Development and Comparison of Determination of Staphylococcus aureus in Different Processed Products of Angelicae Sinensis Radix Based on Real-time PCR Technique / 中国实验方剂学杂志

Objective:To quantitatively analyze the changes of Staphylococcus aureus in different processed products of Angelicae Sinensis Radix. Method:The real-time fluorescence quantitative polymerase chain reaction method （Real-time PCR） was established to quantitatively analyze S. aureus in Angelicae Sinensis Radix decoction pieces which bought from different producing areas， different enterprises and different storage time. The fluorescence quantitative reaction system was SYBR Premix Ex Taq Ⅱ of 10 μL， each of forward primer and reverse primer （10 μmol·L-1） of 0.8 μL， template/genome DNA of 1 μL， double distilled water of 7.4 μL. The reaction conditions of the fluorescence quantitative amplification curve were pre-denaturing for 30 s at 94 ℃， denaturing for 10 s at 94 ℃， annealing for 12 s at 60 ℃， extensing for 30 s at 72 ℃， cycling 45 times， single-point detection signal at 72 ℃. The melting curve was made from 72 ℃， and the step temperature of 0.5 ℃ was kept for 15 s to collect fluorescence. According to the results of Real-time PCR， representative samples were selected from Angelicae Sinensis Radix decoction pieces for comparison between plate counting method and Real-time PCR. Result:The content of S. aureus in different processed products was sorted by rank of raw Angelicae Sinensis Radix>soil-fried Angelicae Sinensis Radix>wine-processed Angelicae Sinensis Radix. The content of S. aureus was the lowest in the samples from Weiyuan area of Gansu province by comparing with other producing areas. Compared with the retail enterprises， the content of S. aureus in raw products and wine-processed products from production and sale enterprises was lower. Different storage time had certain effect on the content of S. aureus in raw products and wine-processed products， and the content of S. aureus increased with the increase of storage time. The detection results of plate counting method were 3-4 orders of magnitude lower than that of Real-time PCR. Conclusion:The established Real-time PCR is superior to plate counting method in specificity， sensitivity， reliability and reporting period， which can provide an effective method for rapid and accurate quantitative detection of S. aureus in different processed products of Angelicae Sinensis Radix.

Technology optimization of Gardeniae Fructus processed with ginger juice and composition changes after processing / 中国中药杂志

To optimize the technology of Gardeniae Fructus processed with ginger juice,establish fingerprints and simultaneously determine seven compounds( geniposidic acid,chlorogenic acid,genipin-1-β-D-gentiobioside,geniposide,rutin,crocin Ⅰ,and crocin Ⅱ) by using ultra high performance liquid chromatography( UPLC). Waters ACQUITY UPLC BEH C18( 2. 1 mm×50 mm,1. 7μm) column was used with acetonitrile and 0. 1% formic acid solution as mobile phase for gradient elution at the flow rate of 0. 4 m L·min-1. The data was comprehensively processed and analyzed with similarity evaluation,principal component analysis( PCA) and partial least squares discriminant analysis( PLS-DA) methods. Twenty common peaks were identified in this study,and the similarity of samples was over 0. 97. The results of PCA and PLS-DA showed that there were differences in chemical compositions and contents between the raw Gardeniae Fructus and those processed with ginger juice,with 9 potential differentiated chromatographic peaks. After being processed with ginger juice,the contents of chlorogenic acid,crocin Ⅰ and crocin Ⅱ were less than before and the contents of other four compositions were higher than before. The optimized preparation for Gardeniae Fructus processed with ginger juice was stable and feasible. The methods of UPLC fingerprints and simultaneous determination of seven components can be effectively carried out to distinguish Gardeniae Fructus and Gardeniae Fructus processed with ginger juice.

Quality evaluation of multi-components simultaneous determination and fingerprint of Gardeniae Fructus from different regions / 中国中药杂志

An analysis method was established by UPLC fingerprint and then applied to simultaneous determination of multiple compounds in Gardeniae Fructus from different areas in China. Samples were separated on a Waters Acquity UPLC BEH C18( 2. 1 mm×50 mm,1. 7 μm) column with 0. 1% formic acid-water and acetonitrile solution as gradient mobile phase at a flow rate of 0. 4 m L·min-1 at various wavelengths. The similarity of samples was over 0. 95 with ″Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine( 2012 edition) ″. The UPLC common fingerprints for 32 batches were established with 19 common peaks identified. The samples were divided into 3 groups analyzed by HCA and PCA. Five components were identified as the main compositions which caused the differences of chemical constituents in the samples from different areas with partial least squares discriminant analysis( PLS-DA). The content of the total components in each area was Zhejiang > Fujian > Jiangxi > Sichuan. This method was accurate and viable,could be used to evaluate the quality of Gardeniae Fructus.

Study on quality of standard decoction of Coptidis Rhizoma based on traditional decoction process / 中国中药杂志

In order to determine the quality evaluation method for standard decoction of Coptidis Rhizoma,15 batches of standard decoction of Coptidis Rhizoma were prepared by using standardized process. Parameters such as traits,p H value,indicative component content,fingerprint similarity,composition transfer rate and dry extract rate were selected as the indexes for quality evaluation. Similarity evaluation and cluster analysis were performed for HPLC fingerprint of standard decoction,and mathematical model was used to study the correlation between dry extract rate,berberine content,berberine transfer rate in standard decoction and berberine content in decoction pieces. The results showed that the similarity of fingerprints was greater than 0. 99 for these 15 batches of standard decoctions of Coptidis Rhizoma. In cluster analysis,the standard decoctions of Coptidis Rhizoma from 4 producing areas were classified into 3 categories,consistent with the content determination results,indicating that there were quality differences among different producing areas.R2 in three linear regression mathematical models established was all greater than 0. 9,with significant difference. The validation of three batches of data showed that the models had good accuracy. Therefore,this model can be used to predict the quality of standard decoction prepared from different Coptidis Rhizoma pieces. In the standard decoction process established in this study,the integrity of the traditional process was greatly preserved,and the established quality evaluation method could be used to comprehensively examine the quality of the standard decoction,which can provide a demonstration for the related research of water extraction preparation containing Coptidis Rhizoma pieces.

UPLC fingerprint and multi-components content determination of different processed products of Angelica sinensis / 中国中药杂志

Ten batches of Angelica sinensis from three producing areas( Tuoxiang,Minxian and Weiyuan of Gansu province) were selected as the research objects,and processed into raw A. sinensis,A. sinensis with alcohol,and A. sinensis with soil respectively through the standard processing methods. Ultra-high performance liquid chromatography( UPLC) was used to establish fingerprint for three processed products of A. sinensis,and determine the contents of 9 phenolic acids and phthalide compounds. The similarity was analyzed with Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine,which showed that the chromatographic peaks of the same processed samples of A. sinensis were basically similar,with all similarities greater than 0. 950. The difference between different processed products and their control spectra was not obvious,with all similarities also higher than 0. 950.On the basis of using principal component analysis( PCA) and OPLS-DA to seek the difference components between groups,the improved distance coefficient method can be used to effectively distinguish the three processed products of A. sinensis by fingerprint similarity. At the same time,the determination method of nine phenolic acids and phthalide in A. sinensis was established by UPLC,and the comparison between different processed products was carried out. The results showed that the content of various components was changed as compared with the raw A. sinensis. The contents of coniferyl ferulate and ligustilide in the A. sinensis with alcohol were increased significantly,and the content of coniferyl ferulate was obviously increased in A. sinensis with soil. The method established in this paper can effectively distinguish different processed products of A. sinensis and determine the content of the main components in them.