Objective To investigate the extraction technique for atractylenolide Ⅰ in Atractylodes macrocephala by supercritical CO_2 fluid extraction and develop a method used for determining the content of atractylenolide Ⅰ in the extract by HPLC. Methods The effects of seven facters, such as the extracting pressure, resolving pressure etc, to the extraction rate of atractylenolide Ⅰ in A. macrocephala by supercritical CO_2 extraction were investigated. RP-HPLC was used to determine the content of atractylenolide Ⅰ in extraction of A. macrocephala. The separation was performed on Hypersil ODS2 column with methanol-water (70∶30) as mobile phase. The flow rate was 1.0 mL/min and the wavelength of UV detector was 220 nm. Results The optimal extracting conditions: taking 10% alcohol as entraiter, the particle size of medicinal substances was 60 screen meshes, extracting pressure 25 MPa, resolving pressure 5 MPa, extracting temperature 40 ℃, resolving temperature 30 ℃, and the extracting time 4 h. Conclusion Supercritical extraction is time-shorter and efficient in extracting atractylenolide Ⅰ from A. macrocephala. It is suitable to both trial and industrialized production. The method established to determine the content of atractylenolide Ⅰ of A. macrocephala by supercritical extraction is simple, sensitive, and reliable.

AIM: To prepare the solid dispersions of angelica dahurica coumarins and measure their dissolution characteristics. METHODS: The solid dispersions were obtained by using the melted and dissolved methods with PEG6000, PVP and poloxamer188 as carriers respectively.The existing state of the coumarins in the solid dispersions were identificated by the differential scanning calorimetery. The dissolution characteristics of the solid dispersions in vitro were analyzed by HPLC method under the chromatographic conditions with ThermoC_(18)(150 mm?4.6 mm,5 ?m) as analytial column and methanol/water(70∶30)as mobile phase,using imperatorin as testing index. RESULTS: The melted and dissolved methods can be used to prepare the solid dispersions.The coumarins were completely dispersed in carrier and formed an euctics mixture with the carrier.The dissolution rates of all solid dispersion were increased obviously. CONCLUSION: The solid dispersion prepared with PVP can increase the solubility and dissolution of the coumarins.

BACKGROUND:Chitosan has good filming and viscosity, it contains free amido, and can coordinate and cross-link with gelatin, thus natural semi-interpentrating polymer network structure can be formed among molecules through hydrogen bonds.OBJECTIVE: To prepare sponge-like wound-healing dressing of good porosity, hydrophilia and air permeability by means of frozen chitosan-gelatin mixture induced phase separation.DESIGN: A comparative observation.SETTING: Laboratory of the College of Science, Guangdong Ocean University. MATERIALS: Chitosan was deacetylated by 97.55%, Mη=1.85×106; Gelatin (CP grade) was produced by Shanghai Chemical Dispensing Factory; Glacial acetic acid, NaOH, formaldehyde and glycerin were all CP grade. XL30-EDAX scanning electron microscope (Philips, Dutch); 3365-type universal material testing machine. METHODS: ① By means of frozen chitosan-gelatin mixture induced phase separation chitosan solution of 0.2, 0.4, 0.5, 0.6 and 0.8 (mass fraction) was mixed with gelatin solution, then a small amount of glycerin to prepare sponge-like wound-healing dressing of good porosity, hydrophilia and air permeability. then a small amount of glycerin, and stayed quietly to deaerate. The samples were plated with gold as routine methods, and then the surface and sectional structures were observed under the scanning electron microscope. The effects of different proportion of chitosan and gelatin on the performance parameters (water retention, moisture absorption, avulsion intensity, air permeability rate) of the sponge-like materials were observed. ② Chitosan-gelatin mixtures of 18, 20, 22, 25, 24, 26 and 28 g/L (mass fraction) were used to prepare sponge-like materials, and the effects of different contents on the performances of the materials were observed. ③ The effects of cross-linking agent (formaldehyde) of different dosages (0.002, 0.004, 0.006, 0.008, 0.010, 0.012 and 0.014 in volume fraction) on the avulsion intensity of the materials were observed.MAIN OUTCOME MEASURES: ① Surface and sectional structures of the sponge-like wound-healing dressing; ② Effects of different proportion of chitosan and gelatin on the performance parameters of the sponge-like materials; ③ Effects of different contents on the performances of the chitosan-gelatin sponge-like materials; ④ Effects of cross-linking agent of different dosages on the avulsion intensity of the materials.RESULTS: ① It could be clearly seen from the surface that the chitosan-gelatin sponge-like materials were porous structure, whereas seen from the section, the pores were honey-comb formation or three-dimensional lamellar structure accumulated by porous lamellars. ② When the content of chitosan was greater, the section looked like honey comb;Whereas as the content of chitosan decreased and gelatin content increased, the section tended to parallel lamellar structure, the water content and water retention of corresponding samples had an ascending trend, but the alvusion iintensity increased at first, and then decreased. The mass fraction of 0.5 was suitable for chitosan in the prepared solution by comprehensively analyzing the performance parameters. ③ The lower the total concentration of chitosan and gelatin, the higher the water content, the easier for the formation of bigger self-chips on the surface, the porosity of the prepared materials increased, hydrophilia and water retention ability were increased, but greater cracks formed on the material surface, and avulsion intensity was smaller. As the concentration became higher, the viscosity of the mixture became greater, the excessive viscosity was not good for mixing uniform, thus the material surface was not plain enough, and the porosity was smaller, the hydrophilia and water retention ability were relatively decreased, and the materials were harder. The total concentration should be 22-25 g/L. ④ Once the dosage of cross-linking agent was too low, very few cross-linking points generated, and the intensity was too low; Once the dosage of cross-linking agent was too high, too many cross-linking points generated, and the net space was reduced, then water content and water retention value were decreased; Whereas overdosage cross-linking agent could increase the fragility of the sponge-like materials, manifested as the decrease of avulsion intensity. When formaldehyde of 0.01 in volume fraction, the avulsion intensity was the maximal, thus the dosage of 0.01 in volume fraction was the most suitable.CONCLUSION: The main factors that affect the structures and performances of the sponge-like wound-healing dressing are the proportion of chitosan and gelatin in the mixture, total concentration of the prepared solution, amount of powder and dosage of cross-linkage agent, etc. The best matching iss chitosan of 0.05 (volume fraction)/gelatin of 0.05 (volume fraction), total mass concentration of 22-25 g/L, amount of chitosan-gelatin powder mixture is 1∶1, and the dosage of cross-linking agent is 0.01 (volume fraction).

BACKGROUND:Neural stem cel (NSC) transplantation is a common method for various ischemicencephalopathies, but inability to survive in the transplantation region limits its further use in clinical practice. OBJECTIVE:To explore the effect of vascular endothelial progenitor cel s (VEPCs) on the proliferation and apoptosis of co-cultured NSCs as wel as vascular remodeling in rats with ischemia reperfusion injury. METHODS:125 Sprague-Dawley rats were randomly divided into five groups, 25 rats in each group, including sham operation, ischemia, NSCs, co-culture, and VEPCs groups. Rat models of ischemia reperfusion injury were made in al groups except for the sham operation group, fol owed by corresponding interventions. The proliferation and apoptosis of neural stem cel s were detected, and vascular remolding in the ischemic region was observed in each group. RESULTS AND CONCLUSION:At different time points after transplantation, BrdU positive cel s were not observed in VEPCs, ischemia and sham operation groups;the number of BrdU positive cel s in the co-culture group was significantly higher than that in the NSCs group (P<0.05);BrdU+/Caspase-3+cel were observed in both co-culture and NSCs groups, and the apoptosis rate of the co-culture group was significantly lower than that in the NSCs group (P<0.05);there were new blood vessels in al the groups except for the sham operation group, and the number of new bone vessels was highest in the co-culture group. To conclude, our experimental results show that VEPCs promotes the proliferation of co-cultured NSCs, inhibits cel apoptosis and and promote angiogenesis in the ischemic penumbra of rats with ischemia reperfusion injury.

Objective To establish a UPLC method for detecting content of imperatorin and isoimperatorin in huoxiang zhengqi oral liquid. Methods ACQUITY UPLC BEH C18 (2. 1 mmí50 mm,1. 7 μm) was used and the mobile phase was methanol and water by gradient elution mode. The column temperature was 30 ℃ ,the flow rate was 0. 3 mL·min-1 and the detection wavelength was 248 nm. Results The linear range of imperatorin was 1. 305-13. 050 μg·mL-1 and the regression equation was as follow Y =13 633X+3 976 (r=0. 999 9). The linear range of isoimperatorin was 0. 596-5. 960 μg·mL-1 and the regression equation was Y=10 661X+1 073 (r=0. 999 9). The average recovery was 99. 25% (RSD=0. 74% ) and 98. 94%(RSD = 0. 63% ),respectively. Conclusion The method is accurate, rapid and reliable, and can be used to determine imperatorin and isoimperatorin in huoxiang zhengqi oral liquid.

Objective To observe therapeutic benefits of intravenously transplanted hone marrow mesenchymal stem cells (BMMSCs) on alcohol-associated dementia (AAD) rat model and study its underlying mechanisms.Methods BMMSCs were isolated by the method of differential adhesion and membrane antigens were detected with flowcytometric analysis.To establish AAD model,SD rats were intragastricly administrated with ethanol (20％,8ml/kg) for 28 days.Then BMMSCs were labeled with DAPI and injected into the blood via caudal vein.And animals were evaluated by observing Morris Maze behavior,hippocampal morphology and neuronal apoptosis.The expression of BDNF was detected by the method of immunohistochemistry.And the activities of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) in rat blood serum were also measured.Results The results of flowcytometry analysis indicated BMMSCs were CD29,CD90-positive,and CD45,CD34-negative.And the cells labeled with DAPI were observed in rat hippocampus 3 days after intravenous injection.Compared with PBS group,the escape latency of rats in BMMSC group was apparently shortened((10.17 ±0.71)s vs.(4.71 ± 0.34)s,P ＜0.01).And the morphological structure was repaired and neuronal apoptosis was reduced in rat hippocampus after BMMSC transplanting((72.67 ± 2.73) vs.(55.5 ± 5.14),P＜0.05).Immunohistochemical results showed that the expression of BDNF was significantly increased in the hippocampus of rats in BMMSC group ((71.54 ± 13.71) vs.(135.25 ± 22.20),P ＜0.05).Also,the activity of GSH-Px was apparently improved in the blood serum of rats treated with BMMSC transplanting ((526.89 ± 62.73) vs.(2592.75 ±243.73),P ＜0.01),but no change for that of T-SOD.Conclusion The results provide a novel therapeutic strategy for improving learning and memory function and reducing hippocampal damage induced by ethanol administration,which is closely related to enhance BDNF expression in the hippocampus and improve the activity of antioxidants.

BACKGROUND: Extraction of fungal DNA plays an important role in fungal genetic engineering and molecular biology research.The result of experiment is affected seriously by the efficiency of extracting DNA especially the quality of DNA. OBJECTIVE: To develop a method for extracting genomic DNA of pathogenic fungi and discuss the optimal combination of components in simple sequence repeat PCR (SSR-PCR) system. DESIGN, TIME AND SETTING: A comparative analysis of DNA extraction methods and an orthogonal experiment were conducted in the Mycology Research Lab of Department of Pathogenobiology in Norman Bethune College of Medicine, Jilin University from July 2004 to December 2006.MATERIALS: Clinical specimens were inoculated on Potato dextrose agar, Potato dextrose broth and Yeast extract peptone dextrose and cultivated under the temperature of 28 ℃ for 3-7 days, after which suspectable colonies were selected to be isolated and purified.METHODS: Three kinds of methods of extracting DNA(beading-salt fractionation method, CTAB method and Gene TLE~(TM) extraction method ) were compared in terms of their effects on DNA quality; Experiment was performed with orthogonal design to four factors (Taq DNA polymerase, template DNA, dNTP, primers) in three levels on the basis of L9 (3~4) orthogonal table, The appropriate annealing temperature and cycles were determined through PCR.MAIN OUTCOME MEASURES: The optimal reaction system determined according to the polymorphism and specificity of amplification of banding pattern.RESULTS: The objective fragments were all amplified by Gene TLE~(TM) extraction method, and the banding patterns obtained were clearer and brighter compared with the other two methods. The result of orthogonal experiment on SSR-PCR system showed that,according to the value of R, the significance of factors followed by ascending were template DNA (2.67), Taq DNA polymerase (2.00), dNTP (0.67) and primers (0.33). According to the value of ki, the optimal level of each factor combination was 30 mg/L template DNA, 1U Taq DNA polymerase, 150 μmol/L dNTP, 0.5 μmol/L primer. However, because primers were nonsignificant factors, which was presented by their small R value, we took A level of primer as 0.25 μmol/L. The best reaction condition was 55 ℃ annealing temperature and 35 cycles.CONCLUSION: The Gene TLE~(TM) method shows higher efficiency of extracting DNA and its operation is fast and simplel According to the results of orthogonat experiment, the optimal SSR-PCR system was 30 mg/L template DNA, 1U Taq DNA polymerase, 150 μmol/L dNTP and 0.25 μmol/L primer. The best reaction condition was 55℃ annealing temperature and 35 cycles.

OBJECTIVE:To establish a method for the simultaneous determination of acetic acid and succinic acid in Banxia syrup. METHODS:RP-HPLC was performed on the column of GL InterSustain-C18 with mobile phase of 0.03 mol/L ammonium di-hydrogen phosphate buffer solution(pH2.0)-methanol(gradient elution)at a flow rate of 0.8 ml/min,the detection wavelength was 210 nm,column temperature was 30 ℃,injection volume was 10 μl. RESULTS:The linear range was 0.020 98-0.209 8 μg/ml for acetic acid(0.999 9)and 12.04-120.4 μg/ml for succinic acid(r=0.999 9);limits of quantification were 0.15,18.24 ng,limits of detection were 0.045, 5.53 ng;RSDs of precision, stability and reproducibility tests were lower than 2%;recoveries were 97.45%-101.68%(RSD=1.39%,n=9) and 98.31%-101.08%(RSD=1.01%,n=9). CONCLUSIONS:The method is accurate and rapid,and suitable for the simultaneous determination of acetic acid and succinic acid in Banxia syrup.

An evaluation system including experiment preparation,experiment process,com-prehensive design of experiment,experiment skills and written exam was established in order to adapt to the experimental teaching reform in our school. Experimental preparation assessment was to evaluate‘preview and self-evaluation report’prepared and submitted by students. Experimental process assess-ment was to evaluate students' classroom performance and experiment reports. Assessment of compre-hensive design experiments was to evaluate the overall participation of students. Skill assessment was consisted of oral test and experiment operation test. Final written examination,mainly consisting of subjective questions,emphasized on student's flexible use of knowledge and ability to solve practical problems. The evaluation system of promoting student's learning and teacher's teaching through the examination not only fully arouse student's attention on experiment,but also make teachers more ob-jectively and really understand students' learning situation and the teaching effectiveness.

Background and purpose:The traditional 3 incision surgery is an important means of esophageal cancer treatment, however, accompanied by more postoperative complications and higher mortality. Minimally invasive esophagectomy is a prospective technology with advantages, such as little trauma and quick recovery. This study retrospectively analyzed the perioperative effect of the esophagus cancer patients who accepted thoracoscopic-laparoscopic esophagectomy (TLE), open-laparoscopic esophagectomy (OLE) and open esophagectomy (OE) from Jan. 2013 to Jan. 2015. Methods:In this study, 72 patients received TLE, 76 patients received OLE and 115 patients received OE, respectively. One-way ANOVA, Kruskal Wallis test and Chi-square test were used to compare the differences of general clinical data, perioperative recover index, the number of lymphadenectomy and the postoperative complication among TLE, OLE and OE. Results:There were differences in the area of operative blood loss, duration of ICU stay and first standing time among the 3 groups. Pairwise comparison demonstrated that TLE group was signiifcantly better than OE group (P<0.012 5). The total number of lymphadenectomy among the 3 groups had no differences. However, the number of lymph node of the upper esophagus in the TLE groups was more than those in OLE group and OE group (P<0.001). The laryngeal recurrent nerve injury incidence in TLE group was signiifcantly higher than those in OLE group and OE group (P=0.012, 0.003). The total surgical complication had no differences among 3 groups. In areas of the cardiorespiratory system severe complication, 3 groups had statistical differences. Pairwise comparison showed TLE group was significantly less than OLE and OE group (P<0.0125). The first day and third day incidences of SIRS rate in TLE group were less than those in OLE group and OE group (P<0.0125). Conclusion:Comparing to OE group, with the same safety and feasibility, TLE had more advantages such as trauma, quick recovery, less complications. With the same effect of total lymphadenectomy to OE, TLE had the more advantage in upper mediastinal and recurrent laryngeal nerve lymph node cleaning.