ABSTRACT
OBJECTIVE：To establis h HPLC fingerprint of Chinese gall leaven ，simultaneous determination of 5 components and optimization of distillers ’grains. METHODS ：HPLC method was adopted. The determination was carried out on Waters Symmetry Shield TM RP18 column with mobile phase consisted of acetonitrile- 0.1％ trifluoroacetic acid aqueous （gradient elution ）at the flow rate of 0.8 mL/min. The column temperature was 30 ℃，and detection wavelength was set at 280 nm. The sample size was 5 µL. Using gallic acid as reference ，HPLC fingerprints of 14 batches of sample were drawn. The similarity evaluation was performed by using Similarity Evaluation System of Chromatogram Fingerprint of TCM （2012 edition）to determine common peaks；SPSS 20.0 software was used for cluster analysis. RESULTS ：There were 11 common peaks in 14 batches of samples ，and the similarity range of 14 batches of samples was 0.424-0.998，A1-A5 was less than 0.850；5 components were identified in B 1-B9 sample，i.e. gallic acid ，（－）-epigallocatechin，methyl gallate ，2，4，6-tri-O-galloyl-α-D-glucose and 2，4，6-tri-O-galloyl- β-D-glucose. The results of cluster analysis showed that 14 batches of samples could be grouped into 3 categories，i.e. A1-A5 into one category，B1 and B 6 into one category ，B2-B5 and B 7-B9 into one category. The linear ranges of above 5 components were 29.96-599.2 μg/mL（r＝0.999 6），0.832-416 μg/mL（r＝0.999 6），0.102-51 μg/mL（r＝0.999 8），0.286 4-143.2 μg/mL（r＝0.999 8）， 0.286 4- 143.2 μg/mL（r＝0.999 8），respectively. The limits of quantitation were 0.060，0.104，0.017，0.029，0.057 μg/mL；the limits of detection were 0.018，0.031，0.005，0.009，0.017 μg/mL，respectively. RSDs of precision ，stability，reproducibility and durability tests were all lower than 3％. The recoveries were 97.16％-101.88％（RSD＝1.60％，n＝6），96.98％-99.24％（RSD＝ 0.85％，n＝6），97.7％-101.64％（RSD＝1.54％，n＝6），97.77％-103.08％（RSD＝1.82％，n＝6），98.16％-101.88％（RSD＝1.24％， n＝6），respectively. CONCLUSIONS：The established HPLC fingerprint and cluster analysis can be used to evaluate the quality of Chinese gall leaven. The established method is simple to Δ 基金项目：国家重点研发计划项目（No.2018YFC1707200）；公 operate and can be used to determine the contents of 5 益性行业科研专项项目（No.201507004-03）；河南中医药大学研究生 components simultaneously ；rice husk distillers ’grains were 科研创新基金项目（No.YJS2018B14） ＊硕士研究生 。研究方向 ：中药饮片及新药研究 。E-mail： not suitable for fermented Chinese gall leaven . 401327039@qq.com
ABSTRACT
Objective To study and establish the HPLC fingerprint standard for the quality analysis and compare effects on the chemical composition of gallnut by ferment of Chinese gall leaven. Methods The fingerprint of Chinese gall leaven was built by Waters Symmmetry ShieldTM RP18 (250 mm × 4.6 mm, 5 μm) C18 column, and acetonitrile-0.1% trifluoroacetic acid aqueous in gradient as mobile phase, the flow rate was 0.8 mL/min, and the detecting wavelength was set at 280 nm. The chemical fingerprint similarity of 10 batches of Chinese gall leaven was calculated with the Chromap Chromafinger 2005 beta 0.1 standard substance comparison and HPLC-MS were adopted to identify the common peaks. Results The fingerprint chromatography for the 10 batches of Chinese gall leaven included 10 common peaks, with a good separation at each peak. The relative retention time for common peaks of each batch was less than 1.0%, and the similarities among 10 samples were greater than 0.90. Gallic acid (peak 1), (-)-epigallocatechin (EGC, peak 2), methyl gallate (peak 3), ethyl gallate (peak 5), epigallocatechin gallate (EGCG, peak 6), 2,4,6-tri-O-galloyl-β-D-glucose (peak 7), and 2,4,6-tri-O-galloyl-α-D-glucose (peak 9) were identified. The gallnut fermented made the content of gallic acid and 2,4,6-tri-O-galloyl-α-D-glucose increased and the contents of methyl gallate, ethyl gallate, and (-)-epigallocatechin gallate decreased. It was found that (-)-epigallocatechin and 2,4,6-tri-O-galloyl-β-D-glucose had formed in the process for the first time. Conclusion The processing mechanism of Chinese gall leaven is related to gallnut fermentation process change and create new chemical composition and fingerprint can be used to monitor the quality of fermentation processing of Chinese gall leaven.
ABSTRACT
Objective: To optimize the processing technology for Chinese gall leaven by orthogonal test. Methods: The L9(34) orthogonal test was used taking the main contents of gallic acid, ellagic acid, and in vitro antibacterial activity as indexes of evaluation. The affecting factors contains factor A, the species of zymocyte, such as Rhizopus koji, Angel yeast, and Aspergillus niger, factor B, the species of tea, such as green tea, black tea, and puer tea, factor C, the material ratios of quantity of raw material, yeast, and tea, such as 25:7.5:2.5, 25:6.25:1.1, and 25:2.5:1.9. Multi-index comprehensive weighted mark method was used in the data analysis. Results: The best processing technology was set at A1B2C1, that strain selection was R. koji, with green tea, and the material ratio was 25:7.5:2.5. Conclusion: The studies show that this processing technology is stable and feasible. The research provides the reference data for processing Chinese gall leaven.