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OBJECTIVE：To esta blish a method for si multaneous determination of 5 components in the branch and root of Juglans mandshurica as gallic acid ，ellagic acid ，1，6-di-O-galloyl-β-D-glucose，1，2，6-tri-O-galloyl-β-D-glucose and 1，2，3， 6-tetra-O-galloyl-β-D-glucose，and to analyze the content difference of above 5 components between the branch and root samples. METHODS：HPLC method was adopted. The determination was performed on Agilent Poroshell 120 SB-C18 column with mobile phase consisted of water （containing 0.2％ formic acid ）-acetonitrile （containing 0.2％ formic acid ）. A gradient elution was performed at a flow rate of 0.3 mL/min. The column temperature was 30 ℃ and the detection wavelength was 270 nm. The sample size was 5 μL. Independent samples t-test and partial least squares-discriminant analysis （PLS-DA）were applied for statistical analysis of 5 components. RESULTS ：The linear range of gallic acid ，ellagic acid ，1，6-di-O-galloyl-β-D-glucose，1，2， 6-tri-O-galloyl-β-D-glucose and 1，2，3，6-tetra-O-galloyl-β-D-glucose were 0.989-63.3，1.58-101，1.01-64.7，3.31-212，3.34-214 μg/mL （r≥0.997 3），respectively. RSDs of precision ，reproducibility and stability tests （12 h）were all lower than 3.2％. The average recoveries of the 5 components were 103.2％（RSD＝4.85％），99.1％（RSD＝2.80％），101.5％（RSD＝1.31％），102.9％（RSD＝ 2.73％）and 104.7％（RSD＝1.28％），respectively. The average contents of the above components in the branch of J. mandshurica were 0.296 5，0.621 1，0.562 5，3.111 7 and 3.451 3 mg/g，respectively. The average contents of above components in the root were 0.673 4，2.755 5，0.964 0，2.946 6 and 4.836 4 mg/g，respectively. The total contents of the 5 components in the branch and roo t of J. mandshurica were 8.043 2 and 12.175 9 mg/g，respectively. The contents of gallic acid ，ellagic acid and 1，6-di-O-galloyl- β-D-glucose in roots were significantly higher than those in branches （P＜0.05 or P＜0.01）. There were no significant differences in the contents of the other 2 components and the total contents of the 5 components in branches and roots （P＞0.05）. The cumulative interpretability （R 2X，R 2Y） and cumulative predictability （Q 2） of the model established by PLS-DA were 0.943，0.745，and 0.710 respectively. The model load diagram showed that the distance between the ellagic acid and the origin was the farthest ，and only variable projection importance of the content of the ellagic acid was greater than 1. CONCLUSIONS：The established method can be used for the content determination of 5 components in the branch and root of J. mandshurica . Except for 1，2，6-tri-O-galloyl-β-D-glucose，the contents of other 4 components and total contents of the 5 components in the root of J. mandshurica are higher than those of the branch. Ellagic acid is selected as the potential marker for discriminating the branch and root samples.
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OBJECTIVE:To establish a method for the determination of residuals of petroleum ether,ethanol,xylene and ace-tic acid in ecabet sodium crude drug. METHODS:Capillary GC was performed on the column of PGE-20M capillary column at the flow rate of 1.7 ml/min,detector was hydrogen flame ionization detector,carrier gas was nitrogen with high purity,column temper-ature was 45 ℃,maintaining 4 min,it increased to 80 ℃ with rate of 10 ℃/min,then increased to 135 ℃ with rate of 30 ℃/min,maintaining 3 min,the injection mode was direct injection,inlet temperature was 250 ℃,and the volume injection was 1.0 μl. RESULTS:The mass concentration was 0.050-1.952 g/L for petroleum ether,0.050-1.941 g/L for ethanol,0.024-0.948 g/L for xy-lene and 0.050-1.947g/L for acetic acid(r=0.999 1-0.999 7);RSDs of precision,stability and reproducibility tests were no more than 1.7%;recoveries were 99.3%-101.0%(RSD=0.7%,n=9),102.3%-103.7%(RSD=0.4%,n=9),101.2%-102.1%(RSD=0.3%,n=9) and 100.3%-102.2%(RSD=0.6%,n=9),respectively. CONCLUSIONS:The method is simple and accurate,and can be used for the control of residual of organic solvents in ecabet sodium crude drug.
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Clostridium cellulolyticum, as one of obligate anaerobic bacteria capable of secreting cellulosome, has not been efficiently cultured due to its strict requirement of growing conditions. In this study, culture conditions of C. cellulolyticum were optimized using response surface methodology. Plackett-Burman design was first used to screen the dominant impact factors for the growth of C. cellulolyticum, which were determined as yeast extract concentration, cellobiose concentration and culture temperature. The steepest ascent path design was then applied to gain the suitable range close to the optimal culture conditions for obtaining high cell density. The central composite design and the response surface analysis were finally used to determine the optimal levels of the influential factors, which were 3 g/L for yeast extract concentration, 7 g/L cellobiose concentration and 34 degrees C for culture temperature. The optimized medium was used for flask culture, and OD600 of C. cellulolyticum was increased from 0.303 to 0.586. With a pH-controlled fermentor at batch mode, OD600 reached 3.432, which was 2.8 times higher than elsewhere reported. These results support further study on the high-density culture of C. cellulolyticum and its application.
Subject(s)
Bacteriological Techniques , Methods , Clostridium cellulolyticum , Culture Media , Population DensityABSTRACT
The roots, barks, branches and pericarps of Juglans mandshurica were used as folk medicine in China and reputed for its treatment of several cancers, such as gastric cancer, liver cancer and leukemia. The extracts of the roots, branches, leaves and pericarps of J. mandshurica have been experimentally proved to show anti-tumor activities. Tannins, which exhibited antioxidant and anti-tumor activities, were the main constituents in J. mandshurica. In this paper, a simple spectrophotometric method was developed for the determination of total tannins in the roots, branches, leaves and pericarps of J. mandshurica collected in Dalian and Anshan of Liaoning Province. Gallic acid was used as standard compound and the content of total tannins was calculated as gallic acid equivalent. As a result of the method validation, a good linearity (r = 0.9997, n = 5) and a high recovery of gallic acid (99.02%, RSD 3.7%, n = 9) was achieved. Eight samples including four parts of J. mandshurica collected in two places were analyzed for their total tannins with the established method. In the corresponding parts of J. mandshurica, except the pericarps, the contents of total tannins showed no significant difference between samples collected in Dalian and Anshan, while the content of total tannins in different parts of J. mandshurica were significantly different. The average content of total tannins in the roots, branches, leaves and pericarps of samples collected in Dalian and Anshan was 45.66, 23.40, 58.24, 3.58 mg g(-1), respectively.