Carissa opaca Stapf ex Hanes fruits is traditionally used in the treatment of asthma, hepatitis and microbial infections. The present study was arranged to investigate the antimicrobial, cytotoxic and antitumor activity of various fractions of C. opaca extract and its bioactive metabolites responsible for that activity.C.opaca antibacterial, antifungal, cytotoxic and antitumor assays are used. Eight strains of bacteria including Bacillus subtilis, Enterobactor aerogenes, Escherichia coli, Klebsiella pneumoniae, Micrococcus luteus, Pseudomonas aeroginosa, Salmonella typhy, and Staphylococcus aureus and four strains of fungal viz: Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and Fusarium solani are used. Brine shrimps and potato dics are used for anticancer and antitumor potency of extract. High performance liquid chromatography (HPLC) is utilized for determination of bioactive metabolites responsible for the activity.To characterize various fractions of C. oapca showed significant antibacterial, antitumor and anticancer activity. In case of C. opaca fruit inhibition growth of Aspergillus niger was ranged between 23.2 ± 1.36% to 43.3 ± 2.39%, Aspergillus flavus ranged between 27.6 ± 1.39% to 65.6 ± 3.44%, Aspergillus fumigatus ranged between 13.2 ± 1.00% to 52.4 ± 1.54% and Fusarium solani ranged between 10.5 ± 1.02% to 14.6 ± 1.74%.HPLC chromatogram revealed the presence of orientin, isoquercetin, myricetin and apigenin. Various fractions of C.opaca are accessible source of ethno pharmacy as they are consumed in different areas of Pakistan with ultimate health compensations.It can be concluded that, various fractions of  Carissa opaca Stapf ex Hanes is widely distributed from Punjab-Himalayas up to 6000 ft, in Murree (Pakistan) as well as in some areas of India, Burma and Sri Lanka [C. opaca is well acknowledged, yet very little scientific information about its activity against microorganism is available. Cancer, being a world-wide health problem, is treated with clinical therapies including chemotherapy, surgery, radiation therapy and so on by using synthetic medicines [Agrobacterium tumefaciens is considered as quick, low-priced and consistent prescreening for antitumor agents of botanicals [ri Lanka  traditiori Lanka , cardiacri Lanka , microbiri Lanka , cough, ri Lanka . In spitri Lanka . In the ri Lanka ,9. Henceri Lanka . Antimicedicines . Toxic sedicines  reportededicines . Brine sedicines ,14. Antitanicals . It is aC. opaca were obtained in March-April 2011 from Abbottabad of Northern Pakistan, recognized by their local names and validated by Dr. Mir Ajab Khan, Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan. A voucher specimen with Accession No. 24561 (C. opaca) was deposited at the Herbarium of Pakistan.Fruits of in vitro investigation.The collected plant samples were cleaned to get rid of dust particles and then dried under shade for one to two weeks and made into fine powder. 2 kg of sample was extracted twice with 5 L of 95% methanol at 25°C for 48 h. For filtration Whatman No. 1 filter paper was used and then filtrate was concentrated on rotary evaporator  under reduced pressure at 40°C. In order to resolve the compounds with escalating polarity, a part of the extract was suspended in distilled water and subjected to liquid-liquid partition by using solvents in a sequence of n-hexane, ethyl acetate, chloroform, butanol and water to obtain their soluble fractions. After fractioning, the solvent of respective fractions was also evaporated by rotary evaporator Extract was dried and then stored at 4°C for further C. opaca powder was extracted with 6 ml of 25% hydrochloric acid and 20 ml methanol for 1 hr. The obtained extract was filtered to a volumetric flask. The residue was heated twice with 20 ml of methanol for 20 min to obtain the extract. The combined extract was diluted with methanol to 100 ml. 5 ml portion of the solution was filtered and then was transferred to a volumetric flask and diluted with 10 ml of methanol. The sample (10 μl) was injected into the HPLC apparatus.50 mg of Samples were analyzed on Agilent HPLC system. Separation was carried out through column 20RBAX ECLIPSE, XDB-C18,  with UV-VIS Spectra-Focus detector, injector-auto sampler. Solvent A (0.05% trifluoroacetic acid) and solvent B (0.038% trifluoroacetic acid in 83% acetonitrile (v/v) with the following gradient: 0-5 min, 15% B in A, 5-10 min, 70% B in A, 10-15 min, 70% B in A are used for separation The flow rate was 1 ml/min and injection volume was 10 μl. Eleven standard compounds including rutin, myricetin, vitexin, orientin, hyperoside, isovitexin, isoquercetin, luteolin, apigenin, kaempherol, and luteolin-7-glucoside were run for comparative detection and optimized. The calibration curves were defined for each compound in the range of sample quantity 0.02-0.5 μg. All samples were assayed in triplicate.C. opaca fruits was carried out through protocol of , Enterobactor aerogenes (ATCC 13048), Escherichia coli (ATCC 15224), Klebsiella pneumoniae (MTCC 618), Micrococcus luteus (ATCC 10240), Pseudomonas aeroginosa (ATCC 27853), Salmonella typhy (ATCC 0650), and Staphylococcus aureus (ATCC 6538). Antibacterial activities were calculated as a mean of 3 replicates. Diameter of the clear zones, showing no bacterial growth, around each well was measured with the help of vernier caliper. Triplicate plates were prepared for each sample.The antibacterial potency of various fractions of a et al.  using eiAspergillus flavus (0064), Aspergillus fumigatus (66), Aspergillus niger (0198) and Fusarium solani (0300). Tubes were prepared in triplicate for each fungus species. Percentage inhibition of fungal growth for each concentration of fractions was determined by the following formula;The agar tube dilution method was used for antifungal activity of plant extracts was determined according to the protocol reported by Duraipandiyan and Ignacimuthu  using AsC.opaca were characterized according to Meyer-Alber et al.[’s formula;Various fractions of er et al. using bret al.[The potato disc method was used for anti-tumor activity of plant extracts as reported by Ferrigini et al.. Number 50 was recorded using graph prism pad software.ICin vitro assays recorded were analyzed with help computerized Graph prism pad software to determined EC50, IC50 and LD50.Data of C. opaca fruit. First of all, the experimental conditions were optimized to get the chromatograms with better resolution within a short resolution time and maximum UV absorption of sample. Hence, flavonoid standard compounds and the plant samples were quantified by assimilation of peak areas at 220 nm within runtime of 20 minutes. The standard solutions were injected in duplicate and the curves were constructed with the averages showing good linear correlation coefficient in the concentration range. All the standard flavonoids confirmed good linearity in a bit large concentration range. The conditions used directed towards the good separation of peaks that may be identified in the chromatogram as apigenin (Rt=4.7), myricetin (Rt=18.5), vitexin (Rt=2.5), orientin (Rt=2.75), hyperoside (Rt=12.5), isovitexin (Rt=3.7), isoquercetin (Rt=6), rutin (Rt=8.7), luteolin (Rt=2.01), kaempherol (Rt=3.4), luteolin-7-glucoside (Rt=1.6)  values. The MIC is the lowest concentration of test sample to inhibit visible growth of bacteria. The antibacterial activity was tested against both gram-negative bacteria as well as gram-positive bacteria. Table Staphylococcus aureus was inhibited by MIC value (1 mg/ml) of HFC, MFC, 2.5 mg/ml of CFC, 5 mg/ml of BFC, while rest of the fractions didn’t inhibit the growth of Staphylococcus aureus. On the other hand, Bacillus subtilis was inhibited by HFC, (0.1 mg/ml); CFC, MFC (1 mg/ml); EFC (1.2 mg/ml); AFC, (2.5 mg/ml) while remaining fractions did not inhibit the growth of the respective bacteria. In case of gram negative bacteria Klebsiella pneumoniae’s growth was inhibited by EFC, CFC, MFC, AFC (1 mg/ml) while remaining fractions did not inhibit the growth of the respective bacteria. Pseudomonas aeroginosa was inhibited by HFC (1 mg/ml); CFC, MFC, AFC (10 mg/ml) and rest of the fractions inhibited the growth at 0.1 mg/ml. Growth of Salmonella typhy was inhibited by CFC, AFC (0.5 mg/ml) while rest of the fractions inhibited the growth at 0.1 mg/ml. However, HFC, EFC and BFC inhibited the growth of Enterobacter aerogenes with MIC (1 mg/ml); CFC (0.1 mg/ml); MFC (5 mg/ml) while remaining fractions did not show inhibition of the concerned bacteria. MIC against Micrococcus luteus and Escherichia coli had no value, showing no antibacterial activity of any of the fractions.The in vitro antifungal activity of various fractions of C. opaca fruit was evaluated against four fungal strains as shown in Table C. opaca fruit inhibition in growth of A. niger was ranged between 23.2 ± 1.36% to 43.3 ± 2.39%, A. flavus ranged between 27.6 ± 1.39% to 65.6 ± 3.44%, A. fumigatus ranged between 13.2 ± 1.00% to 52.4 ± 1.54% and F. solani ranged between 10.5 ± 1.02% to 14.6 ± 1.74%.The C. opaca fruit was evaluated against brine shrimps growth under controlled condition as presented in Table 50 was the lowest concentration of test sample at which 50% death of shrimps occurred. Data of present study indicated that the order of LD50 of various fractions of C. opaca fruits was CFC > MFC > AFC > BFC > EFC > HFC.Cytotoxic screening using brine shrimps bioassays was carried out to provide important preliminary data to select plant extracts. Cytotoxic effect of various fractions of C. opaca fruit. This assay has been routinely used due to its rapid, economical and statistically valid prescreening for antitumor activity. In the present study, Agrobacterium tumefaciens (AT 10) was used against three different concentrations  of different fractions of C. opaca fruit. Tumor inhibition was calculated through IC50 value. The IC50 values of antitumor potential of plant samples are summarized in Table 50 of various fractions of C. opaca fruits for antitumor screening was BFC > AFC > CFC > MFC > EFC and HFC.Potato disc assay was performed to reveal the antitumor potential of various fractions of C.apaca showed the presence of important flavonoid i.e., orientin, isoquercetin, myricetin and apigenin. possess anti-disease wealth particularly minimizes the risk of cancer [C. opaca can verify their folkloric use against cancer, skin infections and rheumatism. This study is a preliminary estimation of antimicrobial activity of C. opaca fruits. The results of the antibacterial and antifungal activity vary with the fractions of the plant studied. It could result in the discovery of new chemical classes that may possibly proceeded as biochemical tools to study infectious diseases and endowed with selective agents for antibiotics. Our results suggest that antimicrobial activity of plant fractions doesn’t depends on only phenolics but other secondary metabolites are also involved. Previous studies revealed that plant extracts have bactericidal effects and consequently seems to be promising in for treatment of microorganisms [Micrococcus luteus and the Gram negative bacteria, Escherichia coli and consequently for rest of the strains they were active. Flavonoids are known to possess antibacterial activity and, according to Tsuchiya et al. [et al. [Sorghum and Cicer erythrophyllum, respectively. As previous reports suggest that Gram negative bacteria are additionally resistant as compare to Gram positive bacteria, the non polar fractions were the only ones to show activity against Gram-negative bacteria [et al.[50 value < 1000 ppm) and non-toxic , all tested showed good brine shrimp larvicidal activity. Moreover, Peteros and Uy [50 values < 100 ppm to brine shrimp lethality.Phytochemicals screening is carried out to allow isolation of novel or valuable components with potential activities at the earliest stages thus economically very important. Present study exposed the naturally occurring dietary substances such as alkaloids, flavonoids, saponins, tannins and other bioactive metabolites in various fractions of plant samples. Methanol fraction of f cancer . In litef cancer ,22. Flavf cancer  like antf cancer ,25. Therrganisms . Anothera et al. , dihydro [et al.  and Mart [et al.  who repobacteria . But in bacteria . For thebacteria  who repoa [et al. who cates and Uy  signifieMost of the tested fraction showed substantial antimicrobial, antitumor and anticancer activities, which is in accordance with the spacious use of tested plant samples in primary health care. It can be concluded that, studied plant parts are accessible source of ethno pharmacy as they are consumed in different areas of Pakistan with ultimate health compensations.The authors declare that they have no competing interests.SS made significant contribution to acquisition of data, analysis, drafting of the manuscript. MRK has made substantial contribution to conception and design, interpretation of data, drafting and revising the manuscript for intellectual content. RAK (ORCID ID: 0000-0003-0453-2090) participated in the design and collection of data and analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/13/372/prepub


Ganoderma sp. for 10 d and compared in relation to biochemical parameters, crude protein (CP) content, and nutritional value by calculating the C/N ratio in order to improve the nutritional value of cattle feed.Current research to enrich cattle feed has primarily focused on treatment using white rot fungi, while there are scarce reports using the enzyme tannase, which is discussed only in reviews or in the form of a hypothesis. In this context, the aim of the present study was to evaluate the effect of tannase on wheat straw (WS) and also the effect of lyophilized tannase at concentrations of 0.1%, 0.2%, and 0.3% (w/w) on WS followed by fermentation with Penicillium charlesii, a tannase-producing microorganism, produced 61.4 IU/mL of tannase in 54 h when 2% (w/v) tannic acid (TA) was initially used as a substrate in medium containing (% w/v) sucrose (1.0), NaNO3 (1.0), and MgSO4  in a 300-L fermentor (working volume 220 L), and concomitantly fed with 1.0% (w/v) TA after 24 h. The yield of partially purified and lyophilized tannase was 5.8 IU/mg. The tannin-free myco-straw at 0.1% (w/w) tannase showed 37.8% (w/w) lignin degradation with only a 20.4% (w/w) decrease in cellulose content and the in vitro feed digestibility was 32.2%. An increase in CP content (up to 1.28-fold) along with a lower C/N ratio of 25.0%, as compared to myco-straw, was obtained.The use of tannin-free myco-straw has potential to improve the nutritional content of cattle feed. This biological treatment process was safe, eco-friendly, easy to perform, and was less expensive as compared to other treatment methods. The drastic increase in human population has caused intensified pressure on agricultural land use, which has led to the conversion of farmland into commercial landscapes. Therefore, countries with agriculture-based economies have endured economic constraints that have negatively impacted the health of cattle due to nutritional deficiencies of feed. Therefore, it is of the utmost importance to improve the quality of cattle feed so that it can be easily be digested by ruminants; therefore, producing higher milk yields to benefit the ever-increasing demand for milk and milk products. Therefore, to overcome problems associated with the decrease in available farmland and the increased demand for milk and milk products, efforts are being made to reduce the wide gap between the requirement and availability of feed and fodder, including technological interventions to increase yield, expand areas to raise fodder crops, conserve land for growing feed and fodder, improve the nutritional value of poor quality roughages, and develop unconventional feeds at competitive prices . The maiGlobally, India is the second largest producer of wheat as a cereal crop, which, incidentally, produces a large amount of crop residue. Apart from feed and fodder production, a very limited amount of wheat straw (WS) is used for various other purposes such as the manufacture of ethanol, paper, and fertilizer, while the majority is either removed from the field, burned on site, piled, spread over fields, or used as mulch . HoweverSeveral physical and chemical treatments have been proposed to improve the degradability of WS and subsequent feed intake by cattle. Still, the practical use of these treatments is restricted by safety concerns, cost, and potentially negative environmental impacts . In thisTannin acyl hydrolase, commonly referred to as tannase (E.C. 3.1.1.20), is produced by various micro-organisms, and has been reported to improve the nutritional value of feed by the degradation of tannins. It is also able to hydrolyze ester bonds , as well as depside bonds  of substrates (or complex polyphenols) such as tannic acid (TA), epicatechin gallate, epigallocatechin gallate, and chlorogenic acid . The breGanoderma spp. was examined and compared in relation to biochemical parameters and crude protein (CP) production to improve the nutritional value of cattle feed, as evaluated by the carbon-to-nitrogen (C/N) ratio.In the present study, the effect of tannase-treatment on WS subjected to fungal pellet inoculum of All chemicals used in this study were of commercial/analytical reagent grade and purchased from Sisco Research Laboratories Pvt., Ltd. , Central Drug House Pvt., Ltd.  or SS Fermozyme Pvt. Ltd. .The spectrophotometric method described by Deschamps et al.  was usedThe remaining TA content in the fermented broth was estimated using the methods of Hagerman and Butler  and exprPre-dried WS was treated with different concentration of tannase (0.1%–0.3% (w/w) with an activity of 5.8 IU/mg) to remove tannins. Briefly, lyophilized tannase was dissolved in citrate phosphate buffer and sprayed on WS to obtain a moisture ratio of 1:2 (WS: moisture) and then mixed, incubated at 40°C under static conditions for 6.0 h, and analyzed for remaining tannin content.The untreated and enzyme-treated WS samples were powdered using a laboratory mill  and sieved  for analytical purposes. After enzyme treatment, the WS was considered tannin-free.Ganoderma spp. obtained from a laboratory stock culture collection , were maintained on malt extract agar (MEA), containing (g/L) malt extract, 20.0; KH2PO4, 0.5; MgSO4•7H2O, 0.5; Ca(NO3)2•4H2O, 0.5; and agar, 20.0 (pH 5.5), at 30°C. The obtained culture was stored at 4°C and subcultured every 2 wk. A 250-mL Erlenmeyer flask containing 50 mL of sterile malt extract broth (MEB) was inoculated with two mycelial discs (8 mm in diameter) from the periphery of 7 d-old fungal cultures grown on MEA. The inoculum was incubated at 30°C for 5 d under static conditions. The obtained mycelial mat was further homogenized and then inoculated (4% v/v) in 2-L Erlenmeyer flasks containing 500 mL MEB and incubated at 30°C for 5 d in a rotary incubator at 200 rpm. The fungal mass, obtained in the form of pellets, was separated from the culture broth through filtration using pre-weighed Whatman grade no. 1 filter paper. The formed pellets were used as inocula for further experiments. After being thoroughly washed with deionized water, dried at 55°C until a constant mass was achieved, and weighed.Enamel trays (40 cm × 32 cm × 7.5 cm) were loaded with 500 g of tannin-free or untreated WS (as a control) and moistened with mineral salt solution and sterilized. Each sterilized tray was inoculated with a dry fungal mass of 3.75–4.00 g (0.75% w/w). Sterile mineral salt solution was added to the trays to obtain a final substrate-to-moisture ratio of 1:3 and incubated at 30°C under 60% relative humidity for 10 d. Following incubation, the trays were harvested to determine production of various enzymes and the proximate and chemical composition of the WS. All experiments were performed in three times.The myco-straw  was removed aseptically from the flasks after incubation for 10 d and then suspended in 20 mL of acetate buffer  and agitated at 200 rpm for 1 h. The extrudates were pressed through a muslin cloth to maximize enzyme extraction and centrifuged at 10,000 rpm for 10 min at 4°C. The obtained enzyme solution was assayed to determine the activities of different enzymes.2O2 and MnSO4 to coerulignone. The increase in absorbance at 469 nm was determined at 30°C and the activity was calculated using an ϵ469 of 27,500 /mol/cm for DMP [Laccase (E.C. 1.10.3.2) activity was determined using the method described by Paavola et al. . MnP , Na2HPO4•7H2O (7.0 g); KCl (0.57 g), NaCl (0.47 g), MgSO4•7H2O (0.12 g), and 1.0 mL of CaCl2 (4% w/v). The solution was filtered through a muslin cloth and a 500-mg aliquot of the wheat straw was placed in a 50-mL centrifuge tube and the prepared fecal suspension was added. Then, CO2 gas was flushed into the centrifuge tubes for 48 h in a 39°C water bath. The mixture contained in the centrifuge tubes was centrifuged and the supernatant was discarded. Next, 35 mL of acidified pepsin (6.6 g in 1 L of 0.1 mol/L HCl) was added to the pellet and the tubes were again incubated under similar conditions for 48 h and then centrifuged. The obtained pellet was filtered through tared filter paper and dried. In vitro digestion was expressed as the loss of dry matter mass during incubation under standard assay conditions.r et al.  with somPenicillium charlesii, produced 61.4 IU/mL in 54 h in 220 L of statistically optimized medium composed of (% w/v) TA (2.0), sucrose (1.0), NaNO3 (1.0), and MgSO4  contained in 300-L vessel and then fed with 1.0% (w/v) TA after 24 h of incubation. The tannase obtained from the fermentation broth was partially purified by ultrafiltration (30 kDa), ammonium sulfate precipitation, and then lyophilized with a final yield of 5.8 IU/mg [A previous study reported that the selected soil isolate, identified as .8 IU/mg .2 = 0.999) between enhanced GA production and TA degradation  and tannase-treated WS samples (at 0.1% w/w of tannase concentration) revealed tannin contents of 3.6% and 1.8% (w/w), respectively. On further increase in tannase concentration up to 0.3% (w/w), a 1.8-fold higher reduction in tannin content was noted compared to the results obtained at 0.1% tannase. These results clearly indicated that an increased tannase concentration resulted in a decrease in tannin content  tannase was the optimal concentration for a remarkable increase in fungus-produced lignocellulolytic enzymes. Our results also indicated that at the optimal concentration of 0.1% tannase (w/w) resulted in a 1.12-fold increase in laccase activity, a 1.06-fold increase in MnP activity, and a 1.17-fold increase in xylanase activity after incubation for 10 d compared to raw untreated WS . A higher DML of 14.6% (w/w) was obtained in Ganoderma grown on WS treated with 0.1% (w/w) tannase. By further increasing the tannase concentration beyond 0.1%, a decline in the lignocellulolytic activities of MnP, xylanase, and laccase along with lower DML was observed. Based on these results, we hypothesized that due to the relatively poor growth of Ganoderma, as observed at higher tannin degradation or GA concentrations; lignocellulolytic activity was decreased, indicating a reduction in lignin depolymerization.As in the previous experiment Table , an incrGanoderma-treated WS with 0.1% (w/w) tannase. In addition, the percentage of degraded hemicellulose decreased to 17.9% (w/w), whereas a further increase in tannase concentration  resulted in a lower concentration of degraded lignin  with increased cellulose degradation  and a higher L/C ratio of 0.27. Also, 32.2% of the in vitro digestibility using 0.1% tannase-treated myco-straw; therefore, 0.1% (w/w) tannase-treated myco-straw was selected, as it showed a lower L/C ratio along with a decent percentage of in vitro digestion, which indicated improved quality of cattle feed.The results of WS compositional analysis presented in Table Ganoderma-treated WS (myco-straw) exhibited maximum SSF efficiency (approximately 20%) on incubation day 10. A higher SSF efficiency leads to better nutrient assimilation by fungi and a consistent increase in CP content. It was observed that a higher efficiency of 24.4% was obtained in tannin-free Ganoderma-treated WS  on the 10th day of incubation compared to the untreated control WS and 1.22-fold higher compared to myco-straw. Furthermore, the tannin-free Ganoderma-treated WS at a tannase concentration of 0.1% (w/w) showed a 1.28-fold higher CP content compared to myco-straw and a 25.0% lower C/N ratio in the fermented substrate , as well as WRF (Ganoderma sp.) under SSF, as compared to the digestibility of untreated WS. Similar to our results, Arora and Sharma [in vitro digestibility up to 1.66-fold by WS treatment with the fungus Phlebia brevispora, obtained from northwestern and northeastern regions of India, while a 1.78-fold increase was observed by the use of fungi obtained in central India. In the present investigation, we established that the presence of 0.1% (w/w) tannase enhanced lignin degradation as well as in vitro digestibility.There are several reports in the literature confirming that WS contains appreciable amounts of tannins ,30. Enhad Sharma  reportedGanoderma spp. Therefore, an optimal GA concentration is required, as GA acts as an inducer, not an inhibitor, of the lignocellulytic enzymes produced from WRF. In the present report, the amount of GA produced (1.5 g/100 g) by tannin degradation with 0.1% (w/w) tannase was sufficient to promote growth of Ganoderma spp. and to induce production of lignocellulytic enzymes. Above this concentration, GA acts as an inhibitor of the lignocellulytic enzymes laccase, and MnP produced by WRF, which are primarily responsible for lignin degradation through inhibition of Ganoderma growth. Our results are in accordance with those reported by Gnanamani et al. [Phanerochaete chrysosporium (NCIM 1197). Also, Patel et al. [Regarding fibrolytic enzymes, several researchers have reported that the presence of aromatic compounds was required, to a certain extent, to enhance ligninase production in several WRF for lignin degradation. In the present study, it was noted that at higher tannase concentrations beyond optimal, more toxic phenolic compounds with anti-nutritional properties (tannin) were degraded up to approximately 90% with 0.3% (w/w) tannase along with the synthesis of higher amounts of GA, which is comparably less toxic than NA. However, this increased GA concentration could lead to lower activity of different lignocellulolytic enzymes produced from i et al. , in whicl et al.  reportedl et al.  observedl et al.  postulatGanoderma-treated WS (beyond 0.1% w/w tannase) contains a lower tannin concentration, which is generally associated with or forms complexes with the hemicellulases of WRF, thereby leading to more free sites available for hemicellulases to degrade greater amounts of hemicellulose in WS by the actions of WRF. On compositional analysis of WS, it was observed that greater amounts of hemicellulose were degraded as compared to cellulose at higher tannase concentrations (beyond 0.1% w/w tannase). Similar results have been reported by several researchers who found that tannin-rich agro-material can easily inhibit hemicellulases over cellulases through association, as cellulases are generally associated with the cell walls of WRF, while hemicellulases are extracellular and are, therefore, more sensitive to tannins present in lignocellulosic materials. In this respect, our results are in accordance with those reported by Hervás et al. [In the present study, we found that by increasing the tannase concentration beyond optimal (0.1% w/w), a greater amount of tannin was degraded and converted into GA and glucose in WS. Therefore, tannin-free s et al. , who obsGanoderma-treated WS was 0.18 at a tannase concentration of 0.1% (w/w). Calzada et al. [Ganoderma sp. rckk02 decreased the lignin content of WS by 34.95% (w/w) with a subsequent reduction in cellulose content by 34.33% (w/w) after incubation for 15 d [The lignin to cellulose ratio (L/C ratio) is considered important for cattle feed. This ratio clearly indicates that during treatment greater quantities of lignin are degraded, thereby leaving more cellulose in the residual substrate. Therefore, enzyme-treated feed may contain more cellulose as compared to lignin in residual substrates that can be easily digested and also provides a rich source of energy to ruminants. The L/C ratio of the tannin-free a et al.  observeda et al.  reportedfor 15 d .Ganoderma-treated WS with 0.1% (w/w) tannase as compared to raw WS. While, a 27.3% (w/w) higher CP content was obtained in Ganoderma-treated WS (myco-straw). Various studies on fungal fermentation have reported the enrichment of WS with fungal biomass as the protein source under SSF [Ganoderma sp. rckk02 compared to raw WS (control). The increase in CP content is partially attributable to fungal proteins, particularly chitin [A 78.6% (w/w) increase in CP content was observed in tannin-free nder SSF ,38. Whilnder SSF  reportedy chitin  reportedPenicillium charlesii resulted in complete fermentation over a short period, which indicates the scope of enriched feed production at an industrial scale. The biological treatment processes are safe, eco-friendly, easy to perform, and involves lower operational costs as compared to other treatment methods. Besides, in this process, the inoculum could be raised from existing growing cultures and reused several times to inoculate bulk amounts of residues. While, the use of chemicals is limited because once used, these substances leach into water sources, thereby requiring further treatments involving large amounts of other chemicals. If this technology could be commercialized in the future, then the enzyme tannase (lyophilized or liquid form) along with the Ganoderma sp. (dried or lyophilized form) can be made available to cattle ranchers as enriched feed at costs subsidized by the government.It could be concluded that the use of a combination of various lignocellulytic enzymes present in WRF and tannase obtained from All authors declare that they have no competing interests.SR and SM designed and performed the experiments, including chemical analysis, collected and analyzed the data, and wrote the manuscript. RKS verified the validity of the experimentation and checked the results. All of the authors read and approved the final version of this manuscript.


UD-101 detects wind-blown sand impacting on its surface. The method was devised by considering the results of wind tunnel experiments that were performed using a vertical sediment trap and the UD-101. Field measurements to evaluate the estimation accuracy during the prevalence of unsteady winds were performed on a flat backshore. The results showed that aeolian sand transport rates estimated using the developed method were of the same order as those estimated using the existing method for high transport rates,  Considerable effort has been devoted to understanding sand transport mechanisms, including the aerodynamic entrainment of sand grains, acceleration of sand grains in air streams, and transport of sand by steady winds –16.Aeolian sand transport mechanisms observed in the field under unsteady wind conditions differ significantly from those observed under steady wind conditions. Since the 1990s, high-frequency sampling systems such as piezoelectric mass flux sensors –22, highet al. :t0 is the sampling period of n (= 1 s) and sd is the diameter of the sensor (= 0.012 m). The vertical-distribution-type sand trap was developed by Hotta and Horikawa , and the total sand flux Q [kg m−1 s−1] is shown in et al. [et al. [−1 < u∗ < 1.77 m s−1 and 0.14 × 10−3 m < d50 < 0.68 × 10−3 m. Here, only the data for d50 = 0.25 × 10−3 m are shown. Q was found to be approximately proportional to q(sz), as expressed by:R2 of the approximation was 0.96, 0.99, 1.00, 1.00, 0.99, 0.98, 0.97, and 0.97 for sz = 0.005, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, and 0.14 m, respectively. As shown in A shows a positive relationship with sz, and it can be expressed as:The relationship between the sand flux at n et al.  employed [et al. , with 0.sz and R2 and between sz and A (shown in sz should be set in the range 0.02–0.08.The relationships between B and C show a positive relationship with d50 (The constants with d50 :(8)B=89Finally, the following equation is derived from Q can be estimated only if n, d50, and sz are measured by performing field experiments, although the equation was derived from observations made during 20-m-long wind tunnel experiments performed by Hotta et al. [et al. [u∗ < 1.8 m s−1 and at a sensor height of 0.005 < sz < 0.14.This equation suggests that a et al.  and Kubo [et al.  on a fla3.3.1.d50 is approximately 0.2 × 10−3 m along the backshore. Beach grasses grow around a small embryo dune at y = 50 m and around a longshore dune at y = 70 to 90 m just in front of the coastal forest area, where y is the onshore distance from the average shoreline position in 2004. The measurement point was located in a flat area between the embryo dune and backshore dune.Field measurements were performed at the Hasaki Beach using UD-101. The beach faces the Pacific Ocean and is located in Ibaraki Prefecture [35°50′25″N, 140°45′42″E; u, the wind direction θ, and n [the same data obtained by Udo [u and θ were measured at a height of 0.9 m above the measurement point (z = 0.9 m) using a three-axis ultrasonic anemometer , while n was measured at z = 0.04 m using UD-101. Four UD-101 sensors were deployed, and they were directed toward the north, east, south, and west. The wind direction in the horizontal plane is expressed clockwise from north, from 0° to 360°. The data of u, θ, and n were recorded on a data logger system  at a frequency of 1 Hz and were automatically downloaded on a weekly basis from the logger. The precipitation Pr was measured at 10-min intervals at the Choshi Meteorological Observatory  by the Japan Meteorological Agency.The annual wind direction is predominantly in the north-northeast direction . Winds ad by Udo , Figure sz was measured manually almost every day in order to calculate Q using sz was calculated by linear interpolation. The sz value of the sensor oriented toward the wind direction tended to be large and that of the sensor directed in the opposite direction tended to be small; for example, for north winds, the sz value of the sensor directed northward tended to be large and that of the sensor directed southward tended to be small.In addition, in this study, The measurement results are shown in 3.2.Q value estimated from n was compared with that obtained using the conventional equations for a constant wind velocity in order to examine the accuracy of Q estimated from the UD-101 data. Here, the commonly employed equations of Bagnold [B, QK, QOQ, and LQ, respectively) were used [The  Bagnold , Owen [5 Bagnold   and B, cK, cOc, and Lc are constants with values of 1.8, 2.78, 0.25 + w0/3u∗ , and 4.2, respectively. The sand transport rate was estimated to be zero when u∗ < u∗t. u∗ was estimated from u; u∗t was obtained using w0 was determined from Rubey's [s is the relative density of the sand (= σ/ρ) and ν is the kinematic viscosity of air. These equations were derived for a constant wind velocity, and hence, Q estimated using the UD-101 data was averaged over 10 min. The 10-min mean sand flux meanQ during periods without rainfall was compared to B, QK, QOQ, and LQ.Here,  Rubey's  equationmeanQ values obtained from n with B, QK, QOQ, and LQ values derived from the equations given above, for periods without rainfall. Data for n = 0 and u∗ < u∗t  were excluded. meanQ showed a clear positive relationship with B, QK, QOQ, and LQ, and the estimated values followed the order OQ < BQ < LQ < KQ. The best agreement was observed between meanQ and OQ when 0.02 < meanQ < 0.10 and between meanQ and BQ when meanQ > 0.10.meanQ versus B, QK, QOQ, and LQ for east-northeast (15° < meanθ < 105°), south-southeast (105° < meanθ < 195°), west-southwest (195° < meanθ < 285°), and north-northwest (285° < meanθ < 375°) winds. For small meanQ values, i.e., less than 0.01 kg m−1 s−1, the meanQ value was overestimated by B, QK, QOQ, and LQ. The probable causes for the overestimation are the low measurement accuracy of n for small meanQ, the low estimation accuracy of z0s for small u∗, variations in the wind velocity, and the high moisture content at the beach surface. Raupach [c0 in u∗, especially for small u∗. A decrease in the instantaneous wind velocity below the threshold velocity, even when the mean wind velocity is greater than the threshold velocity, can lead to an overestimation. The moisture content of the backshore is nonzero even during periods without rainfall because of the proximity of the backshore to the sea. Udo et al. [meanq, decreased by more than one order of magnitude when the moisture content increased during rainfall periods. Raupach  showed to et al.  demonstrmeanQ estimated from n and B, QK, QOQ, and LQ for the east-northeast, south-southeast, west-southwest, and north-northwest winds ; however, meanq was low when meanθ was seaward (in late summer and winter) and was even lower when meanθ was landward (in spring and autumn). The differences between meanQ and B, QK, QOQ, and LQ were found to be independent of meanθ.inds see . While as−1. Udo  showed tBc and Kc are 0.5−2.0 and 1.0–3.0, respectively. This result suggests that the actual values of BQ and KQ are possibly one-third of those shown in Hotta and Horikawa  performe4.meanQ > 0.01 kg m−1 s−1, the transport rates estimated using the UD-101 data were of the same order as those estimated using the conventional equations. The best agreement was observed between meanQ and OQ when 0.02 < meanQ < 0.10 and between meanQ and BQ when meanQ > 0.10. It is necessary to develop a method for accurately determining the constant values in the conventional equations. The cause for the disagreement between meanQ and B, QK, QOQ, and LQ for small meanQ should be investigated in a future research. In field analyses, the estimation of large transport rates under strong winds is important and the proposed estimation method is expected to be useful in obtaining the transport rate.In this study, we developed a new method for the estimation of the aeolian sand transport rate and demonstrated the usefulness of UD-101 for field measurements of the aeolian sand transport rate. The transport rates estimated from the UD-101 data were reasonable and showed a clear positive relationship with the rates estimated using the conventional equations. Further, for


Zones were scanned densitometrically at 254 nm. The Rf values of amlodipine besylate, hydrochlorothiazide, and losartan potassium were 0.35, 0.57, and 0.74, respectively. Calibration plots were linear in the ranges 500–3000 ng per spot for losartan potassium, amlodipine and hydrochlorothiazide, the correlation coefficients, r, were 0.998, 0.998, and 0.999, respectively. The suitability of this method for quantitative determination of these compounds was by validation in accordance with the requirements of pharmaceutical regulatory standards. The method can be used for routine analysis of these drugs in bulk and in formulation.A Simple high-performance thin layer chromatography (HPTLC) method for separation and quantitative analysis of losartan potassium, amlodipine, and hydrochlorothiazide in bulk and in pharmaceutical formulations has been established and validated. After extraction with methanol, sample and standard solutions were applied to silica gel plates and developed with chloroform : methanol : acetone : formic acid 7.5 : 1.3 : 0.5 : 0.03 ( Losartan (LOS), 2-n-butyl-4-chloro-5-hydroxy methyl-1-[2′-(1H-tetrazol-5-yl) (biphenyl-4-yl) methyl] imidazole, potassium is a strong nonpeptide antihypertensive agent which exerts its action by specific blocking of angiotensin II receptors . A literature survey reveals that a variety of spectrophotometric and chromatographic methods, including UV derivative, the simultaneous equation method, colorimetric determination, HPLC, ratio derivative and compensation technique, and a stability indicating HPLC method, have been reported for the determination of LOS in pharmaceutical dosage forms in combination with other drugs –14. SpecIn recent years TLC has been improved to incorporate HPTLC-grade stationary phases, automated sample-application devices, a controlled development environment, automated development, forced-flow techniques, computer-controlled densitometry, quantitation, and fully validated procedures. These features result in methods which are not only convenient, rapid, robust, and cost effective but also reproducible, accurate, and reliable. The objective of this investigation was, therefore, to establish an HPTLC method for simultaneous estimation of LOS, AML, and HCZ in bulk and in tablets. Analytically pure samples of losartan potassium, hydrochlorothiazide, and amlodipine besylate were procured from Madras Pharmaceuticals, Chennai, as gift samples and used as working standards, methanol of HPLC grade from Merck , and chloroform, acetone, and formic acid of analytical reagent grade from S.D. Fine Chemicals were used, without purification to prepare the mobile phase. A solution containing 1 mg/mL losartan potassium, amlodipine besylate, and hydrochlorothiazide was prepared by dissolving 10 mg of each standard in 10 mL methanol and was used as working standard solution.μ filter . To this solution known amount of amlodipine standard (20 mg) was added (standard addition method) as its content is very low in the formulation. This solution  was used for assay of losartan potassium, amlodipine, and hydrochlorothiazide in the tablets.Twenty TRILOPACE* tablets by Akums Drugs & Pharmaceuticals Ltd containing 50 mg losartan potassium, 5 mg amlodipine besylate, and 12.5 mg hydrochlorothiazide were weighed and powdered. An amount of powder equivalent to 50 mg of LOS, 5 mg of AML, and 12.5 mg of HCZ was transferred to a 50 mL volumetric flask. After addition of 30 mL of methanol and sonication (30 min), the solution was diluted to volume with the same solvent and filtered through a 0.45 254 (Merck). Samples were applied as 6 mm bands by means of a CAMAG (Muttenz Switzerland) Linomat V automatic sample applicator equipped with a 100 μL syringe . The distance between the bands of 14.0 mm and the spraying rate of 50 nl per second were maintained. Ascending development of the plate up to a distance of 85 mm was performed at 25 ± 2°C, with chloroform-methanol-acetone-formic acid 7.5 : 1.3 : 0.5 : 0.03 (v/v/v/v) as mobile phase. The development was carried out in a CAMAG twin-trough chamber previously saturated with mobile phase vapour for 20 min. The average development time was 30 min. Densitometric scanning at 254 nm was performed with a CAMAG TLC scanner 3 equipped with CAMAG Wincats software version 1.4.4 using deuterium light source. During scanning process, the slit dimensions were fixed at 4.00 mm × 0.30 mm. Chromatography was performed on 10 cm × 10 cm aluminium HPTLC plates coated with 0.2 mm layers of silica gel 60 F The method was validated in accordance with ICH guidelines .μg of LOS, AML, and HCZ per band were applied on the precoated TLC plates. The plates were then developed, dried, and scanned as described above. Calibration plots were constructed by plotting peak areas against the corresponding concentration of drugs (ng per spot). For all three drugs, the detector response was found to be a linear function of amount in the range 500–3000 ng per spot. The correlation coefficients of all the three drugs were found to be 0.9988 for LOS, 0.9985 for AML, and 0.9990 for HCZ, respectively. The average linear regression equations were Y = 4571.56X + 416.48  for LOS, Y = 3229.40X + 300.24 for AML, and Y = 5917.03X + 422.70  for HCZ. Different aliquots of standard solution equivalent to 0.5–3 N/B and LOQ = 10 × N/B, where N is the standard deviation of the peak areas of the drugs, taken as a measure of the noise and B is the slope of the corresponding calibration plot. LOQ and LOD for losartan potassium were found to be 0.382 and 0.121 μg/spot, respectively. For amlodipine they were 0.584 and 0.188 μg/spot, respectively, and for hydrochlorothiazide they were 0.497 and 0.162 μg/spot, respectively. The sensitivity of measurement of LOS, AML, and HCZ was estimated in terms of the limit of quantitation (LOQ). The smallest amount of each drug was also detected under the chromatographic conditions in terms of the limit of detection (LOD). LOQ and LOD were calculated by use of the equations LOD = 3 × n = 3). The results are reported in  The amount of losartan potassium, amlodipine, and hydrochlorothiazide in the pharmaceutical preparation were determined by replicate analysis  was determined by analysis of these solutions three times on the same day. Interday precision  was assessed by analysis of these solutions on three different days over a period of one week. The results of the precision studies are shown in μg/mL of LOS, HCZ, and AML) was spiked with amounts equivalent to 80, 100, and 120% of standard drugs. These solutions were reanalysed, and the recoveries were found to be within the acceptable limits , peak apex (A), and peak end (E) positions. Results from correlation of the spectra were for losartan potassium r = 0.9996 and r = 0.9994, for amlodipine r = 0.9994 and r = 0.9996, and for hydrochlorothiazide r = 0.9998 and r = 0.9997. The results of peak purity ensure the specificity and can conclude that no impurities or degradation products were coeluted. The mobile phase used was found to be effective in resolving the drugs . The RFμL standard drug solution six times on a HPTLC plate. After development of plate, peak height and peak area were recorded for the zones. The CV (%) of peak height and area were calculated and found to be 0.45 and 0.56, respectively, for LOS, 0.34 and 0.43 for AML, and 0.67 and 0.32 for HCZ.  The repeatability of sample preparation was assessed by application of 2  The proposed HPTLC method for simultaneous analysis of losartan potassium, amlodipine, and hydrochlorothiazide in pharmaceutical dosage forms has been established for the first time. Use of HPTLC enables analysis of several samples at the same time. The method is very simple, rapid, and provides accurate and precise results.


These trimester-specific population-based reference ranges are essential in everyday clinical practice for the correct interpretation of thyroid hormone values and accurate classification of thyroid disorders.Estimation and interpretation of thyroid function tests in pregnant women is of utmost importance for maternal, fetal and neonatal health. Our objective was to calculate laboratory- and geography-specific reference intervals for thyroid hormones during pregnancy in an iodine-sufficient area of the Mediterranean, Crete, Greece. This project was performed in the context of “Rhea” mother-child cohort. Fulfillment of extensive questionnaires and estimation of free triiodothyronine (fT3), free thyroxine (fT4), thyroid-stimulating hormone (TSH), and antithyroid antibodies were performed. The reference population was defined using inclusion criteria regarding thyroidal, obstetric, and general medical status of women. Reference interval for TSH was 0.05–2.53  For TSH, inter- and intra-assay variability were <5.3% and <6.4%, respectively, for levels of 0.32–39 mIU/mL. Accordingly, for free T4 these values were <7.8% and <7.1% for the level of 0.51–4.82 ng/dL (6.56–62.03 pmol/L), for free T3 < 9.1% & <10% for the level of 2.5–13 pg/mL (3.84–19.96 pmol/L), for anti-Tg < 4.9% and <5.8%, and for anti-TPO < 7.4% and 7.2%. The proposed reference limits of the manufacturer for normal euthyroid adults were: free T3: 1.8–4.2 pg/mL (2.76–6.45 pmol/L), free T4: 0.89–1.76 ng/dL (11.5–22.7 pmol/L), and TSH: 0.4–4 Q test was applied to the least extreme; if the test rejects the least extreme outlier, then the more extreme outliers are also rejected. Continuously, when the data followed a Gaussian distribution or were transformed to a normal distribution, reference intervals were computed as follows: mean ± 1.96 × standard deviation. If normality was not achieved, even after transformation or after the outlier deletion, a nonparametric method was applied to estimate the reference intervals, by computing the rank numbers of 2.5th and the 97.5th percentiles to estimate the lower and the higher limits of the reference interval, respectively.All data were analyzed by SPSS 17 for windows. The nature of the underlying distribution of free T3, free T4, and TSH for the reference population was examined by inspecting normality tests, histograms and P-plots. In case of a significant variation from normal distribution, a logarithm transformation (log and ln) was applied to achieve normality. Outliers were identified using box plots. For the identified outliers Dixon's U test was used to compare differences for the 2 trimesters for a level of significance of P < 0.05.Thyroid hormones were expressed as mean, median, standard deviation, 2.5th and 97.5th percentile for the 1st and 2nd trimester. The Mann-Whitney Starting from a total cohort population of 1610 pregnant women, 1300 samples were available for thyroid function and antibody analyses. Of them, 35.2% were in the first (<13 weeks), 61% in the second (13–27 weeks), and 3.7% in the third trimester (>28 weeks) of pregnancy . The agen = 22) were excluded from the analysis, since the sample size was not adequate for the estimation of reference intervals to a reasonable degree of precision. The final population (403 women) was used to determine the reference limits and the 95% confidence intervals for TSH, free T4, and free T3 for the first and second trimester of pregnancy.After implementation of the aforementioned exclusion criteria, a total of 875 women were excluded from the study , Table 1μIU/mL, of free T3 1.54–5.22 pg/mL (2.37–8.02 pmol/L), and of free T4 0.95–1.53 ng/dL (12.23–19.69 pmol/L). For the second trimester, respective reference intervals were: 0.18–2.73 μIU/mL for TSH, 1.78–5.29 pg/mL (2.73–8.13 pmol/L) for free T3, and 0.87–1.45 ng/dL (11.20–18.66 pmol/L) for free T4. Median TSH and free T3 values showed a slight increase in 2nd trimester, while median free T4 values fell as gestational age advanced.According to our results Tables  and 3, tU test revealed significant differences between trimesters for free T4 , while respective P values for TSH and free T3 were 0.058 and 0.054. These findings justify the separation of groups into different trimesters  was not adequate for the estimation of reference intervals to a reasonable degree of precision. However, during the second half of gestation TSH levels return to prepregnancy levels and remain stable [n = 12) in less than 8 weeks of gestation, when hCG has a minimal effect on thyroid. Contrary to The National Health and Nutrition Examination Survey in the USA [μg/g Cr [Our study is limited by lack of data concerning the third trimester. Pregnant women were partitioned into trimesters upon entering the study, and thus the resulting sample size (n stable . In addi the USA , some st the USA . In our [μg/g Cr , 53, whi[μg/g Cr . These fData from this study establish reference values for Greek Cretan pregnant women and point out the need for laboratory- and geography-specific reference ranges in an effort to detect overt and subclinical thyroid disorders and to evaluate the risk for both obstetric complications and impaired fetal development.


Streptomyces strains (either as pure or mixed cultures) to remove pentachlorophenol and chlorpyrifos was studied. The antagonism among the strains and their tolerance to the toxic mixture was evaluated. Results revealed that the strains did not have any antagonistic effects and showed tolerance against the pesticides mixture. In fact, the growth of mixed cultures was significantly higher than in pure cultures. Moreover, a pure culture (Streptomyces sp. A5) and a quadruple culture had the highest pentachlorophenol removal percentages , while Streptomyces sp. M7 presented the best chlorpyrifos removal (99.2%). Mixed culture of all Streptomyces spp. when assayed either as free or immobilized cells showed chlorpyrifos removal percentages of 40.17% and 71.05%, respectively, and for pentachlorophenol 5.24% and 14.72%, respectively, suggesting better removal of both pesticides by using immobilized cells. These results reveal that environments contaminated with mixtures of xenobiotics could be successfully cleaned up by using either free or immobilized cultures of Streptomyces, through in situ or ex situ remediation techniques. Pesticides are normally used to control specific pests and to increase the productivity in crops; as a result, soils are contaminated with mixtures of pesticides. In this work, the ability of  The agriculture sector is a very important part of the economy and provides foods and raw materials needed for a sustainable development. For this reason, this sector uses different resources as pesticides, chemical fertilizers, equipment, and machines . Pesticiμg L−1, and in Brazil in superficial water, rivers, and lakes: 0.001–0.174 μg L−1 [Because of the restriction imposed on toxic organophosphate compounds, chlorpyrifos (CP), a broad-spectrum and moderately toxic organophosphate insecticide, has gained the status of one of the most widely used commercial compounds . The wid4 μg L−1 .μg L−1 [Other substances widely used in both agricultural and industrial sector are chlorophenolic compounds, which are applied as broad spectrum biocides . Among tμg L−1 . In Chilμg L−1 .Thus, remediation of CP and PCP-contaminated sites is urgently required. A number of methods, including chemical treatment, volatilization, photodecomposition, incineration, and stockpiling, can be applied for the detoxification of these xenobiotic compounds –17. HoweArthrobacter, Pseudomonas, Sphingobium chlorophenolicum, and Serratia marcescens capable of PCP degradation have been reported [In some early studies, CP was reported to be resistant to biodegradation due to accumulation of the antimicrobial degradation products in soil . Howeverreported –26. Amonreported , 28.Complete mineralization of pesticides or their transformation to nontoxic products is desirable in bioremediation process, which is more reasonable with the use of microbial consortia rather than single cultures . MicrobiChlorpyrifos (CP) (99% pure) and pentachlorophenol (PCP) (98% pure) were purchased from Sigma-Aldrich Co. . All other chemicals used throughout the study were of analytical grade and were purchased from standard manufacturers.Streptomyces spp.  isolated from organochlorine pesticides contaminated Argentinian soils and sediments [Streptomyces spp. AC5 and AC7) isolated from a Chilean soil contaminated with organophosphorus pesticides [Streptomyces spp. A2-A5-A11-M7), known for its lindane biodegradation potential [Streptomyces spp. AC5 and AC7), and the third consortium consisted of the six actinobacteria strains together.Four ediments , 31, andsticides , were usotential ; the sec−1): starch, 10.0; casein, 1.0; K2HPO4, 0.5; agar, 15.0. The pH was adjusted to 7.0 prior to sterilization. Starch-casein medium (SC) was used for antagonism assays, consisting of (g L−1): L-asparagine, 0.5; K2HPO4, 0.5; MgSO4·7H2O, 0.2; FeSO4·7H2O, 0.01 [−1, for tolerance to toxicity of CP and PCP assay. The pH was adjusted to 7.0 prior to sterilization. Minimal medium (MM) was used for growth of the microorganisms and pollutants removal assays. It consisted of : trypticase, 15.0; soy peptone, 3.0; NaCl, 5.0; K2HPO4, 2.5; glucose, 2.5, was used for the inocula preparation for the immobilization technique. The pH was adjusted to 7.3 ± 0.2 prior to sterilization.Tryptic Soy Broth (TSB), containing , a qualitative assay was performed using MM agar plates. Rectangular troughs were cut in the centre of the plate and then filled with a filter-sterilized solution of the mixed pesticides . The actinobacteria strains were inoculated by streaking perpendicularly to the troughs. Microbial growth was used as a qualitative parameter of the tolerance to the pesticides mixture. The Petri dishes were incubated at 30°C for seven days. Growth controls were performed using plates without pesticides [To evaluate the tolerance of sticides .−1 of biomass (wet weight) of the microbial consortia Streptomyces spp. A2-A5-A11-M7 and Streptomyces spp. AC5 and AC7 and the six pure actinobacteria strains were inoculated in different Erlenmeyer flasks containing 30 mL MM spiked with the pesticides mixture (1.66 mg L−1 of each one). Cultures were incubated at 30°C for 72 h on a rotary shaker at 200 rpm and then centrifuged . Ten milliliters of the supernatant were aseptically taken out in each case for residual pesticides determination. Microbial growth was measured as dry weight at 105°C. All experiments were carried out in triplicate, and the results are given as the means.2 g LStreptomyces spp. strains were individually cultured in TSB for 72 h at 30°C and 200 rpm. The cultures were centrifuged at 8,500 ×g for 10 min, and the pellets obtained were then washed with sterile distilled water for the immobilization. For this, the actinobacteria consortium , was mixed with a sodium alginate solution, obtaining a final concentration of 7.5% of biomass in the support  of the mixed culture [2·2H2O solution and incubated at room temperature for 1 h. Then, the beads (3–5 mm diameter) were washed three times with sterile distilled water [All  culture . This mied water . All the−1 of each one) and subsequently incubated for 72 h at 30°C. Samples collected every 24 h were analyzed for residual pesticides concentrations.The immobilized consortium was inoculated into MM containing mixed pesticides , and NPD detector was used. The injection and detector temperatures were set at 280°C and 300°C, respectively. The oven temperature program began at 90°C for 1 min, increased to 180°C at 15°C/min, then increased to 240°C at 5°C/min, and finally increased to 280°C at 15°C/min. The obtained data were analyzed with the program GC Solution Version 2.30.00  [Supernatant samples of the centrifuged cultures were used to determine residual CP and PCP concentrations. For residual CP concentration determination, 1 mL of each sample was diluted to a volume of 10 mL with distilled water, and then it was extracted twice with 10 mL of hexane. The organic extracts were combined and dehydrated with Nahimadzu) . The retμL loop, a Merck-Hitachi L-7455 diode array detector operating at 215 nm and a Hitachi D-7000 data processor. A LiCHrospher 60 RP select B 250 × 4 mm column of 5 μm particle size with a LichroCART 4-4 guard column (Merck) was used. The mobile phase consisted of acetonitrile and phosphoric acid (1% aqueous solution) 1 : 1 (v/v) with a flow rate of 1 mL min−1. In these operative conditions, PCP retention time was 12 min [−1). The recovery of PCP ranged from 97% to 100%.Residual PCP concentration was determined with an HPLC equipped with a Merck-Hitachi L-7100 pump, a Rheodyne 7725 injector with a 20-s 12 min . Method P < 0.05. Professional versions of Infostat and Statistic 6.0 software were used.All the results are the average of three replicates per sample. One-way analysis of variance (ANOVA) and Tukey test were performed to test the significant differences among treatments. When significant differences were found, Tukey post-test was used to separate the effects among treatments. Tests were considered significantly different at Streptomyces spp. strains isolated from Argentinean and Chilean environments, no antagonistic effect on their individual growth was observed  showed a high degree of tolerance to the toxic mixture, while the other two strains (Streptomyces spp. A5 and AC5) presented moderate tolerance, based on qualitative analyses taking into account the degree of growth of each strain in the surroundings of the mixture of pesticides , reaching a biomass of 98.3 mg L−1 for the mixed culture of Streptomyces spp. AC5-AC7 and 101.67 mg L−1 for Streptomyces spp. A2-A5-A11-M7 ; the mixed culture Streptomyces spp. AC5-AC7 only removed 6% of PCP; instead Streptomyces spp. A2, AC5, and AC7 in pure cultures did not show the ability to remove PCP (P < 0.05) when these strains were grown in a coculture. Similar enhanced degradation has been observed in many studies of different pesticide-degrading consortia. For instance, Sørensen et al. [Sphingomonas sp. SRS2 was grown in coculture with SRS1 strain, rather than pure. Other coculture was able to mineralize a 62% of the added diuron (10 mg L−1) in a mineral medium due to the cooperative degradative capacities of Arthrobacter globiformis strain D47 and Variovorax sp. strain SRS16, since neither strain D47 nor strain SRS16 was capable of performing extensive mineralization of the herbicide in pure culture [Streptomyces sp. AC5 nor strain Streptomyces sp AC7 conclusively removed PCP in MM but combined the constructed two-member consortium removed approximately 6% of PCP, demonstrating that synergistic interactions between both strains may be involved in the degradation of PCP.The pesticide removal abilities of the pure and mixed cultures were determined by analyzing CP and PCP residual concentrations. It was observed that move PCP . Comparen et al.  reported culture . In natuStreptomyces sp. M7 in pure culture showed the best CP removal capability (99.2%) but the mixed culture Streptomyces spp. AC5-AC7 also showed high ability for CP removal (91.52%) (−1 of soil), where carbofuran degradation was maximum whereas minimum degradation was observed for lindane, both in the soil phase and in the liquid phase. They also found that in the mixture of these pesticides, the degradation efficiency was minor than the efficiency detected in systems contaminated with one pesticide at a time. This phenomenon was attributed to the lower number of microorganisms available to degrade specific individual pesticides. In the present work, this phenomenon could also explain the low PCP removal obtained in comparison to CP removal, although further studies are required to prove it. On the other hand, Buono et al. [Paracentrotus lividus embryos, demonstrated that the most toxic pesticides were PCP and AZM at EC50 (median toxic effect concentration 50%) level. They also observed that PCP toxic effects were not significant at concentrations below 0.03 mg L−1, but at higher concentrations, such as 0.3 mg L−1; the effects were significant. In this work, the PCP concentration was approximately 5.5 times higher than 0.3 mg L−1, which could be another reason for the minor removal of this compound. On the contrary, Matamoros et al. [−1 of each pollutant. Although in the present study, the CP was the pesticide with the highest removal percentages from the mixture.All the pure and mixed actinobacteria cultures were able to remove CP, reaching removal percentages greater than PCP removal. The strain (91.52%) . In prevo et al. , who stuStreptomyces strains studied at the present work have been previously exposed to different chlorinated pesticides. Thus, these microorganisms could have the enzymatic ability to release chloride ions, favoring its degradation. The cross-adaptation phenomenon suggests that one pesticide may be rapidly degraded in soil in which it has never been applied; provided that the same soil had been previously exposed to a pesticide belonging to the same chemical group [Streptomyces sp. A5 or Streptomyces sp. M7.All al group . This coKocuria sp. CL2 isolated from secondary sludge of pulp and paper mill, able to use PCP as the sole source of carbon and degrade this pesticide, had been reported [Another actinobacteria strain, reported . Different researchers have demonstrated that cell immobilization techniques can significantly increase the removal efficiency of different pesticides compared with free cells , 52. ThuPseudomonas strain immobilized in calcium alginate mineralized a 50% more of phenol than free cells under the same conditions [The results revealed that CP removal was higher than PCP removal, following the same trend observed in previous assays see , both innditions . Also, Ynditions  studied P < 0.05), evidencing the microbial activity. The sorption of different compounds, such as dyes and pesticides, on different immobilization supports was also reported by other researchers [On the other hand, the pesticide sorption phenomenon to the alginate support was registered for both compounds. However, analysis of CP removal showed that the phenomenon of sorption was gradual, increasing up to 72 h, whereas for PCP it remained almost stable from 24 h until the end of the assay (data not shown). The sorption percentage was higher for CP (60.34% ± 0.42%) than for PCP (5.97% ± 2.41%) (data not shown), although the pesticides removal percentages observed by using alginate beads with or without microorganisms were significantly different  and the mixed culture of the six strains immobilized (Streptomyces spp. A2-A5-A11-M7-AC5-AC7) were the consortia with higher ability to removal the toxic mixture   .Streptomyces spp. strains were able to tolerate a mixture of PCP and CP and did not show antagonistic effects among them. These strains were also able to grow and remove mixed pesticides, in pure as well as in mixed cultures. The immobilization of the cells allowed an increase of the removal of both pesticides. Our results reveal that Streptomyces strains could be used in mixed cultures and in immobilized systems as a potential tool for remediation of environments contaminated with multiple xenobiotics.Six


Studies based on laboratory data about thyroid stimulating hormone (TSH) and free thyroxine (FT4) reference interval (RI) show conflicting results regarding the importance of using specific values by age groups with advancing age. Retrospective laboratory data or non-specific criteria in the selection of subjects to be studied may be factors leading to no clear conclusions. The aim of this study is to test the hypothesis that TSH and FT4 have specific RI for subjects over 60 to 80 years.We evaluated prospectively 1200 subjects of both sexes stratified by age groups, initially submitted to a questionnaire to do the first selection to exclude those with factors that could interfere in TSH or FT4 levels. Then, we excluded those subjects with goiter or other abnormalities on physical examination, positive thyroid peroxidase antibodies (TPOAb), thyroglobulin antibodies (TGAb), and other laboratory abnormalities.versus 50–59 years old (p > 0.05), and 60–69 versus 70–79 years old (p > 0.05). Consequently, we achieved different TSH RI for the three major age groups, 20 to 59 years old: 0.4 - 4.3 mU/L, 60 to 79 years old: 0.4 - 5.8 mU/L and 80 years or more: 0.4 - 6.7 mU/L. Conversely, FT4 progressively decreases = significantly with age, but the independent comparison test between the sub-groups showed that after age 60 the same RI was obtained (0.7 - 1.7 ng/dL) although the minimum value was smaller than that defined by manufacturer. In the comparison between TSH data obtained by this study and those defined by the manufacturer (without segmentation by age) 6.5% of subjects between 60 and 79 years and 12.5% with 80 years or more would have a misdiagnosis of elevated TSH.TSH increased with age in the whole group. There was no statistical difference in the analysis of these independent subgroups: 20–49 TSH normal reference range increases with age, justifying the use of different RI in subjects 60 years old and over, while FT4 decreases with age. Using specific-age RI, a significant percentage of elderly will not be misdiagnosed as having subclinical hipothyroidism. In recent decades there has been increased life expectancy of the population and, consequently, of the aging process. Persons older than age 60 comprise 20 per cent of the world population in the more developed regions, and from 5 to 8 per cent in the less developed regions. The oldest old, persons aged 80 years or older, is the fastest growing segment of the older population and by 2050 the number of this group is projected to be five times as large as at present .Several aspects of the aging process affect the endocrine system and stimulate the use of screening programs for the detection of hormonal changes and drug interventions with hormone replacement therapies to provide better quality of life for the elderly. Evaluation of thyroid function in normal elderly is difficult, since the prevalence of non-thyroid disease and the use of medications that interfere with thyroid function is greater than in young people. As a result, questions about the meaning of functional changes observed in the elderly are relatively common .Data interpretation of thyroid function in the elderly has been changing over the past decades. In a study conducted in 1995 in a non-selected population, the authors considered that subjects of any age with some degree of TSH elevation had some grade of thyroid gland failure . HoweverThe International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Committee developed the theory of reference values for the “Reference Intervals and Decision Limits (CRIDL)” . In 1995Recent studies have shown conflicting results regarding the decision to use reference intervals (RI) of TSH suitable for the elderly or not ,8. On stLikewise, as FT4 is the test of choice for the confirmation of thyroid disease, it is also relevant to assess whether there is a need for RI specific for subjects above 60 years and 80 years or older.The aim of this study was to test the hypothesis that it is necessary to use specific RI for TSH and FT4 for subjects over 60 years old, and over 80 years old, compare the values with those of young subjects, and analyze the impact of using specific TSH RI for these age groups in the screening of thyroid dysfunction.Between March and December 2012, 1200 subjects of both sexes were evaluated prospectively distributed as follows: 120 males and 120 females in each of the following age ranges: 20–49, 50–59, 60–69, 70–79 and above 80 years, who attended a clinical laboratory to collect routine tests for which function evaluation and/or thyroid autoimmunity had not been requested. They were invited to participate in order to arrive at the laboratory on pre-determined weekdays, according to availability of the researchers. A questionnaire was applied by one of the researchers, through direct interviews with participating candidates, which consisted of: 1) Identification data, address and phone number of the participants, as well as the name and telephone number of their physician for communication in case of abnormal results; characterization of their color or race according to themselves; if they consider themselves as healthy individuals; personal or familial thyroid disease in the present or past; current medications , smoking, hospitalization due to illness or accident in the last six months (and what the disease was), and pregnancy . Subjects were from the metropolitan area of Rio de Janeiro, belonging to middle and upper social class, as declared income, 77% white, 18% mulatto and 5% black. Subjects with negative thyroid peroxidase antibodies (TPOAb) and thyroglobulin antibodies (TGAb), normal lipid profile, ultrasensitive C-reactive protein level (CRP), blood count, renal function, and absence of goiter on palpation were in the inclusion criteria. The measurement of urinary iodine was not done since the salt iodization in Brazil is determined by federal law . In addiExclusion criteria included past or present history of thyroid disease, previous thyroid surgery, family history of thyroid disease, TSH <0.1 mU/L or > 10.0 mU/L, as these results indicate a high probability of thyroid dysfunction , palpabl2-3-dimercatopropanol, 2-4-dinitrophenol, 5-fluorouracil, 5-hydroxytryptophanAcetazolamide, acetylsalicylic acid, alpha adrenergic blockers, aminoglutethimide, aminotriazole, amiodarone, androgens and other anabolic steroids, anphenona, anphetamines, antipyrineBenserazide, beta adrenergic blockers, bexarotene, bromine, brompheniramineCadmium, carbamazepine, chromate, chromium picolinate, cimetidine, clofibrate, clomiphene, clomipramine, cobalt, complex anions, corticosteroids, cytostaticsDanazol, diphenylhydantoin, dinitrophenol, dobutamine, domperidone, dopamine and its agonists, other dopaminergic agentsErythrosine, estrogens, ethionamideFenclofenac, fenoldopam, flunarizine, fluor, furosemide, fusaric acidGrowth hormone (GH), GH-Releasing hormoneHalofenate, heroin, heparinInterleukins, iopanoic acid, other radiological contrasts, and other iodine-containing substances and drugs (potassium iodide and others), insulin-like Growth Factor-1, interferonKetoconazoleL-asparaginase, L-dopa inhibitors, levothyroxine, lithium, lovastatin,Mefenamic acid, melatonin, metformin, methadone, methimazole, metoclopramide, mitotaneNevirapine, niacin, nicotinic acid, nifedipine, NSAIDs, nitrateO, p’-DDD, orphenadrine, opioids, oxcarbazepinePara-aminobenzoic acid, perchlorate, perphenazine, phenidone, phenylbutazone, phenobarbital, pimozide, prazozin, primidone, propylthiouracil, pyridoxineQuetiapineRaloxifen, resorcinol, rifampicin, ritonavir, rubidiumSalsalate, serotonergics antagonists, somatostatin and its analogues, spironolactone, St. John’s Wort, stavudine, sulfonamides, sulfonylureas, sulpiride, steroids hormonesTamoxifen, thiocyanate, thyroid hormones and their analogs, troleandomycin, tyrosine kinase inhibitorsValproic acidDrugs are listed in alphabetical order and not in possible importance as interfering in TSH or thyroid hormone values.The study was approved by the Research Ethics Committee of the Hospital Clementino Fraga Filho, Universidade Federal do Rio de Janeiro (HUCFF/UFRJ) and individuals agreed to participate by signing the informed consent form.Serum was collected in the morning, in same species standard sampling tubes with separator gel. The measurements were done on the same day in primary tubes, after blood centrifugation at 3200 RPM for 15 min.Serum TSH, FT4, TPOAb and TGAb were measured by electrochemiluminescence immunoassays on the Roche Modular Analytics® E170 .Serum TSH concentrations were measured by an immunometric method, with an intra-assay percentage coefficient of variation (% CV) of 3.0% at concentrations of 0.040 ± 0.001 mU/L, 2.7% at 0.092 ± 0.002 mU/L and 1.1% at 9.4 ± 0.1 mU/L. The reference interval provided by the manufacturer is 0.3- 4.2 mU/L. Serum FT4, TPOAb and TGAb concentrations were measured by competitive assays. For FT4, the % CV was 1.4% at FT4 concentrations of 0.7 ± 0.01 ng/dL, 1.8% at 1.3 ± 0.02 and 2.0% at 2.7 ± 0.1 ng/dL. The reference interval provided by the manufacturer is 0.9 -1.7 ng/dL. For TPOAb the intra-assay % CV is 6.3% at TPO concentrations of 21.3 ± 1.34 IU/mL, 5.1% at 51.2 ± 2.6 IU/mL and 2.7% at 473 ± 12.7 IU/mL. According to the manufacturer, individuals without thyroid disease score lower than 34 IU/mL. For TGAb the intra-assay % CV is 4.9% at TG concentrations of 47.2 ± 2.3 IU/mL, 1.3% at 588 ± 7.4, and 1.3 at 3289 ± 42.0 IU/mL TGAb concentrations. According to the manufacturer, individuals without thyroid disease score lower than <115 IU/mL.Data were analyzed using the program GraphPad Prism®, version 6.0 . In order to assess the Normal distribution of both data series (TSH and FT4) Kolmogorov-Smirnov tests were performed. Logarithmic 10 was used for analysis. TSH and FT4 were calculated for each subgroup and gender from 20 to 49 years, then, by 10-year age range until 80 years; all those aged older than 80 were grouped together. Descriptive analysis of serum TSH was reported as medians and 25% and 75% percentiles because it is not normally distributed. Two tailed Mann–Whitney test and Kruskal-Wallis test were used to compare the nonparametric TSH distributions in different subpopulations. Evaluation between two subgroups was made by independent test of Dunn multiple comparisons. Means and standard deviations were calculated for FT4, since it has showed a normal distribution. For comparing FT4 between all groups, two-way ANOVA tests were used when more than two data series were analyzed, and Student test in the case of two data series.Outlying observations were calculated using the test proposed by Dixon. In all cases, the level of significance used was 0.05 . For theTo calculate correlations between TSH and FT4 levels in all age groups, TSH data were transformed into logarithmic 10. With normal distribution the two-tailed Pearson test was used; it was considered statistically significant if a p-value was less than 0.05. Pearson test was also used to analyze, individually, correlations between TSH and age and FT4 and age.versus 50–59 years old (p > 0.05), and 60–69 years old versus 70–79 years old (p > 0.05), showed no statistically significant difference. These data confirm that different RI for three major age groups should be used: 20 to 59 years, 60–79 years and 80 years or more. RI calculated for each sub-group are shown in Table The reference group was comprised of 50% females and 50% males. The mean age by gender in each age subgroups are in Table FT4 exhibited a Gaussian distribution. Data of FT4 measurements are shown in Figure Regarding the correlation between TSH and FT4, a high level of significance was observed in all age sub-groups, independently analyzed (p < 0.0001), 20–49 years old: Pearson r = -0.4641, 95% confidence interval (CI) = -0.4961 to -0.2926, R squared = 0.1652; 50–59 years old: Pearson r = -0.3862, 95% CI = -0.4881 to -0.2739, R squared = 0.1492; 60–69 years old: Pearson r = -0.4653, 95% CI = -0.5583 to -0.3607, R squared = 0.2165; 70–79 years old: Pearson r = -0.4946, 95% CI = -0.5839 to -0.3934, R squared = 0.2446; 80 years old and over: Pearson r = -0.3951, 95% confidence interval = -0.4961 to -0.2835, R squared = 0.1561.In order to assess whether the RI obtained in this study had clinical impact regarding the use of RI defined by the manufacturer in screening for thyroid disease, we compare the percentage of subjects who had TSH below or above the RI defined by age range obtained from this study with the values without segmentation by age. The results are showed in Table Usually, for the interpretation of a laboratory test, clinical laboratories use RI provided by the manufacturer of lab kits. The result of a patient’s test is then compared to this RI in order to diagnose whether it is normal or not. TSH concentration is the most sensitive test to reliably detect thyroid function abnormalities and is used as the screening test for studying thyroid function because of the inverse log-linear relationship between circulating TSH and FT4 concentrations ,28.This study, conducted prospectively with a reference population, showed that TSH increases progressively and significantly with age. The value of z less than 3.0 in all age groups indicates that the same RI should be adopted for both genders. On the other hand, FT4 tends to decrease with age. In the age groups above 60 years, FT4 values are equal between males and females. In subjects younger than 60, the value of z pointed that FT4 is lower in males than in females. A study of Kratzsch et al. also reports lower FT4 in males than in females . HoweverThe lower TSH limit defined in this study of 0.4 mU/L is the same for all age groups over 60 years old, and does not differ from the lower limit of younger people. This value is in accordance with previous study using third generation immunometric TSH methodology, that refers to the lower TSH reference limit as approximately 0.3 to 0.4 mU/L, irrespective of the population studied or the method used . The valAssociation between TSH and age is highly significant. The median is 1.5 mU/L in people under 60 years old, increases to 1.7 mU/L from 60 to 79 years old, and to 2.0 mU/L for those aged 80 or more. Upper TSH limit increases from 4.3 mU/L under 60 years old, to 5.8 mU/L in the range of 60 to 79 years old and to 6.7 mU/L in those very old subjects, over 80. So, although the manufacturer’s TSH kit is suitable for subjects less than 60 years of age, the same is not true for those 60 years or more, in which the limits are significantly higher.Within the age ranges of 60–79 years and 80 years or over, a significant percentage of subjects are reclassified as having not elevated TSH if age-specific RI is adopted. The same does not occur in young subjects. According to the RI for each age group obtained in this study, 6.5% of subjects between 60 and 70 years and 12.5% of those with 80 years or more have less misdiagnosis of elevated TSH, leading to 19% reclassification from hypothyroidism to normal, using these criteria. With regard to the effects of age on serum TSH levels, other epidemiological studies indicate that the population’s mean TSH levels increase with age ,31,32. TFailure to find these differences in RI between the young and the elderly reported in other studies may be due to retrospective studies based, or selection choosing populations to be reference for which strict selection criteria were not applied through specific questionnaires and appropriate physical examination to exclude factors such as thyroid dysfunction in the subject or in the family, interfering medications, other illnesses, recent hospitalizations, smoking habits and goiter.Like TSH, FT4 has the same minimum reference value for all age groups. This value of 0.7 ng/dL however, is lower in relation to that defined by the manufacturer (0.9 ng/dL) in all groups. In relation to the maximum reference value, the level of 1.7 ng/dL is suitable to be used for subjects 60 years old and over. However, although this is not the scope of this study, the results obtained by us suggest that a higher reference superior value (1.9 ng/dL) should be used for young individuals.Previous study demonstrated that FT4 remained relatively unchanged with age . These dWe consider relevant the data obtained in this age-related prospective study, once it is very important to distinguish between normal and mildly elevated serum TSH concentrations in elderly subjects. Elevations of TSH in young individuals even light and without decreasing FT4 characterize subclinical hypothyroidism or minimal thyroid dysfunction and are related to comorbidities such as dyslipidemia, adverse obstetric events, impact on cognition, quality of life, cardiovascular events, and evolution to clinical hypothyroidism . These eConcluding, this data shows that prevalence of subclinical hypothyroidism is overestimated in the elderly, in almost 20% of subjects, unless age-specific RI is used. This might improve diagnostic accuracy and reduce the need of confirmatory unnecessary tests.The author’s declare that they have no competing interests.RF did the study design, conducted the field study, made the final selection of study participants; computed the data and wrote the text. CRC made the initial guidance on the statistical requirements for the study, led and oversaw all statistical work. FA did the statistical field study under the supervision of CRC, being assisted during the course of the study also by the statistical mentioned in the acknowledgments. MV was the general supervisor of the study. He had discussions with the staff and the necessary modifications to the original design of the study; he revised, and made necessary adaptions to the final text and made the final approval of the text submitted to the journal. All authors read and approved the final manuscript.


The retention time (tR) for ACF, PCM and TRM were found to be 14.567 ± 0.02, 3.133 ± 0.01 and 7.858 ± 0.02 min, respectively. The validation of the proposed method was carried out for linearity, precision, robustness, limit of detection, limit of quantitation, speci city, accuracy and system suitability. The linear dynamic ranges were from 40–160 μg mL−1 for ACF, 130–520 μg mL−1 for PCM and 15–60 μg mL−1 for TRM. The developed method can be used for routine quality control analysis of titled drugs in pharmaceutical dosage form.A simple, precise and accurate reversed-phase liquid chromatographic method has been developed for the simultaneous estimation of aceclofenac (ACF), paracetamol (PCM) and tramadol hydrochloride (TRM) in pharmaceutical dosage form. The chromatographic separation was achieved on a HiQ-Sil™ HS C18 column , kromatek analytical column at ambient temperature. The mobile phase consisted of 40: 60 (v/v); phosphate buffer (pH 6.0): methanol. The flow rate was set to 1.0 mL min Aceclofenac (ACF) , . The chromatographic conditions were also optimized by using different buffers like phosphate, acetate and citrate for mobile phase preparation. After a series of screening experiments, it was concluded that phosphate buffers gave better peak shapes than their acetate and citrate counterparts. Further to decrease the tR of ACF, gradient mobile phase consisting of phosphate buffer and methanol was also tried in various ratios but was ruled out because of the appearance of hump in baseline. Finally, in isocratic system different ratios of phosphate buffer and methanol at different pH were tried. To improve the peak shape, triethylamine was added. The optimum mobile phase was found to consist of 40: 60 (v/v); phosphate buffer (pH6.0): Methanol. Phosphate buffer (pH 6.0) was prepared by dissolving 1.56 g sodium dihydrogen ortho-phosphate dihydrate and 0.35 g disodium hydrogen phosphate dihydrate in 1,000 mL LC-grade water. Triethylamine (1 mL) was added and the pH adjusted to 6.0 by addition of ortho-phosphoric acid. The flow rate was set to 1.0 mL min−1 and UV detection was carried out at 270 nm. The retention time (tR) for ACF, PCM and TRM were found to be 14.650, 3.133 and 7.842 min, respectively. Acceptable retention time (tR), plates, asymmetry and good resolution for ACF, PCM and TRM were obtained.The HPLC procedure was optimized with a view to develop a simultaneous assay method for ACF, PCM and TRM, respectively. The stock standard solution was diluted with diluent to a concentration of 100 μg mL−1, 130–520 μg mL−1 and 15–60 μg mL−1, respectively. The corresponding linear regression equation was y = 43113 x 85483, y = 16544 x + 2993979 and y = 7579 x – 2200, with square of correlation coefficient (r2) of 0.9998, 0.9987 and 0.9999 for ACF, PCM and TRM, respectively. Residual analysis was performed to ascertain linearity. The linearity of calibration graphs and adherence of the system to Beer’s law was validated by high value of correlation coefficient. No significant difference was observed in the slopes of standard curves  to examine was changed at three levels . One factor at the time was changed to estimate the effect. Thus, replicate injections (n = 6) of mixed standard solution at three concentration levels were performed under small changes of chromatographic parameters (factors). Results, presented in −1, respectively, for ACF, 0.57 and 1.74μg mL−1, respectively, for PCM and 0.31 and 0.95 μg mL−1, respectively, for TRM.The LOD and LOQ were found to be 0.69 and 2.09 μg mLtR) ± S.D. for ACF, PCM and TRM was found to be 14.567 ± 0.02, 3.133 ± 0.01 and 7.858 ± 0.02 min, respectively, for six replicates. Tailing factor for peaks of ACF, PCM and TRM was less than 2 (T ≤ 2) and resolution was satisfactory (Rs ≥ 2). The peaks obtained were sharp and have clear baseline separation.There is no peak interference of blank and placebo at the retention time of ACF, PCM and TRM, which indicates that the method is specific for the analysis in their pharmaceutical dosage form. The specificity of the method is illustrated in T), capacity factor (K’), selectivity (α) and resolution (Rs) between ACF, PCM and TRM peaks were calculated and summarized in System suitability parameters including theoretical plates, peak asymmetry was carried out. Satisfactory results were obtained for the drugs in a good agreement with the label claims thereby suggesting that there is no interference from any of the excipients which are normally present in tablets. The recovery % ± R.S.D. % of six replicate determinations was 99.76 ± 0.29 for ACF, 99.57 ± 0.37 for PCM, and 99.18 ± 0.57 for TRM.The developed HPLC technique is precise, specific, robust and accurate. Statistical analysis proves that the method is suitable for routine analysis of ACF, PCM and TRM in pharmaceutical dosage form.


Under optimized headspace conditions, equilibration temperature of 120°C, equilibration time of 5 min, and sample size of 50 μL, the regression of the peak area ratios of benzene and toluene to IS on the concentrations of analytes fitted a linear relationship well at the concentration levels ranging from 3.2 g/L to 16.0 g/L. Standard additions of benzene and toluene to blank different matrix solutions 1ead to recoveries of 100.1%–109.5% with a relative standard deviation (RSD) of 0.3%–8.1%. The method presented here stands out as simple and easily applicable, which provides a way for the determination of toxic volatile adjuvant in liquid pesticide formulations.The toxic inert ingredients in pesticide formulations are strictly regulated in many countries. In this paper, a simple and efficient headspace-gas chromatography-mass spectrometry (HSGC-MS) method using fluorobenzene as an internal standard (IS) for rapid simultaneous determination of benzene and toluene in pesticide emulsifiable concentrate (EC) was established. The headspace and GC-MS conditions were investigated and developed. A nonpolar fused silica Rtx-5 capillary column (30 m × 0.20 mm i.d. and 0.25  Emulsifiable concentrate (EC) formulations, which are the blend of active ingredient, organic solvent, and surfactants, are the major pesticide formulations currently used in many developing countries. Compared to other formulations, advantages of EC include higher concentration of active ingredient and being simple to manufacture and low in cost, relatively easy to handle, transport, and store, not abrasive, and uniformly spreading and wetting under normal spray and weather conditions. However, the disadvantages of EC cannot be overlooked. The large amount of the organic solvent makes the EC formulations flammable and corrosive. More seriously, the hidden toxic inert ingredients in EC, such as benzene and toluene, might cause adverse effects to humans and the environment. Benzene and toluene are ubiquitous environmental pollutants. The American Petroleum Institute (API) stated in 1948 that “it is generally considered that the only absolutely safe concentration for benzene is zero” . AlthougRegarding the complex matrix of samples, for the volatile toxic organic chemicals, headspace-gas chromatography (HSGC) analysis which allows direct injection of specimens, is without doubt the most common procedure used . In receThe advantages of HSGC-MS are threefold . As a hoTaking chlorpyrifos EC as a model and fluorobenzene as an internal standard, the objective of the present work was to develop a simple and efficient HSGC-MS method for rapid simultaneous determination of benzene and toluene in pesticide EC. The headspace parameters  and method validation were also explored.Benzene, toluene, and xylene, all of analytical grade with purity ≥99.0%, were purchased from Beijing Chemical Works . Analytical purity fluorobenzene was supplied by Sinopharm Chemical Reagent Co., Ltd. . Chlorpyrifos technical with purity 98.0% was provided by Dow AgroSciences (USA). Tween-80 (polysorbate) was purchased from Xilong Chemical Co., Ltd. . Beta-Cypermethrin technical with purity 95.0% was provided by Nanjing Ronch Chemical Co., Ltd.μm film thickness . Data were acquired through GC-MS solution software . Automated analysis was performed using a CTC Combi Pal Headspace AOC-5000 autosampler  controlled by a Cycle Composer software. GC-MS analysis was performed by a GCMS-QP2010 gas chromatography mass spectrometer , equipped with a split/splitless injector and nonpolar fused silica Rtx-5 capillary column (30 m × 0.20 mm i.d.) with a 0.25-μL sample solutions were added to 20 mL screw cap headspace vials. The vials then were placed in the headspace sample tray under the following operating conditions: equilibration time of 5 min with agitation, incubation temperature of 120°C; 100 μL of the vapor in vials was transferred to GC injection port with 1.0 mL syringe heated at 140°C by AOC-5000 robotic arm for GC-MS analysis.Aliquots of 50 The injection temperature was 200°C and operated in split mode (1 : 100). Helium was used as the carrier gas with a flow rate of 0.9 mL/min at a constant linear velocity (34.3 cm/s). Initial column temperature was 40°C held for 5 min, programmed at 45°C/min to 230°C, and finally held at 230°C for 5 min. Total run time was 14.7 min.For the mass spectrometry detection experiments, electron ionization was used with temperatures of 200°C and 250°C for ion source and interface, respectively. The scan range of mass-to-charge ratio (m/z) of ion was 45–110, and quantification analysis was acquired in selected ion monitoring (SIM) mode. Fragment ions were monitored for each analyte as specified in the following: m/z 78, 77, and 52 (benzene); 96, 70, and 50 (fluorobenzene); 91, 92, and 65 (toluene). Stock solution A: accurately weighed 200.0 g of chlorpyrifos technical, 50.0 g of Tween-80, and 250.0 g of xylene to 1000 mL of erlenmeyer flask were dissolved under ultrasound to afford 40.0% (w/w) chlorpyrifos EC. Stock solution B: accurately weighed 80.0 g of chlorpyrifos technical and 120.0 g of xylene to 500 mL of erlenmeyer flask were dissolved under ultrasound to afford 40.0% (w/w) chlorpyrifos solution in xylene. Stock solution C: accurately weighed 20.0 g of Tween-80, and 180.0 g of xylene to 500 mL of erlenmeyer flask were dissolved under ultrasound to afford 10.0% (w/w) Tween-80 solution in xylene. Stock solution D: accurately weighed 5.0 g of beta-cypermethrin technical, 20.0 g of Tween-80, and 175.0 g of xylene to 500 mL of erlenmeyer flask were dissolved under ultrasound to afford 2.5% (w/w) beta-cypermethrin EC. Stock internal standard (IS) solution: accurately weighed 5.0 g of fluorobenzene into 50.0 mL volumetric flask was made up to constant volume with stock solution A to afford 0.1 g/mL stock IS solution.The effect of the matrix composition has long been regarded as a crucial problem in quantitative headspace analysis. In order to compensate for this effect, and to obtain accurate results, analysts often use the internal standard technique for calibration . Later, Equilibration is the most important headspace sampling step. Careful attention toward choosing appropriate equilibration temperatures and times during method development helps ensuring a robust and long-lived procedure. Headspace sampling temperature dependencies lead to the general use of elevated equilibration temperatures. Higher equilibration temperatures are desirable for both greater headspace sensitivity and shorter equilibration times. What is more important is that higher equilibration temperatures can decrease the matrix effect to some extent. Based on this principle, full evaporation HS-GC technique, which adopts an adequate small sample size, was developed to overcome the matrix effect . (benzene)/Area(IS) and Area(toluene)/Area(IS) also reach constant value after 120°C. Accurately weighed 46.8 mg of benzene, 42.5 mg of toluene, and 47.3 mg of fluorobenzene to 10 mL of volumetric flask were made up to constant volume with stock solution A to afford solution for optimization of headspace conditions. To choose the optimal temperature of analysis, five different equilibration temperatures of 60, 80, 100, 120, and 140°C were tested. The equilibration time was set to 10 min. In When the incubation temperature was set to 120°C, the effect of five equilibration times of 5, 10, 15, 20, and 25 min on headspace analysis was screened. Our finding demonstrates that vapor-liquid equilibrium of the analytes and IS can be achieved within 5 min at the given temperature (120°C). Equilibration times longer than 5 min do not yield the expected increase in signal . Long-tiy = 0.095x + 0.2387  and y = 0.1057x + 0.1848 , where y stands for peak area ratio of analyte to IS and x represents concentration of analyte in sample (in g/L). Accurately weighed benzene  and toluene  to 10 mL of volumetric flask, spiked with 1.0 mL of stock IS solution each were, made up to constant volume with stock solution A to afford calibration standard solutions. Under the optimal headspace and chromatographic conditions, separate calibration curves of benzene and toluene were prepared by plotting relative peak area ratios of benzene and toluene to IS against the analytes concentration. The response linearity within the 3.2–16.0 g/L range for benzene and 3.6–16.0 g/L range for toluene was good, and it was characterized by a correlation coefficient of 0.998 and 0.997, respectively. The regression equations for benzene and toluene were as follows: n = 5) of the method for toxic benzene determination were very satisfactory. For another analyte toluene, the results were generally satisfied.The recovery and precision tests of this analytical procedure were performed by spiking blank different matrix solutions with accurate benzene and toluene. The general procedure was as follows: accurately weighed benzene and toluene to 10 mL of volumetric flask, spiked with 1.0 mL of stock IS solution each, were made up to constant volume with different stock solutions to afford spiked solutions for tests. Five replicates were analysed for each spiking level. To our delight, as shown in The matrix effect is a very common case encountered in HSGC analysis, since the matrix markedly influences the vapor-liquid equilibrium of the analyte in the headspace of closed vial. The diversity of the active ingredient, organic solvent, and surfactants makes considerable complex matrix for pesticide EC. In our previous report for HSGC determination of methanol in pesticide EC, the recovery was not acceptable for different matrix under low equilibration temperature . In the μL) and a high incubation temperature (120°C), a near-complete mass transfer of benzene and toluene from the liquid sample to the vapor phase was achieved within 5 min. More importantly, the matrix effect can be basically eliminated under the optimized conditions, which renders the present method applicable for all kinds of pesticide EC, even for other liquid pesticide formulations quality control or sample screening. When the concentrations of benzene and toluene in pesticide EC fall into the range 3.2–16.0 g/L, the regression equation can be used directly. If the concentrations are beyond 16.0 g/L, dilution of the formulation with xylene can be adopted to expand the application scope of the developed method, since the xylene as a matrix has little influence on the assay of benzene and toluene as shown in Integrating the advantages of both automated headspace and fast GC-MS, a simple, reliable, and efficient HSGC-MS method was established for rapid simultaneous determination of benzene and toluene in pesticide EC. By choosing a very small sample size (50