Studies Of Detect Method For Nereistoxin And Application

Objective: Cardial and cerebral vascular thrombosis disease is a threat against human's health in modern time, it will lead to sudden death and nerves functional disturbance. The principal method of clinical treatment is to cut down fibrinogen in the blood and to dissolve internal clot at earlier period as soon as possible. It is the important topic to study and research this kind of drug in the medicine domain. N-V protease is a kind of protease abstracted from some marine life, N-V protease have the function of hydrolyzing plasminogen and dissolving internal clot. We erect a series of method that detect NTX contained in N-V protease. And using ultraviolet spectrophotometry,high performance thin-layer,high-performance liquid chromatography to detect the NTX of the specimen. Method:1 Using ultraviolet spectrophotometry detect the NTX:Through scanning entire wave length ,It is ascertained that the absorption maximum is 220nm.So we detect standard substance and erect equation of linear regression and draw standard curve. The linear equation:y=0.031201137x+0.006263682γ=0.998452656(n=4). The detection limit was 1.25μg/mL. Its recovery rate is 96.7%-100%. It is concluded that the NTX does not exist in the N-V protease. The method is so sensitive and operate easily that become the effect method of NTX detection.2 Using high performance thin-layer silica gel G to detect the NTX.The high performance thin-layer chromatography method to detect the related substances NTX is convenient manipulate and fast to get result. The developing agent is methanol: acetoacetate: water (5:4:1), the chromogenic agent is calcein-palladium chloride, detect under uviol lamp, limit of quantitation is 0.5μg/mL. No NTX in N-V protease, there is NTX in the offcuts by the fist step of protein purification. A highly sensitive method such as mass spectrum is needed for further study.3 Using high-performance liquid chromatography to detect NTX. The detect condition is ascertained. chromatographic column: ZORBAX×Eclipse×DB-C18(4.6×150mm,5μm); mobile phase: Columbian spirit /warter:85%/15%(V/V). flow rate: 1mL/min; column temperature: 20℃. sample size: 5μL/ dowel; linearity speed: 0.170cm/sec; detect wave: 227nm. On the detect condition, we detect standard substance and sample. The linear relationship of calibration culibration curve was good in the range of 2.5-250μg/mL for NTX(R=0.9995). The linear equation: y = -0.6263+7.0493x R=0.9995(n=5)。The detection limit was 40ng, and the recovery was 98.5%- 100%, the relative standard deviation was 2.23-2.87%. The method is simple, accurate and sensitive. It can be adopted to quantification of NTX..Result:It is no NTX existed in the N-V protease by the series of detect method .This methods could be used widely in the varies of territory. Such as agriculture,foodstuff industry and so on..