组会汇报

1. 组会汇报 汇报人：翟丽娟 指导教师：薛照辉副教授

2. “Identification of the absorbed components and metabolites in rat plasma after oral administration of Rhizoma Chuanxiong decoction by HPLC-ESI-MS/MS” From: Journal of Pharmaceutical and Biomedical Analysis Authors: Aihua Zuo, Li Wang, Hongbin Xiao, Limin Li

3. Contents Introduction Materials and Method Results and discussion Conclusions

4. Introduction • Rhizoma Chuanxiongis widely prescribed for curing cardiovascular diseases for centuries • Several types of components including alkaloid, phenolic acid and phthalides have been isolated and identified during the past decades. • Among them, tetramethylpyrazine(TMP), ferulic acid and various phthalides were reported to be the bioactive components. • Up to now, studies of Chuanxiong mainly focus on the absorption, distribution, metabolism and excretion of a few isolated bioactive compounds,suchas TMP, ferulic acid, ligustilide, senkyunolide A

5. Introduction ADME Absorbed into plasma Excreted from the organism Distributed to tissues Metabolism

6. Introduction Reactions dehydrogenation oxidation hydroxylation conjugation • One or a few bioactive components used for in vivo studies are not sufficient to represent and reflect the overall efficacy of Chuanxiong. • It was reported that ferulicacid exhibited distinctly different absorption after oral administration of pure ferulic acid and Chuanxiong extract at the equal dose. • Similarly, the clearance and metabolism of ligustilide after oral administration of Chuanxiong extract was significantly different from that dosed in its pure form

7. Materials and method 2.1 Preparation of Rhizoma Chuanxiong decoction 240g dry powder Immersed in 1.8L of 70% ethanol overnight The mixture Extracted by reflux for 2h at 80℃ After filtered Extracted with 1.6L of 70% ethanol for 1h Supernatants Evaporated to dryness at 60℃ Dried residue dissolved in water Obtain an oral solution

8. Materials and method 2.2 Instrument and conditions HPLC-UV analysis • Equipped with diode-array detecter • ODS-3 column, temperature 35℃ • A: acetonitrile B: 0.1% acetic acid • A gradient program: • 5-10% A from 0—5 min • 10-70% A from 5—45 min • 75-100% A from 45—60 min • From 210nm to 400nm

9. Materials and method 2.2 Instrument and conditions MS experiments • A TSQ triple-quadrupole mass spectrometer • Full scan over mass range of m/z 100-1000 • Positive and negative ionization mode

10. Materials and method 2.3 In vivo study • Eight SD rats (200-300g) • Administered orally to the rats of group A at a dose of 10 mL RCD/kg body weight. Equal dose of distilled water was orally administered to the rats of group B. • The rats were continuously administered twice a day. • Sixty minutes after the fifth drug administration, the animals were sacrificed by decapitation. Group B: N=3, control group for blank rat plasma Group A: N=5, drug group for dosed rat plasma

11. Materials and method 2.4 Sample preparation Extracted Vortexing Evaporated Dissolved 0.25g plasma samples were added into a 10mL polypropylene test tube and extracted with 4mL methanol–ethyl acetate (1:1, v/v)for three times Extraction was performed by vortexing for 20s, ultrasonicating for 10min and centrifuging at 12,000 rpm for 5min. The supernatants were combined and evaporated to dryness under a stream of nitrogen at 40◦C Then the dried residue was dissolved in 0.5mL methanol–water(1:1,v/v) and centrifuged at 12,000 rpm for 10 min

12. Results and discussion

13. Results and discussion • Alkaloid—1 • Phenolic—6 • Alkyl phthalides—8 • Hydroxylatedphthalides—15 • Dimericphthalides--10

14. Results and discussion

15. Results and discussion

16. Results and discussion

17. Results and discussion

18. Results and discussion

19. Results and discussion 441[M+H−glycine]+ 284[M+H−GSH]+ 387[M+H−glutamic acid]+ 439—glycine 385—glutamic acid 282--C5H12N2O6 207--GSH

20. Results and discussion

21. Conclusions • 13 compounds • were absorbed into rat plasma in prototype and simultaneously • identified by comparing their MS, MS/MS, UV spectra and retention behaviors obtained • from rat plasma with that obtained from RCD. • The in vivo metabolic pathways of chemical constituents in rat plasma were predicatively proposed. • By scanning • all possible • metabolites • in EICs mode, 12conjugated metabolites were identified

22. Study insight • More thinking on the design of experiments, for example, how to make control groups. • Enrich the knowledge of HPLC-MS • More profound understanding of the complexity of the reaction in the body, and lay the foundation for future experiments.

23. Plans • 进入了复习阶段 • 应用概率统计考试 • 最优化方法考试 • 英语考试 • 食品化学考试 • 看文献 • 整理文章 • 整理实验数据

24. Thank you