Quality Evaluation of Tremella fuciformis Polysaccharides from Different Sources / 中国实验方剂学杂志

Objective:To establish a method for the determination of polysaccharide and monosaccharide composition of Tremella fuciformis, and to analyze the difference of polysaccharide content in T. fuciformis from different sources and cultivation methods, so as to provide reference for the quality determination.Method:High performance size exclusion chromatography coupled with multi-angle laser light scattering and refractive index detection (HPSEC-MALLS-RID) was employed to determine the content and relative molecular weight distribution of T. fuciformis polysaccharides. The monosaccharide types and proportions of T. fuciformis polysaccharides were analyzed by 1-phenyl-3-methyl-5-pyrazolone (PMP) precolumn derivative high performance liquid chromatography (HPLC).Result:The weight-average relative molecular weight (Mw) and the content of polysaccharides in T. fuciformis cultivated by cut-log from different sources were distributed in 2.618×106-3.503×106 Da and 307.12-609.06 g·kg-1, respectively. These two parameters of polysaccharides in T. fuciformis with substitute cultivation from different sources were 2.723×106-3.886×106 Da and 366.38-647.37 g·kg-1, respectively. The T. fuciformis polysaccharides mainly consisted of mannose, glucuronic acid, glucose, galactose, xylose and fucose, their ratios in samples with cut-log and substitute cultivation were 4.4∶0.7∶1.0∶0.2∶1.4∶1.6 and 4.4∶0.8∶1.0∶0.1∶1.5∶1.5, respectively. The contents of the above six monosaccharides in 39 batches of T. fuciformis from different sources were mannose of 36.71-191.31 g·kg-1, glucose of 10.46-76.10 g·kg-1, galactose of 1.00-6.72 g·kg-1, xylose of 16.73-70.54 g·kg-1, glucuronic acid of 9.74-32.12 g·kg-1, fucose of 17.16-68.20 g·kg-1.Conclusion:The content of polysaccharides in T. fuciformis from different sources has a certain difference, the developed method can be used as a routine method for the quality evaluation of polysaccharides in T. fuciformis.

Determination of Molecular Weight and Content of Bletilla striata Polysaccharide by HPSEC-ELSD / 中国药房

OBJECTIVE:To set up a method for determination of molecular weight and content of polysaccharide in Bletilla striata. METHODS:HPSEC-ELSD method was adopted. The determination was performed on TSK-GEL G4000 PWXL column with mobile phase consisted of pure water at the flow rate of 0.6 mL/min.The column temperature was 30 ℃,and sample size was 20 μL. Evaporative light scattering detector was used.The molecular weight and content of polysaccharide in 3 batches of B. striata were established. RESULTS:The linear range of weight average molecular weight of B. striata polysaccharide were 24.17-178.00 kD(R2=0.985 5). The linear range of BT07 content determination were 0.508 0-5.080 mg/mL(R2=0.998 4). The limits of detection and quantitation were 0.116 5 and 0.274 0 mg/mL. RSDs of precision,stability and reproducibility tests were all lower than 2%(n=6 or n=7).Average recoveries rate were 99.16%-100.20%(RSD=0.39%-0.64%,n=9).Average retention time of 3 batches of samples was 14.28 min(RSD=4.35%,n=3),and weight average molecular weight was 23.54 kD(RSD=3.78%, n=3). Polydispersity coefficient D,MW/Mn)ranged 1.463-1.578. Average content of sample was 93.4%(RSD=4.22%,n=3). CONCLUSIONS:HPSEC-ELSD method is simple,rapid,accurate and reliable,which can be used for simultaneous determination of B.striata polysaccharide.

Conversão enzimática de triacilgliceróis em mono e diacilgliceróis de interesse industrial / Enzymatic conversion of triacylglycerols to mono and diacilglycerols of industrial interest

Mono e diacilgliceróis são produtos empregados na indústria alimentícia, farmacêutica, cosmética e química como emulsificantes e melhoradores de viscosidade de produtos alimentícios, cosméticos e farmacêuticos. No entanto, a forma mais usual de obtê-los é por síntese química, o que acaba rendendo produtos finais caros e com atributos de qualidade, rendimento e de aplicabilidade tecnológica inferiores aos esperados. A busca por formas de obtenção mais racionais, eficientes e com melhor padrão de qualidade destes produtos foi o objetivo principal do trabalho, por meio de hidrólise parcial enzimática, que necessita de condições de reação mais brandas. Foram avaliadas a hidrólise enzimática descontína, empregando como substrato a trioleína técnica, e a hidrólise enzimática descontínua-alimentada, usando como substrato o óleo de girassol médio oléico. Foi utilizada, em ambos processsos, a lipase imobilizada sn-1,3 específica Lipozyme RM IM (de Rhizomucor miehei). A caracterização dos padrões e dos substrados, bem como o acompanhamento da formação dos produtos da hidrólise enzimática foram feitos por determinação da porcentagem de hidrólise, cromatografia em camada delgada (TLC), dos perfis das curvas de fusão e cristalização por calorimetria diferencial de varredura (DSC), cromatografia gasosa (CG) e cromatografia de exclusão de tamanho de alto desempenho (HPSEC). Os parâmetros de hidrólise descontínua foram o tempo de reação, a temperatura e a concentração inicial de substrato. Os parâmetros de hidrólise descontínua-alimentada foram tempo de enchimento e intervalo de alimentação de substrato. Para as respostas analíticas de porcentagem de hidrólise e de composição de frações lipídicas foi aplicado um modelo de regressão múltipla com base em metodologia de superfície de resposta. Os resultados experimentais observados nas reações de hidrólise enzimática descontínua de trioleína técnica mostraram de 24,7 a 34,2% de mono e diacilgliceróis (para 5% de óleo na emulsão) e de 21,4 a 33,6% de mono e diacilgliceróis (para 20% de óleo na emulsão). Os resultados experimentais observados nas reações de hidrólise enzimática descontínua-alimentada de óleo de girassol médio oléico (para 15% de óleo na emulsão), mostraram de 7,9 a 31,8% de mono e diacilgliceróis. Os modelos de superfície de resposta foram considerados significativos e preditivos. As hidrólises obtidas no formato descontínuo e descontínuo-alimentado permitiram efetivamente a obtenção de frações de mono/ diacilgliceróis com vários graus de eficiência de conversão e com corretas identificação e quantificação das frações de lipídios procuradas. As correlações feitas entre porcentagem de hidrólise e entalpias de cristalização e fusão, corroboradas com os resultados qualitativos e/ou quantitativos diretos obtidos na cromatografia de camada delgada (TLC) e de HPSEC, demonstraram que estes atributos podem positivamente indicar a ocorrência efetiva de reação de hidrólise, além de auferir uma escala de desempenho de reação alinhada com o previsto na literatura, à medida que são aumentadas a temperatura, o tempo de hidrólise e a porcentagem inicial de substrato oleoso, sob regime descontínuo, e que puderam ser melhoradas, de forma inovadora, sob parâmetros de tempo total de alimentação e de intervalo de alimentação, sob regime descontínuo-alimentado. A hidrólise parcial enzimática de triacilgliceróis utilizando lipase imobilizada sn-1,3 específica pode ser considerada uma alternativa às vias químicas para a produção de misturas de mono e diacilgliceróis para utilização como aditivos químicos.

Mono and diacylglycerols are products used in the food, pharmaceutical, cosmetic and chemical industries as emulsifiers and viscosity improvers for food products, cosmetics and pharmaceuticals. However, the most usual forms of obtaining them are by chemical synthesis, which ends up yielding expensive final products with attributes of quality, yield and technological applicability lower than expected. The search for more rational, efficient and better quality standards of these products was the aim of the work, through partial enzymatic hydrolysis, which requires milder reaction conditions. Discontinuous enzymatic hydrolysis was evaluated using technical triolein as substrate and discontinuous-fed enzymatic hydrolysis using as the substrate the mid oleic sunflower oil. In both processes, immobilized lipase sn-1,3 specific Lipozyme RM IM (from Rhizomucor miehei) was used. The characterization of the patterns and substrates, as well as the monitoring of the formation of the products from the enzymatic hydrolysis were made by determining the percentage of hydrolysis, thin layer chromatography (TLC), profiles of the melting and crystallization curves by differential scanning calorimetry ( DSC), gas chromatography (GC) and high performance size exclusion chromatography (HPSEC). The parameters of discontinuous hydrolysis were the reaction time, the temperature and the initial substrate concentration. The parameters of discontinuous-fed hydrolysis were filling time and substrate feed interval. For the analytical responses of hydrolysis percentage and composition of lipid fractions a multiple regression model was applied based on response surface methodology. The experimental results observed in the reactions of discontinuous enzymatic hydrolysis of technical triolein indicated amounts of mono- and diacylglycerols from 24.7 to 34.2% (for 5% of oil in the emulsion) and from 21.4 to 33.6% for mono and diacylglycerols with 20% oil in the emulsion. The experimental results observed in the reactions of discontinuous-fed enzymatic hydrolysis of mid oleic sunflower oil (for 15% oil in the emulsion), showed from 7.9 to 31.8% of mono and diacylglycerols. Response surface models were considered significant and viii predictive. The hydrolysis obtained in the discontinuous and discontinuous-fed form allowed to obtain fractions of mono / diacylglycerols with various degrees of conversion efficiency and with correct identification and quantification of the lipid fractions sought. The correlations between the percentage of hydrolysis and enthalpies of crystallization and fusion, corroborated with the qualitative and / or quantitative direct results obtained in thin layer chromatography (TLC) and HPSEC, showed that these attributes can positively indicate the effective occurrence of reaction of Hydrolysis, in addition to achieving a reaction performance scale in line with the literature, as the temperature rate, the hydrolysis time and the initial percentage of oily substrate are increased under a discontinuous regime and can be improved, in a innovative form, under parameters of total filling time and feeding interval, under a fed-batch regime. The partial enzymatic hydrolysis of triacylglycerols using specific sn-1,3-specific immobilized lipase may be considered an alternative to the chemical pathways for the production of mono- and diacylglycerol blends for use as chemical additives.

Content Determination of the Polymer in Cefotaxime Sodium and Tazobactam Sodium(6:1)for Injection by HPSEC / 中国药房

OBJECTIVE:To establish a method for the content determination of polymer in Cefotaxime sodium and tazobactam sodium(6∶1)for injection. METHODS:High performance size exclusion chromatography(HPSEC)was performed on the column of TSK-GEL G2000SWXL with mobile phase of 0.01 mol/L phosphate buffer (pH 7.0) at a flow rate of 1.0 ml/min,detection wavelength was 254 nm,injection volume was 10 μl and the column temperature was 25 ℃. RESULTS:Cefotaxime and the poly-mers were well-separated;the linear range of cefotaxime was 2.3-226.4 ng(r=0.999 9);RSDs of precision and reproducibility tests were no more than 0.79%;results of durability test showed the changes of column temperature,flow rate,wavelength,pH and mobile phase salt concentration had little effects on the separation among different polymer peaks and between polymer peaks and main peaks. CONCLUSIONS:The method is simple,specific and sensitive,and can be used for the content determination of poly-mer in Cefotaxime sodium and tazobactam sodium(6∶1)for injuction.

Research on the determination of molecular weight distribution of anti-HBV placenta transfer factor injection / 中国生化药物杂志

Objective To establish the molecular weight distribution of anti-HBV placenta transfer factor injection (PSTF) by electrophoresis, HPLC and MS.Methods Using the methods of SDS-PAGE, HPSEC, MALDI-TOF-MS to test the molecular of PSTF.Results The Molecular was 8000 Da by SDS-PAGE.There were 5026.67,6783.44,7496.42,8736.55 Da components in PSTF by HPSEC.The main component molecular was 2972 Da and the maximum molecular component was 8194 Da.Conclusion HPSEC is simple and rapid to determine the maximum component molecular of PSTF.

Structure and molar mass analysis of pneumococcal capsular polysaccharides by 1 H NMR and HPSEC-MALLS / 中华微生物学和免疫学杂志

Objective To analyze the structures and molecular weight distributions of the capsular polysaccharides from 6 serotypes of pneumococcus .Methods The structures of pneumococcal capsular pol-ysaccharides of 6 serotypes were analyzed by 1 H nuclear magnetic resonance ( NMR) .Chemical shifts of all characteristic protons were investigated to analyze polysaccharide integrity and inter -assay consistency .High performance size exclusion chromatography-multi angle laser light scattering ( HPSEC-MALLS) was used to measure the molecular weights .Results The chemical shifts of all characteristic protons of the pneumococ-cal capsular polysaccharides of 6 serotypes were consistent with the standard chemical shift .The weight-aver-age molecular mass of the pneumococcal capsular polysaccharides ranged from 7.182×104 g/mol(for serotype 19A) to 1.273×106 g/mol(for serotype 9V)examined by HPSEC-MALLS.Conclusion The structures and molecular weight distributions of pneumococcal capsular polysaccharides could be rapidly and effectively ana -lyzed by 1 H NMR and HPSEC-MALLS.Moreover, C-PS and acetate contained in capsular polysaccharides could also be detected .HPSEC-MALLS is an applicable method for the quantitative analysis of molar mass distributions in different serotypes of pneumococcal capsular polysaccharides . Although 1 H NMR and HPSEC-MALLS have been accepted as the quality control measurements by WHO , to use them as the re-placements of the traditional QC method still needs further investigation .