A study on the standard of influenza neuraminidase inhibition assay / 药学学报

In present study, standard method and standard operation practice for measuring the activities of influenza neuraminidase and its inhibitors have been established. The accuracy and stability of the method has been evaluated. Standard operation is as following: 10 microL sample, 30 microL neuraminidase and 60 microL substrate are added to one well of a 96-well plate, and then incubated at 37 degrees C for 1 h. The reaction was stopped with NaOH before fluorescence intensity determination. One unit of neuraminidase is defined as the amount of enzyme that produces 1 nmol 4-MU in 1 h under above conditions. The inhibition accuracy is indicated by an uncertainty measurement of 6.51 x 10(-12), and its stability was reaffirmed by determination of oseltamivir acid. In this study, systematic assessment of neuraminidase inhibitory assay not only provided theoretical basis of its application in drug discovery, but also made preliminary attempt to use uncertainty measurement as a parameter in biological measurement.

Efficacy of 4-methyl-7-hydroxy coumarin derivatives against vectors Aedes aegypti and Culex quinquefasciatus.

4-Methyl-7-hydroxy coumarin is considered as a lead molecule as a biopesticide. Its mono bromo and tribromo derivatives were synthesized. Two more derivatives were synthesized by acylation. Compound 1 (3,6,8-tribromo-7-hydroxy-4-methyl-chromen-2-one) was found to be the most potent against IVth instar larvae of C. quinquefasciatus and A. aegypti the LC50 being 1.49 and 2.23 ppm respectively. It showed 100% larval mortality at 25 ppm against A. aegypti and at 10 ppm against C. quinquefasciatus. Compounds 1 and 2 (3,6,8-tribromo-7-hydroxy-4-methyl-chromen-2'-oxo-2H-chromen-7-yl acetate) showed remarkable ovicidal activity. Significant reduction of 80-85% hatching of eggs of both mosquito species was observed at the highest dose of 100 ppm. The hatched larvae showed 100% mortality in the successive instars. Compounds 3 and 4 (3-bromo-7-hydroxy-4-methyl-chromen-2-one and 3-bromo-4-methyl-2'-oxo-2H-chromen-7-yl acetate) showed moderate activity against both mosquito species.

Pancreatic lipase inhibitory activity of taraxacum officinale in vitro and in vivo

Obesity has become a worldwide health problem. Orlistat, an inhibitor of pancreatic lipase, is currently approved as an anti-obesity drug. However, gastrointestinal side effects caused by Orlistat may limit its use. In this study the inhibitory activities of dandelion (Taraxacum officinale) against pancreatic lipase in vitro and in vivo were measured to determine its possible use as a natural anti-obesity agent. The inhibitory activities of the 95% ethanol extract of T. officinale and Orlistat were measured using 4-methylumbelliferyl oleate (4-MU oleate) as a substrate at concentrations of 250, 125, 100, 25, 12.5 and 4 microgram/ml. To determine pancreatic lipase inhibitory activity in vivo, mice (n=16) were orally administered with corn oil emulsion (5 ml/kg) alone or with the 95% ethanol extract of T. officinale (400 mg/kg) following an overnight fast. Plasma triglyceride levels were measured at 0, 90, 180, and 240 min after treatment and incremental areas under the response curves (AUC) were calculated. The 95% ethanol extract of T. officinale and Orlistat, inhibited, porcine pancreatic lipase activity by 86.3% and 95.7% at a concentration of 250 microg/ml, respectively. T. officinale extract showed dose-dependent inhibition with the IC50 of 78.2 microg/ml. A single oral dose of the extract significantly inhibited increases in plasma triglyceride levels at 90 and 180 min and reduced AUC of plasma triglyceride response curve (p<0.05). The results indicate that T. officinale exhibits inhibitory activities against pancreatic lipase in vitro and in vivo. Further studies to elucidate anti-obesity effects of chronic consumption of T. officinale and to identify the active components responsible for inhibitory activity against pancreatic lipase are necessary.

Postnatal and prenatal diagnosis of mucopolysaccharidosis type II (Hunter syndrome) / 中华儿科杂志

<p><b>OBJECTIVE</b>Mucopolysaccharidosis type II (MPS II, Hunter syndrome, OMIM 309900) is an X-linked recessive lysosomal storage disease resulting from a deficiency of iduronte-2-sulphate sulphatase (IDS). The present study aimed to establish an enzyme assay method for IDS activity for carrying out postnatal and prenatal diagnosis of MPS II by means of IDS activity assay on plasma, uncultured chorionic villi (CV) and cultured amniotic fluid cells (AF cell) using a new synthesized substrate.</p><p><b>METHODS</b>A fluorigenic substrate (4-methylumbelliferyl-alpha-iduronate-2-sulphate, MU-alpha-Idu-2S) was used for the assay of IDS activity. IDS activity in plasma was determined for diagnosis of the proband. Prenatal diagnosis in 10 pregnancies at risk was carried out according to IDS activity on uncultured CV at 11th week or on cultured AF cell at 18th week of gestation. At the same time, IDS activity was also determined in the maternal plasmas to observe the change of IDS activity in pregnancy. The fetal sex determination was performed by PCR amplification of the ZFX/ZFY genes.</p><p><b>RESULT</b>The IDS activity in plasma of normal controls and obligate heterozygotes were 240.2 - 668.2 nmol/(4 hxml) and 88.7 - 547.9 nmol/(4 hxml), respectively, while the enzyme activities in plasmas were in the range of 0.3 - 18.6 nmol/(4 hxml) in affected male. The IDS activities were 37.2 - 54.9 nmol/(4 hxmg protein) and 21.4 - 74.4 nmol/(4 hxmg protein) in CV and cultured AF cells respectively. Out of 50 suspected cases, 46 were diagnosed as having MPS II and 4 were excluded. Prenatal diagnosis was performed on 10 pregnancies at risk. Four of 5 male fetuses [IDS activity were 4.7, 1.8, 7.0 nmol/(4hxmg protein) in CV, 0.6 nmol/(4 hxmg protein) in AF cell] were diagnosed as having MPS II and the other 5 fetuses were normal females [IDS activity were: 48.7, 5.9, 25.2 nmol/(4 hxmg protein) in CV, 55.2, 40.9 nmol/(4 hxmg protein) in AF cell]. Increased IDS activity was observed in plasma of the pregnant women with unaffected fetuses, while the IDS activity decreased in pregnancies with affected fetuses. IDS activity of one female fetus was very low [5.9 nmol/(4 hxmg protein)], but the IDS activity in maternal plasmas increased, this fetus was a normal female.</p><p><b>CONCLUSIONS</b>The method using a synthesized fluorigenic 4-methylumbelliferyl-substrate was a sensitive, rapid and convenient assay of IDS activity and was reliable for early prenatal diagnosis. Determination of fetal sex would be helpful in excluding the female fetus with low IDS activity from being considered as an affected male fetus. It would be further helpful if IDS activity in maternal plasma was taken into account.</p>

Rapid identification of Salmonella typhimurium in stools by using 4-methylumbelliferone derivatives.

A total of 150 stool samples were collected from the Paediatric ward and screened for the presence of Salmonella typhimurium. A comparison of standard biochemical tests and rapid fluorescence method was done. The fluorescence method was found to be rapid, less time consuming and easy to perform whereas identification of Salmonella typhimurium using standard biochemical tests taken 18 to 24 hours.