ABSTRACT
Objective: To establish a method for the analysis of constituents of Pheretima aspergillum by UPLC-Q-TOF-MS. Methods: The analysis was performed on an Acquity UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm) by gradient elution. The mobile phase consist of 0.1% formic acid-acetonitrile and 0.1% formic acid-water at a flow rate of 0.3 mL/min. The column temperature was at 35 ℃. The information of the compounds was acquired in positive and negative mode. Results: Total 83 compounds were inferred, contains 11 free amino acids, 26 organic acids, 11 necleosides, 5 dipeptides and cyclic dipeptideds and other 21 nitrogen-containing substances, the rest on a total of 10. Conclusion: The method is accurate, rapid, and sensitive, and provide a basis for further clarifying the material basis of its efficacy and the selection of quality control indicators.
ABSTRACT
Objective: To lay a foundation for attenuating the heavy metal accumulation in Pheretima aspergillum by means of genetic engineering technology in further research, we revealed the transcriptional regulation mechanism of MT-2 gene. Methods: The coding sequence of MT-2 gene was amplified by PCR with specific primers, which were designed according to their known cDNA sequences, and the outcomes were contrastively analyzed after the sequencing process. Prior to the isolation of 5' promoter sequence by genome walking technology, three specific primers were designed based on MT-2 cDNA sequence. Meanwhile, the cis-acting elements of MT-2 gene were analyzed by Promoter Prediction online software. Results: After PCR and sequencing processes, a 2 826 bp coding sequence of MT-2 gene were obtained, four exons and four introns were found to compose the coding area by comparing with the known MT-2 cDNA sequence (accession No.KC787373.1). Besides, after genome walking and Promoter Prediction online analysing, a 1 534 bp promoter region of MT-2 was isolated, which contained not only CAAT box, TAAT box, and other core promoter elements, but also three MRE elements which specifically response to heavy metal involved in regulating the MT-2 expression. Conclusion: The expression of MT-2 gene in P. aspergillum can be induced by heavy metal, and the transcriptional level is achieved by MRE regulatory elements located in MT-2 gene promoter region.
ABSTRACT
Objective To screen out the polymerase chain reaction ( PCR) primers for specifically identifying Pheretima aspergillum (E. Perrier) , so as to establish a rapid and accurate method to identify the origin of Pheretima aspergillum. Methods Four kinds of earthworms recorded in pharmacopoeia and six kinds of their adulterants commonly seen in the market were collected. The 12SrRNA sequences related to earthworm were downloaded from GenBank database. PCR amplification for ten kinds of samples was performed using the universal primers, and then the products were sequenced. According to the differences between the above sequences, three pairs of specific primers in the non-conservative district were designed for identification of Pheretima aspergillum. Results High-specificity PCR amplification for Pheretima aspergillum with 12St/12Stf primer occurred when the annealing temperature was increased to 64℃, and only Pheretima aspergillum had single amplification strip, while no strips were found in the other adulterants under the same condition. The achieved specific primers could be well verified in 15 kinds of medicinal materials of earthworm purchased in the market, which had the same morphological features showed by the traditional identifying method. Conclusion With 12St/12Stf primer as a specific marker, the identifica tion of Pheretima aspergillum is rapid and accurate in related species of Pheretima aspergillum, and avoids the effect of some factors such as integrity of experimental materials and drying process.
ABSTRACT
Objective To establish an effective method for the determining hypoxanthine, xanthine, uridine and uracil in earthworm in Shanghai Pheretima and Pheretima aspergillum (E. Perrier), contributing to quality control of the medicinal material. Methods Hypoxanthine, xanthine, uridine and uracil were extracted from the earthworms with 0.9% NaCl by ultrasonic and determined by HPLC. The chromatographic conditions were: SORBAX SB-Aq column (250 mm×4.6 mm, 5 μm, Aglient Co.,Ltd), 5 mmol/L KH2PO4 (pH 2.9) as the mobile phase with a flow rate of 1 mL/min,the detection wavelength was 254nm, the column temperature was set at 30℃, and the injection volume was 10 μL. Hypoxanthine, xanthine, uridine and uracil were determined by HPLC at the same time. Results The linear range was 0.5-100 μg (r=0.999 9) for hypoxanthine, with the average recovery being 99.37%, RSD=1.36% (n=6). The linear range was 0.5-100 μg (r=0.993 1) for xanthine, with the average recovery being 91.57%, RSD=1.40% (n=6). The linear range was 0.5-100 μg (r=0.999 9) for uridine, with the average recovery being 95.31%, RSD=1.64% (n=6). The linear range was 0.5-100 μg (r=0.999 9) for uracil, with the average recovery being 100.21%, RSD=1.98% (n=6). Conclusion The current method is reproducible and has satisfactory recovery, and it can be used to determine hypoxanthine, xanthine, uridine and uracil in earthworm medicinal material.