Streptomyces are efficient producers of various bioactive compounds, which are mostly synthesized by their secondary metabolite biosynthetic gene clusters (smBGCs). The smBGCs are tightly controlled by complex regulatory systems at transcriptional and translational levels to effectively utilize precursors that are supplied by primary metabolism. Thus, dynamic changes in gene expression in response to cellular status at both the transcriptional and translational levels should be elucidated to directly reflect protein levels, rapid downstream responses, and cellular energy costs. In this study, RNA-Seq and ribosome profiling were performed for five industrially important Streptomyces species at different growth phases, for the deep sequencing of total mRNA, and only those mRNA fragments that are protected by translating ribosomes, respectively. Herein, 12.0 to 763.8 million raw reads were sufficiently obtained with high quality of more than 80% for the Phred score Q30 and high reproducibility. These data provide a comprehensive understanding of the transcriptional and translational landscape across the Streptomyces species and contribute to facilitating the rational engineering of secondary metabolite production. Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.12045603 Streptomyces, which comprise the largest genus of Actinobacteria, are huge natural reservoir of secondary metabolites, including antibiotics, immunosuppressants, and other medicinal compounds6. Recent advancements in high-throughput sequencing have led to the development of the genome mining approach, which implicates that the genome of each Streptomyces species has more than 30 secondary metabolite biosynthetic gene clusters (smBGCs) with potential to produce various unexplored secondary metabolites2. These secondary metabolites are synthesized by a series of enzymatic reactions, which depend on the supply of precursor molecules from primary metabolism, such as acetyl-coenzyme A and amino acids7. After active growth terminates, an overall metabolic transition occurs, which leads to the activation of secondary metabolite production9; this metabolic transition from primary to secondary metabolism is governed by multi-layered regulatory mechanisms at transcriptional, translational, and post-translational levels11. Thus, understanding the complex regulatory systems of the metabolic transition is important to enhance secondary metabolite production. The overall metabolic transition encompasses diverse genome-wide gene expression changes, which are regulated by signaling cascades from the pleiotropic regulators to pathway-specific regulators13. To understand the underlying molecular mechanisms of metabolic transitions, transcriptional changes that occur between growth phases have been studied15. For example, the time-series transcriptome analysis of Streptomyces coelicolor demonstrated that coherent genes that are involved in specific metabolism and their regulatory genes exhibit similar expression patterns during metabolic transitions; this suggests that primary metabolism-related genes are functionally connected to the smBGC genes through regulatory gene expression. Based on this suggestion, putative regulatory genes and their interconnected networks could be identified by screening genes that have similar expression patterns13.17. For example, Escherichia coli proteome analysis revealed that only approximately half of protein abundance is determined by transcriptional regulation, which indicates the existence of various post-transcriptional regulation18. In this regard, deciphering translational dynamics is important to understanding post-transcriptional regulations that are closely related to cellular protein levels19. Recently, ribosome profiling has been used to measure translational levels by deep sequencing of the ribosome-protected mRNA fragments (RPFs) at the position of the translating ribosome20. Several ribosome profiling studies in Streptomyces have been reported by our research group for S. coelicolor, S. clavuligerus, and S. lividans, which revealed translational buffering of secondary metabolism-related genes at a later growth phase and that translational abundance is more consistently maintained than transcript abundance22. Translational regulations are advantageous for the tight control of secondary metabolite biosynthesis, as translation requires the highest energy costs among all cellular reactions19. Moreover, the expression of smBGC-associated genes can be more rapidly regulated at the translational level than at the transcriptional level in response to dynamic environmental changes23. Given the dynamic relationship between transcription and translation, as exhibited by translational buffering11, integrative analysis at both levels should unravel complex regulations in Streptomyces. However, transcriptomic and translatomic data have covered only a small portion of approximately 350 reported Streptomyces genomes, which have not been systematically validated at the multi-species level.Bacteria can fine-tune gene expression both at the transcriptional and translational levelsStreptomyces species at four different growth phases, followed by validation of the read quality. The species were S. avermitilis MA-4680, S. clavuligerus ATCC27064, S. lividans TK24, S. venezuelae ATCC15439, and S. tsukubaensis NRRL 18488, which are industrial strains that produce antifungal avermectin, β-lactamase inhibitor clavulanic acid, and immunosuppressant FK506, respectively26. S. lividans and S. venezuelae were characterized by their fast growth and ease of genetic manipulation, and have been employed as heterologous expression hosts30. An overview of the preparation of transcriptomic and translatomic data is illustrated in Fig. S. clavuligerus21 and S. lividans22) among the five species were already reported in previous studies by our research group, this study provided a uniformly processed and mapped dataset of all five species. This facilitates the efficiency of the comparative transcriptome and translatome analysis at multi-time points between multi-species. Further, understanding the transcriptional and translational regulatory mechanisms and developing regulatory synthetic parts, such as promoters, ribosome-binding sequences, 5′ untranslated regions, and terminators4 from the dataset allows rational genome engineering for efficient secondary metabolite production by Streptomyces11.In this study, we provide RNA-Seq and ribosome profiling data of five Streptomyces strains were inoculated from their 20% glycerol stock of spores into 50 mL of R5− liquid medium with 8 g of glass beads (3 ± 0.3 mm diameter) in a 250 mL baffled flask, grown at 30 °C, and pre-cultured at 250 rpm. The R5− liquid medium consists of 103 g L−1 sucrose, 0.25 g L−1 K2SO4, 10.12 g L−1 MgCl2∙6H2O, 10 g L−1 glucose, 0.1 g L−1 casamino acids, 5 g L−1 yeast extract, 5.73 g L−1 TES (pH 7.2), 0.08 mg L−1 ZnCl2, 0.4 mg L−1 FeCl3∙6H2O, 0.02 mg L−1 CuCl2∙2H2O, 0.02 mg L−1 MnCl2∙4H2O, 0.02 mg L−1 Na2B4O7∙10H2O, 0.02 mg L−1 (NH4)6Mo7O24∙4H2O, and 0.28 g L−1 NaOH. The grown mycelium was inoculated to fresh R5− medium with an initial optical density of 0.05 at 600 nm for the main culture as biological duplicates and grown under the previously mentioned conditions. The cells were sampled at four different time points based on the growth profile of each strain, as follows: early-exponential (E), transition (T), late-exponential (L), and stationary (S) phases. The E, T, L, and S time points were 13, 17, 19.5, and 33.5 h for S. avermitilis, 26, 80, 105.5, and 125 h for S. clavuligerus, 9.5, 14, 16, and 20 h for S. lividans, 12.5, 24.5, 30.5, and 48.5 h for S. venezuelae, and 15, 18.5, 28, and 48 h for S. tsukubaensis after inoculation, respectively , and then resuspended with 500 μL lysis buffer . The resuspended cells were frozen with liquid nitrogen and grounded using a mortar and pestle. The ground mycelium was thawed and centrifuged at 4 °C for 10 min at 16,000 × g. The supernatant was collected and stored at −80 °C. Following the preparation of lysates from four growth phases as biological duplicates, the lysates were mixed with a solution of phenol:chloroform:isoamyl alcohol , and the mixtures were separated by centrifugation. DNA in the extracted RNA samples were removed by treatment with 2 μL DNase I , 5 μL 10 × DNase I buffer, and 1 μL SUPERase-In RNase Inhibitor . Lastly, the DNase I-treated RNA samples were purified using phenol:chloroform:isoamyl alcohol  and ethanol precipitation. To eliminate rRNAs in the recovered RNA samples, the Ribo-Zero rRNA Removal Kit for Bacteria  was used according to the manufacturer’s instructions. The quality of rRNA-depleted RNA samples was checked using 2% agarose gel electrophoresis. The suitable RNA samples were then used to construct RNA sequencing libraries using the TruSeq Stranded mRNA Library Prep Kit . The size distributions of the final libraries were checked using the Agilent 2200 TapeStation System . The constructed libraries were sequenced on the HiSeq. 2500 platform using either a 100-bp  or 50-bp (S. tsukubaensis) single-end read recipe , S. clavuligerus ATCC27064 , S. lividans TK24 (NZ_CP009124), S. venezuelae ATCC15439 (CP013129), and S. tsukubaensis NRRL18488 . The statistics pertaining to quality trimming and reference mapping are summarized in Table 33.Raw FASTQ files were processed using the CLC Genomics Workbench . Raw reads were trimmed by their overall quality  and length (minimum length: 15 nucleotides). The filtered reads were mapped to each reference genome sequence with the default parameters . The accession number of each reference genome is as follows: 21. The mycelium that was treated by thiostrepton was collected by centrifugation at 4 °C for 10 min at 3,000 × g, and the cell pellet was washed with 2 mL of polysome buffer that was composed of 20 mM Tris-HCl (pH 7.5), 140 mM NaCl, and 5 mM MgCl2 with 20 μM thiostrepton. The washed pellet was re-suspended in 1 mL of lysis buffer composed of 950 μL of polysome buffer and 50 μL of 20% Triton X-100 with 20 μM thiostrepton. The resuspended cells were dripped into a mortar filled with liquid nitrogen and then grounded with a pestle. The cell debris was removed by centrifugation at 4 °C for 5 min at 3,000 × g. The supernatant was further clarified and collected by centrifugation at 4 °C for 10 min at 16,000 × g. To digest RNA in the lysate (containing 50 μg RNA), the S. avermitilis and S. tsukubaensis samples were treated with 750 U of RNase I  at 37 °C for 45 min, and the remaining strains were treated with 400 U of Micrococcal Nuclease (MNase) (NEB), 20 μl of 10× MNase buffer, and 2 μl of 100× Bovine Serum Albumin (BSA) (NEB) at 37 °C for 2 h. The samples were then loaded onto Illustra MicroSpin S-400 HR Columns  that were previously washed three times with 500 μL of washing buffer, which was composed of 50 mM Tris-HCl (pH 8.0), 250 mM NaCl, 50 mM MgCl2, 25 mM EGTA, and 1% Triton X-100. The column was centrifuged at 4 °C for 2 min at 400 × g, and the flow-through was further purified by a phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation. rRNA was depleted with the Ribo-Zero rRNA Removal Kit (Epicentre) according to the manufacturer’s instructions. The ribosome-protected RNA fragments (RPF) of between 26 and 34 bp were separated by electrophoresis for 65 min at 200 V using 15% polyacrylamide TBE-urea gel (Invitrogen), and eluted in 400 μL of RNA gel extraction buffer, which was composed of 300 mM sodium acetate pH 5.5, 1 mM EDTA, and 0.25% (w/v) SDS. The samples were frozen for 30 min at −80 °C and then incubated at 37 °C for 4 h. The eluted RNAs were isolated by ethanol precipitation and purified once again with the RNeasy MinElute Column  using the manufacturer’s protocol. The enriched RPFs were then denatured for 90 s at 80 °C and incubated for 1 h at 37 °C with 5 mL of 10× T4 Polynucleotide Kinase (PNK) buffer (NEB), 20 U of SUPERase-In RNase Inhibitor, and 10 U of T4 PNK (NEB) to dephosphorylate the 3′ end. The dephosphorylated RNAs were purified using the RNeasy MinElute Column (Qiagen). The sequencing library was constructed from the end-repaired RPFs using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB) according to the manufacturer’s instructions. The final library of approximately 150–160 bp was size-selected by gel electrophoresis for 90 min at 100 V using a 2% agarose gel that was dyed with SYBR Gold Nucleic Acid Gel Stain . The concentration of the final library was measured using a Qubit 2.0 Fluorometer (Invitrogen) and the Qubit dsDNA HS Kit. The size distribution was assessed using the Agilent 2200 TapeStation System (Agilent). The constructed library was sequenced on the Illumina HiSeq. 2500 platform using the 50-bp single-end read recipe  were deposited at the National Center for Biotechnology Information Sequence Read Archive (NCBI SRA)37. Raw read FASTQ files of RNA-Seq and ribosome profiling of S. lividans were deposited at the European Nucleotide Archive (ENA)38. Raw read FASTQ files of RNA-Seq of S. venezuelae were deposited at the ENA39. Raw read FASTQ files of ribosome profiling of S. venezuelae were deposited at the NCBI SRA47. Trimmed read FASTQ files and mapped read BAM files of the raw read FASTQ files in the NCBI SRA  were deposited at the ENA with a new accession48. Trimmed read FASTQ files and mapped read BAM files of the raw read FASTQ files in the ENA  were also deposited at the ENA with the same accession as each corresponding raw read FASTQ file39. The gene expression profile as raw read counts of RNA-Seq and ribosome profiling data of S. avermitilis49, S. clavuligerus50, S. tsukubaensis51, and ribosome profiling data of S. venezuelae52 are available in a text file format in the Gene Expression Omnibus (GEO) database. Also, the gene expression profiles of all datasets (RNA-Seq and ribosome profiling of the five species), including raw read counts, DESeq. 2 normalized values, fold change values between growth phases, and p-values for the fold changes, are available in a text file format in the Figshare53. The raw read FASTQ data of S. clavuligerus in NCBI SRA36 was published in the previous study21. Also, the raw read FASTQ data of S. lividans in ENA38 was published in the previous study22. Note that the ribosome profiling data of Streptomyces griseus was uploaded under the same accession with those of S. venezuelae, but they are not described in this study.Raw read FASTQ files, trimmed read FASTQ files, mapped read BAM files, and the gene expression text files of all samples were uploaded to the public databases Tables  and 2. RS. avermitilis), 13,961,155 reads (S. clavuligerus), 15,686,025 reads (S. lividans), 12,899,493 reads (S. venezuelae), and 51,144,650 reads (S. tsukubaensis). After trimming the sequencing reads by quality score and nucleotide length, more than 99.8% of the sequencing reads remained, which indicated high-sequencing quality. The remaining reads were used as input to generate sequencing QC reports in the CLC Genomics Workbench to validate the quality of the reads. At first, the overall read lengths were extremely long, corresponding to the sequencing read recipe , 98.9% (S. clavuligerus), 98.9% (S. lividans), 98.9% (S. venezuelae), and 96.4% (S. tsukubaensis) of the total reads exhibited an average Phred score of greater than 30, which indicates 99.9% base call accuracy , 97.6% (S. clavuligerus), 97.8% (S. lividans), 98.3% (S. venezuelae), and 95.4% (S. tsukubaensis) at the last position, respectively , 52.19% to 90.88% (S. clavuligerus), 60.15% to 87.08% (S. lividans), 67.35% to 86.23% (S. venezuelae), and 76.33% to 96.26% (S. tsukubaensis) (Table 1). The number of uniquely mapped reads at each gene was counted and normalized using the DESeq. 2 package in R33 to reduce variation between samples. Using the normalized values, principal component analysis (PCA) was performed, which validated the high reproducibility of the sequencing data  broadly ranged from 0 to 20 in the different growth phase samples . The S. clavuligerus E2 sample showed a relatively lower mapped number  compared to other S. clavuligerus samples. S. venezuelae showed 34.2 to 69 million mapped reads (average 13.9% of the trimmed reads), respectively. The two early samples of S. lividans showed 97.2 and 81.1 million mapped reads (46 and 43.8% of the trimmed reads), respectively, while other samples showed low numbers . S. tsukubaensis showed various ranges for the mapping read number; L2 and S2 samples showed 56 and 103 million mapped reads (35 and 53.9% of the trimmed reads), respectively, while other samples showed a lower number of mapped reads . S. avermitilis showed the lowest number of mapped reads and ratio among the five species, with 2 to 13.1 million mapped reads (1.3 to 5.9% of the trimmed reads). Although the minimum mapped read number among the samples was 1.8 million, the numbers obtained are, based on several bacterial transcriptome studies, considered sufficient for analysis of the whole translational profile and differential expression levels of genes, as 1 to 5 million reads are suggested for high statistical significance59. Among the uniquely mapped reads, some reads were mapped within RNA genes, rather than protein-coding genes, which mostly corresponded to tRNA genes. These reads may be the fragments of tRNA and rRNA that were bound to the ribosome and then enriched during monosome recovery60. Therefore, further validation was performed using only the mapped reads of the protein-coding genes. A total of 1.1 to 19.5 million reads were mapped to protein-coding genes that were 4.3 to 81.0% of the uniquely mapped genes, which indicates a high ratio of tRNA gene-mapped reads  broadly ranged from 0 to 20, which was considered significant to analyze the translational abundance in different growth phases  were mapped when the non-specifically matched reads were randomly assigned to one of the mapped positions, while 1.8 to 103.9 million reads (1.3 to 54.9% of the reads) were uniquely mapped when the non-specifically matched reads were excluded Table . These rds Table . The mapses Fig. . In conc


To compare the effect of telmisartan and vitamin E on liver histopathology of non‐alcoholic steatohepatitis (NASH) patients.This noninferiority clinical trial was conducted for 1 year. Fatty liver patients with non‐alcoholic fatty liver disease (NAFLD) activity score (NAS) ≥ 5 (in liver biopsy) were selected. All methods were in accordance with the Declaration of Helsinki. Patients who received telmisartan and vitamin E were denoted as Group‐T and Group‐E, respectively. Forty patients >18 years old were assigned and divided into two groups (20 in each group). Histological improvements were primary outcome measures.P = 0.00 and Group T [GT]: 5.6 ± 0.7to 4.9 ± 1.2; P = 0.03). Fibrosis score improved from 1.6 ± 0.5 to 1.5 ± 0.5 in GE and from 1.7 ± 0.9 to 1.5 ± 0.7 in GT . Steatosis improved in GE from 2.07 ± 0.6 to 1.14 ± 0.66 (P = 0.00) and in GT from 1.94 ± 0.57 to 1.56 ± 0.8 (P = 0.05). Lobular inflammation improved from 2.0 ± 0.4 to 1.6 ± 0.5 in GE (P = 0.02) and from 1.9 ± 0.3 to 1.8 ± 0.4 in GT (P = 0.58). Ballooning score in GE decreased from 1.9 ± 0.3 to 1.7 ± 0.5 (P = 0.03), and in GT, it reduced from 1.9 ± 0.1 to 1.5 ± 0.5 (P = 0.19). NAS improvement was similar in GE (1.6 ± 1.2) and GT  when controlled for weight reduction.Significant improvement in NAS score was noted in both groups , but this study proved that telmisartan is also comparable to vitamin E for NASH with few admitting limitations. Non‐alcoholic fatty liver disease (NAFLD), a hepatic manifestation of metabolic syndrome, affects 20–30% of the general population.In adults, NAFLD is typically classified into two categories: non‐alcoholic fatty liver (NAFL) and non‐alcoholic steatohepatitis (NASH).As oxidative stress has been implicated in the pathogenesis of NAFLD, the role of antioxidants such as vitamin E, which is known to react with reactive oxygen species (ROS), blocking the propagation of free radical reactions in a wide range of oxidative stress situations, has been consequently tested on several occasions.et al.Telmisartan, a potent antagonist of the angiotensin II type‐1 (AT1) receptor, usually indicated for essential hypertension, has recently been proven to act against oxidative stress, insulin resistance, and hepatic fibrogenesis.This randomized, open‐label, noninferiority clinical trial was conducted in the Department of Hepatology, Bangabandhu Sheikh Mujib Medical University (BSMMU), for the first time from July 2016 to June 2017. Before commencement of the study, it was ethically approved by Institutional Review Board (IRB) of BSMMU. The clinical trial was registered on the Sri Lanka Clinical Trials Registry . Patients with ultrasonogramme (USG)‐proven NAFLD admitted to the Department of Hepatology were screened and approached for index biopsy of the liver. Evidence of steatosis, lobular inflammation, and hepatocellular ballooning in biopsy were considered essential components for the diagnosis of NASH. Patients aged >18 years with NAS ≥5 in liver histology were considered for inclusion in the study. On the other hand, patients with a history of alcohol intake >20 g/day; a history of consumption of drugs that can cause fatty liver, that is, tamoxifen, valproic acid, amiodarone, and methotrexate; or a history of drugs that have shown benefit in previous NASH pilot studies, that is, metformin, thiazolidinediones, and fibrates, were considered for exclusion. Moreover, patients with chronic liver disease (CLD) with known etiology (due to hepatitis B virus (HBV), hepatitis C virus (HCV), Wilson's disease, drug‐induced liver injury etc.); pregnancy; comorbid conditions such as chronic obstructive pulmonary disease (COPD), chronic kidney disease (CKD), and congestive cardiac failure (CCF); and history of recent myocardial infarction (MI) were also excluded from the study. Randomization was carried out by lottery method. For the convenience of the study, total study population was divided into two groups (1:1), named: Group T and Group E. Group T patients received 40 mg of telmisartan once daily, and Group E received 800 IU vitamin E for a similar duration. Besides the standard treatment, lifestyle modification was advised for both groups of patients. Patient were advised to avoid saturated fat, excessive sugar‐containing diet, soft drinks, fast food, and refined carbohydrates and were also encouraged to perform moderate exercise . Diabetic patients were treated with lifestyle modification and, if required, with oral sulphonylureas—gliclazide, glimeperide—or with insulin. Dyslipidemia was managed with statin, and for hypertension, antihypertensive drugs, except angiotensin converting enzyme (ACE) inhibitor, ARB, and calcium channel blocker (Diltiazem), were used.Within the study period, 76 NAFLD patients underwent liver biopsy, and 40 were diagnosed with NASH with an NAS score ≥5. Then, they were randomly allocated to the two previously mentioned groups, with 20 patients in each group. Each patient was advised to attend monthly follow up for 3 months and then follow up every 3 months for the next 9 months. At the end of 1‐year follow up, the total number of attritions was 10 (4 patients in Group T and 6 patients in Group E) due to unwillingness to undergo liver biopsy and lack of interest , 2 h after breakfast (2HABF) and fasting lipid profile for diabetic and dyslipidemia patients were assessed according to need. At the end of 1 year, liver biopsy was repeated in both groups. The primary parameters compared between the first and last visits were vital parameters, blood profile and NAS , and fibrosis score.Informed consent was obtained from all patients during enrollment of the study and before liver biopsy. Ethical measures were confirmed to be in concordance with the Helsinki declaration.t test was used. Comparison between the two groups were performed using an unpaired t test. Analysis of covariance (ANCOVA) was used to control for the effect of weight loss on changes in NAS and fibrosis score. The study was conducted with 95% confidence level at 5% acceptable error level, and P value <0.05 was considered statistically significant. To account for the decreased power associated with small sample size, we calculated the between‐subject effect size (Cohen's d) for the improvement parameter of NAS. A large effect size of >0.8 was found, making our statistical inference relevant. Data analysis was performed using SPSS 23 .Quantitative data were presented as mean ± SD, and qualitative data were presented in percentage. To determine associations between the categorical variables, chi‐square test was used, and in case of continuous variables, the paired This study was reviewed and approved by the IRB. BSMMU/2016/3054.Forty NASH patients were selected initially. Six patients of Group E were lost to follow up due to lack of interest, and four patients of Group T did not agree to second biopsy Fig. . Thirty P = 0.262). Mean fibrosis score was 1.57 ± 0.51 in Group E and 1.69 ± 0.70 in Group T (P = 0.615)  within the obese range (according to Asian criteria). Baseline liver histology was also statistically similar between groups. Mean NAS was 5.93 ± 0.73 in Group E and 5.63 ± 0.72 in Group T (Table P < 0.001) and Group T . In Group E, histology improved significantly in all components of NAS except ballooning, where the improvement was not statistically significant. In Group T, histologic improvement was noted in all components, but significant improvement was noted only in ballooning. Fibrosis score also improved in both groups. Therefore, Group E patients showed significant improvement in steatosis, lobular inflammation, and overall NAS, and Group T showed significant improvement in ballooning and overall NAS . Comparison of individual components of the NAS score between the treatment groups shows that improvement in steatosis was significantly higher in Group E than Group T (P = 0.033). However, in ballooning and lobular inflammation scores, mean improvement did not differ significantly between Groups E and T . In Group E, mean fibrosis score improvement was 0.1 ± 0.6, whereas in Group T, it was 0.1 ± 0.9. The difference in fibrosis score improvement between Groups E and T was not statistically significant (P = 0.580) . Mean improvement in BMI was 1.7 ± 0.4 in Group E and 1.02 ± 1.38 in Group T (P = 0.89)  and aspartate aminotransferase (AST), but reduction in gamma‐glutamyltrasferase (GGT) level was not statistically significant. Group T also showed reduction in ALT and AST, with reduction being significant in the latter. While GGT level increased in Group T Table , mean chFBS reduced statistically significantly from baseline in both groups after treatment. Homeostasis model assessment insulin resistance (HOMA‐IR) showed nonsignificant improvement in both groups. All components of the serum lipid profile showed improvement with treatment in both groups, with serum cholesterol level showing significant decrease in patients receiving telmisartan Table .P = 0.204).NAS ≥2 improvement without worsening of the fibrosis score was considered a histological responder. Accordingly, 7 patients (23.3%) were responders, and 23 patients (76.7%) were nonresponders. Among responders, five patients (71.4%) were in Group E, and two patients (28.6%) were in Group T. The difference in responses between Groups E and T was not statistically significant (P = 0.045). Baseline lipid profile was similar between responders and nonresponders, except high‐density lipoprotein (HDL). Mean HDL differed significantly between responders and nonresponders . Baseline liver function tests were also statistically similar among responders and nonresponders. Mean NAS score at baseline was 6 ± 0.8 and 5.7 ± 0.7 in responders and nonresponders, respectively. Mean fibrosis score at baseline was 1.5 ± 0.5 in responders and 1.6 ± 0.6 in nonresponders.Table 2 and 1.2 ± 1.3 kg/m2 (P = 0.154), and mean waist circumference improvement was 4.2 ± 2.6 cm and 2.3 ± 2.1 cm (P = 0.060) in responders and nonresponders, respectively. Weight loss of ≥5% occurred in 13 (43.3%) of 30 patients. Five patients (71.1%) among histological responders and eight patients (34.8%) among histological nonresponders had weight reduction of ≥5%. The difference was not statistically significant (P = 0.087). On the other hand, ≥7% weight loss was achieved in eight patients (26.7%). Among them, three patients were histological responders (42.9%), and five were histological nonresponders (21.7%). Thus, weight improvement of ≥7% was similar across groups (P = 0.300). None of the patients in this study achieved 10% weight loss.Table The present open‐label, randomized, noninferiority clinical trial investigated the effects of 1 year of treatment with telmisartan and vitamin E on liver histology in NASH patients. To our knowledge, this study is the first of its kind among biopsy‐proven NASH patients.et al.,et al.,The most significant changes in hepatic histology induced by vitamin E in this study were reflected by the attenuation of steatosis, lobular inflammation, and NAS. These findings are consistent with Sanyal P = 0.029). This finding was consistent with previous studies, such as the one by Georgescu and Ionescuet al.et al.,et al. observed that telmisartan improves all components of NAS, as well as fibrosis score.et al.,With regard to telmisartan, this study demonstrated statistically significant NAS improvement . These findings are consistent with several previous studies.et al.,In Group E, the improvement in ALT and AST at the end of 12 months from baseline was statistically significant  in the telmisartan group. Other parameters of lipid profile did not improve significantly in either Group E or Group T.In the telmisartan group, baseline ALT and AST improved after 12 months of treatment, with the latter reaching statistical significance. Similarly, Alam P value 0.072 and 0.738, respectively). As lifestyle intervention was adhered to by patients in both groups, and improvement in BMI and weight circumference occurred in both groups, these factors were weighted in the analysis of improvement in the histological activity between these groups.In our study, on direct head‐to‐head comparison of the improvement of dynamic factors between vitamin E and telmisartan, we found that telmisartan was similar to vitamin E with regard to improvement of overall NAS and fibrosis score (The improvement in NAS in both groups could be attributed to weight reduction in both groups. In order to adjust the effect of weight reduction, we used ANCOVA for statistical analysis of histological improvement and found that mean improvement in NAS was statistically similar across groups. Therefore, vitamin E and telmisartan both showed similar histological improvement in NASH.The study limitations were the small sample size, the high number of dropouts, and single center‐based inclusion of subjects. It is very difficult to predict treatment response confidently with a small sample size. The present study also suffered from a lack of multicenter, multiethnic categories of patients.In conclusion, vitamin E and telmisartan improve NAS but not fibrosis score. Telmisartan was similar to vitamin E in improving the overall histology of NASH patients. We recommend conducting large multicenter, double‐blinded randomized controlled trials to consolidate the findings of this study.


Juvenile idiopathic arthritis (JIA) may seriously affects patients’ quality of life (QoL), but it was rarely focused and studied in China, so we explore JIA children’s QoL using Chinese version of the PedsQL4.0 Generic Core and PedsQL3.0 Rheumatology Module scale, and analyzed the psychometric properties of these two Scales among Chinese JIA children.We recruited 180 JIA patients from Children's Hospital Affiliated to Capital Institute of Pediatrics and Hebei Yanda Hospital from July 2018 to August 2019. The questionnaires include information related on JIA, PedsQL4.0 generic core and PedsQL3.0 Rheumatology Module scales. According to the disease type, onset age of and course of JIA, we divided them into different groups, then compared the QoL status among different groups. Moreover, we analyzed the reliability and validity of these two scales in these 180 JIA children.The mean score of PedsQL4.0 generic core scale on these 180 patients was 82.85 ± 14.82, for these in active period was 72.05 ± 15.29, in remission period was 89.77 ± 9.23; the QoL score of systemic, polyarticular and oligoarticular JIA patients were 77.05 ± 19.11, 84.33 ± 12.46 and 87.12 ± 10.23. The mean score of PedsQL3.0 Rheumatology Module scale on 180 patients was 91.22 ± 9.45, for these in active period was 84.70 ± 11.37, in remission period was 95.43 ± 4.48; the QoL score of systemic, polyarticular and oligoarticular JIA patients were 89.41 ± 11.54, 89.38 ± 10.08 and 93.71 ± 6.92. In the PedsQL 4.0 Generic Core scale, the α coefficients of total scale and almost every dimension are all greater than 0.8 except for the school activity dimension of 0.589; the correlation coefficients of 22 items’ scores  with the scores of dimensions they belong to are greater than 0.5 , and the other one is 0.406. In PedsQL3.0 Rheumatology Module scale, except for the treatment and worry dimensions of 0.652 and 0.635, the α coefficients of other dimensions and the total scale are all greater than 0.7; the correlation coefficients of all items’ score were greater than 0.5 .The QoL of Chinese JIA children is worse than their healthy peers, these in active period and diagnosed as systemic type were undergoing worst quality of life. The reliability and validity of PedsQL 4.0 Generic Core and PedsQL3.0 Rheumatology Module scale in Chinese JIA children are satisfactory, and can be used in clinical and scientific researches. Juvenile idiopathic arthritis (JIA) is a systemic chronic inflammatory disease occurred in children below 16 years, featured on chronic joint synovitis and extra articular organs’ damages. It is the most common type of rheumatic diseases in children with prevalence varies from 4.01 to 70 ‰ . Althoug) can be applied to all children, and the PedsQL3.0 Rheumatology Module (PedsQL3.0-RM) was specifically tailored for pediatric rheumatology [The QoL scale for JIA patients and related studies were very mature in many countries –5, but tmatology . These tmatology , 8, but All subjects were recruited from rheumatic immune clinic of Children's Hospital Affiliated to Capital Institute of Pediatrics and Pediatric clinic of Hebei Yanda Hospital from July 2018 to August 2019. Inclusion criteria: (1) JIA patients diagnosed in line with the standard of International Association of Rheumatology [Questionnaire consisted of general information such as gender, age, onset age of JIA, diagnosis age, disease type, treatment history, disease activity, etc.Chinese Version of the PedsQL 4.0-GC and PedsQL3.0-RM was applied to evaluate children’s QoL. PedsQL 4.0-GC scale was composed of 23 items which divided into 4 dimensions, physical health (8 items), emotional functioning (5 items), social functioning (5 items) and school functioning (5 items). PedsQL3.0-RM composed of 22 items which divided into 5 dimensions, pain and hurt (4 items), daily activities (5 items), treatment (7 items), worry (3 items), and communication (3 items).The questionnaire and PedsQL scales were all interview-administered one by one. In this paper, we only studied the child self-reports of PedsQL 4.0-GC and PedsQL3.0-RM which were filled solely on children’s answers.In order to analyze the influencing factors of JIA patients’ QoL, we divided all subjects into different groups: according the onset age of JIA to four groups ; according the course to three groups ; according to the disease type to three groups ; according to activity of JIA to two groups (active and remission). Criteria for determining the disease in remission period were no active joint inflammation; no fever, rash, serositis and other systemic symptoms; no active ophthalmic diseases; ESR and CRP are in the normal range; the overall assessment of the disease by pediatric rheumatologist concluded that it is not in active period .P < 0.05 was considered statistically significant. The reliability and validity of PedsQL 4.0-GC and PedsQL3.0-RM scale were calculated by Cronbach’sαcoefficients and Pearson correlation coefficients. In general, a Cronbach’s α coefficient ranging from 0.70 to 0.84 is regarded as satisfactory inter consistence. Pearson’s Correlation coefficient effect sizes are designated as small (0.10–0.29), medium (0.30–0.49), and large (0.50 or more) in magnitude.The data were analyzed by SPSS 16.0 software. The total score and each dimension’s score of PedsQL 4.0-GC and PedsQL3.0-RM were described by mean ± SD. The differences of scores among different groups were analyzed using con-variable test or independent sample t-tests. This study was approved by Ethics Committee of Capital institute of pediatrics.A total of 180 JIA children (102 boys and 78 girls) aged 8 ~ 18 years participated in this survey. The general information such as age, onset age of disease, course and drug application was showed in Table P < 0.05).We calculate the total score and each dimension of PedsQL 4.0-GC and PedsQL3.0-RM for these 180 children, and QoL scores of patients in active and remission period respectively. The results are shown in Table Table F = 1.091, P = 0.355). The scores of children whose course longer than 5 years were slightly lower than other groups, and the difference also no statistical significance . In Fig. F = 2.709, P = 0.047), and there was no significant difference between groups with different course of JIA .According to the classification of the onset age and course of JIA, in this survey, 45 children with onset age younger than 5 years, 56 children’s onset age are between 5 and 8 years, 53 children between 8 and 12 years, 6 children older than 12 years; 101 children’s course shorter than 3 years, 36 children’s courses between 3 and 5 years and 32 children’s course longer than 5 years. We calculated the QoL scores of different kinds of JIA patients. As shown in Fig. The Cronbach's αcoefficient is used to evaluate the reliability of the scale. In the PedsQL 4.0-GC scale, theαcoefficients of total scale and almost every dimension are all greater than 0.8 except for the school activity dimension of 0.589; In PedsQL3.0-RM scale, except for the treatment and worry dimensions of 0.652 and 0.635, theαcoefficients of other dimensions and the total scale are all greater than 0.7, so the reliability of the scale is satisfactory.The validity of the scale is expressed by the correlation between each item and the dimension it belonged. In PedsQL 4.0-GC scale, the correlation coefficients of 22 items are greater than 0.5 , and the other one is 0.406. In PedsQL3.0-RM scale, the correlation coefficients of all items is greater than 0.5 , which are all highly correlated.The QoL of JIA children is worse than healthy children, and those in active period is significantly worse than those in remission period especially in dimensions of physiological function, school function and pain. In this study, the average score of PedsQL 4.0-GC scale is 82.85, the score of school function dimension is 74.28, which are lower than the scores of healthy children (87.36 and 81.29) in previous survey . This isThe type of disease also can affect the QoL of JIA children. The QoL of patients in Oligoarticular type is the best, followed by patients in Polyarticular type, and those in systemic type is the worst. Table For children with older onset age of JIA, their QoL were better than those with younger onset age. This is confirmed by the fact that the QoL score of children with onset age older than 12 years are significantly higher than other groups , and chIn conclusion, the QoL of JIA children in China is worse than their healthy peers, especially in physiological function and school function. The activity and type of JIA are important factors affecting those children’s QoL. The reliability and validity of PedsQL 4.0-GC and PedsQL3.0-RM scale in Chinese JIA children are satisfactory, and can be used in clinical and scientific research. This may help pediatric rheumatologist to pay more attention to the QoL of children when they treating their basic diseases, and provide basis for development of multi-disciplinary individual intervention programs in order to practical improve the QoL of JIA children in China.


Dyspeptic syndrome is particularly common in postmenopausal women in the form of epigastric pain. The aim of the study was to assess the role of melatonin in chronic dyspepsia in this group of women, and examine the role of Helicobacter infection.H.pylori infection (Group II), and 64 women with H. pylori infection with chronic dyspepsia (Group III). Endoscopic examination was performed, as well as histological assessment of gastric end duodenal mucosa, urease breath test (UBT-13C), and immunoenzymatic assessment of serum 17-β-estradiol, follicle stimulating hormone and melatonin, and urinary 6-sulfatoxymelatonin. In Group III, 14-day antibacterial treatment was introduced with pantoprazole, amoxicillin and levofloxacin followed a six-month treatment with placebo in 32 women (Group IIIa), and melatonin 1 mg/morning and 3 mg/at bedtime in the other 32 women (Group IIIb).The study comprised 152 subjects including 30 healthy women (Group I), 60 women with asymptomatic p > 0,05). The level was significantly lower in Group III . Eradication of H.pylori was obtained in 75.0% women in Group IIIa, and in 84.3% in Group IIIb (p > 0.05). After six months, dyspeptic symptoms resolved in 43.7% patients in Group IIIa and 84.3% in Group IIIb (p < 0.001).No significant differences were found between serum level of female hormone. Serum melatonin levels were similar between Group I (12.5 ± 2.72 pg/ml) and Group II (10.5 ± 3.73 pg/ml; H. pylori-associated dyspepsia, particularly in postmenopausal women with lower levels of this hormone.Melatonin supplementation is useful in treating NCT04352062, date of registration: 15.04.2020. Following the menopause, many women can demonstrate range of psychosomatic disorders. The severity of climacteric symptoms, typically hot flushes, excessive sweating, sleep disorders, irritability, depressive mood, fatigue, headache, vertigo, myalgia, palpitation and formication, can be assessed using the Menopause Rating Scale . HoweverHowever, dyspeptic problems are not relieved by hormone replacement therapy . This maHelicobacter pylori infection.The aim of the present study was to assess the role of melatonin in the pathogenesis of chronic dyspepsia in postmenopausal women, and to examine the effect of The study comprised 152 women aged 49–64 years (mean age 56.3 ± 8.3 years). The group included 62 women who developed dyspeptic problems for the first time after menopause. The study took place in the years 2011–2018.Helicobacter pylori infection; Group II—60 women with asymptomatic Helicobacter pylori infection; Group III—62 women with chronic dyspepsia and Helicobacter pylori infection.Three groups were distinguished: Group I—30 women without dyspeptic complaints and without H. pylori—associated dyspepsia was based on the Kyoto Global Consensus [Diagnosis of onsensus .Helicobacter pylori infection, the UBT-13C urea breath test was performed using FANci-2 System .The main symptoms reported in Group III were epigastric pain of a hunger nature and pain at night, as well as increased appetite. The severity of dyspeptic symptoms was evaluated using the Visual Analogue Scale. All subjects underwent endoscopic examination of the upper gastrointestinal tract and histological assessment was performed using hematoxylin–eosin and Giemsa staining. To confirm The following women were excluded from the study: those with other functional or inflammatory diseases of the gastrointestinal tract, liver and pancreas; those with metabolic, allergic or mental illness; those who were receiving hormone replacement therapy.The following routine laboratory examinations were performed: blood cell count, C-reactive protein, glycosylated hemoglobin, bilirubin, alanine and aspartate aminotransferase, amylase, lipase, urea, creatinine, cholesterol HDL and LDL, triglyceride assay.Immunoenzymatic assay of 17-β-estradiol  and follicle-stimulating hormone  was also performed. Serum melatonin level and urinary 6-sulfatoxymelatonin level were measured by ELISA with IBL antibodies  and Expert 99 MicroWin 2000 Reader .Blood samples were drawn from the antecubital vein at 9:00 a.m. and were frozen at minus 70°C. On the same day, samples of urine were taken over 24 h. Next morning 20 ml volume of urine samples were frozen at minus 70°C.The subjects followed the same diet for seven days prior to the evaluations. On the day of the study, all patients consumed the same liquid diet (Nutridrink—Nutricia), containing 18.9 g carbohydrate, 6.0 g protein and 5.8 g lipid per ml. Three 400 ml meals were taken, with a total caloric value of 1800 kcal, together with 1500 ml of isotonic water.In group III, the following 14-day antibacterial treatment was introduced: pantoprazole (2 × 40 mg), amoxicillin (2 × 1000 mg) and levofloxacin (2 × 500 mg).Afterward, the patients were randomly divided into two equally-sized groups. Group IIIa (n = 32) was administered placebo  as two tablets a day, and Group IIIb (n = 32) received melatonin at a dose of 1 mg/morning and 3 mg/at bedtime, for six months. In this period, the patients applied the same balanced diet with a total caloric value of 1600 kcal.Follow-up clinical examinations were performed after one, three and six months, and the UBT-13C test was performed after three and six months.p value of < 0.05 was considered statistically significant. Statistica 13.3  and MS Excel  were used for statistical calculations.Normally distributed data was compared using Student’s t-test, and non-normal data by the Kruskal–Wallis and post hoc tests. Data were expressed as mean and standard deviation. Therapeutic effects after melatonin supplementation were evaluated using the chi-square test. A H. pylori infection in the control group.The general characteristics of the investigated women are shown in Table The serum levels of 17-β-estradiol were 15.1 ± 4.64 pg/ml in Group I, 14.4 ± 5.27 pg/ml in Group II and 11.9 ± 3.72 pg/ml Group III. These differences were not statistically significant . However lower levels were found in women with symptomatic H. pylori infection: 5.27 ± 1.42 pg/ml  and Group II , and 7.93 ± 2.27 µg/ml in Group III  in Group IIIa, and in 27 women (84.3%) in Group IIIb (p > 0.05).Eradication of p < 0.001, Table After six months, dyspeptic symptoms resolved in 14 women (43.7%) in Group IIIa, and in 27 (84.3%) in Group IIIb (Melatonin was well tolerated: only four women (12.5%) reported increased fatigue in the morning, and two patients (6.2%) headache in the first week of the treatment. No cases required discontinuation of therapy or dose reduction.H. pylori infection, those with both H. pylori infection and with low melatonin levels were more likely to suffer from dyspepsia. In the latter cases, there were indications for antibiotic therapy; however, eradication of H. pylori only eliminated complaints in some patients. Hence, it appears that dyspeptic symptoms may be associated with low secretion of melatonin in the gastric mucosa, and melatonin may have a protective effect in asymptomatic infections. Many studies have shown that even asymptomatic infection induces destructive changes in the gastric mucosa [In humans, melatonin secretion decreases with age . These cc mucosa , 27 and c mucosa , 29.Melatonin demonstrates many beneficial effects in the combined therapy of many gastrointestinal diseases, such as esophageal reflux disease , functio1/2 53.7 min [Melatonin demonstrates good tolerability and safety, due to its pharmacokinetic properties. Oral administration of 10 mg melatonin resulted in a maximum serum concentration of 3550 pg/ml at T53.7 min . Similar53.7 min  and 80 m53.7 min . Thus, tOur study has same limitations. The most significant one was the small size of the study group; even so, the population was relatively homogeneous and well characterized.H. pylori-associated dyspepsia, particularly in patients with reduced melatonin secretion, such as postmenopausal women.Melatonin supplementation can play a significant role in complex therapy of


Non-alcoholic fatty liver is one of the most common comorbidities of diabetes. It can cause disturbance of glucose and lipid metabolism in the body, gradually develop into liver fibrosis, and even cause liver cirrhosis. Mangiferin has a variety of pharmacological activities, especially for the improvement of glycolipid metabolism and liver injury. However, its poor oral absorption and low bioavailability limit its further clinical development and application. The modification of mangiferin derivatives is the current research hotspot to solve this problem.The plasma pharmacokinetic of mangiferin calcium salt (MCS) and mangiferin were monitored by HPLC. The urine metabolomics of MCS were conducted by UPLC-Q-TOF-MS.The pharmacokinetic parameters of MCS have been varied, and the oral absorption effect of MCS was better than mangiferin. Also MCS had a good therapeutic effect on type 2 diabetes and NAFLD rats by regulating glucose and lipid metabolism. Sixteen potential biomarkers had been identified based on metabolomics which were related to the corresponding pathways including Pantothenate and CoA biosynthesis, fatty acid biosynthesis, citric acid cycle, arginine biosynthesis, tryptophan metabolism, etc.The present study validated the favorable pharmacokinetic profiles of MCS and the biochemical mechanisms of MCS in treating type 2 diabetes and NAFLD. Diabetes is one of the most common chronic metabolic diseases, and its incidence is gradually increasing. According to the International Diabetes Federation, the 463 million people with diabetes worldwide account for about 9.3% of the global population in 2019, of which 80% come from low- and middle-income countries. It is estimated that 700 million people will account for 10.9% of the world population by 2045 . IR can Mangifera indica L. and some other medical plants including Anemarrhena asphodeloides Bge., Belamcanda chinensis (L.) DC etc. , 2423, 2Mangiferin calcium salt , Mangiferin  was provided by Changzhou Deze Pharmaceutical Research Co. Ltd. . Mangiferin (purity: 98.1%), rutin (purity: 91.9%) as reference substance were obtained from the National Institute for the Control of Pharmaceutical and Biological Products . Heparin sodium was obtained from Shanghai Huishi biochemical reagent Co., Ltd. . Acetonitrile Methanol and formic acid (HPLC grade) were obtained from Tedia Company, Inc. . Ultrapure water was produced using a Milli-Q plus  water purification system. Leucine enkephalin was obtained from Waters . Xanthurenic acid, 5-L-Glutamyl-taurine, Citric acid, Pantothenic acid, Uric acid, Riboflavin and 3-Hydroxyanthranilic acid were obtained from Sigma-Aldrich .2 inhalation. The study complied with the guidelines of the research commitment institution and its administrative region, as the Jilin Province Experimental Animal Management Ordinance and Changchun University of Chinese Medicine Laboratory Animal Management Measures. All experiments were approved by the Laboratory Animals Ethics Committee, Changchun University of Chinese Medicine.Sprague-Dawley rats  were obtained from Changchun Yisi Laboratory Animal Technology Co., Ltd. . Rats were housed with free access to food and water under standard conditions . All experimental animals were finally euthanized by COMCS and mangiferin were given by gavages according to 60 mg/kg, 240 mg/kg, 960 mg/kg doses as single administration. Rats were fasted 12 h before the experiment and water was taken freely. In the experiment day the administration was according to the predetermined dose. Serial blood samples were collected from the orbital venous plexus (0.3–0.5 mL) at 0 h, 0.5 h, 1.0 h, 1.5 h, 2.0 h, 3.0 h, 4.0 h, 6.0 h, 10.0 h, 12.0 h, 24.0 h after administration.MCS and mangiferin were given by gavages according to 240 mg/kg dose, once a day for 7 days as multiple administrations. Blood samples were collected from the orbital venous plexus (0.3–0.5 mL) on 1, 2, 3, 4, 5, 6 days before dosing. For the last administration, Serial blood samples were collected at 0 h, 0.5 h, 1.0 h, 1.5 h, 2.0 h, 3.0 h, 4.0 h, 6.0 h, 10.0 h, 12.0 h, 24.0 h.The blood samples placed in a centrifuge tube with heparin, 10,000 rpm centrifuge 10 min. After the centrifugation, reserve the plasma in − 20 °C refrigerator.The 200 μL of plasma sample was placed in a 1.5 mL centrifuge tube, added internal standard solution (10 μg/mL rutin standard solution) 25 μL, (0 h plasma used methanol 25 μL to instead), methanol 25 μL (added mangiferin standard solution 25 μL), added 0.9 mL Acetonitrile-acetic acid (9: 1), swirl mixed 3 min, 6000 rpm centrifuged 10 min, supernatant was dried in vacuum at 50 °C, added mobile phase 100 μL to the residue, swirl mixed 2 min, 6000 rpm centrifuged 10 min, the supernatant was injected into High performance liquid chromatography (HPLC). Chromatographic separations were achieved using a Discovery C18 column . The mobile phase used for the separation consisted of Acetonitrile and 0.10% phosphoric acid  delivered at 1 ml/min flow rate. The detection wavelength was set at 318 nm and all measurements were performed at 30 °C.The pharmacokinetic parameters were calculated using DAS software, and select the weighting factors to fit the atrioventricular model.n = 7), model control group  were administered with distilled water intragastrically. MCS High-dose group , Medium dose group , Low-dose group  were administered intragastrically with MCS at doses of 480 mg/kg, 240 mg/kg, 120 mg/kg.The SD rats were fed high-fat feed . At the end of the 12th week, streptozotocin (STZ) (30 mg/kg) was intraperitoneally injected into rats to induce type 2 diabetes complicated with NAFLD model. The rats were randomly divided into the following four groups: Blank control group , fasting insulin (FINS), triglyceride (TG), total cholesterol (TC), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and gamma-glutamyl transpeptadase (GGT) content in rat serum. The rat liver was taken stained with hematoxylin and eosin (H&E).Urine samples were collected and centrifuged at 10,000 rpm for 10 min, filtered through a 0.22 μm filter membrane. Supernatant was transferred to fresh vials for ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) analysis. For metabolomics analysis, the samples (each 5 μL) were injected onto a Waters ACQUITY UPLC BEH C18 Column  kept at 30 °C and at a flow rate of 0.4 mL/min using a Waters ACQUITY UPLC system coupled with a Q-TOF SYNAPT G2 High Definition Mass Spectrometer . Acetonitrile (A) and 0.1% aqueous formic acid (v/v) (B) were used as gradient mobile phase. The gradient elution of A was performed as follows: 5–30% A at 0–6 min, 30–60% A at 6–10 min, 60–100% A at 10–12 min, 100–5% A at 12–12.1 min and then kept at 5% A for 3 min. The positive and negative ion (ESI) modes were used in MS analysis. The source temperature was set to 120 °C. The desolvation gas temperature was set to 400 °C and the flow was set to 800 L/h. The capillary, cone and extraction cone voltages were 3.0 kV, 35 V, 5.0 V in positive ion mode and 2.0 kV, 35 V, 5.0 V in negative ion mode. The full-scan mode was from 100 to 1000 Da. Accurate mass was maintained by Leucine enkephalin. MSE was applied for the MS/MS analysis with the high collision energy on 25-35 eV and the low collision energy on 4 eV.The quality control (QC) samples were used for method validation, which were obtained by mixing 100 μL of each sample. In order to avoid errors during the entire analysis process, the QC samples were run once every 5 samples to measure the stability of the system.p < 0.05)using SPSS statistics 13.0 software.The sample was detected by UPLC-Q-TOF-MS to obtain the total ion current chromatogram of the sample. The raw data files were processed with MassLynx V4.1 and MarkerLynx Application Manager  for peak detection, alignment and normalization. Multivariate analysis was performed by principal component analysis (PCA) and orthogonal projection to latent structures squares-discriminant analysis (OPLS-DA) with the EZinfo 2.0 software. All values are expressed as the mean ± SD. An independent sample t-test between groups was used to evaluate the significant difference (P < 0.05), Tmax (2.99 h) is significantly decreased (P < 0.05). MCS has better oral absorption than mangiferin.The mean plasma concentration-time curves of MCS and mangiferin in different dosage are showed in Fig. P < 0.05), Tmax (3.02 h) is significantly decreased (P < 0.05). In addition, the main pharmacokinetic parameters of multiple and single administration of MCS have no significant difference, indicating that the absorption of MCS in rats is constant basically, and don’t change with continuous administration. MCS almost has no accumulation in the body after multiple doses of administration.The comparison of mean plasma concentration-time curves of MCS and mangiferin after multiple oral administration in dosage of 240 mg/kg are showed in Fig. P < 0.01). Compared with MG, the level of serum FBG, FINS in MHG and MMG decreased significantly after treated with MCS (P < 0.05). It revealed that MCS could better improve insulin resistance. Dyslipidemia is also one of the important clinical manifestations of type 2 diabetes patients with NAFLD. Compared with BG, significant increase could be observed in serum TG, TC level in MG (p < 0.01). After the treatment with MCS, the concretion of serum TG, TC in MHG and MMG decreased significantly (p < 0.05). It revealed that MCS could reduce the blood lipid in model rats. ALT, AST, GGT are the most significant diagnostic indicator for patients with NAFLD. Compared with BG, serum ALT and GGT activities of MG increased significantly (p < 0.01). After the treatment with MCS, the activities of serum ALT and GGT in MHG and MMG decreased significantly . It revealed that MCS could improve abnormal liver function in model rats.Type 2 diabetes patients with NAFLD often suffered from glucose and lipid metabolism disorder, and present with abnormally high fasting blood glucose, fasting insulin and HOMA-IR . As our Histological analysis showed that livers of the MG rats had lobular structures with blurred boundaries, Irregular cell cords, and hepatic sinusoidal compression became smaller or disappears, liver cells showed diffuse fat-like changes, a large number of inflammatory cells infiltration could be seen in the liver lobule, even several inflammatory necrosis merged with each other Fig. .Fig. 5TThe system of UPLC-Q-TOF-MS is used for urinary sample separation and data collection. Metabolic profiling was acquired in the ESI+ and ESI- modes. The representative based peak intensity (BPI) chromatograms in positive and negative ion modes are showed in Fig. p-value< 0.05 between DG, MG and MHG are used as the criterion for another biomarker. Finally, 16 endogenous metabolites were identified as potential biomarkers (Table http://metpa.metabolomics.ca/) analysis, including Taurine and hypotaurine metabolism, Pantothenate and CoA biosynthesis, Alanine, aspartate and glutamate metabolism, Riboflavin metabolism, Arginine biosynthesis, Citrate cycle (TCA cycle), Glyoxylate and dicarboxylate metabolism, Tryptophan metabolism, Primary bile acid biosynthesis, Fatty acid biosynthesis and Purine metabolism (Fig. OPLS-DA analysis was performed on the MG and MHG to find biomarkers for MCS treatment of type 2 diabetes with NAFLD. The OPLS-DA model is of good quality, and the model evaluation indexes in positive ion mode are R2Y = 0.95, Q2 = 0.83, and the model evaluation indexes in negative ion mode are R2Y = 0.91, Q2 = 0.85. In the OPLS-DA score chart Fig. a, b, theMangiferin is widely found in many edible and medicinal plants and has many pharmacological activities, such as antitussive, expectorant, antiasthmatic, central depression, anti-diabetic, antioxidant, anti-inflammatory, bacteriostatic, anti-viral, anti-tumor, choleretic and immunomodulatory, so it has attracted the attention of researchers , 19. EspTherefore, we detected the blood drug concentration of MCS and mangiferin in single and multiple doses, and calculated their pharmacokinetic parameters in different time. Compared with mangiferin, the Tmax of MCS was advanced, and the AUC, Cmax of MCS increased significantly indicating that the degree of oral absorption of MCS was improved.Shorter peak time showed that the rate of absorption of MCS was faster than the monomer of mangiferin. Moreover MCS has higher bioavailability than mangiferin. Compared the pharmacokinetic parameters between single and multiple dose oral administration of MCS, MRT and T1/2 had no significant change, which indicated that the absorption of MCS in rats is basically constant, and it will not change with continuous administration. These results showed that compared to mangiferin, MCS had a faster absorption rate, better absorption degree and its absorption was more constant.IR plays a very key role in the pathogenesis of type 2 diabetes and NAFLD . IR causAmong these metabolites, D-Pantothenoyl-L-cysteine is involved in the biosynthetic pathway of pantothenic acid and CoA, and is a synthetic precursor of Pantothenic acid that is a water-soluble vitamin required for life support. It is involved in the synthesis of acetyl-CoA and plays an important role in the metabolism of protein, fat, and sugar in the body . RiboflaXanthurenic acid, 3-Methyldioxyindole, 3-Hydroxyanthranilic acid are metabolite of tryptophan metabolism. Tryptophan and its metabolites play an important role in various physiological processes in the body, which mainly affect the immune system and nervous system. It is closely related to various diseases such as autoimmune diseases, abnormal liver function, CNS diseases and cancer . 5-L-GluThese metabolites are closely related to the occurrence and development of type 2 diabetes and NAFLD. In this study, MCS can exert its therapeutic effect by regulating the above metabolites.In summary, our results showed that the pharmacokinetic profiles of MCS were better than mangiferin. Also MCS had a good therapeutic effect on type 2 diabetes with NAFLD rats by regulating glycolipid metabolism. The metabolomics could provide effective information for metabolic changes in model rats after administration of MCS in urine. However the animal models do not fully reflect human NAFLD, and there are still some debates about the occurrence of NAFLD in T2DM. Our results might help to provide useful evidence for mechanism and clinical applications of MCS acting on type 2 diabetes and NAFLD.


Ziziphus mucronata and its interactions with antibiotics against bacteria of clinical importance were investigated. While its phytochemicals and antioxidant activities were determined by free radical scavenging assays, the antibacterial activities of the extract and its interactions with the antibiotics were determined by macrobroth dilution and the checkerboard methods. From the results, total phenolic content was 29.67 ± 1.90 mg GAE/100 g, total flavonoid content was 8.72 ± 0.08 mg QE/100 g, and total proanthocyanidin content was 1.94 ± 0.00 mg CE/100 g of dry plant material. The inhibition concentration 50% (IC50) of DPPH, BHT, and ascorbic acid was equal to 0.04 ± 0.02 mg/ml, respectively. Those of the ABTS, BHT, and ascorbic acid were equal to 0.02 ± 0.02, 0.04 ± 0.03, and 0.04 ± 0.02 mg/ml, respectively. The checkerboard assay showed that combining the extract with different antibiotics resulted in synergistic (38.75%), indifferent (30%), additive (28.75%), and antagonistic (2.5%) interactions. The interactions between the extract and antibiotics resulting in enhanced antibacterial activities could have resulted from the antioxidant activities of the extract mopping up the ROS generated by the antibiotics or the ability of both extract and antibiotics simultaneously producing reactive oxygen species with deleterious effects resulting in synergistic antibacterial effects.With the increased incidence of antibacterial resistance in microorganisms, combining natural products from plants with antibiotics may be considered interesting alternatives for synergy to attain multitarget effects. In this study, the antioxidant activity of the methanol extract of  Many plZiziphus mucronata subsp. mucronata, commonly known as buffalo thorn, is a small to medium-sized tree. The plant can be identified during the winter months by the presence of its berry-like fruit ripening from March to September. During the flowering months, nectar-loving creatures visit these flowers in search of nectar. While its stem bark and roots are used for the treatment of rheumatism, syphilis, gonorrhea, gastrointestinal disorders such as dysentery and diarrhoea, and snake bites [Ziziphus mucronata subsp. mucronata on the antibacterial activities of some antibiotics against different bacterial species in vitro to indicate the possible effects of ROS produced as a result of combining the extract with the antibiotics.ke bites , 17, varke bites . To furtZ. mucronata subsp. mucronata were collected from the University of Fort Hare campus in Alice, air-dried at room temperature, authenticated by Prof. D.S. Grierson, and pulverized with a milling machine. One hundred grams of the pulverized sample was extracted with 500 ml of methanol for 72 h with shaking. The extract was filtered with Whatman No. 1 filter paper and concentrated under reduced pressure at 40°C using a rotary evaporator. After the extraction, the crude extract was redissolved in the extracting solvent to the required concentration for bioassay analysis. A voucher specimen (OLAJ/2010/ZM/01) was prepared and deposited in the Griffin's Herbarium of the University.The stem bark of 3, vanillin, Folin–Ciocalteu phenol reagent, and sodium carbonate—and the solvents were of analytical grade. Antibiotic powders of amoxicillin (AMX), chloramphenicol (CHL), ciprofloxacin (CIP), erythromycin (ERY), tetracycline hydrochloride (TET), metronidazole (MET), kanamycin (KAN), and nalidixic acid (NAL) were prepared and used according to the manufacturers' instructions.All chemicals used—2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) diammonium salt, 1,1-diphenyl-2-picrylhydrazyl (DPPH), butylated hydroxytoluene (BHT), gallic acid, rutin, ascorbic acid (VC), quercetin and FeClStaphylococcus aureus ATCC 6538, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Enterobacter cloacae ATCC 13047, Klebsiella pneumoniae ATCC 10031, Proteus vulgaris ATCC 6830, Shigella sonnei ATCC 29930, Bacillus subtilis KZN, Proteus vulgaris KZN, and Enterococcus faecalis KZN were used in this study. They were obtained from the Department of Biochemistry and Microbiology, University of Fort Hare, Alice, South Africa. The antibacterial assays were carried out using Mueller-Hinton II Agar (MHA) (Biolab) and broth. The inocula of the test bacteria were prepared using the colony suspension method [n method . Colonie3 ethanol solution was added to 0.5 mL of extract and allowed to stand for 60 min at room temperature before the absorbance was measured at 420 nm using an AJI-C03 UV-VIS spectrophotometer.Total flavonoids were estimated using the method of Marinova et al. . Here, 0oC for colour to develop. The absorbance was measured in triplicate at 765 nm using an AJI-C03 UV-VIS spectrophotometer.The total phenolic content of ZMM was determined by the modified Folin–Ciocalteu method . Here, tThe proanthocyanidin content of ZMM was determined by the modified method of Sun et al. . A volum3Fe(CN)6) (1% w/v). The mixture was incubated at 50oC for 20 min after which 2.5 mL of trichloroacetic acid (TCA) (10% w/v) was added to the mixture before being centrifuged at 1000 rpm for 10 min. The supernatant of the mixture (2.5 mL) was then mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% w/v FeCl3. The absorbance was measured at 700 nm in a AJI-C03 UV-VIS spectrophotometer.A modified spectrophotometric method of Ferreira et al.  was usedTo determine the free radical scavenging activity of ZMM against DPPH, the method of Liyana-Pathirana and Shahidi  was adop+ solution with 60 ml of methanol to obtain an absorbance of 0.708 ± 0.001 units at 734 nm using AJI-C03 UV-VIS spectrophotometer.For ABTS radical scavenging activity of the methanol extract, the modified method of Johnstone et al.  was adopμg/mL. While CIP concentration ranged between 0.005 and 5 μg/mL, the concentration of ERY was between 0.049 and 50 μg/mL and that of ZMM was between 4.88 and 5000 μg/mL. The antibiotic concentrations were prepared by serial dilution in double-strength MHB. To determine their combinatorial effects, combinations of different concentrations ranging from 1/2 X MIC to 8 X MIC of each antibiotic and those of the extract were prepared in double-strength MHB. Each tube was inoculated with 100 µL of each of the bacterial strains. Blank Mueller-Hinton broth was used as a negative control. The bacterial containing tubes were incubated at 37°C for 24 h. The MIC was defined as the lowest concentration that showed no growth in the Mueller-Hinton broth. Each combination assay was performed in duplicate.The minimum inhibitory concentrations (MICs) for ZMM and the antibiotics were determined in duplicate by the macrobroth dilution method in Mueller-Hinton broth (MHB) according to CLSI  . To deteThe interactions between the extract and the antibiotics were determined using the checkerboard assay as previously described . The frap values < 0.05 were regarded to be significant. The Pearson correlation analysis was performed between antioxidant activity and total phenolic content.Data were expressed as means ± standard deviations (SDs) of three replicate determinations and then analyzed by SPSS V.16 . One-way analysis of variance (ANOVA) and Duncan's new multiple-range test were used to determine the differences among the means. Z. mucronata (ZMM) is presented in In this study, the quantity of total phenolic content, flavonoids, and proanthocyanidins in the methanol extract of Z. mucronata.The absorbance of the reducing ability of ZMM determined by FRAP method was measured with spectrophotometer at 700 nm as shown in 50 of the extract [50 value, the higher the free radical scavenging ability/antioxidant power of the medicinal plant. In this study, the concentration-dependent percentage inhibition of DPPH free radicals by the extract and BHT and ascorbic acid are as shown in in vitro 50% inhibition concentration (IC50) values obtained for the DPPH inhibition of ZMM, BHT, and ascorbic acid were found to be 0.043 ± 0.02, 0.042 ± 0.03, and 0.040 ± 0.02 mg/mL, respectively.The free radical scavenging or hydrogen donor potentials and evaluation of the antioxidative activity of medicinal plant extracts have been widely tested with DPPH radical  and are  extract . The hig50 of ZMM was 0.023 ± 0.02 mg/ml and was significantly different from those of BHT (0.041 ± 0.03 mg/mL) and ascorbic acid (0.042 ± 0.02 mg/mL), those of BHT and ascorbic acid were not significantly different from each other. The lower IC50 of the extract showed that it possesses stronger radical scavenging activity than BHT and ascorbic acid used as controls.The effect of ZMM, BHT, and ascorbic acid on ABTS radical cation scavenging activity is presented in µg/mL, those of CIP (0.018 and 0.284 µg/mL), ERY (0.089 and 22.73 µg/mL), TET (0.44 and 14.20 µg/mL), CHL (0.89 and 28.41 µg/mL), AMX (0.89 and 454.55 µg/mL), NAL (1.78 and 56.82 µg/mL), KAN (1.78 and 454.55 µg/mL), and MET (14.20 and 113.64 µg/mL) varied. The MICs of ZMM are higher than those of the different antibiotics. According to the MIC breakpoints recommended by EUCAST [Enterococcus species, Enterobacter species, Staphylococcus species, and Gram-positive aerobes having MIC values of ≤0.25 µg/mL for ERY, ≤0.5 µg/mL for CIP, ≤1 µg/mL for TET, ≤4 µg/mL for MET, ≤4 µg/mL for AMX, ≤8 µg/mL for KAN, ≤ 8 µg/mL for CHL, and ≤16 µg/mL for NAL are classified as being susceptible. Based on MIC breakpoints for the antibiotics as indicated by EUCAST [E. faecalis KZN, S. sonnei ATCC 29930, S. aureus OK2a, and K. pneumoniae ATCC 10031 which were susceptible to ERY, K. pneumoniae ATCC 10031, B. subtilis KZN, and S. aureus OK2a which were susceptible to TET, K. pneumoniae ATCC 10031, P. vulgaris KZN, and E. faecalis KZN which were susceptible to AMX, S. aureus ATCC 6538, B. subtilis KZN, and P. vulgaris KZN which were susceptible to KAN while K. pneumoniae ATCC 10031, P. vulgaris ATCC 6830, B. subtilis KZN, and S. sonnei ATCC 29930 were susceptible to NAL. Combining TET with ZMM resulted in the reduction of MICs of the ZMM to concentrations ranging between 0.0.0005 and 284.10 µg/mL while that of the TET ranged between 0.028 and 14.20 µg/mL. AMX combined with ZMM resulted in the MICs of the ZMM being reduced to concentrations ranging between 0.0005 and 568.18 µg/mL while that of the AMX was reduced to concentrations ranging between 0.03 and 14.20 µg/mL. Bacillus subtilis KZN was not affected by the herbal-drug combination. ERY combined with ZMM resulted in the reduction of the concentrations of the ZMM to concentrations between 0.028 and 568.18 µg/mL and those of ERY to concentrations ranging between 0.003 and 568.18 µg/mL, with the exception of P. vulgaris ATCC 6830 and S. aureus ATCC 6538 not affected by ERY combined with the ZMM. CIP combined with ZMM resulted in the reduction of the MICs of the ZMM to concentrations ranging between 8.88 and 284.1 µg/mL while those of CIP were reduced to concentrations ranging between 0.018 and 0.0.284 µg/mL. While the combination of NAL with ZMM did not result in a significant reduction in the MICs of the ZMM against most of the isolates, CHL reduced the MICs of ZMM significantly. Combining CHL with ZMM resulted in MICs of the ZMM ranging between 7.10 and 284.10 µg/mL and those of CHL ranging 0.44 and 14.20 µg/mL. With the exception of P. vulgaris KZN and E. faecalis KZN to which the combination of KAN and ZMM had no effect, KAN reduced the MICs of the ZMM to concentrations ranging between 7.10 and 568.18 µg/mL and that of this antibiotic was reduced to 0.444 and 28.41 µg/mL. Generally, the combination of the antibiotics with the extract resulted in reduction in the MICs of antibiotics and the extract significantly as shown in From the macrobroth dilution, ZMM and the antibiotics exerted a varied degree of inhibitory and interactions against the test organisms. While the extract had minimum inhibitory concentrations (MICs) ranged between 141.82 and 568.18 y EUCAST  and BSACy EUCAST , strainsy EUCAST  and BSACy EUCAST , the MICThe fractional inhibitory concentration (FIC) index of the ZMM combined with each of the antibiotics resulted in synergistic (38.75%), indifferent (30%), additive (28.75%), and antagonistic interactions (2.5%). While the fractional inhibitory concentration index (FICI) for the synergistic interaction was between 0.0004 and 0.50, the FICI for the additive interaction was between 0.531 and 1.0, that of indifference was between 1.063 and 2.5, and that of antagonistic interaction was between 3.0 and 18 .β-hydroxyacyl-acyl carrier protein dehydratase activities [In medicinal plants, pharmacological activities of extracts are due to polyphenolic compounds such as alkaloids, flavonoids, and phenolic compounds. The biological and pharmacological importance of these substances has been reported. Tannins possess antimicrobial activities able to damage bacterial membranes or delay bacterial growth for sufficient time for bacterial elimination and for the host to develop its immune system . Flavonotivities  to inhibtivities . The phativities . In thistivities  and the tivities .In treating microbial infections, while oxidative stress in bacteria caused by xenobiotics –39 produEscherichia coli MG1655, Enterococcus faecalis, and Staphylococcus aureus [β-lactams against E. coli [On the other hand, since antioxidants defend physiologically active system against ROS and polyphenols could take part in the generation of ROS and act as prooxidants , the exts aureus . They cos aureus  but augm E. coli . Althoug E. coli  and poly E. coli , the ROSThere were varied degrees of interactions between ZMM and different antibiotics commonly used in the treatment of microbial infections. The antibacterial effects resulting from different synergistic interaction between ZMM and the antibiotics could be attributed to their ability to produce ROS simultaneously to effect bactericidal action while antibacterial effects resulting from other interactions between ZMM and the antibiotics could be attributed to the ability of the extract to act as antioxidant mopping up the ROS generated by the antibiotics and vice versa against the bacterial isolates. The modulating effect of each of the phytochemicals on the susceptibility of each bacterial species to each antibiotic requires further investigations that may be of significant application in the treatment of bacterial infections in disease situations.