Objective:To establish a method for the separation and purification of haemocoagulase from agkistrodon blomhoffii in Changbai Mountain. Methods: An enzyme component with clotting activity was isolated from the venin of agkistrodon blomhoffii in Changbai Mountain by gel filtration, anion-exchange chromatography and heparin-Sepharose affinity chromatography. SDS-Page and RP-HPLC were used to determine its purity, SDS-Page was applied to study its action mode with bovine fibrinogen, HPSEC was used to determine the molecular weight, an IEF method was employed to detect its isoelectric point, and Lowry method was used for the deter-mination of protein concentration. Results:One haemocoagulase was purified from agkistrodon in Changbai Mountain. SDS-Page dis-played one band, and HPLC showed one single chromatographic peak. The haemocoagulase acted only on α chain of fibrinogen. Its molecular weight was 32. 2kD with isoelectric point of 5. 21. The enzyme had clotting activity in vitro. Conclusion:The method can be used for the separation and purification of haemocoagulase from agkistrodon blomhoffii in Changbai Mountain.

OBJECTIVE:To establish a method for the determination of related substances in Methylphenidate hydrochloride for injection. METHODS:HPLC method was adopted. The determination was performed on Waters symmetry C18 column with mo-bile phase consisted of methanol-0.01 mol/L potassium dihydrogen phosphate solution(60:40,V/V)at the flow rate of 1.0 mL/min. The detection wavelength was set at 210 nm,the column temperature was 35 ℃ and sample size was 10 μL. RESULTS:The linear range of impurity A and B were 0.02-3.0 μg/mL(r=0.9998). The limits of quantitation were 0.2,0.6 ng,and the limits of detec-tion were 0.06,0.2 ng,respectively. RSDs of precision,stability and reproducibility were all lower than 2.0%;recoveries were 98.2%-100.0%(RSD=0.56%,n=9),98.0%-100.3%(RSD=0.70%,n=9),respectively. CONCLUSIONS:The method is sim-ple,accurate and suitable for the determination of related substance in Methylphenidate hydrochloride for injection.

Objective:To establish a method for the determination of content and related substances of troxipite tablets by HPLC. Methods:A Waters Symmetry-C18 (150 mm × 4. 6 mm,5 μm) column was used. The mobile phase was methanol -0. 4% phosphoric acid solution (50∶50). The flow rate was 1. 0 ml·min-1. The detection wavelength was 260nm. The column temperature was 35℃and the injection volume was 20 μl. Results:The linear range of troxipite was 3-75 μg·ml-1(r=0.999 7). The average recovery was 99. 7%,RSD=0. 95%(n=9). Conclusion:The method is simple, accurate and specific, and can be used in the quality control of troxipite tablets.

Objective To investigate the effect of isolated carotid sinus perfusion with propofol on plasma endothelin and nitric oxide levels in diabetic rats.Methods Healthy adult Wistar rats weighing 180-220 g were used in this study.Diabetes mellitus was induced by intraperitoneal streptozotocin 30 mg/kg and confirmed by blood glucose ≥ 16.7 mmol/L.Thirty-six rats in which diabetes mellitus model was successfully established were randomly divided into 3 groups ( n =12):control group (group DC) ;low dose propofol group (group DP1 ) and high dose propofol group (group DP2 ).Another 36 non-diabetic rats were also randomly divided into 3 group ( n =12 each):control group (group NC) ; low dose propofol group group (group NP1 ) and high dose propofol (group NP2 ).After 30 min isolated carotic sinus perfusion with K-H solution (control groups),50 μmol/L propofol in K-H solution (low dose propofol groups) or 100 μmol/L propofol in K-H solution (high dose propofol groups),blood samples were taken for determination of plasma endothelin and nitric oxide concentrations.Results The plasma endothelin concentration was lower and nitric oxide concentration higher in groups DP1 and DP2 than in group DC,and in group DP1 than in group DP2 ( P ＜ 0.05).The plasma endothelin concentration was higher and nitric oxide concentration lower in group NPi than in group DP1,and in group NP2 than in group DP2 ( P ＜ 0.05 ).Conclusion Propofol can decrease endothelin level and increase nitric oxide level in diabetic or non-diabetic rats through local effect on carotid sinus,and the effect is stronger in diabetic rats than in non-diabetic rats.

Objective:To evaluate the relationship between the mechanism of propofol inhibiting carotid sinus baroreflex (CSR) and GluR2 subunit-containing AMPA receptors in the nucleus ambiguus of rats with type 2 diabetes mellitus (T2DM).Methods:SPF healthy male Sprague-Dawley rats, aged 3 weeks, were selected and fed a high glucose and high fat diet for 6 weeks, and then streptozotocin 30 mg/kg was intraperitoneally injected to prepare a T2DM model of rats. Twenty-four T2DM rats were divided into 4 groups ( n=6 each) using a random number table method: diabetes mellitus-normal saline group (DN group), diabetes mellitus-propofol group (DP group), AMPA receptor agonist-normal saline group (AN group), and AMPA receptor agonist-propofol group (AP group). Another 12 normal rats were selected and divided into 2 groups ( n=6 each) using a random number table method: normal-normal saline group (NN group) and normal-propofol group (NP group). AMPA receptor agonist mibamitor 1 nmol/L (50 nl) was injected into the nucleus ambiguus using a micropipette at 30 min before perfusion of isolated carotid sinus in AN and AP groups. Propofol 45 mg·kg -11·h -1 was infused for 2 h via the femoral vein in NP group, DP group and AP group, and the equal volume of normal saline was given instead in the other groups. A model for perfusing isolated carotid sinus was developed at 20 min after infusion of propofol or normal saline, the intracarotid sinus pressure (ISP)-mean arterial blood pressure (MAP) curve was drawn, and CSR parameters such as maximum slope (PS), threshold pressure (TP), saturation pressure (SP), equilibrium pressure (EP), maximum decrease in MAP reflexivity (RD), and carotid sinus baroreceptor operating range (OR) were recorded. Brain tissues were taken at the end of perfusion, and the expression of GluR2 subunit in the nucleus ambiguus was detected by Western blot and immunofluorescence. Results:Compared with the corresponding normal saline groups (NN group, DN group, AN group), PS and RD were significantly decreased, TP, SP and OR were increased ( P<0.05), and the ISP-MAP curve was shifted upward in propofol groups (NP group, DP group, AP group), the expression of GluR2 subunit in the nucleus ambiguus was down-regulated in NP and DP groups ( P<0.05), and no significant change was found in the expression of GluR2 subunit in the nucleus ambiguus in AP group ( P>0.05). Compared with NP group, PS and RD were significantly decreased, TP, SP and OR were increased ( P<0.05), the ISP-MAP curve was shifted upward, and the expression of GluR2 subunit in the nucleus ambiguus was down-regulated in DP group ( P<0.05). Compared with DP group, PS and RD were significantly increased, TP, SP and OR were decreased ( P<0.05), the ISP-MAP curve was shifted downward, and the expression of GluR2 subunit in the nucleus ambiguus was up-regulated in AP group ( P<0.05). Conclusions:The mechanism by which propofol inhibits CSR may be related to down-regulation of the expression of GluR2 subunits-containing AMPA receptors in the nucleus ambiguus of rats with T2DM.