Context: Chlorogenic acid (ChA) is the major compound in Shuang-Huang-Lian (SHL), which is mainly composed of ChA, baicalin, and Forsythia suspense Thunb Vahl.Objective: The effects of co-existing compounds in SHL and Lonicera japanica Thunb on the absorption of ChA was investigated.Materials and methods: According to 3 × 3 Latin-square test, ChA alone, the extracts of Lonicera japanica, or the mixture of ChA, baicalin and Forsythia suspense (ChA effective doses is 60 mg/kg) was separately given to six beagles for seven days. The oral pharmacokinetic parameters of ChA in plasma, urine and faeces were quantified by HPLC/UV and analyzed.Results: The pharmacokinetic parameters of ChA alone, the extracts of Lonicera japanica, and the mixture of ChA, baicalin, and Forsythia suspense were as followed: Cmax , AUC0-∞ , t1/2 , and Tmax . Accumulative fraction excretion of ChA in urine were 0.73 ± 0.55, 1.25 ± 1.23, 1.05 ± 0.96%, while that in faeces were 0.68 ± 0.94, 0.19 ± 0.40, and 1.76 ± 3.57%.Discussion and conclusion: Co-existing compounds in SHL have no effect on the absorption of ChA, while the concomitant compounds in Lonicera japanica could decrease that of ChA. ChA in Beagles might have high biological transformation. Lonicera japanica Thunb, Scutellaria baicalensis Georgi and Forsythia suspense Thunb Vahl. ChA is a major phenolic compound (no less than 1.5%) and therapeutic ingredient existed in Lonicera japanica of SHL. Furthermore, ChA shows a wide range of biological activities, such as killing Escherichia coli by hypochlorous acid  have gained worldwide popularity for their complementary therapeutic effects to the western drugs, their various pharmacological effects and low side effects Normile . As a coLonicera japanica and the mixture of ChA, baicalin and Forsythia suspense were compared via oral in beagles in order to reveal the effects of co-existing compounds in SHL and Lonicera japanica on the absorption of ChA. So, the research results have important significance to reveal the mechanism of the prescription of ChA.In this work, the pharmacokinetic properties in plasma, urine and feces of ChA were quantified by the HPLC/UV method  were gift samples from Chenguang Research Institute of Chemical Engineering . Extracts of Lonicera japanica (containing 37.2% ChA) and extracts of Forsythia suspense Vahl (containing about 1.4% forsythin and 9.0% forsythoside) were bought from Sichuan Mingbo Pharmaceutical Co., LTD . Extracts of Scutellaria baicalensis  were bought from Sichuan Sunnyhope Pharmaceutical Co., LTD . Methanol and acetonitrile (HPLC grade) were purchased from Dikma Technologies . Phosphoric acid, acetic acid, trichloroacetic acid, disodium hydrogen phosphate were of analytical grade and obtained from the Chengdu Reagent Company . Water was prepared by an ultra-pure water system . All other chemicals used in the study were of analytical grade at least.ChA and internal standard puerarin were supplied by National Institute for the Control of Pharmaceutical and Biological Products . Extracts of The Sichuan University animal ethical experimentation committee, according to the requirement of the National Act on the Use of Experimental Animal (People’s Republic of China), approved all procedures of the animal study (number: GPT 5-1). Beagles weighting 9.0–11.2 kg   were separately added into 0.5 mL of blank plasma. And then the mixture was vortex-mixed thoroughly to get the final standard solution with the concentrations of 0.404, 0.808, 1.615, 3.230, 6.460, 12.920, and 25.840 μg/mL. In the same manner, the quality control (QC) samples were also prepared at the concentrations of 0.404, 0.808, 6.460 and 25.840 μg/mL.To prepare a series of standard urine samples, 50 μL of ChA standard solution with different concentrations was respectively added into 0.5 mL of blank urine, followed by vortex mixture to get final concentrations of 1.292, 6.460, 12.92, 64.60, and 129.2 μg/mL. QC samples were also prepared at concentrations of 1.292, 6.460, 12.92, and 129.2 μg/mL in the same manner.A series of standard faeces samples and QC samples were prepared by the same method as that of urine samples .All the above solutions were stored at 4 °C before use for no longer than one month.To 0.5 mL of plasma sample, 100 μL of internal standard solution, 100 μL of methanol:0.2% phosphoric acid  and 0.5 mL of 10% solution of trichloroacetic were successively added into a centrifuge tube. Subsequently, the mixture was vortex-mixed for 2 min , and then centrifuged at 12,000 rpm for 10 min . Twenty microliters of supernatant was manually injected into the HPLC for analysis.To 0.5 mL of urine samples or faeces homogenates, 50 μL of methanol:0.2% phosphoric acid  and 0.5 mL of 10% solution of trichloroacetic were added into a centrifuge tube, and other procedures were the same as that of plasma sample.The HPLC apparatus included a Series III Pump, a Model 500 UV/VIS detector and a 7725i injection valve. Data collection and integration were accomplished by Shimadzu EZ Start 7.1.1 program software.18 column  . ChA in plasma was separated by mobile phase consisted of methanol, acetonitrile, and disodium hydrogen phosphate  ; ChA in urine or faeces was separated by mobile phase consisted of methanol, acetonitrile, and disodium hydrogen phosphate with a different volume ratio of 6:8:88. Separation was carried out isocratically at 35 °C with a flow rate of 1.0 mL/min, and UV detection was performed at 322 nm  within 15%.1) or puerarin (A2) in plasma samples was separately obtained. The control group, water, was also post-processed and detected by the same method as above, and then the peak areas of ChA (A1′) and puerarin (A2′) were acquired. The extraction recovery was calculated by the following formula: ChA in plasma samples (%) = A1/A1′; puerarin in plasma samples (%) = A2/A2′.The recovery of ChA was assessed by the six replications of QC samples in plasma. Firstly, plasma samples containing ChA or puerarin were post-processed by the following steps. Concretely, 100 μL of internal standards, 0.5 mL of 10% solution of trichloroacetic, and 100 μL of methanol:0.2% phosphoric acid  were added into 0.5 mL of plasma samples and then vortexed for 2 min. Secondly, solution was centrifuged, and 20 μL of supernatant was injected into HPLC. Finally, the peak area of ChA .t1/2). Cmax and Tmax were obtained from the experimental data. What’s more, accumulative excretion of ChA in urine was accessed by variance analysis, and that in faeces was accessed by rank sum test. And a p-value >0.05 was considered as no significant.Pharmacokinetic data were subsequently processed by the Drug and Statistics ver 1.0 Program , such as area under the plasma concentration–time curve (AUC) and half-life  for this method defined as a signal-to-noise ratio of 3:1 was 0.053 μg/mL. The lower limit of quantitation (LLOQ) defined as the lowest drug concentration, which can be determined with an intra-day relative standard deviation ≤20%, was estimated as 0.105 μg/mL.The result of stability  indicaten = 6) from plasma.The mean extraction efficiency was 88.57% for ChA, and 89.81% for the internal standard (Lonicera japanica (containing 37.23% ChA), that in plasma of beagles dosed with ChA alone showed higher Cmax (2.350 ± 0.483 μg/mL), AUC0-t (5.591 ± 1.406 μg·h/mL), AUC0-∞ (6.324 ± 1.853 μg·h/mL), t1/2 (0.911 ± 0.187 h), and Tmax (1.861 ± 0.499 h). Nevertheless, in beagles co-dosed with ChA, baicalin, and Forsythia suspense, the ChA of plasma showed the similar Cmax (2.332 ± 0.606 versus 2.350 ± 0.483 μg/mL), AUC0-t (5.325 ± 1.341 versus 5.591 ± 1.406 μg·h/mL), AUC0-∞ (6.074 ± 1.473 versus 6.324 ± 1.853 μg·h/mL), t1/2 (1.094 ± 0.193 versus 0.911 ± 0.187 h), and Tmax (1.833 ± 0.279 versus 1.861 ± 0.499) with that of beagles dosed with ChA alone.Mean plasma concentration-time profile and the corresponding pharmacokinetic parameters of ChA after oral administration was separately shown in Accumulative fraction excretion of ChA in urine of R, T1, or T2 groups was, respectively, 0.73%, 1.25%, and 1.05%, as well as that of ChA in faeces was, respectively, 0.68%, 0.19%, and 1.76%, which suggested that ChA without being absorbed was almost metabolized in gastrointestinal tract of beagles.Lonicera japanica (containing 37.23% ChA) by oral in beagles. In the extracts of Lonicera japanica, the other concomitant compounds also existed except for ChA, and they might have the same uptake pathway as ChA. Cmax of ChA decreased in extracts of Lonicera japanica (containing 37.23% ChA) and relative bioavailability of it was only 66.7%, suggesting that concomitant compounds in extracts of Lonicera japanica interfered with the absorption of ChA. Therefore, it’s best to choose the extracts with higher content of ChA for preparation.The pharmacokinetic parameters had remarkable differences between ChA alone and extracts of Scutellaria baicalensis  and Forsythia suspense (containing 1.4% forsythin and 9.0% forsythoside), there was the same absorption and elimination profile of ChA as those of ChA alone. Thereby, co-existing compounds in SHL had no effects on the absorption of ChA. In TCM, Scutellaria baicalensis and Forsythia suspense are usually combined with ChA. Moreover, combination preparation has better clinic efficacy than ChA alone. However, whether ChA effects on the in vivo absorption of Scutellaria baicalensis and Forsythia suspense is not clear and still need further research.To the mixture of ChA, In addition, in R, T1, or T2 groups, accumulative fraction excretions of ChA were very low in urine or faeces, which indicated that ChA could be bio-transformed before exerted from body. ChA is vulnerable to oxidation, because it is a selective hydroxyl free radical scavenger and oxygen free radical scavenger. Therefore, ChA in Beagle dogs might have high biological transformation, which led to very few prototype drugs detected.Lonicera japanica made the absorption ChA less. Therefore, high-purity ChA should be used for preparation. In addition, there was no remarkable difference of accumulative fraction excretions of ChA in urine or faeces of beagles dosed in R, T1, or T2 groups. Furthermore, a trace amount of ChA in the parent form was detected in the urine and faeces. Further studies are ongoing in our laboratory in order to investigate the in vivo process of ChA with co-existing components.In conclusion, other co-existing compounds in SHL had no effects on the absorption of ChA, but the concomitant compounds in extracts of


In order to study the candidate biomarkers in autoimmune cirrhosis (AIC).Isobaric tags are first implemented for relative and absolute quantitation technology on proteins prepared from serum obtained from AIC and normal controls. Proteins found to be differentially expressed are identified with liquid chromatography electrospray ionization tandem mass spectrometry by using a Q Exactive classic ion trap mass spectrometer.108 proteins (32 upregulated and 76 downregulated proteins) are identified from AIC samples, compared with the normal controls. Gene Ontology enrichment analysis, KEGG pathway analysis, and protein–protein interaction map by STRING show that they associate with multiple functional groups, including ion binding activity, peptidase activity, and enzyme regulator activity. Finally, the von Willebrand factor, insulin‐like growth factor‐binding protein complex acid labile subunit, transthyretin, adiponectin proteins are identified with western blot as candidate biomarkers for AIC.These findings offer a comprehensive profile of the AIC proteome about candidate biomarkers and provide a useful basis for further analysis of the pathogenic mechanism of AIC. The peak incidence of disease occurs in the fourth or fifth decade of life.The diagnosis of AIC disease, remains challenging,Currently, the diagnosis of autoimmune cirrhosis (AIC) disease remains challenging, there are no specific diagnostic markers for AIC. In the present study, we first implemented isobaric tags for relative and absolute quantitation technique to identify the differentially expressed proteins in the serum of AIC compared to the normal controls. One hundred and eight differentially expressed proteins were identified and most of them were found to be involved in multiple functional groups, including ion binding activity, peptidase activity, and enzyme regulator activity. Moreover, we identified von Willebrand factor, insulin‐like growth factor‐binding protein complex acid labile subunit, transthyretin, adiponectin proteins as candidate biomarkers for AIC. These results are very helpful to elucidate the pathogenic mechanism of AIC.Liver biopsy is still the most accurate and widely used method, by which cirrhosis can be diagnosed and staged. There are, however, notable disadvantages to this method of examination, including cost, risk of bleeding, and sampling error.Isobaric tags for relative and absolute quantitation (iTRAQ) technology has been currently used as a powerful tool for global evaluation of protein expression about specific diseases or disease stages, which shows significantly improved sensitivity and repeatability when compared with the traditional 2D electrophoresis , and has been widely applied in studying biomarkers for various diseases.2Preparation of Serum Samples: Blood samples were collected from 16 patients (12 women and 4 men) with autoimmune cirrhosis diagnosed according to the following criteriag for 30 min. Aliquots of serum were collected and stored at −80 °C. To reduce the individual differences, aliquots of serum samples from four randomly selected individuals in each group were mixed to create eight pools. Groups n1, n2, n3, n4 , p1, p2, p3, and p4 (p = patient) were formed.m TEAB buffer was added and centrifuged at 12 000 × g for 20 min for three times. Trypsin  was added to the samples for incubating at 37 °C for 16 h.Serum samples were processed using the ProteoPrep Blue Albumin Depletion Kit , which selectively removes albumin and IgG from the serum sample according to the manufacturer's instructions. In order to depurate the protein extraction and determine the final protein concentration, the 2D Clean‐up Kit  and 2D Quant Kit  were used sequentially, following the manufacturer's instructions. Proteins were digested according to the FASP method.Protein Labeling with iTRAQ: iTRAQ labeling was performed according to the manufacturer's instructions . Groups n1, n2, n3, n4, p1, p2, p3, and p4 were individually labeled with iTRAQ reagent .The labeled samples were allowed to incubate at room temperature for 2 h, then all the serum samples were mixed together and vacuum dried.High pH Reverse Phase Fractionation: High pH reverse phase fractionation chromatography was carried out using a Dionex UltiMate 3000 HPLC system. The iTRAQ‐tagged peptides were reconstituted and loaded onto Gemini‐NX C18 columns . The peptides were eluted with a linear gradient formed by buffer A  and buffer B  for 100 min at a flow rate of 200 μL min−1. Twenty‐four fractions were collected at 1 min intervals, based on UV detection wavelength at 214/280 nm. Then all the fractions were dried individually and dissolved in the sample solution (0.1% formic acid and 2% acetonitrile) for nano LC–MS /MS analysis.LC–MS/MS Analysis by Q Exactive: The nano LC–MS/MS was carried out using Q Exactive (Thermo Scientific) with the peptide recognition mode enabled. The peptide mixture was separated on the chromatographic column  at a flow rate of 300 nL min−1. And then peptides were eluted from the HPLC column through the application of a linear gradient from 4% to 50 % solution  for 40 min. Finally, the eluted peptides were detected by Q Exactive and MS data were acquired using a data‐dependent top 20 method, dynamically choosing the most abundant precursor ions from the survey scan  for high‐energy collisional dissociation  fragmentation with each component analysis for 60 min.Protein Identification and Data Analysis: First, the raw files were converted to .mgf files by Proteome Discoverer 1.4 (Thermo Fisher Scientific). Then, Protein Pilot 5.0  was used for protein identification and quantification analysis. Database searching parameters were as follows. Sample type: iTRAQ 8plex (Peptide Labeled), Cys alkylation: MMTS, ID focus: biological modification, digestion: trypsin, Search effort: thorough ID, FDR <1%, T‐test was used to identify significant differences (p < 0.05) in means between AIC and control with an average ratio‐fold change ≥1.5 or ≤0.66, a minimum of two peptide matches in common was confidently considered as differential expression of proteins. The coefficient of variation was used to evaluate the dispersion of the replicates within groups, and detected proteins with coefficient of variation ≤ 0.5 were considered reliable.www.string-db.org) which is a database of known and predicted protein interactions, including direct  and indirect  associations.Bioinformatics Analysis: Bioinformatics analysis was carried out in order to better study the biological function of significantly altered proteins. GO annotation and enrichment analysis (http://www. geneontology.org) which includes three main modules—biological process, cellular component, and molecular function—was employed to categorize proteins into families and subfamilies with shared functions. Pathway analysis using KGEE (http://www.genome.jp/kegg/pathway.html) was used to take advantage of the current knowledge of biochemical pathways and protein–protein interaction networks using STRING database  in the gels were transferred onto PVDF membranes (Roche). After blocking for nonspecific binding with 5% non‐fat milk, the membranes were incubated with primary antibodies against apoE , apoA1 , von Willebrand factor , insulin‐like growth factor‐binding protein complex acid labile subunit , gelsolin , adiponectin , transthyretin , pIgR  at 4 °C overnight, followed by the appropriate horse‐radish peroxidase‐conjugated secondary antibodies. All blots were visualized using ECL (Amersham Biosciences).Quantification was performed using ImageJ software.Statistical Data and Graphics: The statistical analyses were performed using GraphPad Prism version 6.01 for Windows . Student's t‐test was applied for comparisons of quantitative data. The receiver operating characteristic (ROC) analysis with SPSS Statistics 20.0 was performed to evaluate sensitivity and specificity of each marker and their combination in AIC and AIH. For all analysis, p value < 0.05 was considered to indicate statistical significance.33.1The goal of this study was to look for potential biomarkers for diagnosis of AIC using iTRAQ. A total of 133 398 spectra, 46 131 spectra identified, 12 773 distinct peptides, 1954 proteins before grouping, and 385 proteins were acquired through the analysis with ProteinPilot Software 5.0.1 search engine , enzyme regulator activity (17%), and lipid binding , systemic lupus erythematosus (3.8%), regulation of actin cytoskeleton (3.3%), focal adhesion (3.3%), and African trypanosomiasis (2.7%).As shown in Table 3.5Biological systems can be regulated as complex network systems with many interactions among the components in different pathways. In order to better understand the pathogenic mechanisms in AIC, the protein interaction network for the identified variable proteins was constructed by STRING. From the network diagram Figure , many pr3.6p < 0.0001), 2.82 (p < 0.0001), 1.66 (p < 0.0001) in AIC, 1.39 (p < 0.001), 1.46 (p < 0.001), 2.72 (p < 0.0001) in AIH, 1.27 (p < 0.001), 1.21 (p < 0.05), 1.28 (p < 0.01) in HBV and 1.38 (p < 0.001), 1.40 (p < 0.001), 1.30 (p < 0.01) in HCV. However, the ratio of IFGALS, TTR, apoA1, apoE, gelsolin was, respectively, decreased to 0.63 (p < 0.0001), 0.68 (p < 0.0001), 0.78 (p < 0.01), 0.70 (p < 0.001), 0.83 (p < 0.001) in AIC, 0.89 (p < 0.01), 0.85 (p < 0.0001), 0.90 (p > 0.05), 0.95 (p > 0.05), 0.75 (p < 0.0001) in AIH, 0.86 (p < 0.001), 0.90 (p < 0.01), 0.88 (p < 0.01), 0.75 (p < 0.001), 0.88 (p < 0.01) in HBV and 0.98 (p > 0.05), 0.95 (p < 0.01), 0.95 (p > 0.05), 0.94 (p < 0.05), 0.93 (p < 0.01) in HCV  were verified further using western blot Table , their M3.7To evaluate the specificity and sensitivity of each marker and their combination in the diagnosis of AIC and AIH, we conducted ROC analysis. The accuracy of the area under the ROC curve was assessed as follows: 0.9–1 = excellent, 0.8–0.9 = good, 0.7–0.8 = fair, 0.6–0.7 = poor, and <0.6 = not useful. In AIC, ROC analysis results are as follows: vWF , adiponectin , pIgR , IFGALS , TTR , apoA1 , apoE , gelsolin . The combination of these eight proteins displays a significant predictive value for AIC   to form a heterologous protein complex, which can prolong the half‐life of IGFs in the blood.Autoimmune hepatitis (AIH) is one of the autoimmune diseases and is characterized by increases in the levels of γ‐globulins and immunoglobulin G in the blood.TTR, known as prealbumin, has a major role in the transport of thyroxine and retinol as a transporter.Apolipoprotein is mostly synthesized in the liver,2+‐regulated actin filament severing and capping protein, is a highly conserved, polyfunctional regulator of cell structure and metabolism.Gelsolin, a CaIn the study, we first implemented iTRAQ technique to identify vWF, adiponectin, IFGALS, and TTR as candidate biomarkers in AIC, which will provide a useful basis for further analysis of the pathogenic mechanism.The authors declare no conflict of interest.


Saxifraga sinomontana (Saxifragaceae) is a widespread alpine species in the Qinghai–Tibetan Plateau and its flanking mountains. We developed a set of expressed sequence tag–simple sequence repeat (EST‐SSR) markers to investigate the genetic diversity and evolutionary history of the species.EST‐SSR markers based on transcriptome data of S. sinomontana. Nineteen of 50 loci (38%) were successfully amplified, 13 of which were polymorphic. These were tested on 71 individuals from four populations. Three to 18 alleles per locus were detected, and the levels of observed and expected heterozygosity ranged from 0.2817 to 0.9155 and 0.2585 to 0.8495, respectively. In addition, cross‐amplification was successful for all 13 loci in three congeneric species, S. tangutica, S. heleonastes, and S. congestiflora.We initially designed 50 EST‐SSR markers will be useful for studying the genetic diversity of S. sinomontana and disentangling the phylogenetic relationships of related species.These  Saxifraga L. (Saxifragaceae) consists of approximately 500 species that are mainly distributed in the mountainous regions of Europe and Asia . Reads were filtered by trimming adapters and removing ambiguous reads (N > 10%) and low‐quality reads  using in‐house perl scripts, yielding 90,311,228 clean reads. Clean reads were assembled into 176,110 transcripts using Trinity v2012‐10‐05 , and 2.5 μL of 10 mM dNTPs. The PCR profile included an initial pretreatment of 10 min at 94°C; followed by 35 cycles of 1 min denaturation at 94°C, 50 s at locus‐specific annealing temperatures (Table S. sinomontana with the same protocol described above (Table A), observed heterozygosity, and expected heterozygosity were calculated with POPGENE version 1.32 . The Dingri population had the lowest mean values (A = 2) among the four populations. Levels of observed and expected heterozygosity varied from 0.2817 to 0.9155 and 0.2585 to 0.8495, respectively, which indicates that genetic diversity is relatively high in this species. Additionally, a few loci showed significant deviations from Hardy–Weinberg equilibrium: three in the Aba population, two in the Chengduo population, and seven in the Dingri population (P < 0.05; Table Among the 13 polymorphic loci, lues A =  among thS. tangutica, S. heleonastes, and S. congestiflora, using the same PCR protocol as for S. sinomontana (Table All 13 EST‐SSR markers also amplified successfully in S. sinomontana and high cross‐species amplification success. Hence, these are valuable loci for investigating genetic diversity, population structure, and evolutionary history in S. sinomontana and throughout Saxifraga.The 13 EST‐SSR markers developed here showed high polymorphism in


We compared these effect sizes with those from intervention groups in randomized controlled trials (RCTs), with populations similar to our study sample. These RCTs reported significant effects of MBSR compared to control condition. MBSR was delivered in three different Danish cities by ten different MBSR teachers with various professional backgrounds and MBSR teaching experience. One hundred and thirty-two participants were included in the study: 79% were women, mean age 45 ± 10.4 years, 75% of the participants had more than 15 years of education, 38% had a Perceived Stress Scale (PSS) score≥18, and 27% had a history of mental disorder. Post MBSR, the proportion of participants with a PSS≥18 decreased by 16% points (95%CI −26 to −6), p = 0.0032. Within-group effect sizes for (i) the total study population (ii) the subgroup with PSS≥18 at baseline (iii) intervention group in reference RCTs were as follows: PSS: d = 0.50:1.47:1.00, Symptom Check List 5: d = 0.48:0.81:0.77, Five Facet Mindfulness Questionnaire: d = 0.67:1.09:1.00. Our results showed that MBSR was effective. The effects were largest among the participants reporting highest perceived stress level at baseline. Our participants were mainly women who were middle-aged, with high educational levels, and more perceived stress and a greater history of mental disorder than the general population, and who were able to seek out and pay for an MBSR course. Reaching vulnerable groups with a clear need for stress management will, however, require other implementation strategies.We aimed to evaluate the effectiveness of Mindfulness-Based Stress Reduction (MBSR) when implemented in a community setting as a self-referred and self-paid course. Pre-post changes and Cohen’s  Long-term stress is an increasing public health problem and is associated with impaired psychological and physical functioning  examined the accumulating evidence for the effects of MBSR  program was developed by Jon Kabat-Zinn in 1979 at the University of Massachusetts Medical School. The program components and structure are well described   could be replicated in routine health care , psychologist (n = 6), occupational therapist (n = 1), anthropologist (n = 1), and other (n = 1); and their experience of teaching MBSR ranged from 1 to 10 years, with a median of 2 years.The MBSR program was delivered as recommended in 2.5-h weekly group sessions over 8 weeks with one 7-h silence retreat day and 45–60 min of daily homework 6 days a week  mandatory school (1 year of preschool class and up to and including 9. grade) and an optional 11th year of school at the most (≤ 11 years), (2) secondary education and/or vocational training < 3 years at the most (>11 < 16 years) and, (3) secondary education and vocational training ≥ 3 years or tertiary education (≥ 16 years).The Five Facet Mindfulness Questionnaire (FFMQ-15) was used to measure participants’ mindfulness  was used to measure the degree to which an individual has perceived life as unpredictable, uncontrollable, and overloading during the previous month p values using paired t-tests, both among the total study population and among the pre-defined subgroup with PSS ≥ 18 at baseline. An alpha value of 0.05 was used. Furthermore, Cohen’s d were calculated according to the formula: (M post – M pre) / ((sd post + sd pre)/2)  gender, (ii) in participants with self-reported current or previous mental disorder vs. no self-reported mental disorder, and (iii) in participants with tracked attendance data vs. no tracked attendance data.Differences in the pre and post scores of FFMQ, PSS, and SCL-5 were calculated with 95% confidence intervals and ) Lakens , for ourp = 0.0032). Among the group with PSS ≥ 18 at baseline, the PSS score was significantly reduced by 6.4 score points from a pre-mean score of 22.6 ± 3.6 . The majority of post course questionnaire non-responders (43 of 45 who provided data on education level at baseline) reported ≥ 16 years of education. In our study population, 91 of 121 who provided baseline data on education level reported ≥ 16 years of education. There was more missing data on education level for the non-responding group than the responding group (35 vs. 8%). In a sensitivity analysis assuming no change from baseline post MBSR among questionnaire non-responders, the results were as follows: total group, mean change (post minus pre) (95%CI): PSS −2.0 (−2.6 to −1.3), p < 0.0000, SCL-5 −0.2 (−0.2 to −0.1), p < 0.0000, FFMQ-15 3.8 (2.8 to 4.7), p < 0.0000. Subgroup with PSS ≥ 18 at baseline, mean change (post minus pre) (95%CI): PSS −4.1 (−5.4 to −2.8), p < 0.0000, SCL-5 −0.3 (−0.4 to −0.2), p < 0.0000, FFMQ-15: 4.8 (3.1 to 6.4), p < 0.0000.The 69 participants that did not complete the post course questionnaires had higher education levels than the rest of the study population , p = 0.64, FFMQ-15 2.5 (−0.13 to 5.16), p = 0.06. Subgroup with PSS ≥ 18 at baseline, mean difference in pre-post changes (group 2 - group 1) (95%CI): PSS 0.6 (−2.8 to 4.1), p = 0.71, SCL-5 −0.01 (−0.39 to 0.37), p = 0.97, FFMQ-15 1.6 (−3.2 to 6.3), p = 0.52.A total of 53 (40%) had participated in a course delivered by one of the two most experienced MBSR teachers . Comparing this group (1) with the 79 participants (60%) (2) that had participated in the courses delivered by the other eight teachers, the results were as follows: total group, mean difference in pre-post changes (group 2 - group 1) (95%CI); PSS 0.5 (−1.6 to 2.7), n = 88) minus mean changes in men (n = 24) (95%CI) were as follows: PSS 0.8 (−1.7 to 3.3), p = 0.51, SCL-5 0.17 (−0.08 to 0.42), p = 0.17, FFMQ-15 0.5 (−2.8 to 3.8), p = 0.77.Mean changes in women (n = 79) minus mean changes in participants with self-reported disorder (n = 28) (95%CI) were as follows: PSS −2.8 (−5.1 to −0.6), p = 0.01, SCL-5 0.02 (−0.20 to 0.24), p = 0.86, FFMQ-15 −1.2 (−4.3 to 1.9), p = 0.45.Mean changes in participants with no self-reported mental disorder , p = 0.65, FFMQ-15 −0.8 (−3.6 to 2.0), p = 0.56.In 6 out of the 15 courses provided in 2016, participant attendance was systematically tracked. Attendance data was therefore available for 49 of the 132 study participants (37%). Among these 49 participants, median attendance was 8 (range 5–8) out of 8 sessions. Only 1 out of the 49 participants (2%) had participated in less than 6 out of the 8 sessions. Forty-two of the 49 with tracked course attendance (86%) had participated in the retreat day. Mean differences in pre-post changes in the groups divided by tracked course attendance (non-tracked minus tracked) were: PSS −0.9 (−3.0 to 1.3), This study evaluated the effects of attending a self-paid MBSR course in a community setting, delivered by MBSR teachers trained to international standards in Denmark. The results showed statistically significant changes, indicating moderate effect sizes of MBSR on mindfulness, perceived stress, and symptoms of anxiety and depression. For all outcomes, the effects were largest among the subgroup with most perceived stress at baseline. In comparison with within-intervention group effects from reference RCTs that had shown moderate between-group effects, the effects in our total study population were somewhat smaller. This is often seen when translating effects from RCTs to “the real world” with for example more heterogeneous study populations  as well as more heterogeneous intervention providers . Moreover, at baseline the participants in our study had reported a higher level of mindfulness and less perceived stress than the comparison groups from the reference RCTs. This may have meant a ceiling effect for potential changes. Among our pre-defined subgroup with PSS ≥ 18 at baseline, which was more similar to the comparison groups, we found superior effects, especially with regard to PSS Table . Hence, g 0.55) and in between-group analyses (Hedge’s g 0.53) were found  mental health difficulties have more difficulty engaging with the practices that are integral to the program. Although we did not track participants’ home practice, this may be an important avenue for future work , a psychological theory on motivation, proposes that persistent high-quality motivation of behavior requires A strength of the current study was that we evaluated MBSR in geographically different settings, delivered by different MBSR teachers with various professional backgrounds and experience teaching MBSR. Two of the MBSR teachers were co-authors of this paper, but the effect of their courses did not differ significantly from the effect of the courses delivered by the other MBSR teachers. The courses were delivered within typical community settings in Denmark. There were no stringent inclusion/exclusion criteria for participation, or randomization and monitoring of adherence among MBSR teachers and participants. The data collection did not exceed usual quality monitoring of the MBSR courses. Another strength of the current study was the use of intervention groups from reference RCTs for comparison. The effect sizes in our study population were compared with effect sizes in other intervention groups from RCTs with populations similar to our study sample. These RCTs reported significant effects of MBSR compared to control conditions. We suggest that this comparison adds to our initial pre-post analysis, where “regression towards the mean” is a core problem. To our knowledge, this study design has only been used in diabetes prevention implementation research (Absetz et al. A clear difference between our study and the reference RCTs was in self-payment. Self-pay is seldom used in RCTs and can be an indicator of a high level of treatment buy in or commitment. Participation in RCTs is often also associated with high motivation. Related to this, we did not systematically track participants’ course attendance, which is a clear limitation of our study. However, the available data showed a high attendance rate, and the course effectiveness did not differ between the groups divided by tracked course attendance. The effectiveness analysis in our study is based on all participants that referred themselves, self-paid a MBSR course delivered by Danish Centre for Mindfulness in 2016 and completed pre- and post measures. We suggest that these results are generalizable to “the real world” and indicate the effectiveness of MBSR in a community setting.The finding of a higher educational level among the non-responding participants in comparison with the study population should be interpreted with caution, due to the high amount of missing data in the data on education among the non-responding group. Assuming that the non-responding group did not benefit from MBSR participation, the effects for all outcomes remained statistically significant.This study demonstrated the positive effects of implementing MBSR as a self-referred and self-paid course in a Danish community setting delivered by a group of MBSR teachers. The effects were largest among the participants reporting highest stress at baseline, and the results were comparable with reference RCTs. The participants were mainly women who were middle-aged, with high educational levels, and more perceived stress and a greater history of mental disorder than the general population. Reaching vulnerable, low-resource groups with a clear need for stress management will, however, require other implementation strategies.


Citrullus colocynthis (L.), Cannabis indica (L.) and Artemisia argyi (L.) against Brevicoryne brassicae and to conduct biochemical analysis by gas chromatography-mass spectrometry (GC-MS). The results suggested that when using ethanol, C. colocynthis produced a high dry yield (12.45%), followed by that of C. indica and A. argyi, which were 12.37% and 10.95%, respectively. The toxicity results showed that A. argyi was toxic to B. brassicae with an LC50 of 3.91 mg mL−1, followed by the toxicity of C. colocynthis and C. indica, exhibiting LC50 values of 6.26 and 10.04 mg mL−1, respectively, which were obtained via a residual assay; with a contact assay, the LC50 values of C. colocynthis, C. indica and A. argyi were 0.22 mg mL−1, 1.96 and 2.87 mg mL−1, respectively. The interaction of plant extracts, concentration and time revealed that the maximum mortality based on a concentration of 20 mg L−1 was 55.50%, the time-based mortality was 55% at 72 h of exposure, and the treatment-based mortality was 44.13% for A. argyi via the residual assay. On the other hand, the maximum concentration-based mortality was 74.44% at 20 mg mL−1, the time-based mortality was 66.38% after 72 h of exposure, and 57.30% treatment-based mortality was afforded by A. argyi via the contact assay. The biochemical analysis presented ten constituents in both the A. argyi and C. colocynthis extracts and twenty in that of C. indica, corresponding to 99.80%, 99.99% and 97% of the total extracts, respectively. Moreover, the detected caryophylleneonides (sesquiterpenes), α-bisabolol and dronabinol (Δ9-THC) from C. indica and erucylamide and octasiloxane hexamethyl from C. colocynthis exhibited insecticidal properties, which might be responsible for aphid mortality. However, A. argyi was evaluated for the first time against B. brassicae. It was concluded that all the plant extracts possessed significant insecticidal properties and could be introduced as botanical insecticides after field evaluations.Plant extracts contain many active compounds, which are tremendously fruitful for plant defence against several insect pests. The prime objectives of the present study were to calculate the extraction yield and to evaluate the leaf extracts of  Brevicoryne brassicae L. (Hemiptera: Aphididae), is a serious pest native to Europe but now found globally2, and it causes significant losses to economically important crops, including broccoli, oilseed rape, brussels sprouts, cauliflower, black and white mustard, toria, Chinese cabbage, kale, and other field crops1. It also causes yellowing, stunting, and plant wilting and acts as a vector of several viral diseases in crucifers, including cauliflower mosaic virus and turnip mosaic virus4. In the case of severe infestation, plants become covered with aphids, resulting in leaf surfaces roofed with black mould due to honey secretion, which can ultimately cause plant death7. Synthetic chemical insecticides are available sources for controlling this destructive pest, which is a key success for modern agricultural practices and enhances crop yield. However, indiscriminate use of synthetic pesticides for crop production and protection poses poisonous effects through contact, inhalation, and dietary exposure and has become a cause of carcinogenesis, fertility problems and mutagenesis in humans11.The cabbage aphid, 12. Many insecticides derived from botanical sources are available and are easily affordable and accessible to the farming community; they are safer for human beings and for the environment with minimal residual effect, and they are target-specific and less toxic to vertebrates, pollinators and fish17. Studies have demonstrated the aphicidal activity of Chenopodium ficifolium extracts and its isolated phospholipids, the alkaloids from Corydalis turtschaninovii tubers and Macleaya cordata seeds, the oil from Jatropha curcas and rhamnolipid from Pseudomonas species20. Other plant-based products, such as Melia azedarach seed extracts, Lantana camorra leaf and seed extracts and Mentha piperita leaf extracts, possess promising insecticidal properties against B. brassicae23.These circumstances led towards searching for effective and eco-friendly pest control alternatives, especially from natural plant resourcesCitrullus colocynthis (Cucurbitaceae), Cannabis indica (Cannabaceae) and Artemisia argyi (Asteraceae) were selected for the present study, as they have been reported as having insecticidal potential against different stored grains and other field crop pests. The selected plants also possess a natural ability to deter insect attack. Among them, Cannabis has been reported as a repellent against vertebrates and invertebrates. It also showed better results when grown as a companion crop with other crops to prevent the infestation of weedy plants, fungi, insects and nematodes, while the extract from the dried leaves and flowers possess repellent and killing potential against the above-mentioned pests. However, pure cannabinoids extracted from this plants have been reported to kill or repel mites, insects, nematodes, fungi and protozoa24. Moreover, phytoconstituents analysis reported major terpenes, such as β-myrecene and β-caryophyllene, and the profusion of cannabidiolic acid (CBDA) and cannabidiol (CBD) from an extract of C. sativa (hemp) inflorescences by GC-MS, GC-FID and HPLC25. In a recent study, the activity of hemp essential oil from inflorescences was explored against Myzus persicae (Hemiptera), Culex quinquefasciatus (Diptera), Spodoptera littoralis (Lepidoptera) and Musca domestica (Diptera) and showed high toxicity against M. domestica and M. persicae, moderate toxicity against the larvae of S. littoralis and sparse toxicity against C. quinquefasciatus26.Therefore, C. colocynthis has gained the attention of researchers as an alternative botanical insecticide, and the effectiveness of its extracts and isolated compounds has been assayed against economically important insect pest species. This plant showed antifeedant, deterrent and infertility activities against several pests27. The toxicity effects of C. colocynthis fruit, leaf, stem and root extracts were evaluated against Rhopalosiphum padi L. The results revealed the high effectiveness of the stem extract compared to that of the other part extracts against this pest28. Moreover, the identified biocompounds from C. colocynthis fruits, i.e., 7,8-benzoquinoline, were the most efficient against Tetranychus urticae, while quinoline, 2-methylquinoline and 8-hydroxyquinoline were effective against Sitophilus zeamais and Sitophilus oryzae29.Similarly, A. argyi, is generally known as Argyi wormwood or Chinese mugwort and is commonly found in China. Different Artemisia species are known for their pharmacological and insecticidal properties. The ethanol extracts from A. argyi possess a strong repellent effect against Drosophila melanogaster, which increased with increasing concentration and extended exposure time30. Furthermore, the bioactivity of isolated compounds from Artemisia judaica essential oil was evaluated against Spodoptera littoralis at the third larval instar, and against plant pathogenic fungi showed prominent insecticidal, antifeedant and antifungal activity. However, the antifeedant activity was concentration-dependent31. The antifungal activity of compounds isolated by GC-MS, such as β-thujone, α-thujone, camphor, verbinol and davanone, showed high inhibition effects against Rhizoctonia solani, Fusarium solani, and Sclerotinia sclerotiorum whereas, exhibited slight effectiveness against Fusarium proliferatum and Fusarium oxysporum32.As a medicinal plant, C. colocynthis, C. indica and A. argyi against B. brassicae. To investigate safe alternatives for the management of this pest, the present study was conducted to evaluate the insecticidal effects of crude ethanol extracts of C. colocynthis, C. indica and A. argyi against the cabbage aphid B. brassicae by residual/leaf dip and contact/aphid dip bioassays under laboratory conditions at the Biopesticides Lab of Shenyang Agricultural University, P.R. China.Due to issues concerning the use of synthetic pesticides and the increasing resistance in pest species, pests can be managed by introducing botanical insecticides, especially against soft-bodied insects such as aphids. However, to our knowledge, few studies have been conducted regarding the aphicidal activity of the leaf extracts of P < 0.05), the extract yield afforded by C. colocynthis was 12.45%, followed by that of A. argyi and C. indica (12.37% and 10.95%) respectively. However, the extract yield produced by all the plant samples was not much different. The extract obtained from C. colocynthis and C. indica was sticky and fluidic in consistency with a brownish-black to blackish colour and a shiny in appearance. While, the extract from A. argyi was waxy in consistency with a yellowish-brown colour.The extract yield obtained by solvent extraction using ethanol showed that in comparison,  but decreased to 40.00 ± 2.23% after 48 h of exposure at the same concentration. The mortality of B. brassicae by A. argyi (35.00 ± 3.87%) was also higher after 24 h of exposure than that of 24 h of exposure to C. colocynthis and C. indica extracts . The control treatment showed no mortality after 24 h and 48 h and negligible mortality (1.67 ± 1.29%) after 72 h, whereas imidacloprid was the positive control and showed the highest (P < 0.05) mortality, i.e., 98.33 ± 2.58, 91.67 ± 2.58 and 86.67 ± 1.29% after 72 h, 48 h and 24 h, respectively.The mortality data presented in Table P < 0.001) model fitness with  intercept, concentration and time. Moreover, the interaction between the mortality (%) of the aphids versus time and concentration exhibited a highly significant (P < 0.001) positive correlation (F = 8.62). The two-way analysis of variance provided information regarding the interaction between mortality (%), treatment and time and highlighted highly significant results (F = 7.25). Interaction/correlation between treatments × concentration × time and the total variance was firmly trait-specific and significantly correlated with mortality (P < 0.001 and with F = 2.75).The results described in Table −1, with 50.56 ± 7.92% followed by 43.52 ± 7.52, 35.37 ± 7.02, 28.89 ± 6.12% and 20.19 ± 3.16% at 15, 10, 5 and 2.5 mg mL−1, respectively. The maximum mean mortality interaction based on time was recorded as 55.00 ± 6.79% at 72 h exposure, followed by 36.35 ± 6.02 and 23.89 ± 5.79% at 48 and 24 h, respectively. On the other hand, mortality interaction among treatments was evaluated as high (44.13 ± 7.14%) by A. argyi followed by that of C. indica and C. colocynthis . Moreover, the positive control showed a maximum 90.19 ± 2.09% mortality, and the negative control showed a minimum mortality of 0.19 ± 0.18%. Probit analysis showed the LC50 values, slope, chi-square, and fiducial limits at the 95% confidence interval. Although B. brassicae showed sensitivity to the different plant extracts, A. argyi was the most toxic, followed by C. colocynthis and C. indica, and this mortality response is presented in Table However, data for interaction among plant species, concentration and post-treatment time revealed that the maximum mean mortality based on concentration by different treatments/plant species was recorded at 20 mg mLB. brassicae by the contact toxicity method. The results showed that maximum mortality was recorded after 72 h at 20 mg mL−1. However, the highest mortality caused by the crude extract of A. argyi was 93.33 ± 3.14%, followed by that of C. colocynthis and C. indica . High mortality after 48 h of exposure to 15 mg mL−1 was also observed in A. argyi (83.33 ± 1.29%), followed by that of C. colocynthis and C. indica, which had the same mortality values of 81.67 ± 3.41% and 81.67 ± 1.67%, respectively. Additionally, the mortality of B. brassicae by the contact assay was also high for exposure to extracts of C. indica even at low concentrations . The same trend of mortality was observed for C. colocynthis and A. argyi. The negative control treatment showed similar results for mortality in all three experiments for each treatment in both bioassay methods, while imidacloprid showed significantly (P < 0.05) high mortality  for leaf dip and contact bioassay, which was also the highest mortality.The data presented in Table P < 0.001) model fitness with  intercept, concentration and time. However, the interaction between mortality (%) of the aphid versus time and concentration recorded a highly significant (P < 0.001) positive correlation (F = 1.58). The two-way analysis of variance provided information regarding the interaction between mortality (%) versus treatment and time highlighted highly significant results (F = 10.07). The interaction/correlation between treatments × concentration × time and total variance was strongly trait-specific and highlighted significant correlation with mortality (P < 0.001 with F = 1.79). However, data for the interaction among plant species, concentrations and post-treatment time revealed that the maximum mean mortality based on concentration by different treatments/plant species was recorded at 20 mg mL−1, with 74.44 ± 3.74% followed by 70 ± 3.58, 63.33 ± 3.08, 56.48 ± 4.34 and 47.59 ± 4.74% at 15, 10, 5 and 2.5 mg mL−1, respectively. The maximum mean mortality interaction based on time was recorded as 66.83 ± 6.63% after 72 h of exposure, followed by 56.27 ± 6.19% and 51.51 ± 6.82% after 48 and 24 h, respectively. The mortality interaction among treatments was 57.30 ± 6.31% by A. argyi followed by 62.22 ± 7.14% and 55.08 ± 6.62% by C. colocynthis and C. indica, respectively. Moreover, the positive control treatment showed a maximum of 95 ± 1.14% mortality, and a 0.56 ± 0.27% mortality was exhibited by the negative control.The results described in Table 50 values, slope, chi-square, and fiducial limits at the 95% confidence interval for C. colocynthis, C. indica and A. argyi. Mortality response using different plant extracts showed high sensitivity of B. brassicae to A. argyi followed by C. colocynthis and C. indica in the contact assay, which is summarized in Table Probit analysis of the data revealed the LCC. colocynthis, C. indica and A. argyi were evaluated by conducting GC-MS analysis. The principal active compounds, molecular weight , molecular formula (M.F.), retention time (R.T.) and peak area (%) are presented in Tables The presence of biologically active components from the ethanol extracts of C. colocynthis leaves showed the presence of ten compounds corresponding to 99.99% of the total extract; however, 4-nitro-4′-chlorodiphenylsulfoxide (22.94%), erucylamide (22.38%), heptasiloxane,1,1,3,3,5,5,7,7,9,9,11,11,13,13-tetradecamethyl- (21.33%), 5-methyl-2-phenylindolizine (17.85%), and octasiloxane,1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-hexadecamethyl- (7.06%) were the major compounds, and other minor compounds were present in low quantities, with relative peak areas ranging from 0.98–3.57%.The crude extract of C. indica revealed the presence of twenty compounds corresponding to 97% of the total extract. Dronabinol (57.29%), benzenesulfonic acid, 4-nitro- (25.54%), cannabinol (2.29%), and cyclobarbital (2.19%) were the main compounds, while sixteen other minor compounds were reported, with relative peak areas ranging from 0.29–2.00%.However, the results observed through GC-MS analysis of A. argyi revealed the presence of ten compounds corresponding to 99.80% of the total extract; however, neodecanoic acid (43.60%), 2-trimethylsilyl-1,3-dithiane (21.61%), cyclotrisiloxane, hexamethyl- (10.62%), 2-isopropenyl-2,3-dihydrofurochromen-7-one (6.28%), and N-isopropyl-3-phenylpropanamide (4.37%) were the major compounds, and five other minor compounds with lower relative peak areas ranging from 1.44–3.81% were identified.The chemical profiling obtained through GC-MS analysis of B. brassicae, which is becoming problematic in cabbage growing areas of the world. Plant extracts or essential oils are commonly applied for pest control measures because of their effectiveness against different life stages of the pests. However, the selected plants  are naturally repellent to some extent to insect attack, which indicates that they could be suitable candidates against cabbage aphids, B. brassicae. An investigation was performed to detect the phytochemical constituents of the extracts of the above-mentioned plants, and their insecticidal activity was evaluated by residual and contact toxicity methods because of the feeding style of aphids33.Due to the problems concerning the use of synthetic chemicals for pest management, there is an urgent need to introduce natural products, mainly from plant sources, for use against insect pests, especially Melastoma malabathricum34. Furthermore, a relatively high extract yield from C. colocynthis and C. sativa using ethanol as an extraction solvent was reported35.However, compound extraction from plant parts depends upon the type of plant material and solvent used. High-polarity solvents produced a higher yield than that of low-polarity solvents. In our results, ethanol afforded high extract yields during solvent extraction, which was due to its high polarity. Previously, it was reported that ethanol yielded more than other solvents from leaves of B. brassicae using plant extracts increased with increasing concentrations and exposure periods. However, the physical and chemical properties of the essential oils exhibited different persistent levels of insecticidal properties and different action mechanisms37. It was reported that extracts of Cassia sophera and Ageratum conyzoides against B. brassicae induced mortality that was equivalent to that of the positive control, the synthetic insecticide emamectin benzoate38. Moreover, a Mentha piperita extract resulted in maximum insecticidal activity against B. brassicae with increased concentration and time exposure39. Similarly, essential oil from Cinnamomum zeylannicum was found to be effective against B. brassicae, exhibiting 8.3 and 7.4 µl mL−1 LC50 values40. The essential oil of another medicinal plant, Elettaria cardamomum, was reported to be effective against this serious pest, exhibiting 79.2 µl mL−1 LC50 value41. There are reports that B. brassicae was sensitive to 1,8 cineole, a chemical compound found in Laurus nobilis, which at LD50 value of 30 µl mL−1 caused 52% mortality42. Additionally, essential oils isolated from natural plants are usually considered as safe for humans and environment which can be a source of new botanical insecticides. Among some of the plant species, Ocimum basilicum, Mentha piperita, Pimpinella anisum, Mentha pulegium and Foeniculum vulgare have shown outstanding effectiveness against aphids species in both contact and fumigation assays and can thus be considered as active materials for developing new botanical agents43. It was also reported that the use of Azadirachta indica oil as a suitable alternative to synthetic chemicals against B. brassicae caused a noticeable reduction in their population44. All these findings are consistent with our results on the mortality of B. brassicae at different concentrations and varying levels of LC50 values.According to our findings, the mortality of C. colocynthis leaf extract to control B. brassicae are exceptional. However, toxicity of C. colocynthis extracts from leaves and fruits against Aphis craccivora was reported45. Cucurbitacin E glycosides isolated from C. colocynthis extract showed strong insecticidal effects against Aphis craccivora46. Limonene contained by Citrus sinensis was reported to be effective against Myzus persicae at LC50 57.7 µl mL−1, whereas its essential oil at a concentration of 3.3 µl mL−1 caused significant mortality of B. brassicae41. All these results support our findings of using C. colocynthis extract against B. brassicae. However, according to our results from the GC-MS analysis, the chemical composition of C. colocynthis was dominated by four of the ten detected compounds. Among these, erucylamide is a high fatty acid amide that can be used as an insecticide, pigment dispersant, ink and paint additive, fibre softener, and in dyes. Octasiloxane hexamethyl from C. colocynthis also exhibits insecticidal properties.The results of the studies using the M. persicae was reported to be highly toxic, with an LC50 of 3.5 mL L−1 and non-toxic to the beneficial organisms26. An important compound, (E)-caryophyllene myrecene, and α-pinene, contained by C. sativa, showed 98.20% mortality at 3.50 µl mL−1 of Aulacorthum solani47, whereas at the same concentration, similar mortality of M. persicae was reported26. Previous studies have reported the concentration and time-dependent mortality of Tetranychus urticae and Aulacorthum solani following the use of hemp essential oil, which supported our findings indicating the effectiveness of the hemp (C. sativa) crude extract against B. brassicae aphids47. However, among the twenty compounds identified in the crude ethanol extract, caryophylleneonides (sesquiterpenes), α-bisabolol and dronabinol (Δ9-THC) possess insecticidal activities, However, caryophyllene oxide identified from Melaleuca styphelioides exhibited strong aphicidal activities against Aphis spiraecola, Aphis gossypii and M. persicae48 while, the other compounds may need to be further explored to determine their biological properties. To date, few studies on the applicability of hemp essential oil against crop pests have been conducted, and the use of hemp essential oil against B. brassicae is very rare in the literature.The insecticidal activity of the essential oil of hemp inflorescences against A. argyi is a famous plant commonly found in China, and many species of the genus Artemisia have long been used in traditional Chinese medicines, including Artemisia argyi, Artemisia capillaris and Artemisia annua49. In the present study, the crude ethanol extract from the leaves of A. argyi was proven to be highly toxic against B. brassicae. Moreover, four compounds from A. argyi, such as camphor, β-pinene, eucalyptol, and β-caryophyllene, showed strong toxic effects against adults of Lasioderma serricorne50. Other studies have showed that sabinene, and β-myrecen contained in Artemisia absinthium, caused significant mortality of M. persicae at 6.9 µl mL−1 concentration51. Similarly, Artemisia seiberi essential oil caused significant mortality of Eriosoma lanigerum at 6.16 µl mL−1 LD50 value52. Another compound, artemisia ketone, showed significant mortality of B. brassicae at 25.0 µl mL−1 concentration53. However, according to our results, reported compounds from A. argyi leaves were evaluated for the first time for their insecticidal potential. The above-described results supported our findings and clearly demonstrated that the A. argyi crude extract exhibited strong and deadly effects against B. brassicae, which justified its great potential for controlling this pest.Similarly, C. colocynthis, C. indica, and A. argyi against B. brassicae were investigated under laboratory conditions, revealing their positive toxicity. A. argyi proved to be the most toxic in both bioassay methods and caused maximum mortality, followed by C. colocynthis and C. indica, in a dose-dependent manner. This insecticidal activity might be due to individual efficacy or the synergistic action of biological compounds present in these plants. A literature survey regarding the potential efficacy of A. argyi showed that there are no studies reporting on its use against B. brassicae. Additionally, data on the insecticidal potential of C. colocynthis and C. indica against this serious pest and their GC-MS profiling are limited to a few studies. Thus, the present unique and novel study was conducted for the first time to investigate the insecticidal potential of crude ethanol extracts by residual and contact toxicity methods against B. brassicae, a serious pest of cabbage and other field crops.In the present study, the toxicity of ethanol extracts of the leaves of C. colocynthis, C. indica and A. argyi possess potential botanical agents as an alternative to synthetic pesticides against B. brassicae. It can be concluded that, B. brassicae, is reasonably sensitive to A. argyi followed by C. colocynthis and C. indica as shown in both bioassay methods, even at low concentrations and exposure times. Moreover, in comparison, the contact assay afforded higher mortality than that of the residual assay. Additionally, GC-MS analysis revealed the presence of valuable biologically active compounds responsible for aphid mortality. However, further studies are required on the purification and identification of active components responsible for aphid mortality and their evaluations against other insect pests.The investigations indicated that C. colocynthis (Colocynthis) leaves at the 15–20 leaf stage were collected from Bahawalnagar  Punjab, Pakistan from the natural habitat of a desert climate from March to April 2018 and authenticated at the Entomological Research Institute, Faisalabad, Pakistan. This area contains a dry climate with average annual precipitation of 204 mm and a temperature range of 12.7–45 °C. C. indica (wild hemp) and A. argyi (Chinese mugwort) leaves (10–15 leaf stage) were collected from Shenyang  Liaoning, China, from a temperate climate zone from May to June 2018. This area receives annual precipitation of 266.6 mm, and the temperature ranges from −28.5 to 36.1 °C. The collected samples were authenticated at the College of Plant Protection, Department of Pesticides Science, Shenyang Agricultural University, Liaoning P.R., China.Aerial parts of the plants tend to contain a wider variety of compounds, and most of the photosynthesis/respiration occurs in the aerial parts. Additionally, most of the secondary metabolites from plants are produced in leaves. For this reason, The collected leaves were allowed to dry in the shade at room temperature after removing impurities by rinsing in tap water. Dried leaves were ground into a fine powder using an electric blender, and 99.7% ethanol was used as a solvent for extraction. Approximately 100 g of ground powder was extracted thrice with 400 mL of ethanol each time for 72 h at a constant temperature in an incubator shaker (ZWY-1102C) at 100 rpm. After each extraction, the material was filtered through filter paper (Whatman No. 1) and concentrated to reduce the volume by using a rotary evaporator (Buchi Switzerland R-210). Finally, the concentrated filtrate was subjected to drying in a fume hood at 25 °C for 12 h. The extraction yield was measured for each extract, and the extracts of each plant were combined to obtain a bulk sample and stored in glass-stoppered vials at 4 °C until use.B. brassicae, were collected from a cabbage field  in the experimental field area of Shenyang Agricultural University, China. Aphids were reared on cabbage plants in the lab under controlled conditions of 20 ± 5 °C and 45 ± 5% R.H., along with a photoperiod of 16:8 (light: dark).Aphids, −1 were prepared in the Tween solution one day before beginning the experiment. To prepare the agar, 10–15 g agar rods were added to one litre of distilled water, boiled for 10 min with continuous stirring, allowed to cool for 10 min and was then added to Petri dishes 90 mm in diameter and 15 mm deep up to a 3–4 mm level.Tween-20 was used to minimize variations among the treatments effect of any adjuvant material. Fifty millilitres of Tween was dissolved in 950 mL distilled water by shaking to prepare a 5% solution. Serial concentrations of crude extract, 2.5, 5, 10, 15 and 20 mg mLTwo different modes of exposure, residual and contact assays were conducted for the toxicity evaluation. The experiment was replicated five times for each exposure method as well as concentration, and the mortality response was recorded at the exposure times of 24 h, 48 h and 72 h.−1) was used as a positive control. All Petri dishes were incubated for 72 h at 65% R.H., 25 °C and with a 16:8 (light: dark) photoperiod.Briefly, cabbage leaf discs 5 cm in diameter were cut with sterilized scissors and dipped for 10 s in the extracts of varying concentration. Then, the leaf discs were left at room temperature for 10 min to dry and put in Petri dishes containing agar. Then, twenty aphids at the second instar were carefully transferred onto the leaf disc contained in the Petri dish by a fine-haired brush, and caution was taken to prevent injury to the aphids during their transfer to the leaf disc. Then, a double layer of muslin cloth was placed on each Petri dish and covered with a manually perforated lid to avoid suffocation and escape of the aphids  was used as a positive control. All Petri dishes were incubated for 72 h at 65% R.H., 25 °C and with a 16:8 (light: dark) photoperiod.A 30 mm diameter plastic Petri dish was cut from the basal portion, and the remaining ring was used as the container. A nematode strainer (mesh size 300 µm) was used as a holding cell for aphids during dipping into the plant extracts of varying concentration Fig. . Then, tB. brassicae to the crude extracts was observed by needle stimulation, and those individuals who displayed no response were considered dead.Mortality data were recorded at the exposure times of 24 h, 48 h and 72 h by microscopic examination. The response of The solvent-free extract was weighed precisely to 0.005 g and dissolved in 1 ml of HPLC-grade methanol. Then, the sample was added to a centrifuge tube, and graphitized carbon blacks (GCB) was added (0.002 mg/tube) and shaken vigorously for one min on a vortex  to remove coloured components. The colourless sample was centrifuged for 10 min at 24 °C and 12000 rpm  to separate the GCB from the sample. The colourless sample was collected by micropipette and transferred to a new centrifuge tube.C. colocynthis, C. indica and A. argyi were analysed via GC-MS using an Agilent 6890–5973N USA gas chromatograph (GC) equipped with an HP1 TG-5MS polydimethylsiloxane capillary column (30 m × 250 µm × 0.25 µm) interfaced with a Hewlett Packard 5973 mass selective detector. Gas chromatographic parameters were as follows: the temperature was fixed at 110 °C for 2 min initially and increased to 200 °C and 280 °C at increasing rates of 10 °C min−1 and 5 °C min−1, respectively. The inlet temperature was 250 °C; a 10:1 split ratio was used; MS quadrupole temperature, 150 °C; thermal aux temperature, 285 °C; ionization current, 60 µA; MS temperature, 230 °C; MS scan range, m/z 40–450; ionization energy, 70 eV; carrier gas, helium with flow rate, 1.0 mL min−1. Compounds were identified by comparison of the GC-MS mass spectra with those in the literature or in databases at Wiley/NIST.98.155. The relative abundance of each compound was assessed based on the raw data peak areas in the spectra with no response factor correction from the FID.The crude ethanol extracts of P = 0.05 level. All statistical processes were administered by different statistical packages with IBM-SPSS version 25.0 software, while LC50 values, slope, chi-square values and fiducial limits were calculated by the EPA Probit analysis program version 1.5.All recorded mortality data were subjected to two-way analysis of variance (ANOVA) for calculating the interaction among plants, concentration and post-treatment time, while the mean difference between treatments was calculated for the significance test by Tukey’s HSD at the supplementary information.


Staphylococcus aureus widespread in hospitals and communities. Hence, the aim of this study was to find the most common types of S. aureus strain circulating in Shiraz via spa and SCCmec typing methods.Molecular typing such as spa typing is used to control and prevent S. aureus isolates were collected from two tertiary hospitals in Shiraz. Isolates were identified by biochemical tests. Antimicrobial susceptibility tests were performed by standard disk diffusion method and then genetic analysis of bacteria was performed using SCCmec and spa typing. In this study 31.4% of the isolates were methicillin-resistant S. aureus. The majority of isolates were SSCmec type III. Spa type t030 was the most prominent type among MRSA strains. For the first time in Iran, spa003, t386, t1877, t314, t186, t1816, t304, t325, t345 were reported in this study. It was shown that there is a possibility that these spa types are native to this region. Our findings showed that SCCmec II, III and IV disseminate from hospital to community and vice versa. Thus, effective monitoring of MRSA in hospital and community is necessary.Total of 159  Staphylococcus aureus is a major human pathogen, causing a wide range of clinical infections such as skin, soft tissue, bone, joints bloodstream, and pneumonia [S. aureus infections is essential [S. aureus (MRSA) was reported in London [neumonia . Also, tneumonia . Antibiossential . Methicissential . Methicissential . In 1961n London .S. aureus [S. aureus strains circulating in Shiraz by spa and SCCmec typing methods.MRSA is one of the most important nosocomial pathogens that can cause healthcare-associated infections , 6. Stap. aureus  The mainS. aureus isolates collected from two tertiary hospitals (Namazi and Faghihi), Shiraz from December 2017 to September 2018. The strains were isolated from clinical samples including pus, wound, blood, sputum and cerebrospinal fluid. All isolates were cultured on the sheep blood agar and were identified by catalase, tube coagulase, mannitol fermentation and DNase tests [The present study was conducted on 159 se tests .S. aureus ATCC 25923 was used as the control strain.Antimicrobial susceptibility tests were performed, using standard disk diffusion method on Mueller–Hinton agar  according to CLSI 2018 standards guidelines . We used antibiotic disks of penicillin (5 μg), ceftaroline (30 μg), clindamycin (2 μg), trimethoprim–sulfamethoxazole (2.5 μg), gentamicin (10 μg), erythromycin 15 μg), azithromycin (15 μg), ciprofloxacin (5 μg), rifampicin (5 μg), chloramphenicol (30 μg), norfloxacin (10 μg), and nitrofurantoin (300 μg) . The minimum inhibitory concentration (MIC) for vancomycin were determined by E-test  method [ μg, azitResistance cefoxitin  was detected by growth on Mueller–Hinton agar  according to the guidelines of Clinical and Laboratory Standard Institute (CLSI) for MRSA strains detection .S. aureus isolates was extracted by a kit Exgene Clinic SV . Then isolates were confirmed for mecA gene by PCR [DNA all cefoxitin-resistant e by PCR .Different types of SCCmec were carried out by the method described by Oliveira et al. . DiffereAmplification of SCCmec genes were subjected to final volume of 25 mL containing 12.5 mL Master mix , 0.2 mL of each primer with concentration of 10 pmol/mL, and 2 mL of DNA template top up to 25 mL. The PCR protocol comprised of an initial denaturation step at 94 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 60 s, annealing 55–59 °C for 60 s, extension at 72 °C for 1 min, and was followed by a final cycle of extension for 5 min at 72 °C. PCR products were detected by electrophoresis, using agarose 2% and stained with SYBER DNA safe stain, and then visualized under UV light.http://www.spaserver.ridom.de).The X region of the spa gene was amplified, using spa-1113f (5-TAA AGA CGA TCC TTC GGT GAG C-3) and spa-1514r (5-CAG CAG TAGTGC CGT TTG CTT-3) primers . AmplifiStatistical analysis was performed by Chi-square test (SPSS v.22) statistics software. A significant difference was considered at P value of < 0.05.S. aureus isolates were collected from different clinical samples including Skin (35.8%), Blood (28.9%), Wound (11.9%), Fluid (7.5%), Nasal (6.9%), Sputum (5%), Auxiliary (1.9%), Eye (1.3%), and Abscess (0.6%) were collected from Faghihi and Nemazee Hospitals in Shiraz. The 159 isolates were collected from 54.1% male and 45.9% female patients. In the present study, 109 (68.5%) isolates were MSSA and 50 (31.4%) were MRSA according to the disc diffusion and the mecA analysis by PCR. The rate of MRSA isolates was 27.9% and 39.5% in Faghihi and Nemazee hospitals, respectively  among isolates was 31.4% of which 24 and 22 resistance profiles were detected among MSAA and MRSA isolates, respectively. The antibiotics resistance results between MSAA and MRSA are presented in Table P > 0.05). The highest rate of MDR was isolated from skin, blood and wound, as well as from dermatology, internal and outpatient wards.The resistance patterns were observed amongst the isolates: penicillin (78.6%), erythromycin (45.9%), azithromycin (45.3%), norfloxacine 28.3%), trimethoprim–sulfamehoxazole (27%), ciprofloxacin (27%), clindamycin (22%), cefaroline (23.3%), gentamicin (10.7%), nitrofurantoin (4.4%), chloramphenicol (3.1%), and rifampicin (3.1%). All the isolates were susceptible to vancomycin according to 8.3%, trispa gene revealed 15 different spa types, and spa type t030  was the most notable type among MRSA strains, followed by types t021 , t386 , t1877 , t037 , t314 , t0790 , t325 , t018, t081, t186, t1816, t304, t003, t345 . Distribution of samples, hospital wards, spa typing, SCCmec types and pattern of antibiotic among MRSA isolates are show in Table Of the 50 MRSA strains, 4 (8%) harbored SCCmec type I, 2 (4%) SCCmec type IA, 6 12%) SCCmec type II, 14 (28%) SCCmec type III, 2 (4%) SCCmec type IIIA, 13 (26%) SCCmec type IV and 6 (12%) of them were nontypeable by Oliveira method. The most prevalent SCCmec MRSA was SCCmec type III. In the outpatient isolates prevalence of SCCmec type IV was 13 (26%). Ultimately, among the 50 MRSA, typing was performed using spa typing method. Sequencing of 2% SCCmecS. aureus is becoming a serious challenge in public health. Recently, therapeutic options against MDR S. aureus have become limited, causing morbidity and mortality in hospitalized patients [The widespread of MDR patients . Inappropatients .S. aurous isolated from outpatients, 12 and 19 isolates were MRSA and MSSA, respectively. At present, resistance to different antibiotics is on the rise, which can lead to inappropriate use of antibiotics in communities, hospitals and agricultural industry. Hence, infective infection control policies in hospitals can be the cause of increased antibiotic resistance in the communities and hospitals [In this study, the maximum resistance of MRSA isolates was to beta lactam antibiotics such as penicillin (92%), followed by erythromycin, azithromycin (66%) and ciprofloxacin (54%). The rate of resistance to antibiotics was higher in MRSA isolates in comparison with MSSA isolates. Rate of resistance to chloramphenicol, rifampicin, nitrofurantoin and gentamicin was lower than the other antibiotics in MRSA and MSSA. These were in line with other studies from Iran , which cospitals .In our study, vancomycin antibiotic was a highly effective against MRSA isolates, which was consistence with other studies in Iran , 20, 22.MRSA isolates are one of the most common causes of threat to public health . In the S. aureus isolates collected from hospitals and communities [SCCmec typing provides useful information about the resistance of genes to methicillin, and to identify the origin of strains , 29. In munities . Rate ofAmong the MRSA isolates, 15 different spa types were revealed, and Spa type t030 was the most frequent type  followed by Spa type t021 n = 10, 20%). In Iran, different types of spa were reported , 20, 32 %. In IraIn different countries, various spa types among clinical isolates were reported. In Europe, spa t032, in Asia, spa type t030, in America, t008, in Africa, t037 and in Australia, t202 were the most common types [As far as we know, in the present study, spa type t003, t386, t1877, t314, t186, t1816, t304, t325, t345 is the first to be reported in Iran. Spa type t003 was reported in European and American countries, and its presence in Iran might be due to the spread of this spa from one continent to another.High rate of MDR among MRSA isolates requires a new policy in order to control infection. In this study, we showed that the diversity of spa types in MRSA isolates was high and spa type t030 was found to be the most frequent. Also, for the first time in Iran spa type’s spa003, t386, t1877, t314, t186, t1816, t304, t325, t345 were reported.A limitation in this study was that we did not carry out typing of MSSA isolates, due to financial constraints.


Enterocytozoon bieneusi is an important pathogen commonly found in humans and animals. Farmed animals with close contact to humans are important hosts of E. bieneusi. The role of goats in the transmission of E. bieneusi, however, remains unclear. In this study, 341 fresh fecal samples of black goats were collected from five locations in Hainan Province, China. Enterocytozoon bieneusi was identified and genotyped by sequences of the internal transcribed spacer (ITS) region. Phylogenetic analysis was performed by constructing a neighbor-joining tree of the ITS gene sequences. The average prevalence of E. bieneusi in black goats was 24.0% (82/341) with rates ranging from 6.3% (4/63) to 37.2% (32/86) across the locations . Eight genotypes of E. bieneusi were identified, including six known genotypes: CHG5 (n = 47); CHG3 (n = 23); CHG2 (n = 4); CM21 (n = 3); D (n = 2); and AHG1 (n = 1), and two novel genotypes termed HNG-I (n = 1) and HNG-II (n = 1). In the phylogenetic tree, genotype D was clustered into Group 1 and the other identified genotypes were included in Group 2. This represents the first report identifying E. bieneusi in black goats from Hainan Province, with a high prevalence and wide occurrence demonstrated. The two new genotypes identified provide additional insights into the genotypic variations in E. bieneusi. Due to the small percentage of zoonotic genotypes in these animals, there is minimal risk of zoonotic transmission of E. bieneusi. Enterocytozoon bieneusi is one of the most common species of microsporidia contributing to human microsporidiosis, which generally causes digestive disorders, including diarrhea, particularly in the young and in adults with immunodeficiency [E. bieneusi is frequently found in numerous animal hosts worldwide, raising public health concerns regarding its zoonotic transmission [E. bieneusi is the fecal-oral route. E. bieneusi spores can be acquired from infected humans or animals through contaminated food and water [. Tracing the sources of contamination and elucidating the transmission routes of E. bieneusi represent important steps to control E. bieneusi infection in humans.ficiency . E. biensmission . The prind water . TracingE. bieneusi genotypes [E. bieneusi were divided into 11 phylogenetic groups (Groups 1–11) by phylogenetic analysis. Up to 90% of human pathogenic genotypes belong to Group 1 or Group 2, and the genotypes within these groups have been reported in a range of diverse hosts, highlighting the zoonotic nature of the disease and cross-species infections [E. bieneusi in different animals.Sequence analysis of the ribosomal internal transcribed spacer (ITS) region is the standard method for the detection and identification of enotypes . To dateenotypes , 15, 26.fections . Howeverfections . The conE. bieneusi have been performed in goats, particularly those in China [E. bieneusi have been identified in goats, 11 of which have been detected in humans [E. bieneusi in goats in China are not fully understood.The goat industry plays a dominant role in animal husbandry in China. In recent years, a number of studies of in China , 22, 28.n humans , 22, 28.E. bieneusi spores represents a threat to public health, but no information on E. bieneusi in these animals is available. The aims of this study were to examine the prevalence and genotyping of E. bieneusi from farmed black goats in Hainan Province, the southernmost region of China.Hainan black goats are native to Hainan, China and represent a common breed due to their tolerance to local hot and wet weather. They are the main goat breed in Hainan, China, where the goat industry represents an important source of poverty alleviation . These an = 52), Chengmai (n = 63), Wenchang (n = 63), Ledong (n = 77), and Wanning (n = 86) in Hainan Province from September 2018 to March 2019 . Extracted DNA was stored at −20 °C prior to PCR analysis.All fecal specimens were sieved through an 8.0-cm-diameter sieve with a pore size of 45 μm, and filtrates were concentrated by centrifugation at 1500 E. bieneusi in all the extracted DNA samples using two pairs of primers, EBITS3 and EBITS4, and EBITS1 and EBITS2.4 for the first and the second amplifications, respectively. The primers and cycling parameters were designed by Buckholt et al., as follows: the outer primers were EBITS3 (5′–GGTCATAGGGATGAAGAG–3′) and EBITS4 (5′–TTCGAGTTCTTTCGCGCTC–3′) and the inner primers were EBITS1 (5′–GCTCTGAATATCTATGGCT–3′) and EBITS2.4 (5′–ATCGCCGACGGATCCAAGTG–3′). The two sets of cycling parameters were 35 cycles of 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 40 s and 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 40 s, with both of them having a final extension step at 72 °C for 10 min. These reactions produced fragments of 435 and 390 bp, respectively [Nested PCRs were performed to amplify a ~390-bp region containing 76 bp of the 3″ end of the SSU rRNA gene, 243 bp of the internal gene, and 70 bp of the 5′ region of the large-subunit (LSU) rRNA gene. The aim was to detect the presence of E. bieneusi were sent for sequencing  and all were sequenced in both directions. The nucleotide sequences obtained were aligned with each other and reference sequences downloaded from the GenBank database using Clustal X 1.83  to determine the genotypes of E. bieneusi isolates. The genotypes of E. bieneusi obtained in this study were given the first published name when identical to known genotypes in GenBank. Meanwhile, the genotypes that produced ITS sequences with single nucleotide substitutions, deletions, or insertions confirmed by DNA sequencing of at least two PCR products were considered novel genotypes. All were given a genotype name through the addition of roman numbers behind the abbreviation HNG (Hainan Goat), according to their order of appearance. All genotypes were named based on a 243 bp region of the ITS gene of E. bieneusi according to the established nomenclature system [All secondary PCR products positive for e system .E. bieneusi obtained, phylogenetic analysis was performed by constructing a neighbor-joining tree using the program Mega X (http://www.megasoftware.net/) based on the evolutionary distances calculated by the Kimura-2-parameter model. The reliability of these trees was assessed using bootstrap analysis with 1000 replicates.To confirm the genogroup designation and to assess the genetic relationships of novel ITS genotypes of p-value = 0.05.Data entry and analysis were performed using Social Sciences (SPSS) 19.0 software. The statistical significance of differences in infection proportions was generally evaluated by Pearson’s Chi-square test. The significant level of all tests was: MN267058 and MN267059.Representative nucleotide sequences obtained in the study were deposited in the GenBank database under accession numbers E. bieneusi by PCR and sequencing analysis. E. bieneusi was identified in all five locations with the highest infection rate of 37.2% (32/86) in Wanning, followed by 36.5% (19/52) in Ding’an, 30.2% (19/63) in Chengmai, 10.4% (8/77) in Ledong, and the lowest infection rate of 6.3% (4/63) in Wenchang. There were significant differences in prevalence among these locations . The prevalence rates were 38.4% (28/73) and 20.1% (54/268) in kids and older goats, respectively, suggesting significant differences between them   of black goat samples were positive for  < 0.01) .E. bieneusi isolates belonged to eight ITS genotypes including six known genotypes , and two novel genotypes (HNG-I and HNG-II). There were 34 polymorphic sites among the eight genotypes identified (MN267058) and HNG-II (MN267059) had the largest similarity with genotype CHG5 (KP262365) with 10 and with one base difference, respectively. Among the genotypes, CHG5  dominated, followed by CHG3 , CHG2 , CM21 , D , and each of the remaining three genotypes AHG1, HNG-I and HNG-II   .E. bieneusi genotypes in animals according to location and age are shown in The distributions of E. bieneusi, all identified genotypes with the exception of genotype D, were in Group 2  which was lower than that in Chongqing  [E. bieneusi and may thus play a key role in the transmission of microsporidiosis caused by E. bieneusi.To date, there have been nine reports on the identification and genotyping of in goats , 22, 28.5%; 5/8)  and Hena5%; 5/8) , 18, 21,5%; 5/8) , 21, Yun5%; 5/8) , Heilong24.0% 82/41 which 5%; 5/8) , Egypt [E. bieneusi in kids  than in older goats  in Hainan , which was consistent with previous findings [E. bieneusi decreased with increasing age [E. bieneusi can be explained through their lower immune status and higher disease susceptibility.In terms of age groups, we observed a significantly higher infection rate of findings , 21. A r kids 38.%; 28/73 E. bieneusi have been identified in goats worldwide, 11 of which have been detected in humans [E. bieneusi infections in goats likely differ by region.To date, 46 genotypes of n humans , 22, 28.E. bieneusi in goats found at least one zoonotic genotype  with a high prevalence, except for the study from Anhui Province. Interestingly, Peru6 and EbpC were commonly found in humans from Yunnan and Heilongjiang Provinces; they were also found in animals including pigs, minks and birds [All the studies on nd birds , 27, 29.nd birds . These fnd birds , 17. In nd birds . It has nd birds , 24, 27.E. bieneusi infection now requires further study in more systematic molecular epidemiological investigations of E. bieneusi in a larger number of hosts.The other five known genotypes  identified were found in sheep or goats in previous studies , 18, 21.E. bieneusi from farmed black goats in Hainan Province, with high prevalence and wide occurrence demonstrated. It is necessary to develop improved farm management to prevent the occurrence of cross-transmission and re-infection of E. bieneusi between different individuals within each goat farm. Although the six known genotypes identified here have already been reported in goats, two novel genotypes were identified and provide novel insights into the genotypic variation in E. bieneusi. However, due to the small percentage of potentially zoonotic genotypes in these animals, there is a minimal risk of zoonotic transmission of E. bieneusi.In conclusion, this is the first report on the identification of


The injection volume was 2 μl. Multiple-reaction monitoring scanning detection was employed for quantification with an electrospray ion source in the negative ion mode. All the six compounds showed good linearities (r ≥ 0.9996). The LODs of the six bioactive compounds were 0.039 ng/ml, 0.092 ng/ml, 3.112 ng/ml, 2.131 ng/ml, 0.099 ng/ml, and 0.071 ng/ml for spinosin, 6‴-feruloylspinosin, jujuboside A, jujuboside B, camelliaside B, and betulinic acid, respectively. The LOQs were 0.118 ng/ml, 0.276 ng/ml, 9.336 ng/ml, 6.393 ng/ml, 0.299 ng/ml, and 0.213 ng/ml for spinosin, 6‴-feruloylspinosin, jujuboside A, jujuboside B, camelliaside B, and betulinic acid, respectively. According to our knowledge, it was the first time to establish a method with high efficiency and accuracy for the quantification of six bioactive components in the decoction of Ziziphi spinosae Semen, which would provide references for quality control and evaluation of Ziziphi spinosae Semen.This paper was conducted to develop a method containing ultrahigh performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry for simultaneous quantification of six bioactive components in the decoction of Ziziphi spinosae Semen. Analysis was performed on an Agilent ZORBAX Extend-C Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou, has been used as a typical sedative-hypnotic traditional Chinese medicine (TCM) in clinic for thousands of years in China [Ziziphi spinosae Semen , the seeds of in China , 2. Therin China –8. It wain China –11. Sevein China , 13. Recin China –17. In fin China , 19, butDecoction was the most common and traditional using form during TCM clinical application . HoweverMethanol (LC-MS grade) and acetonitrile (LC-MS grade) were obtained from E. Merck . Ultrapurified water (18.25 Ω) was daily prepared with a Milli-Q water purification system . All the other regents were of analytical purity and commercially available.Reference standards including spinosin (batch no. S-043-150626), 6‴-feruloylspinosin (batch no. A-011-170731), jujuboside A (batch no. S-045-160809), and jujuboside B (batch no. S-046-151205) were purchased from Chengdu Ruifensi Biological Technology Co. Ltd. Camelliaside B (batch no. 170313-080) and betulinic acid (batch no. JYB 201701) were purchased from JinYiBai Biological Technology Co. Ltd. The purity of these reference standards was all above 98.0%.Ten different batches of ZSS samples collected from Shandong (batch no. 1-5), Shanxi (batch no. 6-7), and Hebei (batch no. 8-10) provinces, which are the main production regions of ZSS in China, were used as experimental materials after a careful quality evaluation according to the Chinese Pharmacopoeia 2015 edition-part one [μg/ml. A series of reference standard solutions with six different concentrations were prepared from the final mixed reference standard solution by diluting 1, 2, 4, 8, 16, and 32 times, respectively. Finally, the concentration ranges of each reference standard in the series of reference standard solutions were 0.197–6.312 μg/ml, 0.184–5.885 μg/ml, 0.031–0.991 μg/ml, 0.021–0.668 μg/ml, 0.020–0.640 μg/ml, and 0.141–4.504 μg/ml, respectively.The stock resolutions of 6 components were independently prepared. Then, the mixed reference standard solution was obtained by moderate dilution of the stock solution with methanol. The concentrations of the 6 components in the final mixed reference standards solution used for identification and quantification were 6.312, 5.885, 0.991, 0.668, 0.640, and 4.504 w/v and 1 : 8, w/v, resp.), each for 1 h. Solutions were filtered with 4-layer meshes and merged together. Water was added to the merged decoction to fix a final volume of 200 ml to obtain the decoction of ZSS. 0.2 ml of the decoction sample was transferred into a 5 ml volumetric flask, and then appropriate methanol was added to the fixed volume and mixed. Appropriate amount of the mixture was removed and centrifuged at 12000 r/min for 5 min. The supernatant liquor was taken out, and 2 μl of which was finally injected into the UHPLC-QQQ-MS/MS system for analysis.According to the experience of clinical application of ZSS, 10 g dried powder of ZSS (60 mesh sieve passed) was accurately weighed and decocted twice with boiling water  was used at 25°C. A linear gradient program was performed with the mobile phases consisting of eluent A (water) and eluent B (acetonitrile) as follows: 0–1.2 min, 5–31% B; 1.2–2.5 min, 31–95% B; 2.5–4.5 min, 95–100% B; and 4.5–6 min, 100–100% B. The flow rate was 0.3 ml/min.A Shimazu 30A UHPLC  equipped with a binary solvent system was used to perform chromatographic separation. An Agilent C2) of 55 psi; auxiliary heating gas of 55 psi; curtain gas of 35 psi; turbospray temperature of 550°C; and ion spray voltage floating of −4500 V. The MRM transitions were tuned to the optimal for meeting the standard of the experiment. Also, declustering potential and collision energy parameters of the six components were debugged to optimal in order to meet the purpose of accurate detection of all the components.An MS/MS system  with an electrospray ionization source (ESI) was used to perform analysis of the six bioactive components in decoction of ZSS. The detection was performed under negative ion mode with an MRM detection way. The acquisition parameters of mass spectrometry are listed as follows: atomization gas  and limitation of detection (LOD) were performed. LOQ was determined as the concentration whose Proper amount of mixed reference standard solution was taken and detected for six times under the testing conditions. The peak areas of the six components were recorded, which could be used to investigate the precision by calculating the RSDs. The results showed that the RSDs of the six components were 0.60%, 2.51%, 2.75%, 2.91%, 1.12%, and 2.62%. The precision of this method was excellent.Six copies of ZSS powder, taken from the same batch, each for 10 g, were used to prepare the trial solution in accordance with the method in Section 2.3. Peak areas of the six components were recorded to investigate the repeatability of the method by calculating RSDs. The results showed that RSDs were 1.20%, 2.64%, 2.60%, 2.21%, 2.98%, and 1.18%, indicating that the repeatability of this method met the requirement.The trial solution was taken from the samples prepared in Section 2.3 randomly and tested at 0, 2, 4, 8, 12, 24, and 48 h under the analytical conditions. The peak areas of the six components were recorded to calculate the RSDs, which were used to investigate the stability of the samples. The result that the RSDs of the six components were 2.96%, 2.53%, 2.82%, 2.10%, 2.31%, and 2.72% indicated that the stability of the samples was good.Dried powder of ZSS  was accurately weighed, and fair level of reference standard solution was added, which would be then prepared as the trial solution according to Section 2.3. Determination was performed under the analytical conditions, and the peak areas of the six components were recorded in order to calculate RSDs and recoveries. The data shown in μg/g (Ten different batches of ZSS samples collected from their main producing regions in China were prepared as the trial solution in accordance with the method in Section 2.3. Determination of the components was performed under the analytical conditions given in Section 2.4, and the peak areas which would be used to calculate the mass fraction were recorded. The contents of the six bioactive constituents of the decoctions of ZSS samples from three different producing regions were in the ranges of 1375.17–2612.84, 297.27–788.05, 60.20–112.75, 47.34–114.60, 10.17–46.13, and 141.78–616.83 μg/g . InteresA rapid and accurate method for determination of six bioactive components in the decoction of ZSS was established for the first time in this paper. Besides accurate and quick quantitation, the mass information and ion fragments of MRM transition could provide meaningful reference for the identification of the analytes. It was hoped that the analytical method developed would provide a reference for the quality control and evaluation of ZSS. At the same time, the results that great differences existed in the contents of the six bioactive components in the samples from different regions indicated that the origin factor might be related to ZSS clinical efficacy.