Purpose To search for a purification method of Grifolan Synthase crude extract from the mycelium of Grifola frondosa and determine its molecular weight. Methods Grifolan Synthase crude extract from the mycelium of Grifola frondosa was purified by recrystallization and native-PAGE. The process is as follows: Grifolan Synthase crude extract was dissolved in buffer solution (pH7.0). 10ml Grifolan Synthase crude extract solution was mixed with ammonium sulfate slowly to the concentration of 60%, and then mixed with 3ml cooling acetone (refrigeration at -18 °C for 12 hours) slowly, after standing for 24 hours, centrifugated at 5000rpm for 10 minutes at 4°C. Material at the interface was collected and air-dried as higher purity Grifolan Synthase. The higher purity Grifolan Synthase was dispersed in the native-PAGE gel, and the active band of native-PAGE gel was cut down and broken by ultrasonic treatment for 1min, then centrifugated at 5000rpm for 10 minutes at 4°C, the supernatant was taken and mixed with ammonium sulfate slowly to the concentration of 60%, after 24 hours on standing, centrifugated at 12000rpm for 10 minutes at 4°C, the pellets was got and the purity was checked by SDS-PAGE. The molecular weight of Grifolan Synthase was determined by SDS polyacrylamide gel electrophoresis (SDS-PAGE). Results Grifolan Synthase purified by recrystallization and native-PAGE was checked to be a single band by SDS-PAGE. The data of molecular weight obtained by SDS-PAGE showed that the molecular weight of Grifolan Synthase was 55000Da. Conclusion A purification method of Grifolan Synthase crude extract from the mycelium of Grifola frondosa was researched out, and the molecular weight of grifolan synthase was studied in this paper. It can lay the foundation for the further study on the structure and function of Grifolan Synthase.

Purpose To screen out the optimal conditions of cross-linking reaction of preparing for an injectable cross-linked sodium hyaluronate gel(CHA-gel) with higher resistance to hyaluronidase and research for its viscoelasticity and anti-enzyme degradation properties. Methods The CHA hydrogels were prepared with different molecular weights of PEG as cross-linking agent, such as PEG400, PEG1000, PEG6000, PEG10000, PEG20000. The optimal preparing conditions were determined by single factor test and orthogonal experiment. The Enzyme-resistant degradation properties in vitro of CHA-gels were analysed by carbazole and spectrophotometry. Its viscoelasticity was also compared with natural HA-gel by Stabinger method. Results the results of range analysis and variance analysis show that the pH of CHA solution and the ratio of cross-linking agent to HA were significant factors. The optimal preparing conditions of the parameters are 1.5% of HA, 0.001mol/L NaOH, at 37°C, reacting 4hr and 1:15 PEG20000/HA (g/g). Under these conditions, the CHA-gel has excellent Enzyme-resistant properties, R=85.1%, an high percentage of enzyme-resistant property. Its viscoelasticity can reach 61.3×104mPa.s, three times as much as natural HA-gel. Conclusion The CHA-gel with excellent physicochemical properties can be prepared under the optimal conditions, which can set foundation for developing better mechanical and Enzyme-resistant properties products of CHA-gel.

Purpose To search for an isolation method of grifolan synthase, and research grifolan synthase partly enzymatic properties. Methods Taking Grifola frondosa as material, the mycelium was broken by ultrasonic, and then centrifugated, the supernatant was collected, GS was precipitated by different concentrations of ethanol, ammonium sulfate and acetone, the suitable isolation method of GS was researched out by taking enzyme activity and protein content as parameters. Reducing sugar, protein content and total sugar content were determined respectively by using 3,5-dinitrosalicylic acid (DNS) method, Coomassie brilliant blue method and phenol-sulfuric acid method. Taking glucose as substrate, GS activity was reflected by the consumption of glucose. Consumption of glucose was measured by DNS method. The optimal temperature and pH of GS enzyme reaction was determined by carrying out the enzyme assay at different temperatures and pH levels.The acid-base stability of GS was determined by subjecting GS to different pH levels for 60 minutes, and the heat stability of GS was determined by subjecting GS to different temperatures for 30 minutes. The direction of GS enzymatic reaction was determined by measuring the consumption of β-glucan in 1 minute. The possibility of GS existing extracellular was judged by determining GS activity in extracellular fermentation liquor. Results The proper isolation method of GS is that the mycelium was collected from fermentation liquor and broken by ultrasonic for 1min, then centrifugated at 5000rpm for 10 minutes and a supernatant was collected. Ammonium sulfate was added to the concentration of 60%, and then centrifugated at 12000rpm for 10 minutes at 4°C, the pellets was collected as GS crude enzyme. Using this method, 93.86mg of crude enzyme which enzyme activity was 5700U/mg was obtained from 100g mycelium with moisture content of 87.28%, extraction rate of crude enzyme was 0.7379%. The optimum pH of GS enzyme reaction was pH=5.0 and the optimum temperature was 15°C, GS was most stable at pH=5.0 and in the range of 30 °C to 50 °C. Just 0.6996μg β-glucan was hydrolyzed in 1 minute by GS which can actually consume 5700μg glucose per minute in the synthetic reaction of β-glucan, considering the error in actual measurement, it can be considered that GS is a one-way enzyme that it can only catalyze the synthesis of β-glucan. GS activity in extracellular Grifola frondosa fermentation liquor was -0.1875U/ml, indicating that GS is one kind of intracellular enzyme and without GS activity in fermentation liquor or extracellular. Conclusion An isolation method of grifolan synthase form the mycelium of Grifola frondosa was researched out, and grifolan synthase partly enzymatic properties were studied in this paper. It can lay the foundation for the further study on the structure and function of GS and grifolan production.

Objective In order to increase the Paeonol dissolution and content, cortex moutan were smashed into nanoparticles, and the dissolution and content were compared by microscopy before and after super-micro-particle pulverization. Methods Super-micro-particle pulverization and general grinding were used to broke Cortex moutan into particles. The microscopic morphous characteristics of the before- and after- ultra-disintegration particles were compared by microscopy. Methods of HPLC was used to determine the content and dissolution of Paeonol with different grinding conditions. Methods of precipitation and funnel way were used to examine the stability and fluidity of cortex moutan nano-particles. Results Cortex moutan powder after super-micro-particle pulverization appears sphere or like-sphere, and its average size is 200nm~300nm. After the superfine grinding Paeonol dissolution increases 76.19% in comparison with without nano pulverization. The nanoparticle rest angle is θ=33°.The precipitation ratio of Cortex moutan powder with general grinding is 0.28 at 24h, and the precipitation ratio of its nano-power has been to 0.98 at 60min. Conclusion Paeonol dissolution, stability and fluidity of Cortex moutan nanoparticles were improved greatly and this nanoparticles is beneficial to industrial production for traditional Chinese medicine.

In this study we present a method for the determination of free polyethylene glycol in a gel. We detect the PEG residues of cross-linked sodium hyaluronate gel by spectrophotometry. The pretreatment of cross-linked sodium hyaluronate gel is special and the plot of working curve is unconventional. The precision, the recovery rate with marker, the reliability and repeatability of this method are tested and verified. The results indicated that the method has an excellent linear relationship in the range of 0-40.5µg mL-1. The regression equation is Y=0.0108X+0.2047 (R2=0.9994). The standard deviation of the detection method is 1.10% (n=9). The recovery rate with marker is 97.8% and the repeatability is perfect. In conclusion, the method possesses the properties of simple operation, quickness, excellent stability. Moreover this method can be applied for the quality control of gel products cross-linked by PEG and liquid samples containing PEG.