[Study on preparation method of reference substance of aristolactam â -deoxyriboside adducts].

A two-step synthetic process of bromination and cross-coupling with aristololactam Ⅰ as raw material was successfully developed. Three aristolactam Ⅰ-deoxyriboside adducts, namely AAⅠ-dA, AAⅠ-dG, and AAⅠ-dC were obtained after a sequential procedure of impurity removal and purification in four different solvents. The yield of the two-step reaction can reach 90%, and the purity of the product is more than 98%, which can meet the requirements of qualitative and quantitative analyses as traditional Chinese medicine chemical reference products. The process has been proven to have good repeatability and scalability, and it features a concise preparation procedure, efficient purification, and high yield and purity, requiring no chromatographic separation. Compared with pre-vious methods, the newly developed process has significant advantages and is suitable for the preparation of chemical reference products of aristolactam Ⅰ-deoxyriboside adducts. This process provides technical support for the preparation of reference products of aristolactam Ⅰ-deoxyriboside adducts and a solid material basis for the related toxicological research.

Applying Four-Step Characteristic Ion Filtering with HPLC-Q-Exactive MS/MS Spectrometer Approach for Rapid Compound Structures Characterization and Major Representative Components Quantification in Modified Tabusen-2 Decoction.

Modified Tabusen-2 decoction (MTBD) is traditional Chinese Mongolia medicine, mainly used to treat osteoporosis. However, the precise material basis of this prescription is not yet fully elucidated. Herein, we establish an HPLC-Q-Exactive MS/MS spectrometer method with four-step characteristic ion filtering (FSCIF) strategy to quickly and effectively identify the structural features of MTBD and determine the representative compounds content. The FSCIF strategy included database establishment, characteristic ions summarization, neutral loss fragments screening, and secondary mass spectrum fragment matching four steps. By using this strategy, a total of 143 compounds were unambiguously or tentatively annotated, including 5 compounds which were first reported in MTBD. Nineteen representative components were simultaneously quantified with the HPLC-Q-Exactive MS/MS spectrometer, and it is suitable for eight batches of MTBD. Methodology analysis showed that the assay method had good repeatability, accuracy, and stability. The method established above was successfully applied to assess the quality of MTBD extracts. Collectively, our findings enhance our molecular understanding of the MTBD formulation and will allow us to control its quality in a better way. At the same time, this study can promote the development and utilization of ethnic medicine.

[Quantification of aristolochic acids and their DNA adducts in mice kidney and liver by HPLC-MS/MS].

This study aims to establish a quantitative method of 4 aristolochic acids-DNA adducts in mice kidney and liver based on high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) for monitoring the content changes of aristolochic acids-DNA adducts. A Shiseido Capcellpak AQ C_(18) column(3 mm×100 mm, 3 µm) was used, with a mixture of 0.2% acetic acid-5 mmol·L~(-1) ammonium acetate as the aqueous phase and methanol as the organic phase for gradient elution. The multiple reaction monitoring(MRM) scanning method under positive mode by electrospray ionization(ESI) was performed for the detection of the aristolochic acids-DNA adducts which formed by combining aristolochic acid Ⅰ/Ⅱ with deoxyadenosine, deoxyguanosine, and deoxycytidine, respectively. Balb/c mice were given Guanmutong extract by gavage, and the relative content of aristolochic acids-DNA adducts in liver and kidney samples were analyzed within 60 days. It was found that the concentration of 4 aristolochic acids-DNA adducts in the kidney was significantly higher than that in the liver, and there were about 15.87 adducts in per 1×10~6 normal deoxynucleosides, which was 4.5-7.5 times than that of the liver. What's more, some adducts can still be detected on the 30 th day after administration. The concentration of the adducts in the liver was highest on the first day after administration, and a second peak appeared during the 7 th to 14 th days. The results indicated that aristolochic acids-DNA adducts are difficult to eliminate in vivo, and it is of great significance to study the mechanism of liver and kidney injury of aristolochic acid.

[Expression of vascular endothelial growth factor C in pancreatic cancer and its effect upon lymph node metastasis].

OBJECTIVE: To investigate the expression of vascular endothelial growth factor C (VEGF-C) and the effect of antisense oligonucleotide (ASODN) upon lymph node metastasis of pancreatic cancer cell. METHODS: We selected 15 cases of human pancreatic cancer and detected the expression of VEGF-C in primary tumor and lymph node metastasis tissues with immunohistochemistry. Meanwhile, the spontaneous lymphatic metastasis model in nude mice was established with orthotopic implantation for the human pancreatic cancer cell line PANC-1, isolation and culture of primary tumor and spontaneous lymphatic metastasis. The effect of VEGF-C special ASODN upon the apoptosis of pancreatic cancer cell derived from primary tumor and spontaneous lymphatic metastasis were detected by reverse transcription polymerase chain reaction (RT-PCR), enzyme linked immunosorbent assay (ELISA), flow cytometer and terminal deoxynucleotidyl transferase mediated-dUTP nick end labeling (TUNEL). RESULTS: In tissues of human pancreatic cancer, the values of VEGF-C on lymph nodes metastasis were more higher than primary tumor (P < 0.05). About the expression of VEGF-C on pancreatic cancer cell derived from spontaneous lymphatic metastasis and primary tumor in nude mice model, the mRNA levels of VEGF-C were 0.87 +/- 0.11 and 0.61 +/- 0.15 respectively, the VEGF-C levels in culture supernatants were (1682 +/- 157) pg/ml and (1404 +/- 128) pg/ml. The expression of VEGF-C on pancreatic cancer cells derived from lymphatic metastasis were also more higher than primary tumor (P < 0.05). In vitro and vivo, transfection of VEGF-C ASODN decreased the expression of VEGF-C in pancreatic cancer cell. In control group, scramble-sense oligonucleotide (SODN) group and ASODN group, the apoptosis rates of pancreatic cancer cells derived from lymph node metastasis were (2.8 +/- 1.0)%, (5.0 +/- 2.1)%, (13.2 +/- 2.2)% respectively in vitro, and were (1.8 +/- 0.5)%, (2.0 +/- 0.7)%, (4.4 +/- 1.0)% respectively in vivo, the apoptosis was increased significantly after transfection of VEGF-C ASODN (all P < 0.01). But pancreatic cancer cells derived from primary tumor were not effected (all P > 0.05). CONCLUSION: In human pancreatic cancer and nude mice model, the expression of VEGF-C on lymphatic metastasis was higher than primary tumor. The apoptosis of pancreatic cancer cells derived from spontaneous lymphatic metastasis were promoted by transfection of VEGF-C ASODN specially.