sahoo . The recFinally, considering the C6 fermentation data, The lactic acid downstream method proposed in this investigation is based on a bipolar membrane electrodialysis. Two fermentation broths were submitted to this process: C2, C3, and C4 mixed broths (to reach a working volume enough to the downstream process), and C6 fermentation broth.42− resulting from the SBP pretreatment, and a considerable amount of Na+ from the lactic acid neutralization during the fermentation process  and Na+ (6.97 g/L). The overall process yield was 46.23% w/w, higher than the 23% found by Pleissner et al. [C2, C3, and C4 mixed broths resulted in 62.90 L of liquid, with 29.84 g/L of lactic acid, 9.88 g/L of residual sugars, a considerable amount of SO process . The micr et al.  and the 42−, and 12,819.43 mg/L of Na+ (w/w. The main impurities were acetic acid (40.00 g/L) and Na+ (8.98 g/L). The overall process yield was 39.72% w/w. As was expected, the increase in the number of steps led to a decrease in the process yield when compared to the previous one.The C6 fermentation broth (88.00 L) was composed of 31.66 g/L of lactic acid, 1808.00 mg/L of SOL of Na+ . In thisw/w presented in this work is the highest achieved using electrodialysis for the separation and purification of 2G-lactic acid produced from SBP. In general terms, membrane downstream processes are efficient for a separation between lactic acid and fermentation sugars because of their significant differences in molecular weight. Additionally, they are environmentally friendly and easy to scale up. For those reasons, bipolar membrane electrodialysis has attracted attention for organic acid separation and purification [Using defined and semi-defined fermentation broth, Pleissner et al.  achievedfication . An impofication ,44,45,46+ was already demonstrated by Pleissner et al. [The use of electrodialysis for lactic acid separation and purification also has the advantage of recycling inputs that are necessary for previous steps, especially NaOH. The recovery and reuse of Nar et al. . The autw/w lactic acid reached in this study was already more than the commercial lactic acid sold as a solution of 88% [The optimization of each step of this process could lead to higher product recovery yields and a decrease in the impurities. However, the average purity of 96.5% n of 88% . Furthern of 88%  proposedn of 88% , reducinThe lactic acid downstream process is a fundamental step in the lactic acid production chain. Many applications require a final lactic acid of high purity and, together with this issue, comes the environmental concerns regarding solvent use and waste production . AccordiBacillus coagulans A166 strain (Leibniz-Institut für Agrartechnik und Bioökonomie e.V. (ATB), Leibniz, Germany) used in the fermentation was isolated from hemp leaves. The microorganism was stored in glycerol 30% v/v in a freezer at −80 °C.The inoculum was grown in 250 mL sterile Erlenmeyer flasks with 60 mL MRS  broth  provided sugar beet pulp (SBP) in the form of dry pellets. The material was composed of 31.6% hemicellulose, 21.5% cellulose, 9.7% proteins, 8.7% free sugars, 8.5% ash, 2.1% lignin, 2.1% fat, and approximately 15% non-identified material.w/w of SBP was pre-treated at 121 °C for 30 min in the presence of H2SO4 0.5% (v/v). Afterward, the pH was adjusted manually to 5.0 ± 0.1 by adding NaOH 20% (w/w). The pre-treated SBP was transferred to a 2 L BIOSTAT bioreactor  in a lab-scale, and to a 50 L bioreactor  in a pilot-scale, both assembled to 50 °C, 150 rpm and pH of 5.0 ± 0.1. In these conditions, pre-treated SBP was enzymatically hydrolyzed for 24 h using the following:0.3 mL of Accellerase 1500/g of cellulose0.114 mL of Pectinase L40/L of pre-treated SBP0.429 mL of Protease Fermgen/L of pre-treated SBPBased on a previous study from Alexandri et al. , 10% w/wThe average sugar concentration in the hydrolyzed SBP was 16.48 ± 2.77 g/L of glucose; 9.62 ± 3.38 g/L of xylose; and 9.98 ± 3.69 g/L of arabinose.w/w) NaOH. Samples were taken regularly for the analysis of sugar , lactic acid, and other organic acid concentrations, as well as for dry cell weight. After cell inactivation for 20 min at 90 °C, samples were stored at −20 °C.Fermentations using SBP hydrolysate were carried out in duplicate at 52 °C, 400 rpm, and pH 6. The bioreactor used was a 2 L BIOSTAT bioreactor (Sartorius AG) with 1 L of working volume. Regulation of pH was carried out by an automatic injection of 20% (w/w) NaOH. Temperature and stir were fixed at 52 °C and 300 rpm. After the consumption of all the glucose present in the hydrolysate, the continuous process was started. The feed composition was sterile SBP. Different dilution rates were evaluated: 0.10; 0.15; 0.20; 0.30 h−1. The level was fixed at 3 L. The cell retention module system was equipped with hollow-fiber filters UMP-1147 , with a pore size of 0.2 µm. A schematic representation of the system is shown in Continuous fermentations were carried out in a sterile 5 L BIOSTAT bioreactor (Sartorius AG). The initial working volume was 3 L. Sterile SBP hydrolysate was used as a carbon and nutrient source for the microorganism. The pH was adjusted to 6 by injection of 20% Softening was carried out using PUROLITE S950 acid chelating resin (Purolite).Mono- and bi-polar electrodialysis were carried out with 11 cation exchange membranes Type II and 10 anion exchange membranes Type II Anion and cation exchange chromatographies were performed using a weak anion exchange resin RELITE EXA 133 and a strong cation exchange resin RELITE EXC 08 Concentration was done by vacuum distillation plant The downstream process was performed in the following sequenced steps:Details about the operational procedures can be found in a previous work by Neu et al. .The dry matter of SBP was determined by weighing a certain amount and then drying it at 105 °C until constant weight. The content of cellulose, hemicellulose, and lignin of SBP was determined using an ANKOM2000 fiber analyzer . The SBPFor dry cell weight, the sample was centrifuged to separate the cells. The cell pellets were washed and placed in a pre-weighed cup, which was dried in an incubator at 80 °C for 24 h. After cooling to room temperature, the cup was weighed again, and cell dry weight was calculated. This procedure was carried out in duplicate. 2SO4. Detection was carried out by a refractive index detector . The total run time was 17 min. Analysis of HMF and furfural were carried out using a column C18 Eurosphere II . The mobile phase was acetonitrile in water (1:8) with 1% v/v of acetic acid. The flow was 1.0 mL/min at 23 °C. Detection was carried out by a UV/VIS-Detektor at 280nm. The isomeric purity was analyzed using HPLC (KNAUER) coupled with a Chiralpak®MA(+) column  and an ultraviolet detector. The mobile phase was 2 mM CuSO4, and the flow rate was 0.8 mL/min.Organic acids and sugars were analyzed using HPLC ; 10 μL of sample volume  was added on a Eurokat H column  and eluted isocratically with 0.8 mL/min of 0.01 M HSBP is a promising substrate for lactic acid production, approaching a sustainable biorefinery production mode. As a substrate to second-generation lactic acid production, it can reconfigure the sugar beet industry, inserting this crop in the modern biorefinery concept of product valorization and sustainability. This biorefinery can become a supplier for different industrial sectors, including traditional food, energy, and feed, to a more complete chain, encompassing lactic acid, phenolic compounds, and pectin production. In addition, bipolar membrane electrodialysis can be adjusted to perfectly fit the sustainable needs of the lactic acid industry, generating zero waste towards a more sustainable process.


Millions of adults have been reported with hyperlipemia in the world. It is still unclear whether the plasma level of essential amino acids (AAs) will be influenced by the hyperlipemia. This study was aimed to investigate the AAs levels and the underlying metabolic relationship in hyperlipidemic subjects.An ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method was developed for the determination of phenylalanine (Phe), valine , histidine (His), tryptophan (Trp), and methionine (Met). Plasma samples (100 μL) were precipitated by acetonitrile (300 μL) and analyzed on a BEH C18  column at 40 °C by gradient elution. The mobile phase composed of 0.1% formic acid and acetonitrile was used with flow rate at 0.2–0.4 ml/0-3 min. Five AAs were determined at positive electrospray ionization (ESI+) at m/z 118.1/72.1 , 150.12/104.02(Met), 156.06/110.05(His), 166.1/120.1(Phe), and 205.2/188.02 (Trp). A total of 75 healthy subjects and 83 hyperlipidemic subjects, who had blood routine test and plasma lipid test were determined by developed UPLC-MS/MS.It was shown that there was good linearity for Val, Met, His, Phe, and Trp within 1–100 μg/mL. The relative standard deviations of precision and accuracy were all within 15%. The level of Val, Phe, Trp, His, and Met were 35.34 ± 15.64, 22.72 ± 9.13, 17.23 ± 4.94, 16.78 ± 13.64, and 6.24 ± 1.97 μg/mL in healthy subjects, while they were 38.04 ± 16.70, 22.41 ± 8.45, 15.62 ± 5.77, 18.35 ± 14.49, and 6.21 ± 1.97 μg/mL in hyperlipidemic subjects respectively. The Spearman’s correlations analysis showed that there were high correlations between Val, Phe, Trp, His, Met and triglyceride in healthy subjects. While, those correlations decreased in hyperlipemia cases.A convenient and sensitive method for simultaneous determination of Val, Phe, Trp, His, and Met in human plasma was developed. There was a high correlation between Val, Phe, Trp, His, Met and triglyceride. Hyperlipemia influences the metabolic balance of His, Phe, Trp, Met and Val. Amino acid exists in two stereoisomeric forms, L forms and D forms, most of which are L-isomers. The amounts of D-AAs are dramatically low in human body . So far,So far, various analytical methods for quantification of AAs have been reported, such as.gas chromatography with mass spectrometry (GC-MS) detection –10, capiHyperlipemia is a primary and major risk factor for the atherosclerotic cardiovascular diseases. Millions of adults have been reported with elevated total cholesterol (TC) or triglyceride (TG) levels in the world , 25. AltPhe, Val, His, Trp, Met and colchicine (purity > 98%) were bought from Sigma-Aldrich  and J&K Scientific LTD . Acetonitrile, methanol, and formic acid were HPLC graded and bought from Merck Company . Ultra-pure water (resistance > 18 mΩ) were provided by a Millipore Milli-Q purification system .The blood samples of hyperlipidemic and healthy subjects were collected from the clinical laboratory of the First Affiliated Hospital of Wenzhou Medical University. The study was carried out in accordance with the Declaration of Helsinki, and approved by the ethics committee of the First Affiliated Hospital of Wenzhou Medical University.All of these subjects received blood routine test (BRT) and lipid test, which were analyzed by the XE-2100 automated hematology analyzer  and the Beckman AU5800 biochemical measurement . After BRT and biochemical test, 100 μL of residual plasma was collected and determined by UPLC-MS/MS method.An ACQUITY UPLC and Xevo TQ-S Micro triple quadrupole mass spectrometer  was used in this study. Chromatographic separations of His, Met, Trp, Val, Phe and colchicine  were separated at a Waters BEH C18 column , which temperature was set at 40 °C. The mobile phase consisted of 0.1% formic acid water (solvent A), which was prepared by added 0.5 mL formic acid into 500 mL water, and acetonitrile (solvent B). The gradient elution of mobile phase began with A: B = 90:10 v/v, which changed as follows: A:B = 80:20 v/v at 1.2 min, A:B = 10: 90 v/v at 2.5 min, A:B = 90:10 v/v at 3.0 min.The flow rate was set at 0.2 mL/min (0–1.2 min), 0.4 mL/min (1.2–2.5 min), 0.2 mL/min (2.5–3.0 min) respectively. The injection volume was 1 μL. His, Met, Trp, Val, Phe and IS were detected in the multiple reaction monitoring (MRM) mode.A total of 100 μL plasma was added into 1.5 mL centrifuge tube. After that, the plasma was precipitated by 300 μL acetonitrile, which contained 0.1 μg/mL of colchicine (IS). The mixture was put under vortex movement for 0.3 min and centrifuged at 15000 rpm for 5 min. Finally, 1 μL supernatant injection was set for UPLC-MS/MS system analysis.The mixed stock solution of His, Met, Trp, Val, and Phe was dissolved with water at 1.00 mg/mL, and diluted to 10-500 μg/mL. The calibration standards were from 1 to 100 μg/mL, which were prepared by spiking 10 μL different concentration of mixed AAs standard solutions into 90 μL of blank plasma. The final concentrations of added AAs in blank plasma were 1, 2.5, 5, 10, 25, 50, 100 μg/mL, which were precipitated by 300 μL acetonitrile as mentioned in sample preparation.The method validation was evaluated by three quality-control (QC) samples at 10, 40, and 80 μg/mL, which were prepared as samples of calibration curve.The intra-day precision and accuracy of His, Met, Trp, Val, and Phe was carried out three replications in a day, and the inter-day precision was evaluated by measuring daily and continuously for 3 days. The concentrations of QC samples were calculated by the calibration curves developed. The precision was expressed as the relative standard deviation (RSD), and the accuracy was expressed as the relative error (RE), which was calculated as: RSD = Mean QC/SD × 100% and RE = (Mean QC - QC)/QC × 100%.The extraction recovery at 10, 40, and 80 μg/mL was evaluated by comparing the peak area to the pure standard solution at the same concentration. The matrix effects were investigated by comparing the peak area of His, Met, Trp, Val and Phe to the extracted sample added with the same concentration of analytes. The stability was evaluated at the room temperature from 2 h to 24 h.The robustness of UPLC-MS/MS method was studied in term of mobile phase, pH, column temperature and flow rate at three QC sample concentrations. Those parameters were interchanged within the range of 1–10% of the developed conditions, while keeping the other parameters unchanged. The peak areas of 5 AAs were recorded. The ruggedness was determined on two different BEH C18 Columns, centrifuges and by two different analysts to perform the overall analysis.All data of BRT, lipids indices and five AAs were expressed as mean and standard deviation. Independent sample t test was used to analyze the difference between hyperlipidemic and healthy subjects. The relationships of 5 AAs were analyzed by bivariate correlation. The statistical analysis was carried out by using the statistical software SPSS 17 .In this study, Val, Met, His, Phe, and Trp all had characteristic daughter ions in positive model. The optimal collision energy of five AAs was from 10 to 15 eV. If the collision energy was over 20 eV, the MS response will decrease. The developed UPLC-MS/MS parameters and mass spectrum are shown in Table According to the selected UPLC-MS/MS condition, the result showed that there was no interfering endogenous substance observed in the UPLC-MS/MS chromatograms Fig. .Fig. 2UThe calibration curve was developed by the linear regression of peak area ratios against the concentrations. Since AAs are endogenous substances, their calibration curves were subtracted with the area of a blank sample. The equation of calibration curves were showed in supplement Table The intra-day and inter-day precision and accuracy of His, Met, Trp, Val, Phe at the three QC levels in plasma are presented in Table The extraction recovery, matrix effect and stability of the five essential AAs are presented in supplement Table The results of robustness and ruggedness revealed there were no significant changes observed in the chromatographic peaks of 5 AAs. The peak RSD values of mobile phase, pH, column temperature and flow rate were all < 15% (Supplement Table P < 0. 001).A total of 75 healthy subjects  and 83 hyperlipidemic subjects , aged between 18 and 60 years, were participated in the study. Their average ages were 37.19 ± 9.66 and 44.77 ± 9.22. The clinical indices of two groups were listed in Table Based on the UPLC-MS/MS method, the levels of 5 essential AAs in the hyperlipidemic subjects and the healthy subjects were determined. The plasma levels of 5 essential AAs were shown in Table The Spearman’s correlations analysis showed that there was a high correlation among Val, Phe, Trp, His, Met in healthy and hyperlipidemic subjects Table . Among tThe further correlations analysis revealed that the plasma level of TG was related to His, Phe, Trp, and Val in healthy subjects. The correlation coefficients of TG-His, TG-Phe, TG-Trp, and TG-Val were 0.24, 0.27, 0.31, and 0.25. Nevertheless, they decreased to 0.03, 0.06, 0.17, 0.09 in hyperlipidemic subjects  column is widely used to analyze amino acids , 26–28. The developed UPLC-MS/MS method was successfully applied to determine the plasma of hyperlipidemic and healthy subjects. Hyperlipemia is a metabolic disease, and whether the AAs metabolism would be influenced in hyperlipemia is poorly studied. Theoretically, it is difficult for essential AAs to maintain dynamic balance when lipid metabolism is disordered. Although the concentration of AAs in plasma can be dynamically maintained in a balance by the release of endogenous proteins and the utilization of tissues , 30, it The Spearman’s correlations analysis showed that the correlation coefficient of Phe-His was the highest, whether in the healthy subjects (0.94) or hyperlipidemic subjects (0.93). Moreover, Phe had high correlations with Trp (0.84) and Val (0.72) in healthy subjects. However, the correlation coefficients were decreased to 0.69 and 0.56 in hyperlipidemic subjects, which indicated that the level of Phe was more easily to be influenced than the other four AAs in dyslipidemia. Although the independent samples T test showed that there were no statistic differences for those 5AAs between healthy subjects and hyperlipidemic subjects. The correlation coefficients of AAs decreased in hyperlipemia, such as Phe-Trp (0.84/0.69), Phe-Val (0.72/0.56), His-Trp (0.75/0.68), and His-Val (0.73/0.56), which indicated that the balance of His, Phe, Trp, and Val had been changed. Therefore, hyperlipemia or dyslipidemia may influence the metabolic balance of His, Phe, Trp, and Val.A convenient, sensitive, and specified UPLC-MS/MS method was developed and validated for the measurement of the five essential AAs in human plasma: His, Phe, Trp, Met and Val, which can be determined within 3.0 min after precipitated by acetonitrile. For hyperlipidemic and healthy people, the level of Val was the highest, then was Phe, Trp, His and Met. The results of 5 AAs were significantly correlated. For healthy subjects, the plasma level of TG is related to that of His, Phe, Trp and Val. However, in hyperlipemia or dyslipidemia patients, a perturbation was uncovered in the metabolic balance of His, Phe, Trp and Val.Additional file 1 Supplement Table 1 The equation of calibration curves of 5 AAs in human plasmaAdditional file 2 Supplement Table 2 The extraction recovery, matrix effect and stability of 5 AA in human plasma (n = 3)Additional file 3 Supplement Table 3 Parameters of UPLC-MS/MS method interchanged within the range of 1–10%Additional file 4 Supplement Table 4 RSD values of ruggedness determined on two different columns and by different analystsAdditional file 5 Supplement Table 5 Peaks area of 5 AAs (10 μg/mL) in pure standard and blood sample determined by Hilic and BEH C18 column


Fusarium onychomycosis is uncommon in the temperate climate zone of Japan. Based on the morphological characteristics and a gene analysis, we diagnosed a patient with ungual hyalohyphomycosis caused by Fusarium cugenangense belonging to the F oxysporum complex. This intractable disease was cured by 6‐month treatment with efinaconazole 10% solution. Fusarium onychomycosis is uncommon in the temperate climate zone of Japan. Based on the morphological characteristics and a gene analysis, we diagnosed a patient with ungual hyalohyphomycosis caused by Fusarium cugenangense belonging to the F oxysporum complex. This intractable disease was cured by 6‐month treatment with efinaconazole 10% solution. More than 70 human‐pathogenic Fusarium species primarily belong to 8 species complexes. Among these, the Fusarium solani and F oxysporum species complexes  account for approximately 60% and 20% of all Fusarium infection cases, respectively.Fusarium species is one of the common nondermatophyte onychomycoses in South America but not in temperate regions.F cugenangense, a member of the FOSC, that was successfully treated with the topical application of an efinaconazole solution.Members of the genus 2A 45‐year‐old male construction worker in Kumamoto, Japan, presented in January 2017 with a whitish discoloration at the base of his left big toenail and painful paronychia that he had first noticed 1 month previously Figure . His fatA direct microscopic examination of the nail specimen revealed acropetal and intercalary chlamydoconidia Figure . A histoF cugenangense CBS 130 304 (GenBank Accession No. MH485012), CBS 130 308 (MH485011), and CBS 131 393 (MH485019). Thus, we identified the isolate as F cugenangense N. Maryani, L. Lombard, and Kema et Crous (MycoBank MB826807) ; the translation elongation factor 1‐alpha (EF1‐α) gene was amplified using the primers EF1 (5′‐ATGGGTAAGGARGACAAGAC‐3′) and EF2 (5′‐GGARGTACCAGTSATCATGTT‐3′)F cugenangense using 3 or more out of these 6 major criteria: identification of mould in the nail by direct microscopy, isolation in culture, repeated isolation in culture, inoculum counting, failure to isolate a dermatophyte in culture, and histology.Fusarium species were not established,Fusarium species.F cugenangense strain was susceptible (MICs: ≤2 µg/mL) to amphotericin B, efinaconazole, micafungin, terbinafine, and voriconazole, and was low or less susceptible (MICs: ≥16 µg/mL) to amorolfine, 5‐fluorocytosine, fluconazole, itraconazole, ketoconazole, and miconazole  have been reported, mainly in immunocompetent individuals . Kumamoto is located in the south end of the Japanese archipelago and has a subtropical climate (Cfa) with an average temperature of 17.2°C and annual precipitation of 1,986 mm (0.78 inches). Geographically, the southwest Japanese archipelago and the islands of Java are both on the edge of Eurasian plate where many earthquakes and volcanic eruptions occur.Fusarium species is resistant to terbinafine or itraconazole. Both itraconazole and terbinafine pulse therapies were only partially effective on Fusarium onychomycosis, and their clinical cure rates were 52% (13/25 nails) and 50% (4/8 nails), respectively.Fusarium species is lower than that of itraconazole.Candida, Aspergillus, and Fusarium species.Onychomycosis caused by None declared.M. Hirose, H. Noguchi, and T. Matsumoto: diagnosed the patient, analyzed the data, and wrote the paper. U. Kimura and M. Hiruma: advised on the mycological results. R. Kano: examined the antifungal susceptibility for the isolate. T. Yaguchi: genetically identified the fungus. N. Fujimoto, T. Satoh, and H. Ihn: edited and supervised the manuscript. All authors: discussed the results and commented on the manuscript.


P = 0.001; 1.397 [95% CI: 1.245-1.569], P < 0.001, respectively) should be corrected to .In the article titled “Fibrinogen as a Prognostic Predictor in Pediatric Patients with Sepsis: A Database Study” , there wThe author explained that the error was introduced during manuscript preparation and confirms that this does not affect the results and conclusions of the article.


Pungentus, one new and one known, collected in natural vegetation and cultivated soils in northwest of Iran, are studied. Pungentus sufiyanensis n. sp. is characterized by its 1.22 to 1.57 mm long body, offset lip region by a constriction and 7 to 9 μm broad, 18 to 21 μm long odontostyle, 304 to 348 μm long neck, 133 to 161 μm long esophageal expansion, mono-opisthodelphic female genital system without anterior uterine sac, slightly backward directed vagina, absence of pars refringens vaginae, V = 47−54, rounded-conoid caudal region  with saccate bodies, and the absence of male. Molecular analysis, based on D2-D3 expansion segments of the 28S rDNA (LSU), confirms the monophyly of the family Nordiidae and suggests the monophyly of the genus Pungentus, with the new species forming a clade with other Iranian species. New data are presented for six Iranian populations of P. engadinensis, and an updated key for the identification of Pungentus species is also provided.Two species of the genus  Pungentus is an interesting dorylaimid genus, often found in forest habitats of the Northern Hemisphere, and with very restricted presence in southern territories. Its taxonomy was updated by inquirendae or incertae sedis and provided a key to their identification as well a compendium of their main morphometrics.The genus Pungentus species from Iran is very limited. P. engadinensis  to explore the dorylaimid diversity of this region. Their study revealed that they belonged to one new and one known species. The objective of this work was to report Pungentus sufiyanensis n. sp. using morphology, morphometric, and molecular methods and provide new data about P. engadinensis.Several Soil samples were collected from the rhizosphere of several crops and orchards of East–West Azarbaijan and Kurdistan provinces, northwest Iran, during the period 2010–2017. Nematodes were extracted following the protocols by ® software version 12. Microphotographs were edited using Adobe® Photoshop® CS software.Mounted specimens were observed under an Olympus BX 41 light microscope equipped with a drawing tube and a DP50 digital camera attached to it. Morphometrics include Demanian indices and the usual measurements and ratios. Line illustrations were prepared using CorelDRAW2O, 12.5 μl master mix , 0.75 μl of each forward and reverse primers, and 1 μl of DNA template. PCR was carried out using a BIO RAD thermocycler machine in accordance with P. sufiyanensis n. sp. were deposited in the GenBank database under accession number MN855359 as indicated on the phylogenetic tree of For the molecular study of the new species, DNA samples were extracted from a live adult nematode, hand-picked, and placed on a clean slide containing a drop of distilled water or worm lysis buffer (WLB) and crushed by a sterilized scalpel. Then, the suspension was transferred to an Eppendorf tube containing 25.65 μl ddH2O, 2.85 μl 10 × PCR buffer and 1.5 μl proteinase K (600 μg/ml) . The tubes were incubated at −80°C (1 h), 65°C (1 h) and 95°C (15 min). The extracted DNA was stored at −20°C until use. The D2-D3 domains of the 28S rDNA were amplified with forward primer D2A (5´-ACAAGTACCGTGAGGGAAAGTTG-3´) and reverse primer D3B (5´-TCGGAAGGAACCAGCTACTA-3´) . In totaParavulvus hartingii a = 40-50) nematodes of medium size, 1.22 to 1.57 mm long. The body cylindrical, tapering toward both ends but more so toward the anterior extreme as the caudal region is short and rounded. Upon fixation, habitus slightly curved ventrad, to an open C-shape. Cuticle three layered, especially distinguishable at caudal region, bearing fine transverse striations, 2 to 3.5 μm thick at anterior region, 3 to 6 μm at mid-body, and 7 to 10 μm at tail. Lateral chords 8 to 11 μm thick or occupying one-fourth to one-third of mid-body diameter. The lip region is somewhat angular, offset by a weak but perceptible constriction, with nearly truncated anterior margin, 2.1 to 2.6 times as wide as height and 21 to 27% of body diameter at neck base; lips mostly amalgamated, with hardly protruding papillae. Amphidial fovea cup-shaped, opening at the level of constriction, with the aperture 4 to 5 μm long or 52 to 60% of lip region diameter. Cheilostom nearly cylindrical, 1.2 to 1.8 times as long as the lip region diameter, with visible sclerotised walls in its anterior half, and bearing four distinct, sclerotized, circumoral platelets. Odontostyle slightly arcuate dorsally, slender, well sclerotized, 2.0 to 2.5 times as long as the diameter of lip region, 1.1 to 1.4% of total body length, and aperture 2 to 3 μm long or occupying 9 to 17% its length. Guiding ring double. Odontophore rod-like, 0.8 to 1.0 times the odontostyle length. Nerve ring situated at 109 to 121 μm or 30 to 35% of the neck length from the anterior end. Pharynx entirely muscular, consisting of an anterior portion enlarging gradually into the basal expansion that is 8.4 to 13 times as long as width, 4.1 to 5.2 times as long as body diameter at neck base and occupies 40 to 45% of total neck length; gland nuclei located as follows: DN = 61−63, S1N1 = 68−70, S1N2 = 77−80, S2N = 89−92 according to pars dilatata with perceptible lumen. Oviduct–uterus junction marked by a sphincter. Uterus a simple tube-like structure, 37 to 51 μm long or 0.9 to 1.5 times the corresponding body diameter long. Sperm in genital tract absent. Vagina slightly directed backward, extending 15 to 20 μm inwards and occupying 39 to 45% of the corresponding body diameter; pars proximalis vaginae 9-12 × 11-15 μm, with nearly sigmoid walls and surrounded by moderately developed, circular musculature and pars distalis 2 to 3.5 μm and pars refringens vaginae obscure in specimens examined. Vulva a nearly equatorial, transverse slit, preceded by a V-shaped depression of body surface. Prerectum 2.1 to 3.2 and rectum 0.6 to 1.0 times as long as the anal body diameter. The caudal region short, rounded-conoid, slightly more straight at the ventral side, where it bears saccate bodies; two pairs of caudal pores are present.Female: slender  and 1.22 to 1.57 mm long body, lip region offset by constriction and 7 to 9 μm broad, odontostyle 18 to 21 μm long, neck 304 to 348 μm long, pharynx expansion 133 to 161 μm long or 40 to 45% of total neck length, female genital system mono-opisthodelphic, without anterior uterine sac, vagina slightly directed backward, pars refringens vaginae absent, V = 47-54 and caudal region rounded-conoid  with saccate bodies. Male absent.P. angulatusP. longidens (c-ratio more than 60). Nevertheless, it differs from P. angulatus, an Indian species also known to occur in Hungary (n = 22), lip region offset by a weak (vs strong) constriction, longer odontostyle (18-21 vs 14-16 μm) and neck (304-348 vs 225 μm), and relatively shorter female tail (c’ = 0.7-1 vs 1.3) with (vs without) saccate bodies. It differs from P. longidens, a poorly known (but apparently close) species originally described from Spain, by its shorter odontostyle , more posterior location of guiding ring (at appreciably more vs less than lip region diameter from the anterior end), and rounded conoid (vs short rounded to hemispheroid) female tail with (vs without) saccate bodies.The new species resembles ongidens  Andrássy Hungary , by haviP. sufiyanensis n. sp. is phylogenetically related to P. azarbaijanensis but can be differentiated by the female genital system (mono-opisthodelphic vs didelphic-amphidelphic).P. sufiyanensis n. sp. showed 96, 96, 99, 96, and 95% of similarity with P. azarbaijanensis (MH346476), P. engadinensis (AY593050),  P. monohystera (MF325343), P. silvestris (AY593052), and Enchodelus macrodorus (AY593054), respectively, with 27, 26, 2, 26, and 26 different nucleotides, respectively, too. As derived from the analysis of the new sequence herein obtained, the evolutionary relationships of the new species with other representatives of the order Dorylaimida are shown in P. azarbaijanensis, another Iranian species. These both species form a clade together with other Pungentus species, suggesting a low supported monophyly of this genus based upon currently available sequences. All the sequences of Nordiidae representatives constitute a highly supported (100%) clade, a fact that confirms the monophyly of this taxon. Leaving aside Pungentus sequences, the remaining ones form together a second clade, which is not well supported, within the family Nordiidae.A Nblast search of the D2-D3 sequence of Type habitat and locality: the habitat and locality type was Northwest Iran, East-Azarbaijan province, Sufiyan, Roodghat area, Zeinabad village , where the specimens were collected from the rhizosphere of black cherry trees (Prunus cerasus L.).Other localities and habitats: samples were collected from two locations in Northwest Iran: East-Azarbaijan province, Marand district, Kondolaj village, from the rhizosphere of almond and walnut trees; West Azarbaijan province, Bokan district, Khorasaneh area  from the rhizosphere of natural vegetation.Type material: female holotype and paratypes were deposited with the Nematode Collection of the Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz, Iran. The new species binomial has been registered in the Zoobank database (zoobank.org) under the identifier B1F2B3F6-558F-4688-BFFC-0F90BD101357.Etymology: the species name refers to the type locality of the new species, Sufiyan, East-Azarbaijan province, northwest of Iran.Pungentus engadinensis (; Table 3Remarks: the six populations of this species herein examined are, morphologically and morphometrically, very similar to each other, but some minor differences have also been noted, which are regarded as intraspecific variations. Anterior uterine sac according to Pars refringens vaginae, consisting of two small sclerotized pieces, were distinguishable in Divandarreh and Maragheh populations, but they were more inconspicuous in other populations. Vagina orientation also displays some differences: backwards directed in Bokan and Sufiyan populations and near perpendicular to body axis in other populations. Present Iranian populations of P. engadinensis fit very well with those previously studied by other authors (for comparative purposes, see Pungentus engadinensis is a widely distributed species, having been recorded in Asia, Europe, and North America, where it mostly inhabits moist soils (st soils . In Iranst soils  from thePungentusKey to species of the genus (Modified after


The Vz, K10 and absolute bioavailability of troxipide were obviously decreased in rats with GU compared with NC rats, and its tissue distribution  was significantly different between the two groups of rats. Additionally, the pharmacodynamic results suggested that the levels of biochemical factors  were significantly increased, the PG-Ӏ level was obviously decreased, and the protein expression levels of HSP-90, C-Cas-3 and C-PARP-1 were markedly increased in rats with GU compared with NC rats. The above results suggested that the therapeutic mechanisms underlying the metabolic, pharmacokinetic and pharmacological properties of troxipide in vivo in rats deserve further attention based on the importance of troxipide in the treatment of GU in this study, and these mechanisms could be targets for future studies.Troxipide is widely used to treat gastric ulcer (GU) in the clinic. However, a lack of systematic metabolic, pharmacokinetic and pharmacological studies limits its clinical use. This study aimed to firstly explore the metabolic, pharmacokinetic and pharmacological mechanisms of troxipide in rats with GU compared to normal control (NC) rats. First, metabolic study was perormed by a highly selective, high-resolution mass spectrometry method. A total of 45 metabolites, including 9 phase I metabolites and 36 phase II metabolites, were identified based on MS/MS spectra. Subsequently, the pharmacokinetics results suggested that the C The frequent occurrence of GU between the cardia and pylorus mainly results form tissue damage caused by digestive juices decomposing the gastric mucosa, especially near the lesser curvature of the stomach and gastric antrum8. Currently, GU is a widely considered multifactorial disease because it is related to gastric acid, gastric pepsin, infection, physical fitness, environment, living habits and so on11. If not treated adequately, GU can lead to serious complications such as perforation and bleeding15. In addition, the acetic acid-induced GU in rats is most similar to GU in humans20. Here, acetic acid-induced GU is more severe than other GU models , and is the most commonly used model for experimental research on GU22. Moreover,this model is reproducible and reliable. Therefore, we selected a rat model of GU to evaluate relative studies of troxipide in the paper.Gastric ulcer, (GU) a common disease in the clinic, is a peptic ulcer that affects humans of all ages. It is associated with morbidity and mortality, and has become a medical-social problem of global importance that has received increased attention25. It is used to alleviate gastric mucosal lesions  in the acute gastritis and acute exacerbation stages of chronic gastritis. Furthermore, troxipide can inhibit neutrophil-mediated inflammation and oxidative stress to increase gastric mucus secretion, increase the contents of mucopolysaccharide and prostaglandin E2 and enhance the positive effect of the gastric mucosa on barrier function28. And, the troxipide is soluble only at acidic pH and is mainly absorbed from the stomach29. However, few researchers have payed attention to the pharmacokinetic and metabolic process of troxipide. So, detailed studies on the metabolites, pharmacokinetics and therapeutic mechanisms of troxipide in rats with GU are lacking, limiting the clinical use of this drug.Troxipide  rats in the present study for further research and development of troxipide in the clinic. To systematically investigate the major metabolites of troxipide in rats with acetic acid-induced GU, we first time evaluated metabolites in the feces and urine of rats with GU after oral administration of troxipide using the highly selective and high-resolution method of ultra-high-performance liquid chromatography quadrupole orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS). Then, we performed ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to study the pharmacokinetics of troxipide in vivo in rats with carbamazepine as an internal standard (IS). This assay has some merits, such as precise sample preparation, good linearity and specificity and negligible carryover. To obtain a systematic view of dissection of the plasma UHPLC-MS-based pharmacokinetics of troxipide, the above method was firstly applied to the pharmacokinetic study of troxipide in vivo in the present study as an effective evaluation strategy for absorption and metabolism. In addition, we have also investigated the expression of relative apoptosis protein on pharmacodynamics in the paper. Overall, our study on the metabolic, pharmacokinetic and pharmacological effects of troxipide provide a foundation for the structural modification and dosage form design of troxipide. The outline of the workflow of this study was shown in Fig. 32. Because the metabolites exhibited similar structures to that of troxipide and because the biologically active conformation of troxipide was optimized during the biotransformation process, the metabolites may have a wide range of pharmacological activities34. To study the structure of troxipide metabolites, Compound Discovery software, Mass Frontier software and manual elucidation were used to evaluate the fragmentation behaviors of troxipide metabolites. As shown in Fig. 15H23N2O4) was protonated [M+H]+ with m/z 295.16 in the ESI–MS spectrum. In the ESI-MS2 spectra, the base peak at m/z 195.07 yielded an [M + H-C5H12N2]+ ion through the loss of 3-aminopiperidine. The molecular ion at m/z 295.16 also produced the product ion at m/z 84.08, which was a piperidine ring resulting from the loss of C10H13O4N. Moreover, minor ions, including the [295.16-NH3]+ ion at m/z 278.14, [295.16-C5H12N2-C2H3O]+ ion at m/z 152.05, [295.16-C5H12N2-C2H3O-CO]+ ion at m/z 124.05, and [295.16-C5H12N2-C2H3O–C3H2O3]+ ion at m/z 109.06, were also observed in the ESI-MS2 spectrum. According to the fragmentation pattern of the parent compound, the two fragmentation pathways of troxipide are shown in Fig. Drug metabolism can produce metabolites with altered functional parameters such as clearance rate, absorption rate, biological activity and toxicity; thus, it is an opportunity to improve the odds of drug discovery35. Many possible metabolites and metabolic pathways were identified by this software by automatically comparing a sample collected after drug administration and a blank sample. Information on the metabolites, such as the formula, m/z, retention time (RT), peak area, and peak height, was provided by the software. In addition, this software provided some metabolic pathways and MS/MS fragments to determine accurate masses of different parts of troxipide for metabolite structural identification. This method shown some features such as shorter run time, sample preparation simplicity and so on, which has been successfully applied for detecting metabolites products of troxipide and could be practical and reliable for clinical, pharmacodynamics and does-dependent pharmacokinetic study of troxipide and its active metabolite.The main metabolite total ion current (TIC) spectra of troxipide in rat plasma, feces and urine samples are shown Fig. Approximately 10,000 metabolites related to precursor ions were identified in the plasma, urine and feces of rats with GU compared with control rat plasma, urine and feces, and more than 2000 metabolites were screened by peak area filtering. Finally, in positive full scan mode, 45 metabolites from the urine and feces were successfully detected and verified by the fragmentation library of Mass Frontier software. The structures of the metabolites were determined based on precise mass measurements, drug biotransformation knowledge, and characteristic fragmentation pathways. The main biochemical reactions were oxidation, reduction, demethylation, glucuronide conjugation, palmitoyl conjugation and so on. For our knowledge, the 45 metabolites was reported for the first time, the LC–MS/MS spectra of these metabolites were showed in Fig. + molecular ion at m/z 295.16, and the product ions were at m/z 195.07 and 84.08, which was consistent with the results for troxipide. Therefore, M0 was confirmed to be troxipide. The [M + H]+ molecular ion of M4, which exhibited a mass that was 84 Da less than that of M0, was detected at m/z 211.21 at 1.43 min. The characteristic MS/MS ions of M4 were at m/z 132.05 and 153.07. Based on the difference between its composition and that of the parent drug, M4 was determined to be a metabolite that was generated from M0 through the loss of pyridine. The [M + H]+ molecular ion of M8, which had a RT of 0.69 min, was detected at m/z 100.10, and the fragment ions were at m/z 84.08 and 56.05. According to its elemental composition and degree of unsaturation, M8 was surmised to be a metabolite resulting from amide bond cleavage of M0, as detailed in Supplementary Results and Discussion.Here, we also showed the identification process on the following key metabolites of troxipide. Metabolites 4 and 8 (M4 and M8) were derivatives of the parent drug and its fragmentation and were detected and identified in the urine and feces of the rats. The results are shown in Figs. 37. But, the metabolic reactions of troxipide in feces weren’t all agreement with that in urine. In the urine, troxipide mainly underwent demethylation, dehydration, desaturation, hydration, reduction, oxidation, methylation, acetylation, sulfation conjugation, glutamine conjugation, ornithine conjugation, glucoside conjugation, glutamine conjugation, glycine conjugation, and glucuronide conjugation. There results may be related to liver metabolism39. Specially, oxidative metabolism is the most common mechanism of drug metabolism and involves a series of biochemical reactions that produce more readily excretable species. Much importance is given to cytochromes P450 (CYPs) in drug metabolism, and some medicinal chemists have prematurely suggested that CYPs are the only significant enzymes in drug metabolism. The actions of CYPs dominate those of other oxidoreductases and account for approximately half of metabolite formation. Hydrolysis may produce approximately 1/10 of all metabolites, and the remaining reactions are attributed to conjugating enzymes. Glucuronosyltransferases are the most important enzymes, providing structural variety and allowing the formation of functional groups to which glucuronic acid can be conjugated, while sulfotransferases and glutathione S-transferases, methyltransferases, N-acetyltransferases, and other coenzyme-A transferases play lesser roles40. On the above, different metabolites of troxipide may result from different metabolic pathways associating with oxidoreductases, hydrolytic enzyme and conjugating enzymes in the study. In addition, the blood was not a good matrix for monitoring exposure to troxipide metabolites in this study. Therefore, the findings of this study regarding troxipide metabolites in the blood have not done the further investigation.The proposed major metabolic pathways were present in the feces and urine of the rats, and small fractions of the compounds were distributed in the blood. In the feces, troxipide mainly underwent demethylation, dehydration, hydration, reduction, oxidation, methylation, acetylation, palmitoyl conjugation, glycine conjugation, glycine conjugation, and arginine conjugation. These findings may be related to the intestinal flora in rats with GUThe method met the pertinent Food and Drug Administration (FDA) and European Medicine Agency (EMA) guidelines, indicating that the proposed analytical method is applicable for the pharmacokinetic study of troxipide. Furthermore, the results showed that this method can significantly improve the response of troxipide. This study is original and practical and will therefore provide a good foundation for further research on troxipide and its drug development in the future, as detailed in Supplementary Results and Discussion.max, Tmax, MRT(0−t), MRT(0−∞), VRT(0−t), VRT(0−∞), t1/2, K10, Vz, AUC(0−t), and AUC(0−∞)) of all groups were calculated by DAS 2.0 software and were shown in Table UHPLC-MS/MS is currently one of the most useful methods for analyzing biological samples due to its high sensitivity and selectivity. The validated UHPLC-MS/MS method was successfully applied to investigate the plasma concentration–time profile of normal control rats and rats with GU that received a single intragastric administration of 20, 40 or 60 mg/kg troxipide  and intragastrically treated gastric ulcer group (GUGIG), respectively). The doses were selected based on the Chinese Guidance for Nonclinical Pharmacokinetics of Medicinal Products. To study the absolute availability, 40 mg/kg troxipide was administered intravenously. The mean plasma concentration–time profiles are presented in Fig. max, Ka, Vz, K10, t1/2, AUC(0−t) and AUC(0−∞) in the NCGIG were significantly different from those of the GUGIG after oral administration of troxipide ; however, no significant differences in the Tmax, MRT(0−t), MRT(0−∞), VRT(0−t) and VRT(0−∞) were found between the groups. The Cmax  of the GUGIG was slightly increased (P < 0.05) compared with the NCGIG . The Ka  of the GUGIG were significantly increased (P < 0.01) compared with the NCGIG . More importantly, from the Concentration–time (C–t) curve the troxipide concentration in the GUGIG was slightly increased compared with that in the NCGIG, suggesting that troxipide was more easily absorbed into the blood of rats with GU than that of NC rats41. The Tmax of the GUGIG was not significantly different from that of the NCGIG, which suggested that the Tmax not related to dose and disease; this result is consistent with the findings of Gao’s report23. The Vz  of the GUGIG was significantly decreased (P < 0.01) compared with the Vz  of the NCGIG, which suggested that the drug was more slowly distributed in GUGIG rats than in NCGIG rats. The K10  of the GUGIG were significantly decreased (P < 0.01) compared with the K10  of the NCGIG. The t1/2  of the GUGIG was significantly decreased (P < 0.01) compared with the t1/2  of the NCGIG. The K10 and t1/2 results indicated that the excretion rate of the drug was decreased in the GUGIG compared with the NCGIG. The AUC(0−t)  and AUC(0−∞)  of the GUGIG were significantly increased (P < 0.05) compared with the AUC(0−t) and AUC(0−∞) of the NCGIG, and these results were consistent with the Cmax results. Furthermore, there were no significant differences in the MRT(0−t), MRT(0−∞), VRT(0−t) or VRT(0−∞) between the GUGIG and the NCGIG.The CP < 0.05) in the Cmax, VRT(0−t), VRT(0−∞), AUC(0−t) and AUC(0−∞) were observed in the NCGIG after oral administration of different doses of troxipide , but no significant differences in the Ka, Vz, K10, t1/2, Tmax, MRT(0−t) or MRT(0−∞) were observed. Moreover, the Cmax, Ka, Tmax, Vz, K10, t1/2, AUC(0−t), AUC(0−∞), MRT(0−t), MRT(0−∞), VRT(0−t) and VRT(0−∞) were similar between the GUGIG and NCGIG after oral administration of different doses of troxipide. Linear regression analysis of the oral administration data (NCGIG and GUGIG) showed that the Cmax was dose-dependent at concentrations between 20 to 60 mg/kg, and the linear equations of the Cmax in the NCGIG and GUGIG were y = 22.565x − 122.58, R2 = 0.9999 and y = 26.795x − 90.478, R2 = 0.9914, respectively  following oral administration of troxipide at the studied doses, and the linear equations of the AUC(0−t) in the NCGIG and GUGIG were y = 10272x − 85986, R2 = 0.9998 and y = 24002x − 351378, R2 = 0.9005, respectively  in the NCGIG and GUGIG were y = 10674x − 90179, R2 = 0.9998 and y = 27825x − 451882, R2 = 0.8844, respectively  and AUC(0−∞) of troxipide were dose-dependent at concentrations between 20 to 60 mg/kg, indicating that the pharmacokinetic behavior of troxipide in rats was linear with respect to dose.In addition, significant differences , AUC(0−∞), Cmax, t1/2, MRT(0−t), MRT(0−∞), VRT(0−t), VRT(0−∞), Vz and K10 of the intravenously injected gastric ulcer group (GUGIV) were significantly different (P < 0.05) from the AUC(0−t), AUC(0−∞), Cmax, t1/2, MRT(0−t), MRT(0−∞), VRT(0−t), VRT(0−∞), Vz and K10 of the intravenously injected normal control group (NCGIV). These results suggested that in this study, the pharmacokinetics of troxipide in were dramatically altered in rats with GU compared with NC rats, which is consistent with the results presented in Fig. After intravenous injection of 40 mg/kg troxipide, the AUC(0−t)/AUC(0−∞) of the NCGIG and GUGIG were 96.40 ± 2.22% and 88.64 ± 8.63% (far higher than 80.0%), respectively, which indicated that the time elapsed before blood sampling was sufficient to detect the pharmacokinetic parameters of troxipide with high accuracy and precision. Additionally, according to the above calculation, absolute bioavailability following oral administration in the NCGIG and GUGIG were 70.51 ± 8.33% and 48.13 ± 4.90%, respectively. The absolute bioavailability in the GUGIG was significantly decreased (P < 0.01) compared with that in the NCGIG, possibly due to the slower distribution and reduced excretion in the GU rats43. This study of the pharmacokinetics of troxipide may provide accurate and detailed support for further development and clinical application of this drug in the near future. Meanwhile, structural modifications, dosage form changes and drug combinations need to be further investigated to prevent adverse reactions to troxipide.The AUC44. The pharmacokinetic behavior of troxipide in the GUGIG was significantly different from that in the NCGIG. For example, in the GUGIG, the highest concentration of troxipide in the liver, lung and kidney was observed at 3 h, but in the NCGIG, the highest troxipide concentration in the liver, lung and kidney was observed at 2 h. These results suggested that GU can cause poor absorption and membrane permeability in the liver, lung and kidney, which is consistent with the findings of Liu et al.45. The above reasons may be due to the absorption and distribution of patients obviously different from that of healthy human.Here, we performed the study of tissue distribution in heart, liver, spleen, lung, kidney, brain, stomach, intestinal, pancreas by the above UHPLC-MS/MS method. The tissues distribution of troxipide in the NCGIG and GUGIG after oral administration (40 mg/kg) are shown in Fig. 52. Before oral administration of troxipide, the plasma levels of cytokines  were significantly increased in the GUG compared to the NCG  in the GUG compared with the NCG, while the PG-Ӏ level was decreased (P < 0.05) in the GUG. These results are consistent with the findings of Suo, Cao and Ghoshal et al.71. The levels of MTL, GAS and PG-II were significantly decreased  and the PG-Ӏ level was significantly increased (P < 0.01) in the troxipide-treated groups compared with the GUG after 2 weeks of troxipide treatment. Consistently, in the GUG, the PG-Ӏ level was low, and the PG-II level was high; thus, the PG-Ӏ/PG-II ratio was reduced in the GUG compared to the NCG  in the GUG compared to the NCG, high-dose troxipide group (THG), medium-dose troxipide group (TMG) and low-dose troxipide group (TLG). Ulcer inhibition levels are shown in Table Before troxipide treatment, there were significant differences between the NCG ang GUG at the macroscopic level Fig. A. In the74. Therefore, the protein expression of HSP-90, C-Cas-3 and C-PARP-1 in the rats was also investigated. HSP-90 has been shown to induce apoptosis in various cells, and C-PARP-1 and C-Cas-3 are major apoptotic markers. HSP-90 is a heat shock protein that can promote the transportation, folding, and rearrangement of other proteins as a “molecular chaperone”76. Many recent studies have reported that HSP-90 is induced when cells are exposed to sublethal stressors, such as inflammation, infection or ischemia78. In our study, a small number of yellow/brown areas were observed in the tissues of rats from the NCG, suggesting low expression levels of HSP-90. Thus, we speculated that HSP-90 levels would be upregulated (P < 0.01) in the GUG compared with the NCG, and the results were in agreement with our hypothesis. C-Cas-3, a protease with multiple functions, plays a key role in cell apoptosis and is responsible for the cleavage of PARP79. Previous studies have shown that inflammation can activate the mitochondrial apoptotic pathway, thereby activating the caspase cascade80. Our results demonstrated that troxipide downregulated (P < 0.01) the expression of C-Cas-3, as shown in Fig. 83. The protein expression rats of C-PARP-1 was significantly upregulated (P < 0.01)  Fig. A,B in thOverall, GU is the result of an interaction between inflammation and apoptosis. On the one hand, inflammatory factors  promote protein expression of HSP-90, C-Cas-3 and C-PARP-1 and further stimulate cell apoptosis to aggravate gastric injury. On the other hand, cell apoptosis also promotes inflammation and further aggravates gastric injury levels. Moreover, GU promotes changes in MTL, GAS, PG-Ӏ, PG-II and PG-Ӏ/PG-II levels Fig. C. Thus, In summary, for the first time, we studied the metabolism and pharmacokinetics of troxipide in rats with 5% acetic acid-induced GU. The drug study on the disease rats was closer to the clinical application of the drug, because the pharmacodynamics mechanism of patients is obviously different from that of healthy human. A total of 45 metabolites of orally administered troxipide were studied in vivo in rats for the first time. The pharmacokinetic parameters observed for troxipide were significantly different in GU rats when compared with NC rats.85. Therefore, the metabolism, pharmacokinetics and pharmacodynamics of troxipide in vivo in rats deserve further attention due to the importance of troxipide in the treatment of GU in this study. Metabolic studies can provide comprehensive insight into safety monitoring and the design of drug delivery systems89. Although the structures of the metabolites could not be determined conclusively by LC–MS/MS alone, the results significantly expanded our understanding of the pharmacological effects and therapeutic mechanism of troxipide and its associated metabolic pathways, and these metabolites could be targets for future studies on the structural modification of troxipide and further development of new drugs. This study provides an available strategy for identifying and characterizing metabolic reactions in the further study. Here, we developed a simple, rapid and sensitive method for quantifying troxipide levels in biosamples, including plasma, urine, and fecal samples, that can be successfully used to study the pharmacokinetics of troxipide and its major metabolites in GU rats after oral administration in accordance with the guidelines of FDA, EMA and ICH . The data were complex and multifarious, and artificial intelligence and big data banks could be used to identify and characterize metabolites in the future.Studying the pharmacokinetics and metabolism of troxipide is helpful for elucidating its potential pharmacological mechanisms and for its development as a drug for clinical applicationThe results of the pharmacokinetic study suggested that the absorption of troxipide was slightly increased, and the distribution and excretion of the drug were decreased in rats with GU compared with NC rats. The absolute bioavailability and tissue distribution of troxipide following oral administration were significantly different between rats with GU and NC rats. This study fills a gap in the literature and provides a theoretical basis for the reasonable use of troxipide for individualized therapy. In addition, this work also identified the main mechanisms by which inflammatory factors and apoptosis are inhibited in the stomachs of rats with GU. Moreover, our macroscopic, microscopic and biochemical analyses demonstrated that enhancing blood circulation and metabolism at the site of GU can alleviate gastric mucosal injury and maintain gastric mucosal homeostasis.Overall, this systematic and comprehensive study of the metabolism, pharmacokinetics and pharmacodynamics of troxipide and its major metabolites in vivo in rats with GU provides important reference information regarding the clinical application of troxipide in the study.The chemicals and reagents of study were detailed shown in Supplementary Materials and Methods.Male, Specific pathogen Free (SPF), 8–12 weeks old Sprague–Dawley (SD) rats of weighing 200 ± 20 g were obtained from the Vital River Laboratory Animal Technology  2016-0006, Beijing, China). All SD rats were maintained in pathogen-free facility standard cages at constant temperature (25 ± 2 °C) and humidity (55 ± 5%) with 12-h light/dark cycle, and can access to water and food ad libitum. Moreover, all animals were acclimated in accordance to the above condition in the laboratory for 1 week prior to the experiments in this study. In all protocols and procedures, maintenance and treatment of the animal experiment were performed in accordance to guideline on administration of lab animals by State Council and National Institute of Health for the Care, and approved by Animal Ethical and Welfare Committee (AEWC) of Hebei University . All in all, all effects were made to alleviate unnecessary suffering and distress of animals.The ultra high performance liquid chromatography TSQ Altis tandem mass spectrometry system (UHPLC TSQ Altis MS) consisted of a Vanquish UHPLC system  and a Thermo Scientific TSQ Altis Triple Quadrupole mass spectrometry . The analytical column was an ACQUITY UHPLC BEH-C18 column . A binary mobile solvent composed of phase A (0.1% formic acid in water) and phase B (acetonitrile) at a flow rate of 0.3 mL/min. The procedure of gradient elution was as follows: 5–5% B at 0–1.0 min, 5–100% B at 1.0–7.0 min, 100–100% at 7.0–10.0 min, 100–5% B at 10.0–10.2 min, 5–5% B at 10.2–12 min. The temperatures of column and auto-sampler room were maintained at 40 °C and 10 °C, respectively. The injection volume of the sample was 2 µL. The mass spectrometer was applied with electrospray ionization (ESI) source in selective reaction monitoring (SRM) mode. The optimized instrument parameters were shown as below: positive ion, 3,500 V; negative ion, 2,500 V; sheath Gas, 50 Arb; Aux gas, 23.2 Arb; sweep Gas, 0 Arb; ion transfer tube temperature, 52 °C; vaporizer temperature, 350 °C. The finalized precursor → product transitions of troxipide and carbamazepine were m/z 295.072 → 195.071 and m/z 237.012 → 179.071, respectively.*The UHPLC-Q-Orbitrap-HRMS system was equipped with a Dionex Ultimate 3,000 UHPLC system  and a Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer  with electrospray ionization (ESI). Chromatographic separation was performed using a ACQUITY UHPLC BEH C18 column  at 40 °C. Acetonitrile (phase A) and 0.1% formic acid in water (phase B) were used as the mobile phases at a flow of 0.35 mL/min. The procedure of gradient elution was as follows: 0–1 min, 5% A; 1–9 min, 5–100% A; 9–12 min, 100% A; 12–12.1 min, 100–5% A; 12.1–15 min 5% A. The injection volume of sample was 5 µL at 4 °C. The Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer was an analytical instruments of rapid, sensitive, precision and accuracy for detecting unknown metabolites in bio-logical samples by the following parameters: sheath gas 30 Arb, auxiliary 10 Arb, sweep gas 0 Arb, positive spray voltage + 3.5 kV, negative spray voltage -2.8 kV, capillary temperature 320 °C, auxiliary gas heater temperature 300 °C and S-lens RF 50 V. The full scan mode of positive and negative were used by the apparatus under nitrogen conditions, and the ranges of full scan mode were from 50 to 1,200 m/z. All data were acquired under preset model, and processed using Compound Discovery  and extracted mass spectrometry using Xcalibur 3.0 software .A 100 µg/ml standard stock solution was prepared by dissolving 1.0 mg of accurately weighed troxipide in methanol in a 10-mL volumetric flask. The standard stock solution was serially diluted with methanol to obtain several standard working solutions with concentrations of 0.05, 0.15, 0.2, 0.4, 0.5, 1, 2, 5, 10, 50, 80 and 100 µg/mL. Plasma and tissue calibration standards were prepared by diluting the corresponding standard working solutions with blank rat plasma and tissue to concentrations of 5, 15, 20, 40, 50, 100, 200, 500, 1,000, 5,000, 8,000 and 10,000 ng/mL troxipide. The concentrations of the QC samples (plasma and tissue) were 15, 400 and 8,000 ng/mL troxipide. A 100 µg/ml IS stock solution was made by transferring approximately 1.0 mg of accurately weighed IS to a 10-mL volumetric flask and dissolving it in methanol. The IS stock solution was further diluted with methanol to obtain a 1 µg/mL IS working solution. All solutions and samples were immediately stored at 4 °C until analysis.Sample preparation of metabolite identification and pharmacokinetic study were detailed shown in Supplementary Materials and Methods.To investigate the major metabolites of troxipide, we performed a metabolic study. For this experiment, a rat model of GU (n = 20) was established successfully as described above by using 5% acetic acid (10 mL/kg/day). Each rat with GU was placed in a metabolic cage for 3 days before oral administration for the adaptation period. Then, the rats with GU were orally administered troxipide (40 mg/kg) (n = 10) or an equivalent volume of saline (n = 10). Blood samples (0.3 mL) from the orbital vein were collected in heparinized tubes before administration and 15, 30, 60, 120, 240, 480, 720, 1,440, and 2,880 min after administration. The samples were centrifuged at 5,000 rpm for 10 min at 4 °C to obtain plasma. The urine and feces of each rat were collected between oral administration 0–48 h, and then the urine was centrifuged at 3,000 rpm at 4 °C for 10 min. The supernatant was transferred to a new tube. All biological samples were stored at -80 °C until analysis.Raw metabolic data were obtained by systematic analysis using UHPLC-Q-Orbitrap-HRMS. In brief, to identify the metabolites of troxipide, the base peak chromatograms of samples from troxipide-treated rats and saline-treated rats were analyzed and compared. The metabolic data were processed by Compound Discovery  software and Mass Frontier software .The methods were based on the FDA and EMA guidelines to determine troxipide in rat plasma and tissue by validating analytical method, as detailed in Supplementary Materials and Methods.110 SD rats were randomly divided into two groups according to the average body weight: NCG (n = 55) and GUG (n = 55). The rats of GUG were orally administrated 5% acetic acid (10 mL/kg/day) at 9.00 am for 2 weeks. Meantime, the rats of NCG were given an equivalent volume of saline.92. The approximately 0.3 mL of plasma was collected from the orbital venous plexus of the NCGIG and GUGIG rats before administration and 15, 30, 60, 120, 180, 240, 360, 480, 600, 720, 1,440, 2,160, 2,880 min after administration .After 2 weeks, NCGIG rats (n = 30) and GUGIG rats (n = 30) were randomly divided into three groups. All rats were fasted for at least 12 h before the administration but were given free access to water. The NCGIG rats and GUGIG rats were weighed and given a single dose of 20 mg/kg, 40 mg/kg, or 60 mg/kg troxipide (10 mL/kg) via intragastric administration. The rat blood collection method used in our study was performed according to ICH guidelines and the guidelines of the Nonclinical Pharmacokinetics of Medicinal Products by National Medical Products Administration (NMPA) and was based on previous reports and our preliminary studiesAfter 2 weeks, NCGIV rats (n = 10) and GUGIV rats (n = 10) were weighed and intravenously injected with a single dose of 40 mg/kg troxipide (10 mL/kg) via the tail vein to evaluate absolute availability. Approximately 0.3 mL of plasma was collected from the NCGIV rats and GUGIV rats before after intravenous injection and 5, 10, 30, 60, 120, 180, 240, 360, 480, 600, 720, and 1,440 min after intravenous injection. After collection, the plasma samples were immediately centrifuged at 3,000 rpm for 10 min at 4 °C, and the resulting supernatants were collected and stored at -80 °C until analysis.NCGIG rats (n = 15) and GUGIG rats (n = 15) were randomly divided into five groups according to execution time. All rats were weighed and orally administered 40 mg/kg troxipide (10 mL/kg). Then, the rats were sacrificed on ice before treatment or 60, 120, 180, 240 min after oral administration. Tissues were quickly excised, carefully rinsed with saline (pH 7.0) to remove blood from the tissue surface, dried with filter paper and weighed. A total of 1.0 g of each tissue sample was accurately weighed  and homogenized with physiological saline (0.33 g/mL) by using a tissue homogenizer. Finally, all tissue homogenates were stored at − 80 °C until analysis.−1) by oral gavage every day at 9:00 am for 2 weeks. The NCG rats were orally administered an equivalent volume of saline. After 2 weeks, to investigate whether the GU model was successfully established, 6 rats were randomly chosen from each group and sacrificed. Then, the GUG rats were randomly divided into the following four groups (n = 10): the GUG, THG, TMG and TLG. The rats in the GUG, THG, TMG and TLG groups were orally administered 5% acetic acid (10 mL kg−1) every day at 9.00 am for another 2 weeks. The rats in the TMG and TLG and THG were orally gavaged with 20 mg kg−1, 40 mg kg−1 or 60 mg kg−1 troxipide every day at 4:00 pm. The rats in the NCG and GUG were orally administered an equivalent volume of saline every day at 4:00 pm. The dose of troxipide was calculated and adjusted according to a previous study and our preliminary experiment. All rats were given a normal diet and fresh water ad libitum throughout the experiment. After 2 weeks of troxipide treatment, the rats were fasted for 12 h and then anesthetized via intraperitoneal (ip) injection of 3% pentobarbital (40 mg/kg). Serum was collected and stored at -80 °C, and stomach tissue samples were collected after the rats were sacrificed, fixed in 10% formalin, rapidly frozen in liquid nitrogen and stored at -80 °C.After a week of acclimatization, Sprague–Dawley (SD) rats were randomly assigned to the NCG (n = 16) or GUG (n = 46). Prior to the initiation of troxipide treatment, the GUG rats were given 5% acetic acid (10 mL kg2), ulcer index and ulcer inhibition (%) in the rats in the study. The stomachs were inflated by injecting 10% formalin solution to fix the tissues overnight, and then opened along the greater curvature and photographed. The GU area (mm2) was calculated by the longest lengthand (d1) and width (d2) of the ulcer using the following equation: 2). And, the gastric tissues were directly observed to determine the ulcerative lesion index according to the severity of GU lesion scoring system. The ulcer index of each rats were calculated in accordance to the scoring criteria of ulcer index  of the rats stomach were used to analyze IHC with polyclonal antibody of HSP-90, CCAS-3 and C-PARP-1. Firstly, the paraffin sections were incubated with primary antibody at 4 °C all night. After washing, the sections were incubated with secondary antibody and the DAB kit  was used to stain tissue sections. Then, the sections in each group (n = 10) were observed with light microscopy . For each slice, at least 20 regions were randomly selected and the correspondin micrographs were captured with magnifications of 100. Subsequently, the positive staining of the images was observed using the software Image Pro Plus 6.0 . Later, the expression of proteins was demonstrated by the ratio of integral optical density (IOD). IOD = average optical density × positive area. Bar graphs in this point were performed with OriginLab Origin Pro.2019b .Enzyme-linked immunosorbent assay (ELISA) kits were utilized for measurement of rats serum of Motilin (MTL), Gastrin (GAS), Pepsinogen I (PG-I), Pepsinogen II (PG-II), Interleukin (IL-17), Interleukin (IL-6), Tumor Necrosis Factor (TNF-α), Interferon-γ (IFN-γ), Activator Protein (AP-1) following the manufacturer’s protocols. The above commercial assay kits were purchased from Jiangsu mbbiology biological technology Co., Ltd. . And, we were investigated the ulcer area  and time to reach Cmax (Tmax) were directly obtained from pharmacokinetic concentration–time data. The area under the concentration–time curve to the last measurable concentration point (AUC(0−t)) was evaluated by the linear trapezoidal rule, and the AUC(0−∞) was calculated as following equation: AUC(0−∞) = AUC(0−t) + Ct/Ke, where the Ct was the last concentration of detectable and the Ke was a constant of terminal elimination. Moreover, the elimination half-life (t1/2), mean residence time (MRT), variance of residence time (VRT), elimination constant (K10) and absorption constant (Ka) of the drug were all determined. The absolute bioavailability (Fa) was calculated using the formula:P < 0.05 or P < 0.01. In additionally, all charts and figures were performed by using OriginLab Origin Pro. 2019b .Pharmacokinetic parameters of troxipide were calculated by the pharmacokinetic software Drug and Statistics 2.1 vision . The maximum concentration (CSupplementary Information.