The lower limits of quantification were 0.1, 0.1, and 0.2 ng/mL for dexmedetomidine, dezocine, and midazolam in rat plasma, respectively. The analytes were determined with selected reaction monitoring under positive ionization mode. The intra- and interday precision and accuracy were all within acceptable limits during the entire validation, and the stability of analytes was acceptable under various storage conditions. The validated method was successfully applied in pharmacokinetic studies of dexmedetomidine, dezocine, and midazolam following intravenous injection.A simple, sensitive, and accurate LC-MS/MS method was established and validated for the simultaneous quantification of dexmedetomidine, dezocine, and midazolam in rat plasma. Chromatographic separation was achieved on a C18 column (50 mm × 2.1 mm, 3  In intensive care unit (ICU), it is becoming increasingly important to measure and manage delirium, pain, and agitation in adult patients. Actually, guidelines for the treatment of delirium, pain, and agitation in critically ill patients do not provide recommendations on pharmacologic treatment , 2. For But up to now, being lack of enough attention and over emphasis on efficacy, the pharmacokinetic characteristics of dexmedetomidine, dezocine, and midazolam combination in ICU patients were still unclear. In the previous studies, they had been reported for the pharmacokinetic study of dexmedetomidine , dezocinThe objective of the present study was to establish a simple, sensitive, and accurate LC-MS/MS method for simultaneous determination of dexmedetomidine, dezocine, and midazolam in rat plasma and investigate the pharmacokinetic characteristics of dexmedetomidine, dezocine, and midazolam in rats. Then, the method would lay the foundations for monitoring of dexmedetomidine, dezocine, and midazolam in patient plasma in ICU.Dexmedetomidine and midazolam were obtained from Sigma-Aldrich Chemical Company . Dezocine and carteolol (IS) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products . HPLC-grade methanol and acetonitrile were obtained from Tedia . HPLC-grade formic acid was purchased from Kermel Chemical Reagent Co., Ltd. . All other chemicals were of analytic grade or better.µm; Thermo Scientific, USA) that was maintained at 25°C. The water (containing 0.1% formic acid and solvent A) and acetonitrile (solvent B) was used as mobile phase with gradient elution. The gradient was as follows: 0 min 5% B, 0.5 min 5% B, 2.0 min 80% B, 2.1 min 5% B, and 3.0 min 5% B, and then stopped. The flow rate was set at 0.4 mL/min. The select reaction monitoring (SRM) conditions (source parameters) were defined as follows: spray voltage 3.5 kV, vaporizer temperature 250°C, and capillary temperature 300°C. The optimized mass spectrometric parameters and mass spectra for all the analytes are shown in A TSQ Quantum Ultra mass spectrometric detector with electrospray ionization (ESI) interface  was coupled with a Dionex Ultimate 3000 ultra-performance liquid chromatography system consisting of an LPG-3400SDN pump, a WPS-3000TSL autosampler, and a TCC-3000RS column compartment. The separation of analytes and IS was achieved by using a Hypersil GOLD C18 column  at final concentrations of 0.1, 0.5, 1.0, 10.0, 20.0, and 100 ng/mL for dexmedetomidine; 0.1, 0.5, 1.0, 10.0, 20.0, and 100 ng/mL for dezocine; and 0.2, 1.0, 2.0, 20.0, 40.0, and 200 ng/mL for midazolam. The quality control (QC) samples were prepared in blank rat plasma at four different concentration levels, lower limit of quantification , low QC (0.2/0.2/0.4 ng/mL), medium QC (5.0/5.0/10.0 ng/mL), and high QC (80.0/80.0/160 ng/mL), for dexmedetomidine, dezocine, and midazolam, respectively.Standard mixed stock solutions of dexmedetomidine, dezocine, and midazolam were prepared in DMSO at 1.0, 1.0, and 2.0 mg/mL, respectively, and then serially diluted with methanol to obtain the working solutions. The carteolol (IS) stock solution of 20 mg/mL was prepared in double-distilled water and further diluted with methanol to 1.0 μL of rat plasma was mixed with 10 μL of IS solution (1.0 μg/mL) and 550 μL of ethyl acetate. Then, the extraction was carried out by vortex mixing for 3 min and centrifuging at 14,000 rpm for 10 min. The organic phases were transferred into clean polythene tubes and evaporated to dryness with a gentle nitrogen stream at 40°C. The residues were reconstituted in 100 μL of methanol followed by vortex mixing. After centrifuging at 14,000 rpm for 10 min at 4°C, 10 μL of the supernatant was injected onto the LC-MS/MS system for analysis.Frozen plasma samples were thawed at room temperature and vortexed for 1 min. 100 A full method validation of the assay for the analyte determination in rat plasma was performed according to the China Food and Drug Administration guidelines for the preclinical pharmacokinetic study with respect to selectivity, linearity, precision, accuracy, recovery, matrix effect, stability, and dilution integrity .Six male Sprague-Dawley rats weighing 180–220 g were supplied by the Shandong Laboratory Animal Center of Shandong Academy of Medical Sciences (certificate number SCXK (Shandong) 2014-0007). Rats were housed in environmentally controlled quarters (about 23°C–25°C) under a 12 h: 12 h light-dark cycle for at least 1 week before experiment. All rats were fasted but with freely available water for 12 h prior to pharmacokinetic study. The animal welfare and experimental procedures were approved by the Animal Ethics Committee at Institute of Materia Medica, Shandong Academy of Medical Sciences .μg/kg), dezocine (80 μg/kg), and midazolam (400 μg/kg) after simultaneous intravenous administration through tail vein to healthy SD rats (n=6). Blood samples were drawn into heparinized tubes before and at 2, 5, 15, 30, 45, 60, 120, 180, 240, 360, and 480 min after a single intravenous administration. The samples were immediately centrifuged at 3500 rpm for 10 min at 4°C. The plasma was separated and stored below −20°C until LC-MS/MS analysis [The established LC-MS/MS method was applied for the plasma concentration determination of dexmedetomidine  was chosen to achieve satisfactory chromatographic results. The mobile phase consisted of water (containing 0.1% formic acid) and acetonitrile with gradient elution, which played a great role in achieving good peak symmetry with appropriate retention time and high MS sensitivity.After lots of preliminary experiments, a Hypersil GOLD C18 column  were 2.14, 2.02, 2.30, and 1.86 min, respectively, and all samples were found to have no interferences from endogenous substances at the retention time of the analytes. The SRM chromatograms of the three analytes and IS are shown in y) versus concentration (x). The LLOQ was determined as the lowest concentration with a signal-to-noise (S/N) ratio of 10, and the accuracy was between 80.00 and 120.00% by analyzing six replicates. The linear ranges, correlation coefficients, weight factors, regression equations, and LLOQs for all analytes are summarized in To evaluate linearity, the calibration curves for each analyte were prepared and assayed from the ratio of the analyte peak area to the IS peak area (n=6), which could cover the whole range of the calibration graph. For dexmedetomidine, dezocine, and midazolam, the intraday and interday accuracy was between 92.60 and 119.00%, and the intraday and interday RSD was <14.28% (The QC samples at four concentration levels (lower limit of quantitation (LLOQ), low, medium, and high) were selected and analyzed to evaluate the intraday and interday precision and accuracy on three consecutive days  with the responses of dexmedetomidine, dezocine, and midazolam from the samples spiked after extraction at the same level. The matrix effect was measured via comparison of MS responses from the samples spiked after extraction with the responses from neat samples at the same concentration. The results showed that they were all within the acceptable limit for extraction recovery, and there was no significant matrix effect on dexmedetomidine, dezocine, and midazolam in this method .The stability of analytes in rat plasma was evaluated at the following temperature and timing conditions: 6 h at ambient temperature, 10 d at −20°C, three freeze-thaw cycles, and 24 h at 4°C in autosampler. The measured concentrations (QC samples) of dexmedetomidine, dezocine, and midazolam were all within acceptable limits during the entire validation .Six replicate samples, with the concentrations of 500, 500, and 1000 ng/mL for dexmedetomidine, dezocine, and midazolam, respectively, were diluted 10-fold with blank rat plasma. The diluted samples were analyzed and the estimated accuracy and precision were compared to the theoretical value. The precision (RSD) was less than 15%, and the accuracy was within 85–115% for all the analytes.(0–8 h), MRT(0–8 h), T1/2z, C2 min, Vd, and CLz/F are summarized in The plasma concentration-time profiles of dexmedetomidine, dezocine, and midazolam following single intravenous administration in SD rats are presented in In the present study, a simple, sensitive, and accurate LC-MS/MS method for the simultaneous quantification of dexmedetomidine, dezocine, and midazolam in rat plasma has been developed and validated, and then the LC-MS/MS assay was successfully applied to the pharmacokinetic studies of dexmedetomidine, dezocine, and midazolam in rats after combination administration. A simple liquid-liquid extraction process was used, and the total run time was no more than 3.0 min. The results were of great importance and valuable reference due to providing preliminary nonclinical evidence from animals after a single dosing. The method was probably used to determine the concentration of dexmedetomidine, dezocine, and midazolam in patient plasma in ICU.


Eryngium caeruleum, Notholirion thomsonianum and Allium consanguineum have been aimed for the scientific verification of their purported traditional uses against various infectious diseases.Herbal medicines have long been used for various ailments in various societies and natural bioactive compounds are gaining more and more importance due to various factors. In this context, three plant species i.e., Enterococcus faecalis, Proteus mirabilis, Escherichia coli, Salmonella typhi, Klebsiella pneumonia and Pseudomonas aeruginosa. The antifungal potential was investigated by dilution method of Muller-Hinton agar media of the plants’ samples. The fungal strains used were Aspergillis fumigatus, Aspergillis flavus and Aspergillis niger. Ceftriaxone and nystatin were used as standard drugs in antibacterial and antifungal assays respectively.In this study, three plants were assayed for antibacterial and antifungal potentials. The antibacterial investigations were performed via well diffusion method and nutrient broth dilution method. The bacterial strains used in the study were N. thomsonianum were tested against five bacterial strains while the samples from A. consanguineum and E. caeruleum were tested against six bacterial strains. All the samples exhibited prominent antibacterial activity against the tested strains. Overall, chloroform and ethyl acetate fractions were found most potent among the three plants’ samples. N. thomsonianum excelled among the three plants in antibacterial activity. Similarly, in antifungal assay, N. thomsonianum exhibited strong antifungal activity against the fungal strains. The chloroform fraction displayed MFCs of 175.67 ± 5.20***, 29.33 ± 5.48*** and 63.00 ± 4.93*** μg/ml against Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger respectively. The whole study demonstrates that all the three plant species were active against tested bacterial and fungal strains.Different fractions from N. thomsonianum, A. consanguineum and E. caeruleum have broad antibacterial and antifungal potentials. In all of the plants’ samples, chloroform and ethyl acetate fractions were more active. Furthermore, being the potent samples, the chloroform and ethyl acetate fractions of these plants can be subjected to column chromatography for the isolation of more effective antimicrobial drugs.It can be concluded from our findings that  Morus alba L., Morus mongolica Schneider, Broussnetia papyrifera (L.) Vent, Sophora flavescens Ait and Echinosophora koreensis Nakai have been reported to possess strong antibacterial potentials [Cananga odorata and Mitrephora diversifolia respectively have also been reported to possess strong antifungal activities [Herbal therapies have long history for their use in various ailments. Being comparatively harmless, the natural products have attracted the focus of innovative researchers in the treatment of various challenging diseases , 2. Micrtentials . Similartivities –24. Varitivities .Eryngium caeruleum belongs to the family Apiaceae. Several species of this genus have been reported to possess antimicrobial activities [Notholirion thomsonianum belongs to the family Liliaceae. This plant has been used ethnomedicinally for the treatment of various infectious diseases especially intestinal [Allium consanguineum belongs to the family Amaryllidaceae. A wide variety of species of Allium genus have been reported to possess notable antimicrobial properties in which the onion and garlic are the prominent candidates [N. thomsonianum and A. consanguineum were exploited for extraction due to ethnomedicinal use of rhizome. Secondly, the rhizomes and underground part of multiple plants of the families of these plants have been reported to be rich sources of numerous bioactive compounds i.e., Allium sativum and Allium cepa [E. caeruleum were employed for extraction. Therefore, the current study was aimed to evaluate the antibacterial and antifungal potentials of Eryngium caeruleum, Notholirion thomsonianum and Allium consanguineum and scientifically validate its folkloric uses.tivities . Similartestinal . Likewisndidates –30. The ium cepa . Likewisium cepa . TherefoE. caeruleum, N. thomsonianum and A. consanguineum were collected from Malakand division, KPK, Pakistan, located at Latitude: 34.5030° N and Longitude: 71.9046° E and authenticated by Dr. Nasrullah at Department of Botany, University of Malakand, Pakistan. Sample of each plant is submitted/stored in the herbarium of University of Malakand, Pakistan. The voucher numbers given by the herbarium officials are H.UOM.BG.109 (E. caeruleum) H.UOM.BG.106 (N. thomsonianum) and H.UOM.BG.158 (A. consanguineum). The rhizomes of the N. thomsonianum and A. consanguineum were isolated from the plants weighing approximately 3 kg each, divided into small pieces and shade dried for 2 to 3 weeks. Likewise, the aerial parts of E. caeruleum were collected and dried in the shade. After drying, each plant materials were cut using a cutter mill into a powder and macerated in 80% methanol for 2 weeks. After individual soaking these plant samples were filtered using Whattman filter paper (Whatman no. 1). The filtrates were evaporated using rotary evaporator  at 40°C under reduced pressure [E. caeruleum, N. thomsonianum and A. consanguineum were obtained weighing approximately 400 g each.The plants used in this research, i.e. pressure , 34. SemE. caeruleum, N. thomsonianum and A. consanguineum (300 g each) were suspended separately in 500 ml of distilled water in separating funnels and diluted with 500 ml of n-hexane. After vigorously shaking, all the three extracts were allowed to separate into two distinct layers. The upper n-hexane layer was collected and the same procedure was repeated until colorless n-hexane layer was obtained. After the collection of n-hexane fraction of each plant, it was fractionated with other solvents with increasing polarity i.e., chloroform, ethyl acetate and at last the aqueous fraction was obtained. The weights obtained for n-hexane, chloroform, ethyl acetate and aqueous fractions of E. caeruleum were 62 g (20. 66%), 37 g (12.33%), 59 g (19.66%) and 110 g (36.66%) respectively. Similarly, the weights obtained for n-hexane, chloroform, ethyl acetate and aqueous fractions of N. thomsonianum were 69 g (23%), 45 g (15%), 51 g (17%) and 81 g (27%) respectively. Likewise, the weights obtained for n-hexane, chloroform, ethyl acetate and aqueous fractions of A. consanguineum were 45 g (15%), 37 g (12.33%), 64 g (21.33%) and 96 g (32%) respectively [The successive solvent-solvent extraction procedure was followed for the fractionation of these plants samples. The crude methanolic extracts of ectively –38.Enterococcus faecalis (stools), Esherichia coli (UTI), Proteus mirabilis (UTI), Pseudomonas aeruginosa (Burn patient skin), Klebsiella pneumoniae (UTI), Salmonella typhi (Stools). All bacterial and fungal strains were kindly donated by Department of Microbiology, Quaid-e-Azam University, Islamabad, Pakistan. These were identified by various biochemical tests and were kept at 4°C in agar slants in freeze-dried condition until later use [Aspergillus fumigates (patient’s sputum), Aspergillus niger (grapes) and Aspergillus flavus (soil) were used.Antibacterial potential of plants’ samples were investigated against ater use . The fun8 CFU/ml were prepare by comparing with McFarland standard No 2 and was later on diluted to a cell density of 1 × 106 CFU/ml through double beam UV-visible spectrophotometer (Thermo electron corporation USA) at 625 nm. Fungal strains were grown at 25°C and suspensions corresponding to 2.5 × 104 cells ml − 1 were prepared by dilution in normal saline. Standardization of fungal strains were done using microscopic enumeration with a cell-counting hematocytometer and optical density method as previously reported [The cultures of bacterial strains were prepared by incubating at 37°C for 24 h. The suspensions of bacterial strains with cell density of 1 × 10reported . Fungal reported .The well-diffusion method was used for the evaluation of antibacterial activity of various samples of the three plants , 43. NutIn the center one, ceftriaxone (1 mg/ml) was added which served as positive control while in the rest of wells, the plant samples were added. The petri dishes were kept at 37°C in BOD incubator for 24 h. After incubation, the zone of inhibition of each sample was measured in mm. All the samples were run in triplicate and the data obtained was expressed as mean ± SEM.For the determination of minimum inhibitory concentration (MICs), the broth dilution method was employed. Briefly, stock solution having the concentration of 50 mg/ml was prepared in sterile distilled water. Various dilutions were prepared from the stock solution ranging from 0.125 to 10 mg/ml. Nutrient broth media was prepared in sterile water and sterilized in autoclave. The media prepared was inoculated with various strains in separate conical flasks. A few ml of inoculated media was transferred aseptically into properly labeled test tubes under laminar flow hood and the test samples were added to them. The test tubes were incubated at 37°C for 24 h. After incubation, the test tubes were observed for turbidity which is directly related to the growth of bacterial strains. Similarly, the MICs of each plant’s sample were recorded as the maximum concentration at which no turbidity was observed –46.A. fumigatus, A. niger and A. flavus. The plants’ samples of various dilutions (62.5 μg/ml to 10 mg/ml) were prepared in DMSO. Fungal media, i.e. muller hinton agar media was prepared in sterile water and autoclaved. A few ml of the prepared media was transferred to the labeled test tubes and 1 ml of plants’ sample was added to each of them. The test tubes were inoculated with test strains and incubated in BOD incubator at 25°C for 8 days. After incubation, the test tubes were observed for fungal growth. The minimum fungicidal concentrations (MFCs) were recorded as the maximum concentration of the sample at which no fungal growth was observed in test tubes. All the procedure was performed in triplicate and the nystatin was employed as positive control [Antifungal activity was carried out for the plants’ samples against  control , 48.P values less than or equal to 0.05 were considered statistically significant. The standard error of mean (SEM) were calculated at 95% confidence intervals.Two-way ANOVA followed by Bonferroni’s multiple comparison test was applied for the comparison of positive control with the test groups. N. thomsonianum exhibited the highest inhibitory zones against the tested strains. The Nt.Cf exhibited 31.67 ± 0.67, 29.33 ± 0.88, 26.33 ± 0.33, 25.67 ± 0.67 and 29.33 ± 0.88 mm zone of inhibitions (ZOI) against P. mirabilis, E. coli, S. typhi, K. pneumonia and P. aeruginosa respectively as shown in Table N. thomsonianum, Nt.EtAc and Nt.Cr also demonstrated notable antibacterial potentials. The least activity is attributed to the Nt.Hex fraction.The well diffusion method was used for the three plants against various bacterial strains. The crude extracts and sub-fractions were analyzed for their antibacterial effect by the determination of their inhibitory zones against each strain. Among the three plants, A. consanguineum also showed notable inhibitory zones against the tested strains. Among different samples of A. consanguineum, the Ac.EtAc and Ac.Cf displayed considerable ZOIs in comparision to the positive control. The Ac.EtAc demonstrated 15.50 ± 0.76, 12.66 ± 0.88, 6.83 ± 0.92, 19.16 ± 0.44, 20.83 ± 0.44 and 10.33 ± 0.60 mm ZOIs against E. faecalis, P. mirabilis, E. coli, S. typhi, K. pneumonia and P. aeruginosa respectively  of N. thomsonianum possess strong antibacterial and antifungal results. The MIC values of positive control and chloroform fraction were going considerably parallel i.e., if we cursorily observe the Table A. niger and A. flavus the chloroform fraction demonstrated MFC values of 63.00 ± 4.93 and 29.33 ± 5.48 μg/ml respectively, which are less than 100 μg/ml, which can be considered to be significant in the context of therapy of various diseases [Allium sativum L. (garlic), Allium ampeloprasum L. (elephant garlic) and Allium cepa have been reported to possess strong antibacterial and antifungal activities [Allium sativum which has been reported to be active against various pathogenic bacterial strains [Eryngium palmatum one of medicinally important specie of Eryngium has been verified to be active against microbes [Staphylococcus aureus strains significantly [E. caeruleum, A. consanguineum and N. thomsonianum are also prominent candidates of these genera. The study reveals that among the three plants, N. thomsonianum was rich in antimicrobial agents and secondly it may also be implied that the chloroform fraction was rich in such secondary metabolites which confer antimicrobial potential to this plant.Microbes are considered as the mainstay and origin of multiple diseases. Microbial infiltration into the body tissues and blood lead to various diseases some of which are difficult to treat and extremely lethal , 50. Thediseases . The restivities . Importa strains . In the microbes . Similarficantly . The resN. thomsonianum, A. consanguineum and E. caeruleum possess considerably sufficient antibacterial and antifungal potentials. It may also be concluded that antimicrobial potential of various samples of these plants might be due to wide variety of compounds present in these plants. Moreover, we observed that specifically the chloroform and upto some extent the ethyl acetate fractions of these plant contain considerably high activity. These two fractions (in each plant) are potent targets to be subjected to bio-guided isolation and exploration of novel natural antimicrobials.Based on our current investigations it can be concluded that


Studies have reported on the arthroscopic technique for release of external snapping hip syndrome. However, no study with large sample size has been reported for arthroscopic surgery.Patients with 229 bilateral and 19 unilateral external snapping hips were treated from January 2012 to June 2013. After locating the contracture position, arthroscopic surgery was performed accordingly. Preoperative and postoperative angles were compared.P < 0.001). Similarly, the flexion angle was also significantly improved for all the four types (P < 0.001). Excellent ratio and satisfaction rate were good in types I and II. All the clinical features were cured after arthroscopic surgery.Comparing range of motion, all patients obtained higher adduction and flexion angles. At postoperative follow-up of 24 months, the adduction angle was improved from −14.4 ± 5.14 to 35.7 ± 4.21 for type I, from −31.2 ± 5.22 to 31.7 ± 2.84 for type II, from −49.0 ± 3.47 to 21.6 ± 3.43 for type III, and from −64.5 ± 4.65 to 18.3 ± 3.10 for type IV (Arthroscopic surgery could be an effective procedure for external snapping hip, due to less operating time, small scar, fast postoperative recovery, and complete contracture release. Snapping hip syndrome (SHS) is characterized with an audible or palpable snap when flexing or extending the hip and sometimes can be associated with pain , 2. BaseCommonly, the first step of management of ESH is conservative. This consists of rest, avoiding movements that provoke snapping, stretching exercise, non-steroidal anti-inflammatory drugs (NSAID), and injections of steroid into the trochantric bursa , 7. OnceRecently, arthroscopic technique to release the ITB in patients with ESH was introduced with excellent contracture release and fast recovery , 11. In This study was approved by the Institutional Review Board of Shanghai Tenth People’s Hospital affiliated to Tongji University. Each subject provided his or her written informed consent.Between January 2012 and June 2013, a total of 248 patients  with ESH syndrome treated in our hospital by arthroscopic surgery were included in this study, based on the inclusion criteria. Patients mainly presented themselves with complaints of snapping, clicking or popping sound heard when squatting from the standing position or during jugging at the lateral upper thigh over the area of GT, sometimes accompanied by pain. Furthermore, all patients experienced this disorder from repeated injection on hip during their childhood. Inclusion criteria were set as follows: (1) all patients diagnosed with ESH by history and physical examination, (2) all patients aged between 16 and 40 years old, (3) all patients received the contracture release under arthroscopy, (4) a minimum of 2-year follow-up, and (5) available data for hip assessment. Exclusion criteria were (1) the presence of intra-articular disease, (2) the presence of bony deformities, and (3) the patients with chronic disease or infections.All patients experienced failed conservative treatment which is used for each patient for a period of at least 3 months before surgery. Passive and active stretching was applied to increase muscle length. Eccentric control is trained to modify neuromuscular control to allow muscle lengthening. Modification of movement patterns (gains) and consistent stretching can be helpful to prevent recurrence. Rest, icing, and anti-inflammatories were advocated to avoid inflammation of tissues.Before surgery, we performed the preoperative evaluation including physical and radiographic examinations. Preoperative range of motion of hip was measured Fig. . A hip MAll the arthroscopy operations were performed under general anesthesia. Patient was placed in lateral decubitus position on a standard operating table. The hip being flexed, adducted, and internally rotated to the maximum possible degree without traction was the position of the operation. Standard sterile draping was done. Two portals were approximately 3–4 cm apart and were marked over the GT. For portals, oblique incision of 3 mm in size was made on the skin and subcutaneous tissue. A standard 30° scope with a diameter of 4 mm was inserted through proximal portal at a 30° angle Fig.  was initAfter surgery, non-steroidal anti-inflammatory drugs and ice therapy were used for pain release for just 3 days postoperatively. The patients were encouraged to flex the hip and knee joint and cross the legs. The rehabilitation program was suggested to achieve rapid recovery until 6 months postoperatively. Postoperative rehabilitation was same for each patient. Patients were evaluated preoperatively and at 3, 6, 12, and 24 months postoperatively. At each follow-up, physical examination and questionnaire were performed. If subjects could obtain completely recovered, they were identified as excellent for type I or an adduction angle of hip that increased >30° for type II, 45° for type III, and 60° for type IV.t test. For ratio comparison, chi-square was performed. A P value <0.05 was considered statistically significant.All statistical analyses were performed using SPSS 19.0 software. All preoperative and postoperative indices were compared by a paired 2 (range 17.7–29). Median duration of symptom was 10 years (range 1 month–30 years). Among the 248 patients, 76 were diagnosed with type I, 83 with type II, 55 with type III, and 34 with type IV.In this study, there were 248 patients who received arthroscopic surgery to treat ESH , and vascular injury. No infections occurred in the series. Moreover, there was no major swelling, hematomas, and wound dehiscence in these cases. All patients could sit with their legs crossed Fig. . NeitherP < 0.05) (Table 5). The excellent ratio in type I [76/76 (100%)] and type II [83/83 (100%)] was higher than in type III [51/55 (92.7%)] and type IV [25/34 (73.5%)]. The satisfaction rate was higher in types II [67/69 (97.1%)] and III [53/55 (96.4%)] than in type I [73/76 (96.1%)]. Although the satisfaction rate of type IV [32/34 (94.1%)] is comparatively lower than in other three types, patient compliance were very good and happy with the results.The outcome of surgery in type I and type II were significantly higher than in type III and IV patients (In the current study, we included a total of 248 patients to investigate the outcomes of treatment of ESH under arthroscopy. After surgery, we found all patients received the contracture released. Although outcomes in types I and II are significantly higher than those in types III and IV, there is no difference with the patients’ satisfaction in four types.ESH syndrome was first reported by Valderrama in 1969 . AlthougCommonly, a program of conservative management for systematic ESH is applied first. In case that conservative therapy failed to treat with ESH, a variety of surgical techniques are attempted with variable success. Zoltan et al.  performeHowever, there are many complications for open techniques, including large scar, wound complication, and slow recovery. Arthroscopic release of the ESH has become more common. Ilizaliturri et al.  reportedSome limitations should be mentioned. First, inherent bias of retrospective analysis might be inevitable. Moreover, a lack of effective assessment for ESH resulted in difficulty in comparison with other studies. Finally, comparison between open and arthroscopic surgery was not performed in this study.In the present study, arthroscopic surgery could be an effective procedure for ESH, due to less operating time, small scar, fast postoperative recovery, and complete contracture release. These promising results of arthroscopic treatment of ESH need randomized trial to compare with open procedures.


Bothrops moojeni snake venom. The toxin was purified by a combination of three chromatographic steps . BmooPAi was found to be a single-chain protein with an apparent molecular mass of 32 kDa on 14% SDS-PAGE, under reducing conditions. Sequencing of BmooPAi by Edman degradation revealed the amino acid sequence LGPDIVPPNELLEVM. The toxin was devoid of proteolytic, haemorrhagic, defibrinating, or coagulant activities and induced no significant oedema or hyperalgesia. BmooPAi showed a rather specific inhibitory effect on ristocetin-induced platelet aggregation in human platelet-rich plasma, whereas it had little or no effect on platelet aggregation induced by collagen and adenosine diphosphate. The results presented in this work suggest that BmooPAi is a toxin comprised of disintegrin-like and cysteine-rich domains, originating from autolysis/proteolysis of PIII SVMPs from B. moojeni snake venom. This toxin may be of medical interest because it is a platelet aggregation inhibitor, which could potentially be developed as a novel therapeutic agent to prevent and/or treat patients with thrombotic disorders.This work reports the purification and functional characterization of BmooPAi, a platelet-aggregation-inhibiting factor from Sever lectins .SVMPs comprise a group of zinc-dependent enzymes of varying molecular mass, widely distributed in Viperidae venoms. They are synthesized as multidomain precursors and stored in the venom gland as inactive zymogens , 9–11. SαIIbβ3 platelet integrin , blocking the last phase of platelet aggregation and clot formation [Several studies have investigated SVMPs as platelet aggregation inhibitors due to their specificity to platelet integrins , 14–18. ormation . Therefoormation . Unlike ormation . Bothrops moojeni snake venom, which showed an inhibitory effect on platelet aggregation.In the present study, we describe the isolation and functional characterization of BmooPAi, a toxin comprised of the dis-cys domain, originating from autolysis/proteolysis of PIII SVMPs from B. moojeni venom was purchased from Bioagents Serpentarium . Acrylamide, ammonium bicarbonate, ammonium persulphate, azocasein, bromophenol blue, ethylenediaminetetracetic acid (EDTA), bovine fibrinogen, glycine, β-mercaptoethanol, N,N′-methylene-bis-acrylamide, sodium dodecyl sulphate (SDS), N,N,N′,N′-tetramethylethylenediamine (TEMED), and Tris were purchased from Sigma Chemical Co. . Molecular weight markers for electrophoresis and all chromatographic media  were purchased from GE Healthcare Technologies . All the agonists used in the platelet aggregation assays , and ristocetin) were purchased from Helena Laboratories . All other reagents used were of analytical grade.DesiccatedMale Swiss mice (20–25 g) and male Wistar rats (200–250 g) were provided by the Centro de Bioterismo e Experimentação Animal (CEBEA) at the Federal University of Uberlândia . The animals were maintained under conditions of controlled temperature (22 ± 2°C) and light/dark cycle (12 hours) with free access to food and water. The experimental protocol was approved by the Committee for Ethics in Animal Experimentation of the Federal University of Uberlândia (CEUA/UFU), Minas Gerais, Brazil (Protocol number 108/12).The experiments were performed in accordance with current guidelines for human research, established by the Committee for Ethics in Human Research of the Federal University of Uberlândia, Minas Gerais, Brazil . Blood was obtained by blood donation from 10 individuals who were invited to participate in the research as volunteer donors. The criteria for the selection of donor volunteers included no signs or symptoms of disease, malnutrition, or dehydration; age between 18 and 65 years; weight more than 50 kg; no use of any medication that interferes with haemostasis; no use of illicit drugs; no alcohol consumption in the last 24 hours preceding the experiment; and no haemostasis disorders. B. moojeni (200 mg) was dissolved in 2.0 mL of 0.05 M ammonium bicarbonate buffer (pH 7.8) and clarified by centrifugation at 10,000 ×g for 10 min. The supernatant was applied to a DEAE-Sephacel column (2.5 × 20 cm) previously equilibrated with 0.05 M ammonium bicarbonate buffer (pH 7.8). Chromatography was carried out at a flow rate of 20 mL/h, with a linear concentration gradient of the same buffer (0.05–0.6 M), and fractions of 3.0 mL/tube were collected. All peaks were monitored by measuring absorbance at 280 nm on a spectrophotometer . The seventh peak, designated D7, was pooled, lyophilised, and applied to a Sephadex G-75 column (1.0 × 100 cm) previously equilibrated with 0.05 M ammonium bicarbonate buffer (pH 7.8). The samples were eluted from this column with the same buffer, at a flow rate of 20 mL/h, and fractions of 3.0 mL/tube were collected. The second fraction, designated D7S2, was pooled, lyophilised, and submitted to the third step of separation using a HiTrap Heparin HP column (5 × 1 mL) in an ÄKTApurifier™ HPLC system, previously equilibrated with 20 mM Tris-HCl buffer (pH 7.0) containing 5 mM calcium chloride. The samples were eluted with an increasing concentration gradient of 20 mM Tris-HCl buffer (pH 7.0) containing 2.0 M sodium chloride. Elution was carried out at a flow rate of 30 mL/h; fractions of 1.0 mL/tube were collected and the absorbance was read at 280 nm. Isolated BmooPAi was concentrated in the major peak. To evaluate the degree of purity, isolated BmooPAi was passed through a reverse-phase C2/C18 column (4.6 × 100 mm) using the ÄKTApurifier HPLC system. The column was equilibrated with solvent A (0.1% trifluoroacetic acid) and eluted with a linear concentration gradient from 0 to 100% of solvent B  at a flow rate of 0.3 mL/min. Absorbance was monitored at 280 nm.Protein isolation was carried out in three steps. The crude venom ofμL) was determined from linear regression calculations based on the values obtained from the standard curve. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) was performed using 14% (w/v) gels. Electrophoresis was carried out at 20 mA/gel in Tris-glycine buffer (pH 8.3) containing 0.01% SDS. The molecular mass standard proteins used included phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and α-lactalbumin (14.4 kDa). Gels were stained with Coomassie blue R-250, 0.2% (w/v). The relative molecular mass of BmooPAi was estimated using Kodak 1D image analysis software.The protein concentration was determined using the method established by Bradford . DetermiThe sequencing was performed in a membrane obtained from the purified protein in solution. The liquid sample was loaded onto an Applied Biosystems Prosorb™ device. The protocol for sample preparation using this device was followed, and the Prosorb membrane was loaded onto the instrument for sequence analysis.μL of bovine fibrinogen  were incubated with 20 μg of BmooPAi for 120 min at 37°C. The reaction was stopped by the addition of a denaturing buffer containing 10% (v/v) glycerol, 10% (v/v) β-mercaptoethanol, 0.2% (w/v) SDS, and 0.001% (w/v) bromophenol blue (pH 6.8). Reaction products were analysed using 14% (w/v) SDS-PAGE.Fibrinogenolytic activity was assayed as previously described , with brμg BmooPAi were supplemented with 200 μL of saline and incubated for 60 min with 800 μL of azocasein solution (1 mg/mL). The reaction was stopped by the addition of 200 μL of 15% trichloroacetic acid to precipitate the undegraded azocasein. The mixture was left standing for 20 min before centrifugation at 10,000 ×g for 10 min. The proteolytic activity was estimated by reading the absorbance of the clear supernatant at 405 nm. One unit of azocaseinolytic activity was defined as the amount of enzyme that produced an absorbance increase of 0.01 units.Samples of 20 μg BmooPAi diluted in 25 μL of saline. After three hours, animals were euthanized by an overdose of ketamine/xylazine, the skin was removed and the diameter of the haemorrhagic spot was measured on the inner surface. Control animals received the same volume of sterile saline.Haemorrhagic activity was determined as previously described , with soμg BmooPAi . Control animals received the same volume of sterile saline. After one hour, mice were euthanized by an overdose of ketamine/xylazine and bled by cardiac puncture. Whole blood was placed in tubes and kept at 25–30°C. Activity was determined by measuring the time to the onset of blood clotting.Defibrinating activity was carried out as previously described , with slμg/25 μL), the same volume of saline (negative control), or 0.2 mol/L calcium chloride (positive control) was added to 200 μL of citrated bovine PRP at 37°C. Clotting activity was determined by measuring the time to fibrin clot onset by a coagulometer (CLO Timer).Coagulant activity was assayed using bovine plasma. The plasma samples were mixed with 3.8% sodium citrate  and centrifuged at 2,500 ×g for 15 min to obtain platelet-rich plasma (PRP). BmooPAi  into the subplantar region of one hind paw. An equal volume of sterile saline was injected into the contralateral paw. The volume of each paw was measured using a plethysmometer  before and 1, 2, 3, 4, 5, 6, and 24 h after the injection. Results were calculated as the difference between each paw and expressed as the percentage increase in paw volume relative to the initial volume.Groups of three male Wistar rats received an intraplantar injection of BmooPAi  into the right hind paw. An equal volume of sterile saline was injected into the left hind paw for control. The weight in grams (g) required to elicit a nociceptive response, that is, paw flexion, was determined as the nociceptive threshold. A cut-off value of 300 g was used to prevent damage to the paws. The nociceptive threshold was measured before and 1, 2, 3, 4, 5, 6, and 24 h after BmooPAi injection using a Randall-Selitto apparatus . Results were calculated as the difference between each paw and expressed as the percentage decrease in nociceptive threshold relative to the initial threshold. To reduce stress, the rats were habituated to the apparatus one day before the experiment.Hyperalgesia was measured according to the paw pressure test, as previously described , with slμL of PRP maintained at 37°C under continuous stirring, in siliconized glass cuvettes. Aggregation was triggered with collagen (10 μg/mL), ADP (20 μM), or ristocetin (1.5 mg/mL) immediately after adding BmooPAi  to human PRP. Finally, 100% aggregation was expressed as the percentage absorbance relative to PPP aggregation. Control experiments were performed using only platelet agonists. All experiments were carried out in triplicate.Platelet aggregation assays were performed as previously described  in human B. moojeni snake venom.In recent years, a number of snake venom proteins that interfere with platelet function have been identified. Most of these proteins belong to the SVMP family, and their effects are probably due to the presence of dis-cys domains which interact with specific protein molecules on platelet membrane surfaces –29. In tα-1 [ B. moojeni snake venom. B. moojeni crude venom (200 mg) was applied to ion-exchange chromatography on a DEAE-Sephacel column, which produced eight main protein peaks, designated D1 to D8 ( B. moojeni). This fraction was devoid of proteolytic, haemorrhagic, defibrinating, or coagulant activities. In addition, BmooPAi did not cause hyperalgesia and oedematogenic responses in the hind paw when rats received a 50 μg intraplantar injection of the toxin. Thus, BmooPAi does not contribute to the local effects caused by B. moojeni envenomation.The toxin was isolated using a three-step procedure, including ion-exchange, gel filtration, and affinity chromatographies. Other toxins have been purified using similar procedures, including BmooFIBMP-I  and Bmooα-1 , both meD1 to D8 . The D7 D1 to D8 . The D7SD1 to D8 . The nonβ-mercaptoethanol , Bothropasin (GI: 209870468), Jararhagin (GI: P30431.1), Leucurolysin-B (GI: P86092.1), Brevilysin-H6 (GI: P0C7B0.2), Atrolysin-A (GI: Q92043.1), and AaH-IV (GI: 255917952) are PIII SVMPs that showed homology with BmooPAi; Jarastatin (GI: Q0NZX5.1), Jararhagin-C (GI: P30431.1), Leucurogin (GI: P0DJ87.1), and Salmosin (GI: O93518.1) are disintegrin or disintegrin-like proteins homologous to BmooPAi . in vitro, such as alkaline pH, low calcium, or the presence of reducing agents, generating intact biologically active polypeptides of ~30 kDa, which correspond to the dis-cys domain [PIII SVMPs comprise high molecular mass proteinases composed of a dis-cys domain following the proteinase domain. Some PIII SVMPs have an additional lectin-like domain linked to the others through a disulphide bond , 13. SVMs domain .Although BmooPAi showed high homology with many PIII SVMPs, it showed no haemorrhagic activity. These data suggest that this toxin originated from the proteolysis/autolysis of a PIII SVMP and comprises only the noncatalytic dis-cys domain. For this reason, BmooPAi was not able to induce haemorrhage due to the absence of the proteinase domain, preventing proteolysis of the capillary components. B. jararaca [ Agkistrodon acutus [Some PIII SVMPs homologous to BmooPAi, such as Bothropasin , Brevilyjararaca  and AaH-n acutus . Similarn acutus , in whicαIIbβ3 integrin (GPIIb-IIIa), α2β1 integrin, and GPVI and also their ligands, von Willebrand factor (vWF), fibrinogen, and/or collagen [μg of BmooPAi inhibited approximately 32% and 72% of ristocetin-induced platelet aggregation, respectively, whereas 50 μg completely inhibited platelet aggregation induced by ristocetin. On the other hand, BmooPAi was not able to inhibit the platelet aggregation induced by ADP and collagen, even at high concentrations (50 μg).The extant literature shows that several PIII SVMPs and processed dis-cys domains are able to interfere with platelet aggregation, including Jararhagin , Jararhacollagen , 45. In  in vitro, even at static conditions. This interaction plays a key role in the initial adhesion of platelets to the subendothelium, which in turn induces a complex cascade of signals which results in platelet aggregation [ in vitro [According to Coller and Gralnick , ristoceregation . Since tin vitro . The impin vitro  and von in vitro . in vitro vWF-GPIb binding)-induced platelet aggregation have been isolated and characterized from the venom of several snake species [A number of haemostatically active proteins that inhibit ristocetin , a toxin comprised of the dis-cys domain, originating from autolysis/proteolysis of PIII SVMPs from


The antifungal properties of O-CSPX-M against Phytophthora capsici (P. capsici), Verticillium alboatrum , Botrytis cinerea (B. cinerea) and Rhizoctonia solani (R. solani) were also assayed. Apparently, chitosan metal complexes showed enhanced antifungal activity against four fungi at the tested concentrations compared to that of chitosan. It was shown that Cu complexes can inhibit the growth of P. capsici 100%, and Ni complexes can inhibit the growth of B. cinerea 77.1% at a concentration of 0.4 mg/mL and 0.2 mg/mL, respectively. The pot experiment also verified the result. In addition, the phytotoxicity experiment showed that O-CSPX-M had no obvious toxicity on wheat leaves. This kind of complexes may represent as an attractive direction for chemical modifications of metal fungicides.Based on a condensation reaction, a chitosan-derivative-bearing amino pyridine group was prepared and subsequently followed by coordination with cupric ions, zinc ions and nickel ions to synthesize chitosan metal complexes. The calculations using the density functional theory (DFT) show that the copper ions and nickel ions underwent dsp After that, the synthesis and application of organometallic compounds have been extensively studied, and considerable progress has been made. For example, oxaliplatin and nedaplatin have been widely used as antitumour drugs, and Ziegler-Natta catalysts greatly simplify the conditions for olefin polymerization. In recent years, organometallic complexes have been widely applied in many new fields, such as energetic materials1, photosensitizers2 and fungicides3.Organic metal complexes are an important branch of organic chemistry research. Many organic ligands exhibit excellent activity after their coordination with metal ions. Early in 1817, the first organometallic compound, Zeise salt  linkage17. Hence, the chitosan Schiff base metal complex with novel characteristic may have significant antifungal activity.Schiff base is a type of organic compound with good biological activity and can be coordinated with a large number of metal ions to form complexesPhytophthora capsici, Verticillium alboatrum, Botrytis cinerea and Rhizoctonia solani are plant pathogens. They can cause diseases to a variety of vegetables, fruits and crops. The control of these threatened fungi is necessary. Therefore, the antifungal properties of metal complexes against these four kinds of fungi were evaluated.1H NMR, 13C NMR, elemental analysis and XRD, and then proofed using Gaussian09.As shown in Fig. −1 to 3500 cm−1 of CS could be considered as -OH and -NH. The characteristic absorbance band of =C-H was observed at 3007 cm−1. The NH2 bending vibrations was shown at 1593 cm−1. In the spectrum of O-CSPX, an extra peak appeared at 1667 cm−1, which is the absorption peak of the stretching vibrations of C=O. The absorption peak of pyridyl appeared at 1525 cm−1, and the C=C was observed at 1730 cm−1. The band at 1409 cm−1 and the band at 1632 cm −1, respectively, display the existence of C-N and C=N. The absorbance bands at 3305 cm−1 and 1209 cm−1 were attributed to the -OH group in the ring system.Figure −1 in the spectrum of O-CSP1-Cu. Due to the coordination of the Cu(II) ions with the p-π conjugate group, the C=C vibrations of metal complexes shifted to a higher wave number in comparison of O-CSPX19. One additional signal was observed at 1397 cm−1 and was attributed to the carbonyl groups of CH3COO−.As shown in Fig. 1H NMR spectra of the CS and O-CSPX are shown in Fig. 21: (1) δ = 3.34 ppm: H2; (2) δ = 3.89–3.95 ppm: H5, H6; (3) δ = 4.07–4.09 ppm: H3, H4; (4) δ = 4.98 ppm: H1; (5) δ = 4.50 ppm: = C-H; (6) δ = 6.75, 6.87, 7.63, 7.76 ppm: pyridyl-H.The 22.24:m is the C/N of the O-CSPX, (C/N)o is the C/N of the CS, and a and n are the amount of nitrogen and carbon introduced after modification, respectively.The degree of deacetylation (DD) of CS was calculated using the equation given belowThe results of elemental analysis are shown in Supplementary Table 13C NMR spectra of the CS and O-CSPX are shown in Fig. The The water solubility of chitosan is poor. This is due to the existence of a large number of hydrogen bonds in the molecule, which restricts the free rotation of the units in each group. From Fig. 25. The results are shown that the acetic acid aqueous solution would not enhance the antifungal efficacy at concentrations lower than 0.125 mg/mL  acetic acid at an original concentration of 8% (w/v). Because acetic acid can enhance the antifungal action, in order to exclude the effect of acetic acid on the experimental results, we determined the inhibitory effect of acetic acid against four fungi under different concentrationsL Figure .P. capsici, V. alboatrum, B. cinerea and R. solani are shown in Table P. capsici and B. cinerea. The inhibition rate of O-CSPX-M against R. solani ranged from 31.84% to 64.80%, and the antifungal activity of O-CSPX-Cu was better than the others. Moreover, its inhibitory effect was much better than that of Cuproxat at low concentrations.The antifungal activities of the metal complexes against P. capsici and V. alboatrum, and the highest antifungal index was 100% and 64.6%, respectively. The inhibition effect of nickel complexes against B. cinerea was relatively high, and the inhibition rate of nickel complexes could be higher than 77.11% against B. cinerea at a concentration of 0.4 mg/mL. The zinc complex showed weaker antifungal activity relative to the other two complexes, but the antifungal index was still higher than that of highly deacetylated chitosan. This shows that the antifungal activity of chitosan derivatives can be strengthened by grafting metal ions. As seen in Table As can be seen from Table 14, this is because the substituent on the pyridine ring is far from the complexation site, and the influence on the dihedral angle in molecular was very small. Hence, a super-conjugated system was formed in O-CSPX-M, and the bond energy of the coordination bond was enhanced.The antifungal results show that the antifungal activity of O-CSPX-M is hardly affected by the types of pyridyl. Combined with our prior research26. The results further confirm that metal ions grafted in the synthesized chitosan derivatives contribute a lot to the antifungal action.The bioassay results show that the O-carboxymethyl chitosan metal complexes can obviously inhibit the growth of fungi. They all show broad-spectrum antifungal activity. The antifungal activity of these metal complexes is attributed to their ability to trigger cell death when they are attached to negatively charged microbial cells causing irreversible damage to their cell membraneThe protective and curative activity of the chitosan derivatives are shown in Table 27. The Schiff base partially counteracts the growth inhibition effect of metal ions and reduces the damage caused by O-CSPX-M to plants.As is shown in Table 30. However, improper use of metal fungicides can affect crop growth. Based on past soil damage caused by pesticide abuse, the development of new organic chemicals with high-efficiency, low-toxicity and low metal content is imminent. In this study, we have proposed a straightforward synthetic route to novel metal complexes coordinated with chitosan Schiff bases. The antifungal effect of the metal complexes against P. capsici, V. alboatrum, B. cinerea and R. solani was evaluated. The results showed that all twelve metal complexes could effectively inhibit the selected fungi. The inhibition rate of some metal complexes is better than that of commercial pesticide Cuproxat at the same dose. Moreover, a phytotoxicity assay of O-CSPX-M was also conducted, and unlike the traditional metal fungicides, the twelve metal complexes were not significantly toxic to the leaves of wheat. O-CSPX-Zn can even increase chlorophyll content in wheat leaves at low concentration. This is mainly because chitosan itself promotes plant growth and counteracts the phytotoxicity of metal ions.Metal fungicides are widely used because of their inhibitory effects on many plant pathogenic fungi32. This is because the inner orbit of transition metal ions after coordination is filled with electrons, displayed as paramagnetism. Therefore, they cannot be analysed using NMR. Thus, in this study, we used density functional theory to calculate the specific configuration of the complexes. The calculations show that the copper ions and nickel ions underwent dsp2 hybridization, the zinc ions underwent sp3 hybridization, and they all formed a coordination bond with the carbon atom in the p-π conjugate group due to steric hindrance. The characterization of metal complexes provides an attractive direction for chemical modifications.For metal complexes, the hybrid forms and ligand fashion of metal ions are an important part of their structural analysis. However, many previous reports have focused on the activities, and few have paid attention to the ligand fashionIn conclusion, this study provides insights into the synthesis of metal fungicides from marine organisms. Compared to the metal fungicides used, the chitosan metal complexes had better antifungal activity, lower phytotoxicity and lower metal content. This kind of complexes may represent as an attractive direction for chemical modifications of metal fungicides.P. capsici, V. alboatrum, B. cinerea and R. solani) were provided by Qingdao Academy of Agricultural Sciences.Chitosan , was purchased from Qingdao Yun Zhou Biochemistry Co., Ltd. 2-Aminopyridine, 2-Amino-5-methylpyridine, 2-Amino-5-chloropyridine and 2-Amino-5-(trifluoromethyl)pyridine were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. The other reagents are all of analytical grade and used without further purification. Four crop-threatening pathogenic fungi  was performed using a Vario EL-III elemental analyser. All geometries were fully optimized at the B3LYP/6–31 + G  level and the B3LYP/Lanl2DZ level of theory with the Gaussian 09 suite of programs.FT-IR spectra were recorded on a Thermo Scientific Nicolet iS10 FT-IR spectrometer with KBr disks. 14 .First, four types of aminopyridine (0.05 mol) and acetylacetone  were added to ethanol (150 mL) and heated at 80 °C for 12 h. The solution was concentrated under reduced pressure. Then the orange crystal solid was washed with cold ethanol for three times, drying in the oven at 50 °C for 12 h14.Highly deacetylated chitosan was prepared as previously described in the literature14, 7.5 g of monochloroacetic acid in 20 mL of ethanol was added dropwise. The mixture was heated at 60 °C for 6 h. Then, the mixture was precipitated by the addition of excess ethanol and the precipitate was filtered. The products were washed with ethanol and dried at 60 °C for 6 h .First, CS (1.5 g) was added to 75 mL distilled water in a three neck flask, 60 mL ethanol and 1.5 mL of acetic acid were added, and the solution was stirred for 1 h. Then, the PX (two-fold that of CS) was dissolved in 50 mL of ethanol, and added dropwise into the three neck flask. After stirring for 15 h at 60 °C3COO) 2·H2O (1.1 g), Zn (CH3COO) 2·2H2O (1.2 g) or Ni (CH3COO) 2·4H2O (1.3 g) in 100 mL water was stirred for 6 h at 60 °C. The solution was precipitated with excess ethanol and centrifuged. The metal complexes were obtained after drying at 50 °C for 6 h. The product yield was 67.1% for O-CSP1-Cu, 66.2% for O-CSP2-Cu, 65.5% for O-CSP3-Cu, 50.1% for O-CSP4-Cu, 65.5% for O-CSP1-Zn, 75.2% for O-CSP2-Zn, 56.1% for O-CSP3-Zn, 54.7% for O-CSP4-Zn, 57.1% for O-CSP1-Ni, 49.9% for O-CSP2-Ni, 56.7% for O-CSP3-Ni and 47.4% for O-CSP4-Ni.A solution of 1.0 g O-CSPX and Cu . For curative activity, pepper seedlings were irrigated with the treatment as above at 24 h after irrigated with spore suspension (3.5 × 103). Then the irrigated plants were kept at 25 °C with 85% humidity for 7 days. The disease index and control efficacy were calculated34. Three pepper seedlings per pot and three pots per concentration were used.The protective and curative activity of O-carboxymethyl chitosan copper complexes against Triticum aestivum L. Jimai 22) seeds. After being sterilized, they were transferred to a Petri dish with moist gauze for germination at 25 °C for 24 h in the dark. Then, 2,736 germinated seeds were individually transplanted to 57 Petri dishes with nylon mesh, and each Petri dish contained 48 seeds. Wheat seedlings were cultivated in a growth incubator with a light intensity of 800 mol m−2 s−1, a day/night cycle of 14 h/10 h at 25 °C/15 °C, respectively, and the relative humidity was controlled at 70%. When the second functional leaf of wheat seedlings was fully expanded, the experiments were randomly divided into 33 groups, including a negative control group (sprayed with distilled water), 6 free metal ion groups , 2 positive control groups  and 24 metal complex groups (sprayed with 0.1 mg/mL and 0.2 mg/mL of O-CSPX-M solution), and each group had three replicates. The volume of distilled water, free metal solutions or O-CSPX-M solutions sprayed on each sample was 45 mL. Additionally, the wheat seedlings were cultured with Hoagland solution, and the solution was renewed every other day.The present study was conducted with wheat , chlorophyll b (Chl-b) and total chlorophyll (a + b) content were determined using a spectrophotometer at 665 nm and 649 nm. All the processes, from extraction to spectral measurement, were performed in dim light to avoid the degradation of the chlorophyll in the samples.In this test, we used chlorophyll contents to represent phytotoxicity. The content of chlorophyll was determined using the method of Sedmak and GrossbergSupplementary Information


Fructus aurantii (FA) decoction. Analysis was performed on Waters BEH (R) C18  at a flow rate of 0.2 mL/min by using (A) acetonitrile and (B) 0.5% acetic acid-water as the mobile phase. The method was well validated on linearity, precision, recoveries, and stability. Then, we used the same UHPLC conditions for quantitative analysis of meranzin hydrate, naringenin, and hesperetin in rat plasma. The method proved to be linear within the concentration ranges of 3.3–3300 ng/mL for meranzin hydrate, 6.95–3555 ng/mL for naringenin, and 1.8–236 ng/mL for hesperetin. The RSD of precision ranged from 1.22% to 9.08%, and the average extraction recovery ranged from 96.49 ± 1.42% to 102.01 ± 3.16%. Besides, we performed a comparative pharmacokinetic study after oral administration of FA decoction at a low dose of 15 g/kg and high dose of 30 g/kg body weight for seven days to rats. The AUCt) method was established for simultaneously detecting naringin, hesperidin, neohesperidin, meranzin hydrate, naringenin, and hesperetin in  Fructus aurantii (FA), also called Zhi Qiao in Chinese, refers to the dried unripe fruit of Citrus aurantium L. or its cultivar (Rutaceae). FA is a supremely important Qi-invigorating herb in China for centuries, and it vigorously modulates the motion of Qi and fortifies the spleen and stomach. Thus, FA is usually taken to remedy indigestion problems, like postprandial fullness, early satiation, abdominal pain, and distension, and other symptoms caused by the obstruction of the Qi activity [activity . In viewactivity , and FA-activity . It is wactivity , 5. Flavactivity –9. Varioactivity , antidepactivity , 11, infactivity , antioxiactivity , anticaractivity , 15, antactivity , and hypactivity .1/2 pathway, and subsequent modulation of brain-derived neurotrophic factor (BDNF) levels [To date, all the published methods were developed for determining components in FA. Zhao et al.  used a H) levels . In funcHerein, a simple and accurate UHPLC detection method for simultaneous determination of six active compounds , and acetic acid buffer solution was from Dingguo Biotechnology Co., Ltd. . FA (crude drug) was collected from Hunan province and bought and authenticated by Professor Sui-Yu Hu .Standards including naringin, hesperidin, neohesperidin, naringenin, and hesperetin (purity >98%) were purchased from Chengdu Pufei De Biotech Co., Ltd. . Meranzin hydrate (purity >98%) was purchased from DIAO Company . Sulfamethoxazole  was taken as an internal standard (IS) and from the National Institute for the Control of Pharmaceutical and Biological Products . Acetonitrile and methanol (HPLC grade) were obtained from Sigma-Aldrich . Glacial acetic acid and ethyl alcohol  were obtained from Heng Xing Chemical Factory , and purified water was from Hangzhou Wahaha Group Co., Ltd. . μm) from Waters Co. (USA) with temperature at 40°C. (A) Acetonitrile and (B) 0.5% acetic acid-water constituted the two parts of the mobile phase at the following gradient elution procedures . The flow rate was controlled at 0.2 mL/min, and the injection volume was 5 µL.Analyses were implemented on a Waters ACQUITY UHPLC system and an ACQUITY PDA . The analytical column was a Waters BEH (R) C18  for 0.5 h at room temperature and thereafter was boiled for 0.5 h. The filtrate was collected, and the residue was boiled again in distilled water  for 0.5 h. The filtrates were mixed together, filtered through a 0.45 µm porous filtration membrane, then farther concentrated in the rotary evaporator to a final concentration equivalent to 1.7 g/mL FA, and stored at −20°C until analyzed. In the experiment, FA decoction (100 µL) was precisely measured, 900 µL methanol was added, then ultrasound was done for 5 min, and it was centrifuged at 15000 rpm (4°C) for 10 min; the supernatant liquor (20 µL) was shifted to a 1.5 mL EP tube, methanol-water (780 µL) was added, and ultrasound was performed, and it was centrifuged at 15000 rpm (4°C) for 10 min again. Finally, 5 µL of the supernatant liquor was injected into the apparatus for detection.The raw herb (200 g) was immersed in distilled water  to establish calibration curves. QC samples were prepared at three concentration levels containing naringin , hesperidin , neohesperidin , meranzin hydrate , naringenin , and hesperetin .The concentrations of standard solutions were 1492 µL sample from the above 7 concentrations of mixed liquor in order (3 replicates per concentration). Method validation was followed with Guidance for Industry Bioanalytical Method Validation by U.S. Food and Drug Administration (FDA). Limit of detection (LOD) under the analytical approach was measured at a signal-to-noise (S/N) ratio of 3. Lower Limit of Quantification (LLOQ) was defined as the lowest concentration of the standard curve that can be measured with a precision within 20% and an accuracy of 80–120%.We precisely injected 5 Intra- and interday variations were for determining the precision of the developed method. Relative standard deviation (RSD) was utilized as a measurement of precision. Intra- and interday repeatability were determined on 5 replicates within 1 day and 5 consecutive days, respectively.The accuracy of the analytical method was evaluated by using the recovery test. Recoveries of 6 compounds were investigated by spiking with the authentic standards to the samples of FA before extraction. Five replicates of FA decoction were tested. Peak areas of each analyte in five FA samples were recorded. Then, the concentrations of the 6 compounds in FA decoction after spiking were calculated according to the peak area using the calibration curve. Average recovery percentage was calculated by the formula: recovery (%) = /spiked amount × 100%.The stability of the analytical solution at environmental temperature was studied by detecting sample solution at 0, 2, 4, 8, 12, and 24 h. The RSD values of peak areas were taken for assessment.n=8): low-dose group (15 g/kg FA decoction) and high-dose group (30 g/kg FA decoction). The two groups were given by gavage twice a day for 7 days in succession. At the 7th day of medication, the rats were prohibited feeding for about 12 hours without restriction of water. On day 8, the rats were orally administered for the last time at 8 am, and then plasma samples (0.5 mL) were collected from the caudal vein at 5, 10, 20, 30, 60, 90, 240, 360, 480, and 600 min into heparinized vacuum tubes, followed by centrifugation at 3500 rpm (4°C) for 10 min. Then, the plasmas were obtained and immediately stored at −20°C until analysis. During the experiment, the rats were injected intraperitoneally with physiological saline for maintaining fluid balance.16 male Sprague-Dawley (SD) rats weighing 200 ± 20 g were from experimental animal center of Central South University and fostered in an environmentally controlled room  for 1 week before the experiment. All experimental procedures were implemented strictly according to the guidelines of the Animal Care and Use Committee, Central South University, China. This research was approved by the Animal Ethics Committee of Central South University. Rats were randomly divided into 2 groups , naringenin , and hesperetin . Plasmas (100 µL) were spiked with β-glucuronidase (5 µL) and acetic acid buffer solution (10 µL), subsequently vortexed for 10 seconds, and incubated for 4 h at 37°C. Then, SMZ (50 µL) and methanol (135 µL) were added, ultrasound was done for 10 min, and centrifugation at 12000 rpm (4°C) for 10 min was performed. The supernatant (50 µL) was transferred, distilled water (50 µL) was added, then ultrasound and centrifugation again. At last, the supernatant of 5 µL was injected into UPLC.Standard stock solutions were prepared by dissolving reference standards in methanol to yield concentrations of 198 (1) Specificity. Specificity refers to analyze different plasma samples for interference at the retention time of the constituent. It was evaluated by comparing the chromatogram of blank plasma, blank plasma with the reference of three compounds, and medicated plasma.(2) Linearity. Blank plasmas of healthy rats  were added with suitable amount of stock solution of meranzin hydrate, naringenin, and hesperetin. Then, 50 μL SMZ was added to each solution and a specific amount of methanol to a constant volume of 300 μL, yielding a series of concentrations of 3300, 825, 206, 51.6, 12.9, 6.5, and 3.3 ng/mL for meranzin hydrate, 3555, 889, 222, 55.5, 27.8, 13.9, and 6.95 ng/mL for naringenin, 236, 59, 29.5, 14.7, 7.3, 3.7, and 1.8 ng/mL for hesperetin.(3) Precision and Accuracy. The intra- and interday precisions were determined by assaying 5 replicates of QC samples at 3 different concentrations on the same day or three consecutive days. Precision was estimated through intra/interday RSD, and accuracy was weighed as a relative error (RE).(4) Extraction Recovery. The extraction recoveries of the three constituents were evaluated by comparing the relative peak areas obtained from plasma samples with the constituents spiked before and after extraction.(5) Stability. QC plasma samples of each concentration at low, medium, and high levels were detected. The freeze and thaw stability were investigated after three freeze (−20°C) and thaw (room temperature) cycles on consecutive days. Long-term stability was studied by storing QC samples at −20°C for 20 days. Short-term stability was assessed by analyzing QC samples kept at room temperature for 4 h.P value of <0.05 was considered statistically significant. The DAS 3.2.8 pharmacokinetic program  was used to process pharmacokinetic parameters.The database was set up with the SPSS 17.0 software package from SPSS Inc., Chicago, Illinois (US). Differences between the two groups were analyzed by one-way analysis of variance. A two-tailed In our study, various mobile phase conditions were tried to obtain optimal responses, suitable retention times, and good peak shapes. Methanol-water, acetonitrile-water, even adding different concentrations of phosphoric acid and acetic acid, 0.5% acetic acid-100% water were all tested as potential mobile phases. The different chromatographic system, gradient elution program, flow rate, and over temperature were also optimized. Eventually, a gradient elution by acetonitrile and 0.5% acetic acid-water with a flow rate of 0.2 mL/min at 40°C was chosen to obtain satisfactory sensitivity and good peak shapes.To achieve the goal of strong absorption and low interference of components, peak area under the 284 nm wavelength was calculated for naringin, hesperidin, neohesperidin, naringenin, hesperetin, and 324 nm for meranzin hydrate. Under the proposed chromatographic conditions, all 6 constituents were sufficiently resolved and successfully separated within 9.0 min without “cross-talk” peaks. Representative chromatograms in blank solvent, standard solution, and FA decoction are shown in (1) Calibration Curves, LOD, and LLOQ. r), linear range, LOD, and LLOQ of 6 components. All calibration curves exhibited good linear relationship, and r were higher than 0.9939.(2) Precision, Recovery, and Stability. Results concerning the precision of this developed method were exhibited in n=5), which demonstrated that the recoveries were consistent, accurate, and reproducible for the measurement.The newly developed analytical method was subsequently applied to determine the six constituents in FA decoction. The contents in FA decoction (per 1.7 g/mL) were naringin 24.84 ± 0.59 mg/mL, hesperidin 5.16 ± 0.18 mg/mL, neohesperidin 15.00 ± 0.76 mg/mL, meranzin hydrate 0.35 ± 0.01 mg/mL, naringenin 0.28 ± 0.01 mg/mL, and hesperetin 0.20 ± 0.01 mg/mL, and the overall RSD was below 5.10% . There wβ-glucuronidase and acetate buffer were tried and added into plasma samples, vortexed, and incubated at 37°C for 30 seconds; then, SMZ solution and methanol were added. The method successfully elicited out the peaks of meranzin hydrate, naringenin, and hesperetin, and endogenous substances did not intervene the target peak. We also measured the extraction effect of incubation for 2 h, 4 h, 6 h, 12 h, and 24 h, respectively, and found that incubation for 4 h had the similar effect as 6 h, 12 h, and 24 h. Eventually, we chose β-glucuronidase and acetate buffer for enzymatic deconjugation, methanol for deproteinization, and incubation for 4 h as the optimal plasma pretreatment.To select the best suitable IS, we tried different chemical compositions, such as caffeine, luteolin, diphenhydramine, and SMZ. Then, SMZ was selected as the IS of the three compounds in plasma sample determination for its stable, high recovery rate, and similarity in the retention. In order to establish an appropriate plasma processing method, different extraction methods of plasma samples—ethyl acetate, methanol, diethyl ether, acetonitrile and water bath, and adding acid or alkali—were all tested to examine the extraction effect of meranzin hydrate, naringenin, and hesperetin in plasma. When utilizing acetonitrile and water bath to eliminate protein of plasma samples, few chromatographic peaks existed, and even the target component did not appear. Similarly, by using methanol to precipitate protein, naringenin and hesperetin could not be detected. Besides, The optimized UHPLC method for content determination in FA decoction with the same mobile phase condition, the same chromatographic system and gradient elution program, and the same flow rate and temperature was also applied to the pharmacokinetic study of meranzin hydrate, naringenin, and hesperetin in rat plasma following oral administration of FA decoction at a dose of 15 g/kg and 30 g/kg body weight.(1) Specificity. The typical chromatograms of blank plasma added with the 3 analytes and IS, and plasma sample after oral administration of FA decoction for 0.5 h (15 g/kg body weight) are represented in (2) Linearity of Calibration Curves and LLOQ. As shown , y = 210.36x + 8.3364 , and y = 29.540x + 3.5492 . LLOQ for the 3 constituents in rat plasma were meranzin hydrate (3.30 ng/mL), naringenin (6.95 ng/mL), and hesperetin (1.80 ng/mL).As shown , the met(3) Precision and Accuracy. In (4) Recovery. The average extraction recovery ranged from (96.49 ± 1.42)% to (102.01 ± 3.16)%, and the RSDs were less than 6.05%  Stability. Results of the stability tests in The validated method was applied to determine the plasma concentrations of meranzin hydrate, naringenin, and hesperetin in rats after oral administration of FA decoction at a dose of 15 g/kg and 30 g/kg body weight. The mean plasma concentration-time profiles of meranzin hydrate, naringenin, and hesperetin are shown in Cmax of meranzin hydrate was inferior to that of naringenin. Also, hesperetin presented quite low concentrations in rat plasma, with the Cmax at 83.90 ± 14.18 μg/mL for the 15 g/kg group and 174.32 ± 39.04 μg/mL for the 30 g/kg group, respectively.As shown, naringenin was the component with the highest concentration among all constituents in rat plasma, and this was identical with the Tong et al. study , for thetz1/2 of meranzin hydrate was 2.67 ± 0.81 h (at the 15 g/kg group) and 2.23 ± 0.59 h (at the 30 g/kg group), and the elimination rate of meranzin hydrate was faster than naringenin  and hesperetin . Compared with the 15 g/kg group, oral administrationof 30 g/kg of FA, the AUCt) in FA decoction. However, naringin, hesperidin, and neohesperidin could not been detected in the rat plasma even after orally administering 30 g/kg FA decoction, which might be associated with the hydrolysis caused by microbial bacterial from the gastrointestinal tract , 25. NarEarlier research of our team has found that meranzin hydrate could accelerate gastrointestinal motility in rats subjected to the forced swimming test and reverse the inhibition effect of the decreased ghrelin levels on gastrointestinal motility, demonstrating that meranzin hydrate possessed antidepressive and prokinetic-like effects . Besidest)(0– and Cmax of meranzin hydrate, naringenin, and hesperetin multiplied significantly with the increase of FA dosage. The t1/2 of meranzin hydrate was faster than that of naringenin and hesperetin in the two groups. This work, on the one hand, demonstrated a detailed research of quantitative determination in FA decoction and rat plasma, as well as pharmacokinetics; on the other hand, it may lay the experimental foundation for pharmacokinetic interactions between FA and other drugs.To the best of our knowledge, this is the first report using the same UHPLC chromatographic condition for determination of naringin, hesperidin, neohesperidin, meranzin hydrate, naringenin, and hesperetin in FA decoction and for detection of meranzin hydrate, naringenin, and hesperetin in rat plasma. Both of the detections were fully and well validated. Most importantly, we further performed the pharmacokinetics after oral administration of 15 g/kg and 30 g/kg FA decoction to rats. The AUC


The growth rate of the isolates in presence of pollutants, chromatography, Gibbs and turbidometric methods were used to assess their biodegradation activity. A total of 39 mycobacterial isolates (43.3%) were recovered from 90 samples that belonged to 21 various species consisting of M. fortuitum; 6 isolates, M. flavescens and M. paragordonae; 4 isolates each, M. monacense, M. fredriksbergense and M. aurum; 2 isolates each, 7 single isolates of M. conceptionense, M. porcinum, M. simiae, M. celeriflavum, M. novocastrense, M. neoaurum, M. obuense and 12 isolates that belonged to 8 unknown potentially novel mycobacterial species. The isolates were categorized in three groups based on their bioremediation activity, i.e., 5 (12.8%) organisms without biodegradation activity, 20 (51.2%) organisms with previously reported biodegradation activity, and 14 (35.9%) organisms that showed biodegradation activity but not previously reported. Our results showed that the Iranian ecosystems harbor a good reservoir of diverse mycobacterial species with biodegrading potentiality for neutralizing environmental chemical pollutants.Anthropogenic origin pollutants including pesticides, heavy metals, pharmaceuticals and industry chemicals impose many risks to human health and environment and bioremediation has been considered the strategy of choice to reduce the risk of hazardous chemicals. In the current study, weaimed to screen and characterize mycobacteria from the diverse range of Iranian aquatic and terrestrial ecosystems with harsh and unfavorable environmental conditions that can be utilized for biodegradation of target pollutants. Mycobacteria were isolated from a collection of 90 environmental samples and identified to the species level using conventional microbiological and molecular methods including the PCR amplification of  During past decades and with the advances in science and technology various chemicals and synthetic products such as petroleum derivatives like petrochemicals and plastic, insecticides and herbicides, radioactive substances and many other similar materials were increasingly introduced into the environment  for 15 min to reduce the number of not-desirable microbial contaminants such as fungi, Protista and other bacteria. Afterwards, the pretreated samples were subjected to vacuum filtration . The filters were rinsed and mashed in tubes containing 15 ml of distilled water. Almost 100 µl aliquots of dissolved samples were transferred into the tubes of Löwenstein–Jensen (LJ) and Sauton’s media Allen  and incug for 20 min at room temperature. The pellets and supernatants were decontaminated in separate tubes by addition of 3% sodium lauryl sulfate and 1% NaOH . The samples were incubated for 3 weeks at temperatures of 25, 30 and 35 °C in an atmosphere of 5% CO2 . The suspension was homogenized, tenfold serial dilutions were prepared in sterile water and 200 μl of each pretreated 10The details of environmental samples tested in the current study are given in Table The isolates were characterized phenotypically by the use of conventional phenotypic and biochemical tests Wayne . The tesg for 10 min. The supernatant was transferred to a sterile microtube and centrifuged at 20,000×g for 10 min. The modified Pitcher method included the lysis of actinomycetes cells obtained with the pretreatment with lipase (2 mg/ml) followed by the cell wall disruption using lysozyme  and proteinase K  in the presence of 3% sodium dodecyl sulfate (SDS). The DNA-containing aqueous phase was purified with phenol–chloroform–isoamyl alcohol  and chloroform-isoamyl alcohol . The precipitation of DNA by either of two methods, was carried out using ammonium acetate (7.5 mol/l) and ethanol at −20 °C. The Precipitated DNA was washed with 70% ethanol and re-suspended in 100 µl of Milli-Q water.Chromosomal DNA was extracted using simple boiling method or the modified Pitcher method . The primers included hsp65F: 5′-CTGGTCAAGGAAGGTCTGCG-3′, and hsp65R: 5′-GATGACACCCTCGTTGCCAAC-3′) as recommended by Khan and Yadav (-AGA GTT TGA TCM TGG CTC AG-3′and 1492R: 5′-CG GTT ACC TTG TTA CGA CTT-3′) , M. simiae (KF028776), M. frederiksbergense (KF019696), M. sacrum like (KU564076), M. vanbaalenii like (KU564074), M. gadium like (KU564078), M. aurum (KU564075), M. tusciae like (KU564073), M. novocastrense (NR_029208), M. obuense (KF028777) and M. phocaicum like (KF019699).The GenBank accession numbers for the 16S rRNA sequencing of the mycobacteria isolated in this study are; M. cookii like (A7) JCM 30922 and CCUG 67561 and M. phocaicum like (A3 and A85) JCM 30989 and CCUG 67787.The culture collection numbers for two potentially novel strains of mycobacteria in this study are listed below: The biodegradation activity of the strains isolated from environmental samples in the current study was evaluated according to Kanaly and Harayama . The det2PO4 (0.42), K2HPO4 (0.375), (NH4)2SO4 (0.244), NaCl (0.015), CaCl2·2H2O (0.015), MgSO4·7H2O (0.05), and FeCl3·6H2O (0.054).PAHs mix solution (1-1)  were purchased from AccuStandard. PAHs stock solution was 0.2 mg/ml in dichloromethane and methanol. Phenol was purchased from Merck, Germany. Phenol stock solution was 10 mg/ml in deionized water. Sodium sulfate was purchased from Merck, Germany. Sodium sulfate stock solution was 10 mg/ml dissolved in deionized water. All other solvents and chemicals used were of reagent grade. Mineral Salt Medium (MSM) contained (g/l), g/l were KH8 CFU/ml) into 100 ml aliquots of % 1 PAHs, % 1 phenol and % 1 sodium sulfate enriched MSM were prepared in 250 ml flask and incubated for 6 days at 30 °C in an orbital shaker (90 rpm). To evaluate the bacterial growth, samples were collected at 24-h intervals and the absorbance at 560 nm was measured by Spectrophotometer.Evaluation of biodegradation activity of isolates was carried out by adding 1 ml of direct colony suspension is made in normal saline and turbidity adjusted to 0.5 McFarland standard . A reversed phase column C18  using mobile phase with acetonitrile/water  was used to separate PAHs  as the extraction solvent, vortexed for 10 s, then centrifuged at 3000×g for 20 min. An amount of 150 µl of the collected supernatant was mixed with 30 µl NaHCO3, and 20 µl of Gibbs reagent  was added to the mixture and shacked for 30 min at room temperature  for the water samples ranged between 600 and 1200 mg/l  in the 16S rRNA , 293–304 (G–T), 307 (C), 328 (T), 614–626), 661–744 (G–C), (A–T), 631 (G), 824–876 (T–A), 825–875 (A–T), 843 (C), and 1122–1151 (A–T) . This followed by M. flavescens and M. paragordonae  and a potentially novel species closely related to uncharacterized species M. sacrum . The other thirteen isolates comprised a single isolates each belonged to seven established species that were M. conceptionense, M. porcinum, M simiae, M. celeriflavum, M. obuense, M. novocastrense and M. neoaurum, as well as six unknown isolates potentially novel species of mycobacteria that were closely related to M. tusciae (isolate A4), M. vanbaalenii (isolate A6), M. gadium (isolate A16), M. pallens (isolate A23), M. cookii (isolate A7), and M. vaccae (isolate A14).Based on morphological, culture and biochemical properties and the genus specific marker, i.e., the presence of a 228-bp fragment of the The relationship between our isolates and the standard established species of mycobacteria was supported by a high bootstrap value in phylogenetic tree based on 16S rRNA gene depicted by MEGA 6. The corresponding value for the isolates that showed substantial nucleotide differences with the known mycobacterial species and named as novel species requiring further investigation found to be rather low Fig. .Fig. 216M. paragordonae, i.e., the isolates A24, A25, A52 and A90 and one isolate of M. simiae that is, the isolate A18 , morpholine, thiomorpholine and piperidin degradation , natural rubber and squalene degradation  was reported  and polybrominated biphenyl (PBB) as well as natural and synthetic rubber (Berekaa and Steinbüchel The isolates A44, A49, A63, A69, A91 and A115 were identified as M. flavescens. The organism was first isolated and characterized from a drug treated tuberculous guinea pig (Bojalil et al. The isolates A53, A83, A88 and A113 were identified as M. monacense which was first isolated from clinical samples (Reischl et al. M. monacense is capable of crude oil and various PAHs degradation (Miller et al. The isolates A55 and A75 were identified as M. celeriflavum. This species is a rapid growing scotochromogenic Mycobacterium that was first isolated and characterized from clinical sample in Iran in 2015 (Shahraki et al. The isolate A8 was identified as M. porcinum which was first isolated from the lymph node of porcine (Tsukamura et al. The isolate A15 was identified as Mycobacterium species. The isolates were evaluated for biodegradation activity against PAHs, phenol and sodium sulfate. All isolates, except for the isolate A7, showed biodegradation activity. The isolates A2, A12, A13 and A28 were found to degrade PAHs and sodium sulfate. The isolates A4, A6, A14 and A23 were found to degrade the PAHs and the isolate A16 was able to degrade sodium sulfate.In our study, ten isolates including A2, A4, A6, A7, A12, A13, A14, A16, A23 and A28, were found to have phenotypic and molecular characteristics of novel gyrB, secA1, and DNA-DNA relatedness.The characterization of these unknown mycobacteria remains to be completed using a thorough phenotypic and molecular assays including cell wall composition analysis, sequence analysis of key genetic markers such as In conclusion, the results of our study showed that the diversity of mycobacteria, particularly prokaryotes, offers the potential for adaptation to various habitats, including environments severely contaminated with hydrocarbons and heavy metals. This broad adaptability has great value for the bioremediation of damaged ecosystems. Our study also confirms the idea that despite being abundant in environment, mycobacteria have been simply ignored for such significant usage. Indeed, there is an untapped potential with regard to bioremedial actinomycetes particularly mycobacteria that has yet to be discovered and administered in bioremediation of hazardous chemicals.