A survey was conducted to evaluate the multi-exposure level and correlation among toxic metal biomarkers . A total of 592 individuals who participated in the survey were residents near an industrial complex in Gwangyang and Yeosu (exposed group) and of Hadong and Namhae (control group) in southern Korea from May 2007 to November 2010. The Gwangyang and Yeosu area exposed groups had slightly higher blood Pb (2.21 and 1.90 µg/dL), urinary Cd observed values (2.20 and 1.46 µg/L), urinary Cd with a urinary creatinine correction (1.43 and 1.25 µg/g Cr), and urinary Hg observed values (2.26 and 0.98 µg/L) in women participants than those in the Hadong and Namhae area (control group). Blood Pb (3.18 and 2.55 µg/dL), urinary Hg observed values (1.14 and 0.92 µg/L), and urinary Hg with a urinary creatinine correction (1.06 and 0.96 µg/L) for male participants were also slightly higher than those in the Hadong and Namhae area (control group). The correlation among urinary Cd, Hg and Pb concentrations in the blood was significant. We suggest that the exposed group of residents were simultaneously exposed to Pb, Cd, and Hg from contaminated ambient air originating from the iron manufacturing industrial complex. National and international environmental pollution and disease problems continue to rise, and the WHO reported that 25%–35% of diseases in developed countries are due to environmental factors . The SouIn this study, we continuously and systematically monitored and evaluated Pb in blood (B-Pb), Cd in urine as an observed value (U-Cd), Cd in urine as urinary creatinine correction (U-Cd cr), Hg in urine as an observed value (U-Hg), and Hg in urine as urinary creatinine correction (U-Hg cr) in residents of the Gwangyang and Yeosu industrial complex areas.The east-west distance across Gwangyang Bay is 27 km, and it is 15 km wide from north to south. The Bay is surrounded by Gwangyang, Yeosu, Namhae, Hadong as a closed-coast; thus, pollutants spread out and become isolated in this area . The winThe participants in the exposed group were residents in the Gwangyang Bay area. Toxic metal  assessments were performed intensively from May 2007 to November 2010, in the Gwangyang and Yeosu National Industrial Complex around Gwangyang Steel works as representative industrial areas in South Korea. Gwangyang and Yeosu aimed the Residents who lived within 5 km with National industrial complex as the center. The Hadong and Namhae aimed the residents who lived over than 5 km with National industrial complex as the center. Response: We have conducted a multiple linear regression analysis, after adding to exposure and control areas. Residents of Hadong and Namhae areas were the control group with limited or no occupational and environmental exposure to Pb, Cd, or Hg. The subjects (n = 592) for the study were collected from those who agreed to participate (n = 2838). Subjects were excluded if they had an unclear address, no biological sample (blood and urine) data, or their and urinary creatinine concentration was out of the range of 0.5–3.0. This study was approved by the Institutional Review Board of Soonchunhyang University.Six ml of whole blood was collected in a Vacutainer  green capped tube containing heparin for the Pb measurements. The samples were carried to the laboratory at 4 °C and frozen at −20 °C. After thawing, we used a roller mixer to re-mix the samples before analysis. Urine was collected in a specimen cup, transferred to a 15 mL conical tube, and delivered refrigerated (4 °C) to the laboratory for the analysis of urinary Cd and Hg. The urine was frozen at −20 °C until analysis, which was performed within 24 h. The final heavy metal concentrations in the urine were revised to µg/g creatinine (cr). A survey of the enrolled subjects was performed by personnel who were trained sufficiently through interviews, and a standardized questionnaire derived from the Korea National Institute of Environmental Research was used. Information, lifestyle, demographic characteristics, dietary habits, diseases and drugs were included in this questionnaire survey.The analysis of B-Pb and U-Cd were performed with a GF-AA instrument . The limits of detection (LODs) were 0.1 and 0.3 µg/dL, respectively. Hg was analyzed with the Milestone DMA-80 direct mercury analyzer , and the LOD was 0.1 µg/dL.Standard Reference Material was used for the internal quality control  product for Pb in the blood. During the test, the analyzing was performed in Lot and almost all results were correct .t-test. Pearson’s correlation coefficients were used to investigate the relationship among factors affecting blood and urinary heavy metal concentrations. For studying the factors, which effected on the concentration of the heavy metal, we carried out a multiple regression analysis. A p < 0.05 was considered significant.We used Excel  for heavy metal data collection and the SPSS ver. 20.0 software package  for the statistical analysis. The general characteristics of the study subjects and the geometric mean concentrations of heavy metals are shown as averages. We compared the average differences in the heavy metal concentrations by Student’s 3. The median B-Pb concentration of the 592 subjects was 2.55 µg/dL and 0.98 and 5.09 µg/dL were the 5th and 95th percentile values. Median U-Cd concentration was 1.77 µg/L (0.33 and 6.98 µg/L were the 5th and 95th percentiles). The U-Cd Cr was 1.32 µg/g Cr (0.67 and 2.26 µg/g Cr were the 5th and 95th percentiles). The U-Hg was 1.18 µg/L (0.21 and 3.13 µg/L were the 5th and 95th percentiles). The U-Hg Cr, 1.07 µg/g cr (0.57 and 1.75 µg/g cr were the 5th and 95th percentiles).The 592 participant’s  characteristics and levels of Pb, Cd, and Hg are shown in p < 0.01) for lood-Pb; 2.20 µg/L in the exposed group and 1.46 µg/L in the control group (p < 0.01) for urinary-Cd;1.43 µg/L in the exposed group and 1.25 µg/L in the control group (p < 0.01) in urinary-Cd creatitnine; and 1.23 µg/L in the exposed group and 0.98 µg/L in the control group (p < 0.05) for urinary-Hg. The geometric mean of urinary-Hg creatinine in women tended to be higher in the exposed group than that in the control group, but the difference was not significant. Blood-Pb, urinary-Hg and urinary-Hg cr were slightly higher in the exposed group of men than those in the control group. The geometric means were 3.18 µg/dL in the exposed group and 2.55 µg/dL in the control group (p < 0.01) for blood-Pb; 1.14 µg/L in the exposed group and 0.92 µg/L in the control group (p < 0.05) for urinary-Hg; and 1.06 µg/L in the exposed group and 0.96 µg/L in the control group (p < 0.05) for urinary-Hg creatinine. The geometric means of urinary-Cd and urinary-Cd creatinine in men of the exposed group tended to be higher than those in the control group, but the difference was not significant.The correlation analysis results between the blood-Pb, urinary-Cd, urinary-Cd creatinine, urinary-Hg, and urinary-Hg creatinine, blood-Pb are shown in This study was the first national cohort study performed with residents who live in South Korea’s industrial complex area. We investigated heavy metal concentrations in biological samples among people who live in the Gwangyang and Yeosu industrial complex area.The average B-Pb concentration was 2.21 µg/dL in women and 3.18 µg/dL in men of the exposed group, which was higher than those values in the control groups of men and women (1.90 µg/dL in women and 2.55 µg/dL in men). The average (control) Korean B-Pb concentration is 1.91 µg/dL , whereasp < 0.01). The U-Cd levels in previous reports of a Korean (control) survey showed 1.23 µg/L for U-Cd and 1.56 µg/g for U-Cd cr [Women in the exposed group had significantly higher U-Cd (2.20 µg/L) and U-Cd cr (1.43 µg/g) than those in the control group (1.46 µg/L for U-Cd and 1.25 µg/g for U-Cd cr)  than the concentration of control group (0.98 and 0.92 µg/L in U-Cd). However, in case of U-Hg, exposed group was significantly higher (1.12 and 1.06 µg/g cr in woman and man candidates) than control group (1.06 and 0.96 in woman and men) in men (p < 0.05), but not in women (p > 0.05). The results of the control group in our study were higher than those found in Germany (0.34 µg/g cr) [Because many factories exist in the industrial complex, the residents could be affected by Hg emissions into the atmosphere. Geometric means that could influence residents of the exposed group were 1.23 µg/L and 1.14 µg/L in U-Hg of women and man candidate. It was slightly higher (µg/g cr) , the Uniµg/g cr) , and Canµg/g cr) . People Pearson’s correlation coefficients were significant for almost all variables except for the correlation between U-Cd and U-Hg cr, and between U-Cd cr and U-Hg. We suppose that these three toxic metals are emitted simultaneously, so that a higher Pb exposure results in higher Cd and Hg exposure. Participants in the exposed group were slightly affected by ambient air exposure, although the main exposure source is from intake of food especially seafood containing these three metals . More stWe suggest that the exposed group of residents were simultaneously exposed to Pb, Cd, and Hg from contaminated ambient air originating from the iron manufacturing industrial complex. Residents who live in the Gwangyang and Yeosu areas should be systematically monitored and continuously evaluated for health effects. We need to research the health damage to residents in industrial areas to provide basic data for expanding and establishing a center for environmental pollution control measures.


Asthma is a disease with high cost for the National Health Service. Two of the most recent LABA/ICS combinations for persistent bronchial asthma are Beclomethasone dipropionate/Formoterol (B/F) delivered via the Nexthaler device and Fluticasone furoate/Vilanterol (F/V) delivered via the Ellipta device. No comparison has been carried out yet in terms of cost analysis in asthma, to our knowledge. Aim of the present monocentric, observational, retrospective study was to calculate and compare the costs of mild-to-moderate asthma patients assuming B/F 100/6 μg b.i.d. to those of patients assuming F/V 92/22 μg once-a-day over a 12-week treatment period from the Italian National Health Service perspective.1 values, number of relapses, healthcare resources as hospitalizations due to asthma relapses, days of hospitalization, general practitioner (GP), specialist visits, and days of inactivity, were recorded over the study period together with the use of extra medications (systemic steroids and antibiotics). In order to compare the outcomes achieved in both groups, the propensity score matching method was used in STATA, and statistical significance was accepted for p < 0.05.Data were obtained automatically and anonymously from the institutional database of the Lung Unit of the Specialist Medical Centre (CEMS), Verona, Italy, UNI EN ISO 9001-2008 validated. FEV1 and comorbidities, returned a cohort of 40 group A patients of the entire cohort matched with 40 patients of group B, fully comparable for demographics and clinical characteristics. In the PS-matched cohort, the mean (±SE) number of relapses per patient during the follow-up was 0.53 (±0.12) in group A and 0.28 (±0.07) in group B. In group A, n = 25 (62.50 %), n = 9 (22.50 %), and n = 6 (15 %) patients had 0, 1, 2 relapses, respectively. In group B, n = 29 (72.50 %), and n = 11 (27.50 %) had 0 and 1 relapse, respectively. Over the study period, the average number of hospitalizations per patient was 0.15 (±0.06), with 0.28 (±0.12) days of hospitalization in group A, and 0.08 (±0.04) with 0.08 (±0.04) days of hospitalization in group B, respectively. The difference between the two groups in terms of FEV1(L) improvement vs baseline was 0.11 in favour of group B (p = 0.007). When results were compared, the improvement in lung function obtained in group B proved significantly higher both in terms of absolute FEV1 and of FEV1 % predicted. Clinical data of 77 patients treated with B/F b.i.d (Group A) and of 40 patients treated with F/V 92/22 μg once-a-day (Group B) were selected. The PS-matching process, designed as matching on the baseline covariates, gender, age, FEVp = 0.9). In particular, the mean (±SE) cost for visits per patient was € 26.82 (±3.73) in group A and € 11.36 (±2.30) in group B (p = 0.002), and the mean cost for rescue medications per patient was € 35.24 (±6.93) in group A, and € 18.73 (±3.38) in group B, respectively (p = 0.05).The mean (±SE) cost of hospitalizations per patient was € 345.30 (±133.23) in group A and € 172.65 (±98.18) in group B, respectively, with a mean not significant difference of - € 172.65 in favour of group B (Even if both ICS/LABA combinations were checked over a limited period of time, they seem characterized by a different profile in terms of effect on lung function and economic impact on mild-to-moderate asthma. The once-daily inhalation of combined Fluticasone furoate/Vilanterol 92/22 μg showed the potential for enhanced clinical outcomes and reduced costs when compared to Beclomethasone dipropionate/Formoterol 100/6 μg b.i.d. Bronchial asthma is a chronic inflammatory disease of the airways. It is characterized by airflow limitation, usually reversible spontaneously or following therapy, bronchial hyper-responsiveness and accelerated decline in lung function, which may progress to irreversible obstruction of the airways .The underlying mechanism responsible for asthma is the excessive presence and activation of inflammatory cells within the mucosal, muscular and vascular structures of the airways, which cause the release of inflammatory mediators and the remodeling of the airways. Clinically it manifests by the appearance and recurrence of cough, dyspnea, wheezing , and chest tightness . The cliAccording to WHO estimates, 235 million people suffer from asthma. The Italian National Institute of Statistics (ISTAT) survey on health and use of health services estimated a prevalence of asthma of 4.2 %  in Italy in 2012  was 82.2 % in group A and 93.3 % in group B, respectively. In other words, 30 doses were skipped (approximately corresponding to 15 days of treatment) in group A, and to 6 doses were skipped in group B (corresponding to 6 days of treatment).1 in litres (L) and FEV1 % predicted values were compared at baseline and at the end of the study period in the PS-matched cohort  value was 2.52 (±0.13) at baseline and 2.62 (±0.12) at the end of the study, and the difference vs baseline was 0.10. In this group, mean FEV1 % pred. was 82.40 % (±1.63) and 87.08 % (±1.58) at baseline and at the end of the study, respectively; the FEV1 % pred. difference vs baseline was 4.68 %.In group A mean (±SE) FEV1 (L) was 2.51 (±0.12) at baseline and 2.72 (±0.13) at the end of the study. The FEV1 difference vs baseline was 0.21. In this group, mean (±SE) FEV1 % pred. was 81.93 % (±2.00) and 89.50 % (±2.64) at baseline and at the end of the study, respectively; the FEV1 % pred. difference vs baseline was 7.57 %.In group B the mean (±SE) FEV1 (L) improvement vs baseline was 0.11 in favour of group B (p = 0.007). The corresponding difference in terms of FEV1 % pred. improvement vs baseline was 2.89 in favour of group B (p = 0.04).The difference between the two groups in terms of FEVIn the PS-matched cohort, the mean (±SE) cost of hospitalizations per patient was € 345.30 (±133.23) in group A and € 172.65 (±98.18) in group B, respectively, with a mean difference of - € 172.65 in favour of group B. The mean (±SE) cost for GP visits per patient was € 12.88(±2.27) in group A and € 5.68(±1.77) in group B, with a mean difference - € 7.20 in favour of group B. The mean (±SE) cost for specialist visits per patient was € 13.95 (±2.27) in group A and € 5.68 (±1.48) in group B, with a mean difference - € 8.26 ; the patient’s adherence to treatment. Also the cost of treatments is progressively regarded as a factor which can significantly contribute to precise the economic profile of different treatments, and then to compare their convenience, particularly from the National Health Service perspective.The present study was an observational, retrospective analysis aimed to compare the costs of mild-to-moderate asthmatic patients assuming Fluticasone furoate/Vilanterol once-a-day or Beclomethasone dipropionate/Formoterol b.i.d over a 12-week period.1 % predicted. When the results obtained in group A and B were compared, the improvement obtained in group B proved significantly higher both in terms of absolute FEV1 and FEV1 % predicted.Both treatments were effective in improving lung function significantly, particularly when the difference calculated from baseline was measured as FEVThe trend in favour of treatment B was also confirmed by the cost analysis.In general terms, the cost analysis showed a tendency towards a clear reduction of mean total costs in patients treated with Fluticasone furoate/Vilanterol 92/22 μg once-a-day. The hospitalization cost, which is the main component of cost, dropped by 50 % with Fluticasone furoate/Vilanterol 92/22 μg once-a-day. Unfortunately, due to both a certain dispersion of data occurring during the limited duration of the study in a small sample of subjects, and to the low frequency of hospitalization events over this short period of time, the comparison between the two groups in terms of hospitalization cost did not reach the statistical significance.In particular, even calculated over this limited period of investigation, the number of GP and Specialist consultations was significantly lower in patients treated with Fluticasone furoate/Vilanterol. Correspondingly, the cost due to visits  and to extra-medications (rescue medications) needed for facing relapses  were also significantly lower in this group, thus confirming the substantial decrease of asthma impact during Fluticasone furoate/Vilanterol treatment.2- receptors in favour of Vilanterol. Basically, these peculiarities are those which consent the 24-h therapeutic effect of the Fluticasone furoate/Vilanterol combination and then the once-a-day assumption, which likely leads to a better patients’ adherence to a long-term therapeutic strategy as confirmed in the present paper over a 12-week period.Even if both ICS/LABA combined treatments are regarded as highly effective and safe in persistent asthma –10, noneThe present study has some limits: the short duration of the investigation (12 weeks only) and the relative small number of subjects included. Moreover, the study is a monocentric investigation. On the other hand, some points of strength consist in the automatic selection of subjects from a general data-base and in the use of the propensity score matching method for assessing and comparing the outcomes, which assured a strictly objective system for comparison between the two subjects’ samples.The present study showed that the once-daily inhalation of combined Fluticasone furoate/Vilanterol 92/22 μg has the clear potential for enhanced clinical outcomes and reduced costs when compared to Beclomethasone dipropionate/Formoterol 100/6 μg b.i.d.


Rumen is an excellent environment for microbial growth consisting of bacteria, fungi and protozoa which are widely known to play important role in the fermentation process of ruminant cattle feed . Cellulaendo--β-D-glucanase (endoglucanases), (2) exo--β-D-glucanase (exoglucanases), and (3) β -glucosidase [Cellulose degradation by cellulolytic bacteria is a product of synergy in a group of cellulase enzymes. Cellulase enzyme system consists of three groups of hydrolytic enzymes, i.e. (1) es), and  β -glucoEndo--β-D-glucanase enzyme hydrolyzes β bonds randomly in a morphous regions of cellulose fibers [Exo--β-D-glucanase enzyme works towards reducing and non-reducing end of polysaccharide chains, especially on crystalline cellulose region, and liberates glucose as the main product resulted by β-glucosidase enzyme. Hydrolysis of crystal line cellulose part can only be done efficiently by exoglucanase enzyme. The synergy between endoglucanases and exoglucanases enzymes produces cellobiose molecules. Cellulose hydrolysis effectively requires an enzyme (β-glucosidase) that breaks down cellobiose into two molecules of glucose [Endo-1,-β-D-gluce fibers . Exo-; dinitrosalicylic acid (C4H4KNaO6.4H2O), (Merck); Natriumhydroxide (NaOH), (Merck); 3,5 - dinitrosalicylic acid, (Sigma); Sodium Sulfite (Na2SO3), citric acid, Na2HPO4.7H2O, NaHPO4, p-nitrophenyl cellobioside (PNPC), carboximethyl cellulose (CMC), p-nitrophenyl-β-D-glukopiranoside (pNPG), p-nitrophenol.Media used in this study were Luria Bertani medium . Optical density (OD) was measured at λ 600 nm by taking as much as 1 ml sampling with interval of 2 h for 30 h . The first sampling was done at 0th h and continued until OD values showed a clear decline. OD was measured with UV-vis spectrophotometer at λ 600 nm. Growth curve was obtained from the result of absorbance measurement on the time.10 ml of cultured E. cloacae WPL 214 was taken and transferred into 10 ml Luria Bertani growth medium in erlenmeyer flask. Culture suspensions were incubated for 24 h in a shaker incubator at 30°C, 35°C, 40°C and pH 4, 5, 6, 7, 8 with shaking at 120 rpm. OD measurement was conductedby taking 1 ml sampling. OD was measured using UV-Vis spectrophotometer at λ 600 nm.1 ml of cultured cellulolytic bacteria isolates of E. cloacae of 98 × 1010 CFU/mlwas put into 20 ml of liquid medium and incubated in a shaker incubator for 16 h at 35°C. Afterwards, culturewas centrifuged at 6000 rpm at 4°C for 15 min. Pellet was discarded while supernatant containing cellulase enzyme was taken to assay endo--β-D-glucanase, exo--β-D-glucanase, and β-glucosidase enzyme activity.Enzyme activity assay was conducted on enzyme produced from optimum growth time and temperature in the following way: Single colony of endo--β-D-glucanase activity was conducted using 3.5-dinitrosalisilat acid (DNS) method by using CMC as a specific substrate. Endo--β-D-glucanase activity was assayed by mixing 100 ml enzyme with 100 ml substrate(1% CMC in 0.1M citratephosphate bufferat pH7) and incubating it in a water bath at 50°C for 30 min. Enzyme activity of an amount of formed reducing sugars was measured using DNS method by adding 600 µl DNS into tube and placing it in a boiling water bath for 15 min together with control  and finally cooling it in ice water for 20 min. Total volume of enzyme activity assay in this study was 800 µl. Absorbance measurement was afterward conducted using cuvettes. Absorbance was read using spectrophotometer at λ 550 nm. One unit of enzyme activity was defined as the amount of enzyme required to form 1 mmol of product per unit time for each ml of enzyme [Assay of exo--β-D-glucanase activity, 100 μl enzyme mixed with 900 μl pNPC (1 mMol pNPC in 10 ml of citrate phosphate buffer at pH5) was incubated for 30 min at optimum temperature 35°C. Reaction was stopped by adding 100 μl 1M Na2CO3. Liberation of p-nitrophenol was read using spectrophotometer at λ 405 nm. A sa blank, 100 μl aquadest and 900 μl substrate that were treated the same as sample condition were used and reaction was stopped by adding 100 μl of 1M Na2CO3. One unit of enzyme activity is equivalent to the amount of enzyme required to produce 1 μmol p-nitrophenol/min [To measure 2CO3 and absorbance was read using UV-Vis spectrophotometer at λ 405 nm.p-nitrophenol standard was made in the range of 0.1-0.5 mM p-nitrophenol from 10 mM p-nitrophenol stock in phosphate citrate buffer solvent pH 5. 100 μl of each standard p-nitrophenol solution was mixed with 900 μl of phosphate citrate buffer pH 6 and incubated at 35°C for 30 min. Reaction was stopped by adding 100 μl of 1 M Naβ-glucocidase activity was assayed using pNPG method. β-glucocidase activity was measured by mixing 100 μl enzyme with 900 μl substrate (1 mM pNPG in 10 ml of phosphate citrate buffer pH5) and incubating it at 35°C for 30 min. Reaction was stopped by adding 100μl of 1M Na2CO3. As blank, 100 μl aquadest and 900 μl substrate that were treated the same as sample condition were used. Liberation of p-nitrophenol was read with spectrophotometer at λ 405 nm and then compared top-nitrophenol standard curve. One unit of enzyme activity was equivalent to the amount of enzyme required to produce 1 μmol p-nitrophenol/min [Measurement of This research was descriptive research so that the duplo data collected and analyzed in descriptive and expressed in the form of narrative and figures.E. cloacae WPL 214 cellulolytic bacterial inoculants is shown in th h with absorbance (λ 600 nm) of 3.122.Growth curve of Incubation temperature was specified at 30°C, 35°C, 40°C and 45°C and phosphate citrate at pH 4, 5, 6, 7, 8. The highest cellulose enzyme activity was obtained at 35°C as much as 0.154 U/ml  and at pnd h  specific substrate. Enzyme activity was determined by measuring the amount of p-nitrophenol released [p-nitrophenol in 1 min (U/ml) in experimental conditions. Cellulase enzyme activity curve was used to determine the optimum time required to obtain maximum cellulase enzyme activity from enzyme production process.In addition to growth curve observation, measurement of cellulase enzyme production was also conducted. Optimum production of cellulase enzyme occurred at the end of stationary phase, at the same time as the peak of microbial growth which occurred at the 22nd h . Celluloreleased . ObservapNPC specific substrate while cellobiase was assayed using pNPG specific substrate. Assays were carried out using the enzyme produced when enzyme activity reached optimum time, at the 22nd h. Assay was also carried out at optimum temperature of 35°C and optimum pH of 5. Data of exoglucanase and cellobiase enzyme activity are presented in Measurement of exoglucanase and β-glucocidase enzyme activity was conducted using p-nitrophenol derivative substrates. Exoglucanase was assayed using Endo--β-D-glucanase enzyme activity was measured simultaneously with the making of the growth curve. Sample was taken every 2 h and the enzyme extracted. Cellulase enzyme activity curve was used to determine the optimum time required to obtain maximum cellulase enzyme activity from enzyme production process. E. cloacae WPL 214 isolates showed that the optimum time to produce cellulose enzyme was at the 22nd h with endoglucanase enzyme activity of 0.09 units/ml (U/ml). Results of enzyme activity measurement using specific substrates of p-nitrophenol derivatives on E. cloacae WPL 214 isolates showed exoglucanase activity of 0.13 U/ml while cellobiose enzyme activity was at 0.10 U/ml. This result indicated that these isolates had activity on endoglucanase, exoglucanase and cellobiose enzymes. Cellulose hydrolysis requires synergistic activity of various cellulase enzymes with different specifications to produce multi enzyme system. It also requires coordination in cellulose molecule breaking, product resulted, and catalytic movement of cellulose chain [Research data of se chain ,13. MultEnterobacter bacteria produce cellulase enzyme. Isolation and identification have also been made from several plants, i.e. 53 endophyticentero bacteria from some plants including citrus, coconut, eucalyptus, sugarcane, and soybean. These studies identified E. cloacae, Pantoea agglomerans, Hafniaalvei, and Pantoea ananatis. The lowest cellulase enzyme production is obtained from coconut plants as much as 20% (2 of 10), equivalent to that produced by citrus crop as much as 20% (4 of 20). The level of cellulase production obtained, from the highest to the lowest, is as follows: Sugarcane, 100% (4 of 4), eucalyptus, 84.6% (11 of 13), and soybean, 83% (5 of 6) [Enterobacter chrysanthemi, E. cloacae, and Proteus mirabilis, Erwinia chrysanthemi, among others. These microorganisms are already known to have cellulolytic activity. Cellulolytic bacteria found in the digestive tract of insects have higher cellulolytic activity ability as they are naturally involved in the digestion of lignocellulosic substrates found in insect feed. Genetic engineering (recombinant) is performed on those cellulolytic bacteria and is further used in its role of converting cellulosic biomass (CMC substrate and sugarcane bagasse) into bioethanol product more quickly and efficiently through process of fermentation at 37°C and pH 7. Cellulosic ethanol products from cellulolytic bacteria aim to reduce dependence on petroleum [Not all (5 of 6) . Isolatietroleum .E. cloacae isolated from pumpkin (Aulacophora atripenis) bees can grow on a medium with different carbon sources, including CMC and 2% Avicel to produce Endo-1,4-β-D-glucanase enzyme. The maximum production of cellulase enzyme is obtained after 96 h of fermentation. The highest endoglucanase enzyme production occurs when grown in medium of 0.75% CMC. Enzymes Endo-1,4-β-D-glucanase is optimum at pH 5.8 and 40°C. The maximum number of enzymes produced on CMC substrates and Avicel 2% is lower, at 0.05 µM/ml, compared to enzyme produced on substrates of 0.75% CMC, 0.9 U/ml [E. cloacae WPL 214 isolate in this study also had 0.09 U/ml endoglucanase enzyme activity obtained at optimum time: 22nd h at pH 5 and temperature 35°C.0.9 U/ml . E. cloaendo--β-D-glucanase, exo--β-D-glucanase and β-glucosidase can be produced from cellulolytic isolates of E. cloacae WPL 214 isolated from bovine rumen fluid waste of Surabaya Abbatoir, Indonesia. This indicates that E. cloacae WPL 214 can be used to hydrolyze fibrous feeding material containing lignocellulose.Based on the research results, it can be concluded that cellulolytic enzyme having activity of WPL designed the research, collected and processed samples. DSN helped in designing the research. ABY carried out the data collection and gathering assay samples. TN and KS assisted in manuscript preparation; WPL, DSN, TN, and KS collected materials for manuscript. All authors have read and approved the final manuscript.


Ziziphora clinopodioides Lam. (Lamlaceae) (Xintahua) showed protective activities against rat acute myocardial ischemia in rats. This study aims to isolate acacetin, a flavonoid, from the aerial part of Z. clinopodioides, to develop an HPLC method for its detection, and to evaluate its protective effects on neonatal rat cardiomyocytes.The total flavonoids from ethanol extract of the aerial part of 1H and 13C NMR spectroscopy. The content of acacetin in Z. clinopodioides collected from three different origins was determined by HPLC. The neonatal rat cardiomyocytes were isolated and cultured in vitro to establish a hypoxia/reoxygenation injury model. The viability of cardiomyocytes was measured by the MTT method. Changes of malondialdehyde (MDA) content in the medium were also determined.Sephadex LH-20 silicagel and pillar layer chromatography silica gel were applied for the isolation and purification of acacetin and its structure was elucidated on the basis of Z. clinopodioides ranged from 45.50 to 47.41 μg/g. Acacetin of 25, 10, 5 μg/mL significantly decreased the MDA content in a model of hypoxia/reoxygenation injury .The acacetin content in various batches of Acacetin protects neonatal cardiomyocytes from the damage induced by hypoxia/reoxygenation stress through reduction of lipid peroxidation and enhancement of the antioxidant activity. Ziziphora clinopodioides Lam. (Lamiaceae) (Xintahua) is among the commonly used herbal drugs in Traditional Uighur Medicine and Traditional Kazak Medicine, with efficacy for the treatment of hypertension, fever, edema, heart disease, neurasthenic, insomnia, tracheitis, lung abscess and hemorrhoids [Z. clinopodioides consist of a large number of iridoids, phenolic and flavonoid compounds, including chrysin 7-O-rutinoside, linarin, diosmin, methyl 4-hydroxy-3,5-dimethoxybenzoate, 7-O-methylsudachitin , thymonin , caffeic acid and luteolin  [orrhoids ,2. The p3) and ethyl acetate (EtOAc) portions from the ethanol extract of the aerial part of Z. clinopodioides showed protective effects on rat acute myocardial ischemia and neonatal rat cardiomyocytes [The trichloromethane . Trypsin was from Amresco LLC . Streptomycin was produced by North China Pharmaceutical Group Corporation . DMEM (high glucose and low glucose) was from Invitrogen Corporation . Ampicillin sodium for injection was produced from China-promise Pharmaceutical Industry . Malondialdehyde (MDA) was provided from the Nanjing Jiancheng Bioengineering Institute . Acacetin was prepared in the Key Laboratory of Xinjiang Uighur Medicine . HPLC grade methanol and acetonitrile were purchased from Fisher Scientific . Water (0.055 μS/cm) was purified by a Milli-Q system from Millipore . Sephadex LH-20 silicagel was from Amersham Pharmacia Biotech (USA). Pillar layer chromatography silica gel (100-200 mesh) was from Qingdao Marine Chemical Plant (China). All other chemicals were of analytical grade.Z. clinopodioides were collected at the Astronomical Observatory  and the Chrysanthemum terrace , South Mountain of Tianshan Mountains in Urumqi, China, in September and August 2010. Another batch was from Xiao Dong Gou  of Altai Mountains in Alaty, China, in August 2010. The plant materials were identified by associate researcher Jiang He , according to Hudaberbi et al. [Two batches of NMR nuclear magnetic resonance was measured using JEOL ECP-500, INOVA400 and INOVA-600 . Melting points were measured with a semi-automatic melting point apparatus  system. A BS124S Electronic Balance  was used for analysis. A DG-5031 ELISA Reader  and Shimadzu UV-2501  were used for cardiomyocyte experiments.Neonatal Sprague-Dawley rats (1-3 days old) of either sex were maintained under standard environmental conditions. All animals were purchased from the Experimental Animal Centre of Xinjiang Medical University . Certificate Number: SCXK (Xin) 2003-2001. The study protocols were approved by the Ethics Committee on Animal Experiments, Xinjiang Material Medica, China (no.20110515).Z. clinopodioides (10 kg) was extracted with water and then the residue was extracted with methanol (MeOH) under reflux. The methanol extract was suspended in water and then successively extracted with CHCl3 and EtOAc. Then, the solution was vacuum-distilled using a rotary vacuum evaporator  to yield the CHCl3 fraction (142.5 g) and EtOAc fraction (138.5 g). The EtOAc fraction was purified on silica gel eluted with a gradient of CHCl3-MeOH. Eluates were combined according to thin layer chromatography (TLC) behavior using two solvent systems CHCl3-MeOH (97:3) to offer compound 1 (320 mg).The air-dried aerial portion of Z. clinopodioides was refluxed in 30 mL of methanol for 1 h after soaking for 20 min. After extraction, solvent was added to the extraction vessel until the final weight was equal to the starting weight to counter solvent loss. The extract was thoroughly mixed on a vortex mixer, and filtered through a 0.45 μm syringe filter prior to HPLC injection.Dried powder (1.0 g) of the aerial portion of All experiments were conducted with a Shimadzu-LC 2010C HPLC system. The mobile phase consisted of acetonitrile (A) and water with 1.0% glacial acetic acid (B), with the proportion of A:B held at 37:63. The chromatographic separation was performed using a YMC-Pack ODS-A  column with a flow rate of 1.0 mL/min. The column temperature was maintained at 35°C. All analytes were monitored at 326 nm.e.g., plotting the peak areas versus the concentrations of each analyte.Stock standard solutions of acacetin were prepared in MeOH and diluted to different concentrations to build calibration curves, Sample was analyzed using the developed method to verify the stability of the sample. The stability test was carried out by analyzing the sample at 0, 2, 4, 8, and 24 h. The relative standard deviations (RSDs) of peak areas at different times were calculated.Intra-day and inter-day variations were used to determine the precision of the developed method. The intra-day precision or inter-day precision was determined by analyzing replicated samples) on 1 day or over 3 consecutive days, respectively.The accuracy of the developed method was evaluated by spike recovery. Acacetin was added into 0.5 g of sample. Then, the mixtures were extracted and analyzed. The spiked recovery was calculated as follows:2 at 37°C) in DMEM supplemented with 2 mmol/L L-glutamine, 10% (v/v) foetal calf serum and penicillin/streptomycin (100 U/mL) to minimise fibroblast contamination. Cardiomyocyte-rich cultures (>90%) were plated onto fibronectin-coated 96-well plates, 100 μL per well, at a final density of 1.00 × 105 cells per cm2 in supplemented DMEM [Primary cultures of neonatal rat cardiomyocytes were prepared from the ventricles of 1 to 3-day-old Sprague-Dawley rats as previously described ,10. The ted DMEM ,12.The culture solution was discarded after 24 h co-cultivation of myocardial cells and different concentrations of acacetin, and then 180 μL DMEM and 20 μL MTT were added to each well for 4 h cultivation. The supernatant was discarded and 150 μL DMSO was added to each well, mixed evenly, and the absorbance (A) was measured at 570 nm within 10 min using a DG-5031 ELISA Reader .2 in advance), and the solution was placed in a hypoxic culture box (99.99% N2) for 120 min. Then, the medium was replaced with one saturated with pure O2, and the cells were exposed to a normoxic atmosphere containing 95% air and 5% CO2 at 37°C (reoxygenation) for 30 min. A thiobarbituric acid (TBA) method was used to determine the content of MDA [After cultivation for 72 h, the medium was exchanged with one that was hypoxic , group II served as the myocardial cell injury control group , groups III, IV and V were treated with three different doses  of acacetin . The inhibition rate was calculated from the absorbance of the medium containing added acacetin over the medium of the control (group I).P values less than 0.05 were considered significant.The results were reported as the mean ± standard derivation (SD) of at least three measurements. The analysis of MDA data was performed with the SPSS 10.0 statistical package , while simple linear regression was performed in Excel . Results with 1H-NMR δ: 12.93 , 10.87 , 8.05 , 7.11 , 6.89 , 6.51 , 6.20 ; 13CNMR δ: 181.8 (C-4), 164.2 (C-7), 163.3 (C-2), 162.3 (C-4′), 161.4(C-5), 157.3 (C-9), 128.3 , 122.8 (C-1′), 114.5 , 103.8 (C-10), 103.5 (C-3), 98.9 (C-6), 94.0 (C-8). Compound 1 was identified as acacetin by comparison of its physical and spectral data with the literature [Compound 1 was yellow needle-like crystals. DMSO-d6, 0 M Hz δ:2 = 0.9999 (P < 0.001). The test range was 1.168-12.848 μg/mL. The LOD (S/N = 3) and LOQ (S/N = 10) was 0.29 μg/mL and 1.17 μg/mL, respectively.The calibration curve for acacetin was as y = 43237 x + 10969, RThe RSD of peak areas at different times were less than 1.13%, indicating that the sample was stable for at least 24 h.Z. clinopodioides.The RSD value of intra-day and inter-day precision was 0.11% and 1.64%, respectively, which suggested that the developed method was precise enough for determining acacetin in Z. clinopodioides   Table .Figure 1The cardiomyocyte viability was greater than 50% when subjected to an acacetin dose less than 12.5 μg/mL Table .Table 3P < 0.001), 10.64 ± 3.54 nmol/L (P < 0.001) and 15.45 ± 14.62 nmol/L (P = 0.033), respectively. These results confirmed that as the treatment concentration of acacetin increased, the MDA content in cardiomyocytes decreased. At 25 and 10 μg/mL acacetin, a significant reduction was observed  (Table The MDA content was significantly increased from 0.14 ± 21.34 to 30.72 ± 1.40 nmol/Lafter myocardial cell injury (Table Flavonoids are polyphenol compounds, which are widely distributed in a variety of plants and have many pharmacological activities associated with cardiovascular protection such as antioxidation , anti-inAcacetin exists in plants of asteraceae –22, and in vitro, free radical injury was involved [In cardiomyocyte injury induced by hypoxia/reoxygenation, which is similar to heart ischemia-reperfusion injury involved . After minvolved . In thisAcacetin protects neonatal cardiomyocytes from the damage induced by hypoxia/reoxygenation stress through reduction of lipid peroxidation and enhancement of the antioxidant activity.


Post-exercise ingestion of a high molecular weight (HMW) carbohydrate (CHO) solution has been shown to result in greater rates of muscle glycogen synthesis, attributed, in part, to the higher rates of gastric emptying compared to a low molecular weight (LMW) CHO solution. Given the higher rate of gastric emptying, a more rapid rise of glucose and insulin would be expected. However, differences have been reported in the pattern and time course of the subsequent insulin and glucose responses following ingestion. These differences have been attributed to timing and technique  of blood sampling. Thus, the current study sought to examine differences in the glucose, insulin, and glucagon response to post-exercise ingestion of a HMW and LMW CHO solution.2 max (37.4 ± 4.3 ml·kg·min-1) was determined prior to testing session 1. In sessions 1-3, subjects completed a glycogen depleting cycling bout of 60 minutes at 70% VO2 max, followed by six, one-minute sprints at 120% VO2 max. Immediately post-exercise subjects ingested a placebo (PLA), or a LMW or HMW CHO solution (10%) providing 1.2kg· bw-1 CHO; assigned randomly. Blood was sampled prior to ingestion and every ten minutes for 120 minutes post-ingestion. A two-factor repeated measures ANOVA was used to determine differences among treatments (p ≤ 0.05).Sixteen resistance trained men  participated in this double-blind, placebo-controlled, randomized crossover study, which consisted of three testing sessions, each separated by one week. VO-1; HMW, 5.2 ± 0.3mmol · L-1; p = 0.086). Insulin remained elevated throughout blood sampling. Peak plasma insulin occurred at 40 minutes . Plasma glucagon declined following CHO ingestion with a more rapid difference following LMW (20 minutes) than HMW (30 minutes) CHO solution. However, no differences were noted between CHO treatments. Glucagon achieved a peak value of 38.7 ± 5.5ng·L-1 after ingestion of the PLA, while the lowest values observed following ingestion of the LMW and HMW CHO solutions were 12.0 ± 1.7ng·L-1 and 11.5 ± 1.4ng·L-1, respectively.Post-exercise ingestion of the LMW and HMW CHO solutions caused an increase in plasma glucose and insulin at 10 minutes. Glucose remained elevated in LMW until 60 minutes; and 70 minutes in HMW. The difference between HMW and LMW at that time approached significance (LMW, 4.7 ± 0.3mmol · L-1 CHO result in similar responses in glucose, insulin, and glucagon. Further study is needed to determine the effect on subsequent performance.These data suggest that when venous blood is sampled, ingestion of HMW and LMW CHO solutions providing 1.2kg· bw


Introduction. Benzamide can specifically bind to melanoma cells. A 18F-labeled benzamide derivative, [18F]N-(2-diethylaminoethyl)-4-[2-(2-(2-fluoroethoxy) ethoxy)ethoxy]benzamide ([18F]FPBZA), was developed as a promising PET probe for primary and metastatic melanoma. Methods. [18F]FPBZA was synthesized via a one-step radiofluorination in this study. The specific uptake of [18F]FPBZA was studied in B16F0 melanoma cells, A375 amelanotic melanoma cells, and NB-DNJ-pretreated B16F0 melanoma cells. The biological characterization of [18F]FPBZA was performed on mice bearing B16F0 melanoma, A375 amelanotic melanoma, or inflammation lesion. Results. [18F]FPBZA can be prepared efficiently with a yield of 40–50%. The uptake of [18F]FPBZA by B16F0 melanoma cells was significantly higher than those by A375 tumor cells and NB-DNJ-pretreated B16F0 melanoma cells. B16F0 melanoma displayed prominent uptake of [18F]FPBZA at 2 h (7.81 ± 0.82 %ID/g), compared with A375 tumor and inflammation lesion . [18F]FPBZA microPET scan clearly delineated B16F0 melanoma but not A375 tumor and inflammation lesion. In mice bearing pulmonary metastases, the lung radioactivity reached 4.77 ± 0.36 %ID/g at 2 h . Conclusions. Our results suggested that [18F]FPBZA PET would provide a promising and specific approach for the detection of primary and metastatic melanoma lesions. Melanoma, with a steadily increasing incidence rate for the past 30 years, has become a serious public health problem worldwide , 2. In tMalignant melanoma is very aggressive and more likely than other skin cancers to spread to other parts of the body, including brain, lungs, liver, and lymph nodes . The pro18F]fluorodeoxyglucose ([18F]FDG). [18F]FDG is actively taken at the cellular level, then phosphorylated, and no longer metabolized, and thus it remains trapped within the cells. [18F]FDG PET was recommended for staging and detecting recurrent melanoma [18F]FDG PET was unacceptably low for the initial assessment of early-stage malignant melanoma [18F]FDG PET scans also failed to detect the pulmonary and brain metastases and should be limited to staging patients with more advanced melanoma [18F]FDG displays relatively poor selectivity for distinguishing tumor from inflammatory tissue. Increased glucose metabolism in inflammatory tissues is often the main cause of false-positive [18F]FDG PET findings in oncology.Positron emission tomography (PET) imaging technique offers unique advantages including high sensitivity as well as deep tissue tomographic and repetitive imaging capabilities. Further, this imaging approach is directly translatable to clinical application. The most generally used radiotracer in clinical practice is fluorobenzamide ([18F]FBZA) exhibited high tumor uptake and was considered as a promising candidate for clinical study [18F]FBZA is conducted via conjugation of a 18F-synthon  to the primary amino group of N,N-diethylethylenediamine (DEDA). This method requires a multistep synthesis and is time-consuming and laborious. Furthermore, it is difficult to make the multistep radiosynthesis fully automatic, which in turn sets a high technical barrier for [18F]FBZA in the clinical use.Primary melanoma most commonly arises from the skin; however, it can also develop in other areas, including the eye and mucosa. A key feature of melanoma is the extensive melanin present in most tumor cells, making it a very attractive target for both diagnosis and treatment. Various radiolabeled benzamide analogues have been developed for targeting melanin with high selectivity and affinity –18. Amonal study , 18. The18F-labeled tracers with an additional fluoroalkyl moiety have higher lipophilicity and tend to display a higher nonspecific binding to the normal organs. Polyethylene glycol (PEG) is a component that has been widely used as a modifier of drugs and has been approved by the food and drug administration of United States of America. This study developed an easy-to-synthesize method to prepare a [18F]fluoroPEGylated radiotracer for melanoma detection. We modified the phenol moiety of benzamide with a short chain PEG and then labeled with 18F to afford [18F]N-(2-diethylaminoethyl)-4-[2-(2-(2-fluoroethoxy)ethoxy)ethoxy]benzamide  with visualization under UV (254 nm). NMR spectra were recorded on a NMR spectrometer  operating at 400 MHz for 1H NMR spectra and 100 MHz for 13C NMR spectra at Instrumentation Resource Center of National Yang-Ming University. All chemical shift values were reported in ppm (δ). Electrospray ionization-mass spectra (ESI-MS) were acquired on a FINNIGAN LCQ mass spectrometer at Instrumentation Resource Center of National Taiwan University. Analytic as well as semipreparative high performance liquid chromatography (HPLC) was performed with a Waters 600E pump equipped with a Waters 2998 photodiode array detector and a flow count radio detector  for gamma ray detection. Radioactivity was assayed using a Capintec CRC-15R dose calibrator  or a γ-scintillation counter .All reagents and solvents were purchased from commercial suppliers and were used without further purification. Triethylene glycol di-terature . Thin la2CO3 . The reaction mixture was stirred at ambient temperature overnight, diluted with H2O and extracted with dichloromethane. The combined organic layers were washed with H2O, dried over MgSO4, and evaporated. Column chromatography of the crude product on silica gel eluting with MeOH/CH2Cl2 (1/10) afforded compound 1  as a yellowish solid. 1H NMR  δ (ppm) 1.03 , 2.58 , 2.65 , 3.44 , and 6.89 , 7.76 . 13C NMR  δ (ppm) 11.6, 37.6, 47.0, 52.0, 114.9, 126.3, 128.8, 160.1, 166.1. ESI-MS m/z calculated for C13H20N2O2: 236.15 found [M + H]+: 237.16.A solution of 4-hydroxybenzoic acid  in thionyl chloride (5 mL) was refluxed for 5 h. Excess thionyl chloride was removed under reduced pressure. The residue was then redissolved in THF (10 mL). To this solution was added DEDA  and K 1  in anhydrous acetonitrile  were added K2CO3  and triethylene glycol di-p-tosylate . The reaction mixture was heated to reflux for 4 h. The solvent was removed by rotary evaporator. The residue was redissolved with H2O and extracted with dichloromethane. The combined organic layers were dried over MgSO4 and concentrated. Column chromatography of the crude product on silica gel eluting with MeOH/CH2Cl2 (1/10) afforded the tosyl precursor  as an yellowish oil. 1H NMR  shows δ (ppm) 1.08 , 2.43 , 2.64 , 2.72 , 3.52 , 3.60–3.71 , 3.83 , 4.13–4.17 , 6.92 , 7.33 , and 7.80–7.75 . 13C NMR  shows δ (ppm) 11.6, 21.6, 37.1, 47.1, 51.6, 67.5, 68.8, 69.2, 69.7, 70.8, 114.3, 127.1, 128.0, 128.7, 129.8, 133.0, 144.8, 161.2, and 166.9. ESI-MS m/z calculated for C26H38N2O7S: 522.24 found [M + H]+: 523.25.To a solution of 2  in THF (4 mL) was added tetrabutylammonium fluoride (TBAF) solution in THF . The resulting mixture was refluxed for 12 h. The solvent was evaporated and the crude product was purified by column chromatography on silica gel eluting with MeOH/CH2Cl2 (1/10) afforded FPBZA  as an yellowish oil. 1H NMR  shows δ (ppm) 1.07 , 2.62 , 2.70 , 3.52 , 3.70–3.80 , 3.88 , 4.17 , 4.49–4.63 , 6.94 , and 7.76 . 13C NMR  shows δ (ppm) 11.6, 37.1, 46.9, 51.6, 67.5, 69.4, 70.4, 70.8, 70.9, 83.1, 114.3, 127.1, 128.7, 161.2, and 166.69. ESI-MS m/z calculated for C19H31FN2O4: 370.23 found [M + H]+: 371.23.To a solution of compound18F]HF was produced by irradiation of 95% 18O-enriched water with a 17-MeV proton beam at 18 μA. At the end of irradiation, [18F]HF was transferred to a vial by helium gas pressure and then transferred through a QMA Sep-Pak cartridge. The [18F]fluoride trapped on the cartridge was desorbed by elution with 0.8 mL of K2CO3/Kryptofix 2.2.2 (K2.2.2) solution  into a reaction vessel. The K[18F]/K2.2.2 solution in the reaction vessel was heated at 100°C under reduced pressure. The azeotropic drying was repeated twice by further addition of anhydrous MeCN (2 × 0.8 mL). To the dry residue was added compound 2 (2.0 mg) in anhydrous dimethyl sulfoxide (DMSO) and the resulting mixture was heated at 100°C for 5 min. After cooling to room temperature, the reaction mixture was diluted with HPLC mobile phase and filtered with a 0.45 μm membrane filter . The crude product was injected into a C18 semipreparative HPLC column . The mobile phase started from 95% solvent A (0.1% trifluoroacetic acid in water) and 5% solvent B (0.1% trifluoroacetic acid in MeCN) and then ramped to 35% solvent A and 65% solvent B at 20 min at a flow rate of 4 mL/min. The desired fractions were combined, evaporated under reduced pressure, redissolved in normal saline, and filtered through a 0.22 μm aseptic membrane filter  to afford the final product. Radiochemical purity of the final product was analyzed by analytic HPLC using a Phenomenex Luna C18 column (4 × 250 mm) with a mobile phase consisting of MeCN/10 mM ammonium formate buffer (21/79) at a flow rate of 1 mL/min.[18F]FPBZA (7.4 MBq) was incubated in 0.5 mL of human serum. At designated time points , 75 μL of the sample was taken and mixed with MeCN (250 μL). The resulting mixture was vortexed intermittently for 1 minute, then followed by centrifugation at 1,000 g for 10 min to pellet the precipitated serum proteins. The radioactivity of the supernatant and the precipitate was measured with a γ-scintillation counter. The radioactive components in the supernatant were assayed on an analytic HPLC as described above.[18F]FPBZA (74 kBq) was added to a mixture of 1-octanol (1 mL) and phosphate buffer saline ; then, the mixture was vigorously vortexed for 5 min. After subsequent centrifugation at 3,000 rpm for 5 min, aliquots (100 μL) were taken from each phase and the radioactivity was determined with a γ-scintillation counter. The partition coefficient was expressed as log P = log10 (counts in 1-octanol/counts in PBS).FPBZA (37 kBq/1.0 mL/well) was added to each well and incubated at 37°C for 5, 15, 30, 60, and 120 min. Triplicates were carried out for each time point. One milliliter of ice-cold PBS was used to intercept the uptake of tracer. The supernatants were aspirated and the cells rinsed twice with 1 mL of ice-cold PBS. Then, cells in each well were harvested with 0.5 mL of trypsin-EDTA and washed twice with 1 mL of ice-cold PBS. A 10 μL of cell suspension was taken to access cell viability with trypan blue and to count the number of viable cells under the microscopy. The radioactivity of the cell suspensions was measured with a γ-scintillation counter and normalized to the number of viable cells. Cellular uptake of [18F]FPBZA was expressed as the percentage of the administered dose that had accumulated per million cells (%AD/106 cells).For cellular uptake assay, B16F0 cells and A375 cells were trypsinized and grown overnight in 6-well culture plates , was conducted FPBZA (37 kBq/1.0 mL/well) was added to each well and incubated at 37°C for 120 min. One milliliter of ice-cold PBS was used to intercept the uptake of tracer. The supernatants were removed and the cells were rinsed twice with 1 mL of ice-cold PBS. The cells in each well were harvested with 0.5 mL of trypsin-EDTA and washed twice with 1 mL of ice-cold PBS. The radioactivity of the cell suspensions was measured with a γ-scintillation counter and normalized to the number of viable cells. The cellular uptake of [18F]FPBZA was expressed as %AD/106 cells.To further demonstrate the specific binding of FPBZA in 0.1 mL of normal saline through lateral tail veins. Five mice were sacrificed with CO2 at each designated time point following injection. Blood samples were obtained by cardiac puncture. Tumor, inflammation lesion, and major organs were excised, and parts of these tissues/organs were weighed and assayed for radioactivity with a γ-scintillation counter. The results were presented as percentage injected dose per gram of tissue (%ID/g). Values were expressed as mean ± SD for a group of five animals.Mice bearing tumor or inflammation lesion were injected with 3.7 MBq of [18F]FPBZA in 0.1 mL of physiologic saline through the lateral tail vein. Animals were scanned for 10 min at each time point , with the long axis of the animal parallel to the long axis of the scanner. All images were reconstructed with the OSEM method, with a 128 × 128 pixel image matrix, 16 subsets, 4 iterations, and use of a Gaussian filter. For data quantitative analysis, a region of interest (ROI) was placed on each tumor, inflammatory lesion and normal organs. The average radioactivity was obtained from the average pixel value within the multiple ROI volume. AsiPro software (Concorde Microsystems) was used for viewing microPET images and for data analysis. The counts in each ROI were converted to radioactivity per gram of tissue (nCi/g), assuming a tissue density of 1 g/mL and were then normalized to percentage of injected dose per gram of tissue (%ID/g).MicroPET images were obtained using the R4 system , and this produced 63 image slices over a 7.89 cm axial field of view (FOV), with a slice thickness of about 1.25 mm. The in-plane spatial resolution at the center of the FOV was about 1.6 mm using the ordered-subsets expectation maximization (OSEM) reconstruction method. Each tumor-bearing mouse was anesthetized with 0.2% isoflurane  using a vaporizer system  and injected with approximately 1.85–3.7 MBq of F/K2.2.2 in dry DMSO at 100°C for 5 min produced [18F]FPBZA with an average radiolabeling efficiency of >90% (n = 5). After purification by semipreparative HPLC, [18F]FPBZA final product was obtained with a radiochemical yield of 40–50%, a radiochemical purity of >97%, and a specific activity of 30–40 GBq/μmol. The synthesis time was ~40 min from the end of bombardment, including the process of purification and formulation. The identity of [18F]FPBZA was confirmed by coinjection with the authentic FPBZA . Defluorination of [18F]FPBZA in human serum was not observed. [18F]FPBZA, with a partition coefficient (log P) of −0.48, is more hydrophilic than the unPEGylated benzamide derivative [18F]FBZA (log P = 1.7) [After treating the [P = 1.7) .18F]FPBZA in B16F0 melanoma cells and A375 amelanotic melanoma cells was conducted to assess the specific binding of [18F]FPBZA toward high melanin-expressing tumor cells. Rapid and significant association of [18F]FPBZA with B16F0 cells was observed early after 5 min of incubation (2.58 ± 0.21 %AD/106 cells) . The uptake of [18F]FPBZA in B16F0 melanoma cells presented a time-dependent increase, while that in A375 cells did not.Cellular uptake study of [6 cells) . Continu18F]FPBZA dramatically decreased from 4.12 ± 0.20 %AD/106 cells to 0.63 ± 0.22 and 0.34 ± 0.23 %AD/106 cells, respectively, after 2 h of incubation  was observed at 0.5 h postinjection (p.i.), then declined slowly to 8.31 ± 1.00 and 7.81 ± 0.82 %ID/g at 1 and 2 h p.i., respectively. Most normal tissues displayed relatively low radioactivity accumulation through the whole study period, compared with that of tumor. The radioactivity remaining in muscle and blood was 2.83 ± 0.46 and 3.78 ± 0.41 %ID/g at 0.5 h p.i., respectively, and reduced to 1.16 ± 0.09 and 2.17 ± 0.10 %ID/g at 2 h p.i., respectively. A prolonged [18F]FPBZA retention in tumor, compared with those in normal tissues, rendered favorable tumor-to-normal tissues ratios for tumor detection. The tumor-to-muscle ratio was 3.72 at 0.5 h p.i., and significantly increased to 6.60 at 2 h p.i.. The tumor-to-liver and tumor-to-blood ratios also reached 4.49 and 3.52 at 2 h p.i., respectively. The bony uptake increased slightly over time and reached 3.21 ± 0.16 %ID/g at 2 h p.i.. The accumulation of [18F]FPBZA in inflammation lesion was low (1.67 ± 0.56 %ID/g) and was only slightly higher than that in muscle .18F]FPBZA was observed in the pigmented eyes of C57BL/6 mice (7.69 ± 1.56 %ID/g), there was a minor uptake in the unpigmented eyes of nude mice (2.91 ± 0.45 %ID/g).In A375 amelanotic melanoma-bearing nude mice, the tumor uptake was low (3.00 ± 0.71 %ID/g) and the tumor-to-muscle ratio was just 2.72 at 2 h p.i.. Although high uptake of [18F]FPBZA microPET imaging of B16F0 melanoma-bearing C57BL/6 mice showed excellent tumor-to-background contrast  at 2 h p.i. . Pulmonary B16F0 metastases can be clearly seen and localized by the coregistration of microPET images with CT.[contrast . The rad2 h p.i. . High tu18F]FPBZA as a specific PET probe for melanoma and presented a preclinical assessment of [18F]FPBZA in melanoma animal models.Melanoma is an increasingly important public health problem around the world. Highly invasive melanoma is currently the fifth most frequently diagnosed cancer in men and the sixth in women in the United States . The sur18F]-fluoro-3-hydroxymethylbutyl)guanine by radiofluorination of its tosyl precursor in DMSO gave higher radiochemical yield than that in acetonitrile [18F-fluorination of compound 2 with K[18F]/K2.2.2 in DMSO afforded noncarrier-added [18F]FPBZA with high radiochemical yield. An average radiolabeling efficiency of >90% was achieved within 5 min (n = 5). [18F]FPBZA preparation via one-step reaction was straightforward, convenient, and reliable, which was a significant advantage over [18F]FBZA.DMSO, a highly polar and aprotic solvent, is very suitable for conducting nucleophilic substitution reaction. In our previous study, preparing 9-, only limited cellular uptake was observed (0.31 ± 0.06 %AD/106 cells) after 2 h of incubation [18F]FPBZA in melanotic B16F0 cells was thirteenfold higher than that in amelanotic A375 cells. To confirm that the specific binding of [18F]FPBZA in B16F0 cells is attributed to cellular melanin, B16F0 melanoma cells were pretreated with various concentrations of NB-DNJ. NB-DNJ can effectively inhibit the cellular tyrosinase activity and results in an appreciable loss of pigmentation in the treated melanoma cells. The uptake of [18F]FPBZA in 1 mM NB-DNJ-pretreated B16F0 melanoma cells dramatically reduced to 0.34 ± 0.23 %AD/106 cells after 2 h of incubation, similar to that in A375 amelanotic cells. Taken together, the results of in vitro studies clearly demonstrated that [18F]FPBZA was a melanin-specific agent and the accumulation of [18F]FPBZA in melanoma cells was correlated with the cellular melanin content.A series of cellular uptake studies revealed a rapid and high uptake of [cubation . Accumul18F]FPBZA in B16F0 melanoma-bearing mice displayed rapid and high tumor uptake and persisted up to 2 h p.i, comparable to those reported for [18F]FBZA and [18F]MEL050  [18F]FPBZA was higher than those of [18F]FBZA and [18F]MEL050, high tumor-to-muscle and tumor-to-blood ratios  suggested favorable pharmacokinetic profile for specific targeting of melanoma. [18F]FPBZA PET would be a sensitive modality for noninvasive imaging of melanoma in vivo. However, the slightly increasing bone radioactivity implied that defluorination of [18F]FPBZA in living subjects seems likely and may interfere the detection of bone metastasis of melanoma.The distribution of FPBZA in these organs. Moreover, high radioactivity accumulation in the pigmented eyes of C57BL/6 mice but not in the unpigmented eyes of BALB/c nude mice indicated the specific binding of [18F]FPBZA to cellular melanin in vivo. Taken together, the [18F]FPBZA accumulation in the B16F0 tumor xenograft was melanin-specific but not sigma receptors-mediated.It has been reported that the density of sigma receptors are high in liver and kidneys , while t18F]FDG is currently the most commonly used PET tracer for the detection and staging of cancers, and also for the evaluation of therapeutic efficacy in the clinic. In comparing the microPET images of the subcutaneous B16F0 tumor model, the tumor-to-muscle ratio for [18F]FPBZA at 2 h p.i. reached 6.53 ± 0.55, which was slightly higher than the tumor-to-background ratio of 5.8 ± 0.5 for [18F]FDG [18F]FDG is not a tumor-specific probe. The poor selectivity of [18F]FDG in distinguishing tumor from inflammation is the major cause of false-positive findings in clinical PET applications. In contrast to [18F]FDG, [18F]FPBZA showed no perceptible accumulation in the inflammatory lesion. [18F]FPBZA PET may be applied for specific detection of melanoma without interference from inflammation.[[18F]FDG . Further18F]FPBZA PET was also employed to monitor metastatic lesions with an experimental pulmonary metastasis mouse model. [18F]FPBZA microPET scan displayed remarkable radioactivity accumulation in lungs of pulmonary metastases-bearing mice compared with those of normal mice. The tracer uptake in muscle and liver was also significantly lower than that in pulmonary tumor lesions, rendering favorable tumor-to-normal tissues ratios. However, significant [18F]FDG uptake in the heart and the interscapular brown fat could interfere with the delineation of pulmonary metastatic lesions [18F]FPBZA was a melanin-specific PET probe for imaging melanoma, either a subcutaneous xenograft or pulmonary metastases. Liver was another common site that readily developed distant metastases from the cutaneous malignant melanoma (15–20%) [18F]FBZA, the low liver uptake and favorable tumor-to-liver ratio of [18F]FPBZA observed in subcutaneous melanoma animal models (1.74 %ID/g versus 2.35% ID and 4.49 versus 2.80) suggested that it may be more suitable for imaging liver metastases of melanoma MEL050 was the positive charge in the nicotinic ring of [18F]MEL050. The binding of the cationic substances to the melanin can be reinforced by ionic interaction [18F]FBZA or [18F]FPBZA was electrically neutral. Thus, the further improvement of 18F-labeled benzamide-derivative radiotracers could focus on the optimization of the pharmacokinetic properties by introducing the cationic moiety.A possible explanation for high melanoma uptake of [eraction . In cont18F-labeled α-melanocyte-stimulating hormone (α-MSH) analogues were developed via the conjugation of [18F]SFB or 4-nitrophenyl 2-[18F]fluoropropionate and proven to be as excellent PET imaging agents for melanoma FPBZA can be readily prepared with high radiochemical yield and radiochemical purity. This preparation method for [18F]FPBZA was very simple and straightforward, compared with [18F]FBZA. [18F]FPBZA showed highly specific uptake in the high melanin-expressing melanoma both in vitro and in vivo studies. Our results suggested that [18F]FPBZA may be a promising PET probe for the diagnosis of primary melanoma and the metastatic lesions.[18F]FPBZA after incubation 2 h incubation in serum , quantitative analysis of [18F]FPBZA microPET images in C57BL/6 mice bearing B16F0 melanoma lung metastasis  and representative microPET/CT images of C57BL/6 mouse bearing B16F0 melanoma lung metastases at 2 h postinjection of [18F]FPBZA .Supplementary material included analytic HPLC chromatogram of [


Curcuma aeruginosa Roxb (Zingiberaceae) has been used as a traditional folk medicine for the treatment of rheumatic disorders in Bangladesh. The aim of the current study was the bioassay-guided isolation and purification of an antinociceptive principle from the methanol extract of C. aeruginosa rhizomes.The rhizome of C. aeruginosa rhizomes. Vacuum Liquid Chromatography (VLC) and open column chromatography were used for separation. Crystallization was used for the purification of the isolated compound germacrone (1). Diclofenac (10 mg/kg) and aspirin (100 mg/kg) were used as positive control and 5 % carboxymethyl cellulose (CMC) in distilled water (10 ml/kg) for negative control were used in the acetic acid induced writhing and formalin induced licking methods.The antinociceptive activity was determined using acetic acid induced writhing and formalin induced licking in the Swiss albino mice to investigate central and peripheral antinociceptive principle of (1), a cyclic sesquiterpene. Germacrone (1) showed 22.66, 34.77 and 51.17 % inhibition of writhing at doses of 10, 20 and 40 mg/kg, respectively; 30.43 and 37.53 % inhibition in the initial phase and 32.27 and 60.96 % inhibition in the second phase of licking at doses of 200 and 400 mg/kg, respectively.The methanol extract exhibited 37.50 and 45.31 % inhibition of writhing; 33.27 and 38.13 % inhibition of licking in the first phase and 69.72, 73.71 % inhibition of licking in the second phase at doses of 200 and 400 mg/kg, respectively. VLC of the extract yielded five fractions (Fr. 1 to Fr. 5). Fr. 1 exhibited 33.98 % inhibition that was comparably higher than other fractions (Fr. 2 to Fr. 5) at a dose of 100 mg/kg. Column chromatography of Fr. 1 generated five fractions (SF. 1 to SF. 5). Fraction SF.3 exhibited 46.88 % inhibition that was most potent among the other fractions at a dose of 50 mg/kg. Crystallization of the fraction SF.3 yielded germacrone 1) showed a potent activity in both writhing and licking methods that indicates the compound as a central and peripheral antinociceptive principle of C. aeruginosa rhizomes with possible anti-inflammatory activity.Germacrone ( Curcuma aeruginosa Roxb . C. aeruginosa is one of the most prominently used medicinal plants in Bangladesh, India, Myanmar, Indonesia, Malaysia, and Thailand. The rhizome is very popular as a folk medicine in Bangladesh for the treatment of pain and inflammation associated with rheumatic diseases. In addition, the different parts of this plant are also commonly used as traditional medicines for the treatment of uterine pain, uterine inflammation and various gastrointestinal disorders such as diarrhea and colic [C. aeruginosa [C. aeruginosa [C. aeruginosa [1) [C. aeruginosa rhizome is still unknown and needs to be identified.As part of our continuing bioassay-guided isolation and purification of the active principles from traditional medicines –3, we innd colic . Pharmacnd colic , antimicnd colic , 7, antind colic , 9 and and colic  have beeruginosa , 11. Zedruginosa . Althougruginosa  and the nosa [1)  have beeC. aeruginosa rhizome and the active principle it contained through bio-assay guided fractionations.On the basis of the use of a plant as the folk and traditional medicine as well as the pharmacological properties reported in the literature, the present study was carried out to investigate the antinociceptive activity of the MeOH extract of 60 F254 with visualization by spraying with 10 % H2SO4 in MeOH followed by heating. VLC was done using Silica gel 60 (0.040–0.005 mm), Merck, Germany. Open column chromatography was done by using Silica gel 60 (0.063–0.020 mm), Merck, Germany. The IR Spectrum was obtained using a Shimadzu IR Prestige-2 FT-IR while the 1H-NMR spectra were recorded on an ultra shield Bruker DPX 400 spectrometer. The NMR spectra were recorded running gradients and using residual solvent peak (δ 7.25 s for 1H-NMR) as an internal reference.Organic solvents were obtained from Merck, Germany; Diclofenac was purchased from Beximco Pharma, Bangladesh; Aspirin was purchased from Square Pharmaceutical Ltd, Bangladesh; Thin Layer Chromatography (TLC) was run on Merck TLC plates precoated with SiC. aeruginosa rhizomes were collected from Panchagarh, Rangpur Division of Bangladesh, in December 23, 2011 and their identity was confirmed by the National Herbarium of Bangladesh, Chiriakhana Road, Mirpur-1, Dhaka-1216, Bangladesh, where a voucher specimen has been deposited (Accession Number 37514). After collection, the rhizomes were washed with water, cut into pieces and sun-dried because aqueous suspensions of sun-dried powder is used in the folk and traditional medicinal preparation. Then, dried rhizomes were powdered by using a Spices Mill. Dried powdered rhizomes (1.5 kg) were extracted with MeOH at room temperature (4000 ml × 72 h × 3 times). The extracting solvent was filtered, and the filtrate was concentrated in vaccuo by a rotary evaporator (45 °C) to get the crude MeOH extract .n-hexane (5 L), chloroform (5 L) , EtOAc (5 L), acetone (5 L) and MeOH (5 L) to obtain Fr. 1 (48 gm), Fr. 2 (29 gm), Fr. 3 (20 gm), Fr. 4(43 gm) and Fr. 5 (17 gm), respectively. On the basis of the analgesic activity, Fr. 1 (48 gm) was subjected to silica gel (300 gm) open column chromatography and eluted with n-hexane-EtOAc step gradients. Following this, 68 fractions of 200 ml each were collected as follows: 1–48 (n-hexane), 49–54 , 55–58 , 59–62  and 63–68 (EtOAc). On the basis of the analytical TLC, these fractions were then pooled into five new fractions (SF. 1 to SF. 5) as follows: SF. 1 , SF. 2 , SF. 3 , SF. 4 , SF. 5 . The analgesic activity of SF. 1 to SF. 5 were evaluated and the most potent fraction  was crystallized from n-hexane to get colorless rod shape crystals of germacrone . The schematic diagram of the bio-assay guided isolation and purification of germacrone (1) from the MeOH extract are shown  was subjected to a silica gel (120 gm) VLC and eluted with 75 gm was1(Germacrone): Rf value on Si-gel TLC with n-hexane: EtOAc (9.5:0.5) as mobile phase is 0.55. IRνmax (KBr) cm−1: 1129, 1289, 1384, 1438, 1672, 2849, 2915, 2983. 1H-NMR (CDCl3) δ (ppm): 1.42 , 1.61 , 1.70 , 1.75 , 2.09 , 2.34 , 2.83 , 2.93 , 3.39 , 4.69 , 4.96 . The chemical structure of germacrone (1) is shown , were used throughout the experiments. All animals were kept in standard environmental conditions, had free access to standard food  and water heir use . The ani1) were administered orally  to the Swiss Albino mice after an overnight fast. Suspensions of all test samples were prepared with 5 % carboxymethyl cellulose (CMC) in H2O for oral administration. Test samples  and a negative control (5 % CMC in H2O) were administered orally 30 min prior to intraperitoneal administration of a 0.7 % v/v acetic acid (100 %) solution (0.1 ml/10gm). Diclofenac was administered 15 min prior to acetic acid injection. Then, the animals were placed on an observation table. Each mouse of all groups was observed individually to count the number of writhing they made in 10 min commencing just 5 min after the intraperitoneal administration of the acetic acid solution. Full writhing was not always observed in the animals as sometimes the animals started to writhe but they did not continue. This incomplete writhing was considered as half-writhing. Accordingly, two half writhing were taken as one full writhing. The number of writhing in each treated group was compared to that of a control group in which diclofenac (10 mg/kg) was used as a reference (positive control).The antinociceptive activity of the samples was evaluated using acetic acid induced writhing in mice –18. In t2O as negative control, 200, 400 mg/kg of the MeOH extract, 100 mg/kg of VLC fractions and 20, 40 mg/kg of germacrone (1) for 1 h before being challenged with formalin, while 100 mg/kg of acetylsalicylic acid (aspirin) was used 30 min prior to formalin injection. All the tested drugs, the negative control, the MeOH extract, fractions, germacrone (1) and aspirin (positive control) were administrated orally by use of a feeding needle. Suspension of all test samples was prepared with 5 % carboxymethyl cellulose (CMC) in H2O for oral administration. The responses were measured for 5 min (first phase) and 15–30 min (second phase) after formalin injection.The experiment was performed with slight modification of the published methods , 18–21. t-test. Comparison of the analgesic activity in all groups was made using one-way ANOVA followed by Dunnett’s multiple comparisons test. The significance level was p < 0.05. All statistical analyses were conducted using SPSS (Version 17) software package for Windows .All data were expressed as mean ± SEM using one-sample Acetic acid induced writhing is an useful method for investigating peripheral antinociceptive drug leads , 18. PaiC. aeruginosa in acetic acid induced writhing exhibited 37.50 % and 45.31 % inhibition (p < 0.001) at doses of 200 and 400 mg/kg, respectively  in the early phase of licking at doses of 200 and 400 mg/kg, respectively; 69.72 and 73.71 % inhibition (p < 0.001) in the late phase of licking at doses of 200 and 400 mg/kg, respectively , by reducing the number of licking at doses of 200 and 400 mg/kg. Thus, the results indicate that the significant pain reduction is might be due to the presence of central and peripheral antinociceptive drugs that are also effective as an anti-inflammatory agents. In this model, aspirin  was used to insure the peripheral antinociceptive and possible anti-inflammatory activity because it is more potent in the late phase. The extract used in formalin induced licking exhibited 33.27 and 38.13 % inhibition  inhibition of acetic acid induced writhing and 23.94 % (p < 0.001) inhibition in the early phase and 62.15 % (p < 0.001) inhibition in the late phase of formalin induced licking. The result of Fr. 1 was more potent in both methods compared to the other fractions at the same dose of 100 mg/kg.The separation of the MeOH extract by VLC eluted 5 fractions (Fr. 1 to Fr. 5). These fractions were screened for the central and peripheral antinociceptive activity using acetic acid induced writhing and formalin induced licking. Fr. 1 was found to have the most activity in both writhing and licking and exhibited 33.98 % (p < 0.001) in acetic acid induced writhing at a dose of 50 mg/kg that showed most activity than the other new fractions with similar dose  and screened to find the most potent fraction using only acetic acid induced writhing. The fraction SF. 3 exhibited the most potent analgesic activity with inhibition of 46.88 % , δ 2.93  and δ 4.96 . The spectrum also demonstrated six methylene proton peaks at δ 2.34 , δ 2.83 , δ 2.93 , δ 3.39 , δ 4.69  and at δ 4.96 , and peaks of four methyl group at δ 1.42 , δ 1.61 , δ 1.70 , and at δ 1.75 . Based on these spectral data analysis, germacrone (1) was identified. Germacrone (1) is a cyclic sesquiterpene, which was previously isolated from this plant [1H-NMR data is very consistent with that of germacrone in the literature [p < 0.001) of writhing at doses of 10, 20 and 40 mg/kg, respectively. Thus, the presence activity of acetic acid induced writhing indicates germacrone (1) as a peripheral antinociceptive principle of C. aeruginosa rhizome. Besides, germacrone (1) indicated no lethal toxicity in mice at a dose of 750 mg/kg in the literature [1) was kept far below the lethal dose 750 mg/kg and was kept consistent with the positive control.The presence of carbonyl group was shown by the strong absorption in IR spectrum at 1672.4 cmterature . It was terature . The sel1) by acetic acid induced writhing was also previously investigated by Ozaki (1990) [C. xanthorrhiza Roxb, where he determined the activity of germacrone (1) in writhing method with 58.08 % inhibition (p < 0.01) at a single dose of 75 mg/kg and used indomethacin (10 mg/kg) as a positive control. Moreover, Ozaki (1990) [1), but did not mention any data or proper citation in support of his claim.The peripheral antinociceptive activity of germacrone (i (1990)  from C. i (1990)  insisted1) showed potent peripheral antinociceptive activity in acetic acid induced writhing method, therefore we investigated the effect of germacrone (1) in formalin induced licking method to clarify the central antinociceptive effect through both phases and peripheral antinociceptive effect through the second phase. The second phase was also used to investigate the possible effect of drug on inflammation. In formalin induced licking method, germacrone (1) showed 30.43 % (p < 0.001) and 37.53 % (p < 0.001) inhibition in the early phase and 32.27 % (p < 0.005) and 60.96 % (p < 0.001) inhibition in the late phase at doses of 20 and 40 mg/kg, respectively. The reduction of both phases of licking indicates germacrone (1) as a central antinociceptive while the inhibition of the second phase indicates that it is a peripheral antinociceptive drug, which might have possible anti-inflammatory activity. This finding of the possible effect on inflammation strongly support the published literature of anti-inflammatory activity of germacrone (1) [1) as a centrally and peripherally acting antinociceptive principle of C. aeruginosa rhizome with possible anti-inflammatory activity.As germacrone (rone (1) , 27. Thu1) showed potent activities in both writhing and licking methods indicating the compound is a central and peripheral antinociceptive principle of C. aeruginosa rhizome with possible anti-inflammatory activity. Thus, the results also strongly support the literatures of the anti-inflammatory activity of germacrone (1). The investigation indicated that C. aeruginosa rhizome is a rational choice as folk and traditional medicines for the treatment of rheumatic disorders.Germacrone (


Thiophene, 2--5-thiophene (17) showed moderate and selective activities. Compounds 3, 6, 8 and 12 displayed minimal inhibitory concentration (MIC) ranged from 4 to 256 μg/mL on all the 14 tested bacteria. MIC values below 10 μg/mL were obtained with 8, 3, 6 and 12 against 50, 42.9, 35.7 and 21.4 % of the tested bacteria. The lowest MIC value of 4 μg/mL was obtained with compound 3 against Klebsiella pneumoniae ATCC11296, Enterobacter cloacae BM47, compound 6 against Escherichia coli ATCC8739, K. pneumoniae ATCC11296, E. cloacae BM47 and compound 8 against K. pneumoniae ATCC11296 and E. cloacae BM47. The activity of flavonoid 3 was better or equal to that of chloramphenicol in all tested K. pneumoniae,Providencia stuartii, E. aerogenes, E. cloacae and Pseudomonas aeruginosa strains. Within isoflavonoids, neobavaisoflavone scaffold was detected as a pharmacophoric moiety. This study indicates that natural products such as 3, 6 and 8 could be explored more to develop antimicrobial drugs to fight MDR bacterial infections.The present study was designed to assess the antimicrobial activity of 19 natural products belonging to terpenoids, alkaloids, thiophenes and phenolics against a panel of 14 Gram-negative multidrug-resistant (MDR) bacteria. The results demonstrated that amongst the studied compounds, alkaloids and terpenoids were less active contrary to flavonoids: neocyclomorusin ( Infectious diseases including bacterial infections continue to be a serious health problem worldwide. Multidrug-resistant (MDR) pathogens considerably increase the mortality and morbidity. In effect, clinically, the continuous emergence of Gram-negative MDR bacteria drastically reduced the efficacy of antibiotic arsenal leading globally to an increase of the frequency of therapeutic failure Rice . Consequ1; yellow solid; m.p. 286.2–287.7 °C)                   , isoflavonoids , benzophenone (16), benzofuran (4), ellagic acid derivative (19), lignan (5), alkaloids , terpenoids  and thiophene (19). They were tested for their antimicrobial activity on a panel of 14 bacterial strains and the results are summarized in Table Compounds tested in the present study Fig.  were pre11) and triterpenoid (15) exhibited very weak activities. Deacetylnomilin (11) and herranone (15) had detectable MIC values against 2/14 (14.3 %) and 5/14 (35.7 %) bacterial strains respectively (Table Both diterpenoid 1 and tri9) and maculine (10) displayed low activity against the tested bacteria. Their inhibitory effects were observed on 1/14 (7.1 %) and 2/14 (14.3 %) microbial strains, respectively, for 9 and 10  showed MIC values below 100 µg/mL against Escherichia coli ATCC 8739, Enterobacter aerogenes ATCC 13048 and EA27, Klebsiella pneumoniae ATCC11296 and Providencia stuartii ATCC29916 -5-thiophene  and candidone (6) as well as isoflavonoids neobavaisoflavone (8) and daidzein (12) had MIC values ranged from 4 to 256 µg/mL on all the 14 tested bacteria. Moreover, MIC values below 10 µg/mL were obtained with 8, 3, 6 and 12 against 7/14 (50 %), 6/14 (42.9 %), 5/14 (35.7 %) and 3/14 (21.4 %) tested bacteria respectively. MIC values below 100 µg/mL were obtained with compound 3 on all tested bacteria. MBC values below 10 µg/mL were also obtained with compound 3 against K. pneumoniae ATCC11296 and Enterobacter cloacae BM47 whilst values ranged from 8 to 256 µg/mL were noted on all tested pathogens. The lowest MIC value of 4 µg/mL was obtained with compound 3 against K. pneumoniae ATCC11296, E. cloacae BM47, compound 6 against E. coli ATCC8739, K. pneumoniae ATCC11296, E. cloacae BM47 and compound 8 against K. pneumoniae ATCC11296 and E. cloacae BM47.The best activities were obtained with phenolic compounds; amongst them, flavonoids neocyclomorusin (16) were not active meanwhile coumarin (14), lignan (5) and benzofuran (4) were also found to be poor antibacterial agents. The best activities were obtained with flavonoids and isoflavonoids. Within flavonoids, it appeared that hydroxylation of compound 1 to yield 2 did not significantly changed the antibacterial activity  was another pharmacophoric moiety  as well as the tested furoquinoline alkaloids were poor antimicrobial compounds. A keen look of the activities of phenolics shows that benzophenone 6 were noPseudomonas aeruginosa tested in the present study, play a key role in multidrug resistance of pathogenic Gram-negative bacteria (Nikaido 3 and isoflavonoid 8) against the problematic MDR strain P. aeruginosa PA124. This value was identical to that of the reference compound chloramphenicol . MDR Enterobacteriaceae, including K. pneumoniae, E. aerogenes, P. stuartii and E. coli, have also been classified as antimicrobial-resistant organisms of concern in healthcare facilities  in many cases, suggesting that bacterial effects of these phytochemicals could be expected  suggests that hemi-synthesis reaction with neobavaisoflavone could be explored in more details for antibacterial drug development. To the best of our knowledge, the antibacterial activity of the best compounds  against MDR bacteria is being reported for the first time. However, several flavonoids and isoflavonoids are known to possess antibacterial activities against both drug-sensitive and MDR Gram-negative phenotypes  and thin layer chromatography (TLC) were performed over silica gel 60H (particle size 90 % <45 mm), 200–300 mesh silica gel silica gel GF254, respectively. 1D- and 2D-NMR spectra were carried out with a Bruker DRX-400 MHz. Melting points were measured by an Electro thermal IA 9000 digital melting point apparatus and are uncorrected.Teclea afzelii Engl. (Rutaceae) (10674/SRF/Cam), Erythrina excelsa Baker (Fabaceae) (61487/HNC), Erythrina senegalensis A. Rich. (Fabaceae) (59409/HNC), Echinops giganteus A. Rich. (Asteraceae) (23647/SRF-Cam), Pachystela msolo Engl. (Sapotaceae) (3849/SRFK), Garcinia ovalifolia Oliv. (Guttiferae) (55523/HNC) and Alchornea laxiflora (Benth) Pax and Hoff (Euphorbiaceae) (45363HNC).Plant species were collected in Yaoundé  and in Dschang  and identified by the specialist of the national herbarium in Yaoundé, Cameroon where their voucher are kept under the registration codes: For the collection of plants, no specific permits were required for the described field studies. For any location/activity, no specific permissions were required. All locations where the plants were collected were not privately-owned or protected in any way and the field studies did not involved endangered or protected species.1–10, 14) or newly isolated . Compounds 1–3 were obtained from the roots of Milicia excelsa whilst 4 was identified from the leaves as previously reported     ; Hence, air-dried roots (7 kg) were macerated in MeOH/DCM  for 48 h and the organic (286 g) solid obtained after evaporation of the solvents in vacuo was further extracted with hexane , ethyl acetate  and MeOH (201 g). The EA fraction was purified by column chromatography on silica gel in gradient conditions of hex/EA. Three compounds were isolated as follows: 9 (6 mg), 10 (5 mg) and 11 (10 mg).Compound 12 was obtained from the roots of Erythrina excelsa (2.2 kg) (Fabaceae); The crude extract obtained from the maceration of the air-dried roots was fractioned by silica gel flash chromatography using hex/EA in gradient conditions. The fraction issued from hex/EA (1:1) was chromatographed using the same condition as above from which 12 (45 mg) was isolated. Similarly, 13 (5 mg) was isolated from the roots of Erythrina senegalensis (Fabaceae) as described for 12. Compound 17 (5.0 mg) was isolated from the roots of Echinops giganteus; the powdered roots of E. giganteus (Asteraceae) was macerated successively in DCM/MeOH  and MeOH for 48 h and 24 h, respectively. The organic solutions were pooled together based on their TLC profile. Eighty-one grams of a red dark crude extract were obtained after evaporation invacuo. Furthermore, the crude extract was poured onto water and extracted with Hex , DCM , EA , n-butanol . Fraction B was purified on silica gel CC using gradient conditions of Hex/EA and 17 was obtained from the Hex/EA . Similarly, bark powder (2.8 kg) of Pachystela msolo Engl. (Sapotaceae) was extracted with DCM/MeOH (1:1), yielding a dark crude extract (30 g). The purification of this later in gradient conditions of Hex/EA afforded 15 (5 mg). The stem bark (2.5 kg) of Garcinia ovalifolia (Guttiferaceae) was air-dried, ground and macerated in MeOH for 48 h. A brown residue (120 g) was obtained after concentrating the organic solution. Vacuum liquid chromatography was used for a first fractionation with gradient of hex/EA and EA/MeOH. Fractions obtained from Hex and Hex/EA  were pooled together (1.22 g) and purified by silica gel CC with gradients of the same mixture of solvents to afford 16 (12 mg). Fractions collected from EA/MeOH (9:1 and 4:1) were also pooled together and purified on silica gel CC with gradients of DCM/MeOH to yield compound 18 (90 mg). The stem bark (1.8 kg) of Alchornea Laxiflora (Euphorbiaceae) was macerated in MeOH. The concentrated methanol crude extract (30 g) was subjected to silica gel flash chromatography using Hex (A), EA (B) and MeOH (C). Fraction C (10 g) was purified by silica gel CC using the gradient of DCM/MeOH. Compound 19 (15 mg) was isolated from sub-fractions eluted with DCM/MeOH (95:5).Compound p-Iodonitrotetrazolium chloride ≥97 %  was used as microbial growth indicator  was used as reference antibiotics (RA) against Gram-negative bacteria. or Eloff .P. aeruginosa, K. pneumoniae, E. aerogenes, E. cloacae, E. coli, P. stuartii, obtained from the American Type Culture Collection. Their bacterial features were previously reported  colorimetric assay according to described methods  determinations on the tested bacteria were conducted using rapid ds Eloff  with somds Eloff , 2009. Tds Eloff , 2008a. 3), candidone (6) and neobavaisoflavone (8) could be potential antimicrobial drugs to fight MDR bacterial infections and could also be used as motifs for developing related antibiotics with strong potency. To explore more the therapeutic values of the studied compounds, a combination with commonly used antibiotics will be further performed. Also, the study of the mechanism of action of the effective compounds will be carried out to better understand their inhibitory effects.Regarding the medical importance of the studied microorganisms, the results obtained and reported in this study interestingly showed how secondary metabolites are still a strong source of inspiration in drug discovery. Thus, the present data provided evidence that neocyclomorusin (