Molecular identification of Uncaria (Gouteng) through DNA barcoding.

BACKGROUND: While DNA barcoding is an important technology for the authentication of the botanical origins of Chinese medicines, the suitable markers for DNA barcoding of the genus Uncaria have not been reported yet. This study aims to determine suitable markers for DNA barcoding of the genus Uncaria (Gouteng). METHODS: Genomic DNA was extracted from the freshly dried leaves of Uncaria plants by a Bioteke's Plant Genomic DNA Extraction Kit. Five candidate DNA barcode sites (ITS2, rbcL, psbA-trnH, ITS, and matK) were amplified by PCR with established primers. The purified PCR products were bidirectionally sequenced with appropriate amplification primers in an ABI-PRISM3730 instrument. The candidate DNA barcodes of 257 accessions of Uncaria in GenBank were aligned by ClustalW. Sequence assembly and consensus sequence generation were performed with CodonCode Aligner 3.7.1. The identification efficiency of the candidate DNA barcodes was evaluated with BLAST and nearest distance methods. The interspecific divergence and intraspecific variation were assessed by the Kimura 2-Parameter model. Genetic distances were computed with Molecular Evolutionary Genetics Analysis 6.0. RESULTS: The accessions of the five candidate DNA barcodes from 11 of 12 species of Uncaria in China and four species from other countries were included in the analysis, while 54 of total accessions were submitted to GenBank. In a comparison of the interspecific genetic distances of the five candidate barcodes, psbA-trnH exhibited the highest interspecific divergence based on interspecific distance, theta prime, and minimum interspecific distance, followed by ITS2. The distribution of the interspecific distance of ITS2 and psbA-trnH was higher than the corresponding intraspecific distance. Additionally, psbA-trnH showed 95.9 % identification efficiency by both the BLAST and nearest distance methods regardless of species or genus level. ITS2 exhibited 92.2 % identification efficiency by the nearest distance method, but 87 % by the BLAST method. CONCLUSION: While psbA-trnH and ITS2 (used alone) were applicable barcodes for species authentication of Uncaria, psbA-trnH was a more suitable barcode for authentication of Uncaria macrophylla.

[Quality standard of zhuang medicine jasmini sambacis flos].

OBJECTIVE: To study the qualitative and quantitative determination of Zhuang medicine Jasmini Sambacis Flos and establish its quality control standard. METHODS: Macroscopic, microscopic and TLC identification were adopted to carry out the qualitative identification, the mensuration of inspection items and extractum were according to the Chinese Pharmacopoeia. The contents of quercetin and kaempferide in Jasmini Sambacis Flos were determined by HPLC. RESULTS: The qualitative identification methods had strong specificity. In HPLC quantitative determination, the linear range of quercetin and kaempferide was in the range of 0.4008 - 3.2064 microg and 0.0403 - 3.2256 microg, respectively. CONCLUSION: These methods are simple, accurate and reproducible, and can be used to control the quality of Jasmini Sambacis Flos effectively.

[Research on quality standards of zhuang medicine Lonicerae dasystylae flos].

OBJECTIVE: To establish quality standard of Zhuang medicine Lonicera dasystyla, and provide scientific basis for the quality control of L. dasystyla. METHOD: Characteristics of materia medica, microscopic features, TLC indentification, inspection, extractum and determination of chlorogenic acid, macranthoidin B, dipsacoside B were carried out through the experience, microscopic, physical and chemical methods, respectively. The standard of quality control was formulated thereafter. RESULT: The characteristics of materia medica, microscopic features, TLC indentification were specified, the average contents of water, total ash, acid-insoluble ash, alcohol-soluble extracts, chlorogenic acid were 11.6%, 6.6%, 0.2% , 24.4%, 1.16%, respectively, the total amount of macranthoidin B and dipsacoside B was 3.13%. Quality standard of L. dasystyla was proposed according to experimental results. CONCLUSION: The quality of L. dasystyla can be controlled effectively with the quality standard.

[The study of dynamic acccumulation of camptothecin in Camptotheca acuminata fruit by TLCS].

OBJECTIVE: To establish TLC scanning method for the determination of camptothecin in Camptotheca acuminata fruit, and analysis the dynamic accumulation of camptothecin in Camptotheca acuminata fruit to find out the best collection period. METHODS: Silica gel H-CMC-Na thin layer plate was adopted in the determination with chloroform-acetone (7 : 3) used as deeloper, Single-wavelength and linear scanning of TLC was used, and the detection wavelength was 360 nm. RESULTS: There was a good linear relationship for Comptothecin within the range of 0.0542 - 0.3252 microg, the average recovery was 97.13%, RSD was 1.76%. CONCLUSION: The method is accurate, simple and reliable, and can be used for the determination of camptothecin in Camptotheca acuminata fruit and dynamic accumulation research.

[Studies on fingerprints of ant nest of Macrotermes annadalei from different areas].

OBJECTIVE: To study comparatively the fingerprint of zhuang medicinal material-ant nest of Macrotermes annadalei from different areas in Guangxi. METHOD: The fingerprint of total amino acid of 12 lots of ant nest of M. annadalei from different areas was determined by high speed amino acid analyzer. Separation was perform on anylytical column (4.6 mm x 40 mm), column temperature at 57 degrees C. The flow rate of buffering solution was 0.4 mL x min(-1), and the flow rate of ninhydrin was 0.3 mL x min(-1). RESULT: 18 characteristic peaks in the fingerprints were identified in 12 lots of samples, the chromatographic overlap rate was 89.4%-100.0%, and the total relative peak area of 7 lots of samples was over 52% in eight main peaks. CONCLUSION: The components of amino acid of ant nest of M. armadalei from different areas in Guangxi are very similar. The qualities of majority samples are good as well. The fingerprints can provide the useful information for the quality evaluation and the identification of ant nest of M. annadalei.

[Studies on the quality standard of xingnao tinctures].

OBJECTIVE: To establish the method for quality control of Xingnao tinctures. METHOD: The Mentholum, Flos Caryophylli and Borneoum Syntheticum in this medicine were identified by TLC method. The content of Mentholum was determined by GC. Conditions of GC were: With Naphthalene as an internal standard, packed glass column, Shimadzu 7G 2.1 mm x 3.2 m, carrier gas N2, flow rate 50 mL.min-1, column temperature. 145 degrees C, ingector and detector temperature. 180 degrees C, injection way of splitless and injection volume 3 microL. RESULT: The spots on TLC plates were clear, with no interference in the blank reference. The precision and repeatability of GC were are good respectively. The average recovery was 98.4%, RSD = 0.94%. CONCLUSION: This method was fast, simple, accurate and suitable for quality control of Xingnao tinctures.