Wei Guo Wang

Determination of Free Polyethylene Glycol in Cross-Linked Sodium Hyaluronate Gel

Abstract: 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.

Preparation of Liuweidihuang Nano-Microcapsules and its Physicochemical Properties

To prepare out a novel preparation of liuweidihuang which effectively preserves Paeonol, Ursolic acid and Polysaccharides, and the preparation process should be scientific, lower-price and simple. The microscopic characteristics and its physicochemical properties of super-micro-particle of liuweidihuang from above process were also discussed. Methods According to the therapeutic and physicochemical properties of materials in liuweidihuang, the processed technology is as follow: Ripe rehmannia glutinosa,Tuckahoe and Alisma digitalis were boiled for 4.5h., the root bark of the peony tree(Paeonia suffruticosa), dogberry and yam were smashed to nanoparticles. Then select the reasonable method to drying the mixture. The microscopic shape characteristics of the before- and after- broken particles was compared. Paeonol content and dissolution were determined by HPLC. The stability and fluidity of liuweidihuang nanoparticles were examined by precipitation and funnel way. Results The approach of prepared liuweidihuang nano-microcapsules is scientific, lower-price and simple. The average size of processed super-micro-particle of liuweidihuang is 400±46nm. The hardness of the table is 9.7kg/cm2. The thickness of the table is 0.6cm. The friability of the tablet is ＜1%. Each tested item complied with standards of troches of Chinese medicine, and it is beneficial to industrial production for Chinese medicine.. Conclusion The nano-microcapsule is dispersive, desiccative, mobilizable, stable and three times concentrated than traditional pills of Liuweidihuang. It also can be used to produce different forms of drugs in the market.

The Changes of Fibrinolytic Activity of Nattokinase in Artificial Gastrointestinal Environment and Human Digestive Juice

Purpose: To search for the change of fibrinolytic activity of different forms of nattokinase when they were treated in human digestive juice, artifical gastric and intestinal juice differently, and to lay the foundation of a proper pharmaceutical dosage form of nattokinase. To provide direct evidences that Nattokinase products can be administered orally and to screen a suitable dosage form for oral administration of nattokinase.Methods: The non-crushed sample was prepared through the solid fermentation of soybean meal at 37°C for 36 h, and evaporation at 70°C for 8 h, The fine and ultra-fine powder were gotten from the non-crushed sample by the micronizer and ultra micronizer. Nattokinase extract was the supernatant liquid of 0.9% saline of non-crush samples. The nattokinase’s fibrinolytic activity of non-crushed sample, fine powder, ultra-fine powder and extract treated in artifical and human digestive were measured by the Fibrin plate method. Results: Three kind of solid form samples still remain about 80% fibrinolytic activity in artificial gastric juice for 4 h, and the fibrinolytic activity of nattokinase extract is 48.7% at the end of treatment. Treated in artificial gastric juice for 4 hours, then handled in artificial intestinal fluid for 4 h, the final fibrinolytic activity of fine and ultra fine powder is 47.0% and 51.0%. The fibrinolytic activity of extract is 79.8% at the end of treatment in human digestive juice for 4 h. At the beginning of treatment in human digestive juice the enzyme activity of fine and ultra fine powder is 130.8U/mL and 132.1U/mL, however, after treatment for 4 h the enzyme activity is 158.7U/mL and159.6U/mL, which is near to the total enzyme activity of sample. Conclusion: Nattokinase products can be administered orally, and solid preparations of nattokinase are more suitable for oral route. The optimum dosage form for oral administration of nattokinase is enteric-coated capsules containing ultra fine powder.

The Purification of Grifolan Synthase and the Determination of its Molecular Weight

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.

Preparation of Grifola frondosa Fermented Beverage without Preservatives

Purpose To prepare Grifola frondosa fermented beverage and study on its production process, ingredients and characteristics. Methods The following process was used to prepare Grifola frondosa fermented beverage: Grifola frondosa original strains →Strain activation →Inclined plane strain →Liquid spawn →Submerged fermentation →Fermentation broth with mycelium →Colloid mill grinding →High pressure homogenization →Homogenate →Add preservatives or not →Package → Sterilizing or not →Finished product. Grifola frondosa mycelium biomass was determined by Weight-Drying method. Total sugar content was measured by Phenol-sulfuric acid method. Reducing sugar content was measured by 3, 5-dinitrosalicylic acid (DNS) method. Protein content was measured by Coomassie brilliant blue method. The quality of Grifola frondosa fermented beverage was evaluated every 10 days from six areas: color, precipitation, transparency, aroma, taste and the bacteria growth situation. Results Mycelia biomass of Grifola frondosa fermentation product is 378.3 ± 7.21mg/100ml. The total sugar content is 1.8995 mg/ml, the reducing sugar content is 0.2539 mg/ml, and grifolan content is 1.6456 mg/ml, protein content 7.971μg/ml. The total number of colonies is 67 CFU/ml and no E. coli in Grifola frondosa fermented beverage, this comply with the requirements of the national food safety standard. The samples of Grifola frondosa fermented beverage without sterilized and preservatives placed at room temperature for 6 months are normal, and no bacteria growth. Conclusion The production process of Grifola frondosa fermented beverage is feasible. In the case without sterilization and preservatives, Grifola frondosa fermented beverage can be placed at room temperature for 6 months without deterioration and still maintain its characteristics of nutrient-rich, pure taste, clean and hygienic, with natural mushroom fragrant. This can provide a reference for the development of Grifola frondosa submerged fermentation products.

Preparation of a Novel Complex Antimicrobial Agent and its Antimicrobial Activity In Vitro

Purpose To develop a novel complex antimicrobial agent and determine the optimal components of the composite antimicrobial agents and its antimicrobial activity in vitro. Methods According to antimicrobial mechanisms，antibacterial spectrums，physical and chemical properties and applicabilities of existing antimicrobial agents in clinical use, select out cefoperazone sodium, sulbactam sodium and cephradine as the basic components to make a novel complex antimicrobial agent. Utilize yeast, staphylococcus aureus and E. coli bacteria as test bacteria. Do the three factors four-level orthogonal experiments by the maximum amount, the middle amount, low amount and Minimum amount of the three-component agent to research the optimum ratio of the drug. Measure the titer of the compound antimicrobial agent by the way of tube-plate method (2 doses). With known contents of Penicillin Sodium for Injection as control, and determine its minimum inhibitory concentration against staphylococcus aureus, E. coli and yeast by using the agar doubling dilution method. The experimental results were analysized by statistical analysis software SPSS16.0. Results The results of the three factors four-level orthogonal experiments indicate the optimum ratios of Cefoperazone Sodium, Sulbactam Sodium and Cephradine against E. coli, yeast and staphylococcus aureus were 2:2:3, 1:2:2 and 2:6:5, their titers were 1353.9U/mg, 982.7U/mg and 1015.5U/mg. With the highest titer proportion 2:2:3 as the composition of the antimicrobial compound. This compound antimicrobial agent had a good antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria and Fungi, its minimal inhibitory concentration (MIC) against staphylococcus aureus, E. coli and yeast were 2.000μg/ml, 0.500μg/ml and 16.000μg/ml. Conclusion This research acquires a composite of antibiotics. This antimicrobial compound has a broader spectrum and higher antimicrobial activity in vitro comparing with traditional common single antibiotics, and it especially has a good antimicrobial activity against fungi. The results set a scientific foundation for enriching clinical medicines.

Preparation of Cortex Moutan Nanoparticles and its Microscopic Characteristics and Physicochemical Properties

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.

Research on the Determination Method of Enzymatic Activity for β-Glucan Synthase (ΒGS)

Purpose To research out a suitable method for the determination of enzymatic activity for β-glucan synthase. Methods According to the methods for the determination of ΒGS in current reports, taking Grifola frondosa as material, a suitable method for the determination of enzymatic activity for ΒGS was researched out. ΒGS was isolated from the mycelium of Grifola frondosa. Taking glucose as substrate, ΒGS activity was reflected by the consumption of glucose. Consumption of glucose was measured by 3,5-dinitrosalicylic acid (DNS) method, the standard curve for glucose content measurement was plotted by determining absorbance values of different glucose concentrations at 540nm. One unit of ΒGS corresponds to the amount of enzyme which incorporated 1μg glucose into β-glucan in 1 minute. The optimum pH and temperature of the determination of enzymatic activity for ΒGS was determined respectively by carrying out the enzyme assay at different pH levels and temperatures. Results The method for the determination of enzymatic activity for ΒGS was a suitable, correct and usable method which can be popularized widely. The regression equation of the standard curve for glucose content measurement was y=0.0008x-0.0247, R2=0.9996. The optimum pH of the determination of enzymatic activity for ΒGS was pH=5.0 and the optimum temperature was 15°C. The suitable determination process for β-glucan synthase enzymatic activity is as follows: 1.0ml of enzyme was added in a test tube followed by 1.0 ml glucose (1.0mg/ml), and then the pH level was adjusted to 5.0. The test tube was incubated at 15°C for 10 minutes. Later, DNS reagent (1.5ml) was added to the test tube kept in boiling water for 5 minutes and cooled in water. A blank was prepared (2.0ml distilled water and 1.5ml DNS solution). Each solution was fixed the volume to 25ml, the color intensity was estimated at 540 nm using spectrophotometry. Conclusion A method for the determination of enzymatic activity for β-glucan synthase was researched out by taking Grifola frondosa as material, and it can be widely extended and applied by its characteristics of simple, rapid, high sensitivity and low cost.

Isolation of Grifolan Synthase (GS) and its Partly Enzymatic Properties

Abstract: 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.

Preparation of Cross-Linked Sodium Hyaluronate Gels with Different Molecular Weights of PEG and Research for its Viscoelasticity and Enzyme-Resistant Properties In Vitro

Abstract: 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.